Methods of Biochemical Analysis: v. 30 (Methods of Biochemical Analysis Series: No)

METHODS OF BIOCHEMICAL ANALYSIS Volume 30

Advisory Board

N.G. ANDERSON, Division of Biologkal and Medical Research, Argonne National Laboratories, Argonne, IL 60439 T . H . BUCHER, Institute of Physiologacal Chemistry, and Physical Biochemistry and Cell Biology, University of Munich, 8000 Munich 2, West Germany W.E. COHN, Oak R d g e Nationul Laboratory, T N 37830 P. DOUZOU, Institute of Physico-Chemical Biology, Edmond de Rothschild Foundation, Paris 75005, France S. GATT, Department of Biochemistry,Hebrew University-Hadassah Medical School, Jerusalem, Israel C. JOLICOEUR, Department of ChemlrtT, University of Sherbrooke, Sherbrooke, Que'bec, J l K 2R1, Canada J.H.R. KAGI, Biochemical Institute, University of Zurich, Zurich 8032, Switzerland R.W. LUMRY, Department of Chemistry, University of Minnesota, Minneapolis, M N 55455 B.G. MALMSTROM, Department of Biochemistry and Biophysics, Chalmers University of Technology, S-412 96 Gotebmg, Sweden A. MEISTER, Department of Biochemistry, Cornell Medical College, New York,N Y 10021 R.S. MELVILLE, Bureau of Medical Services, Food and Drug Administration, Retired, 111 12 Kenilworth, Garrett Park, M D 20896 M. OTTESEN, ChemicalDepartment, The Carlsberg Research Center, DK 2500 Copenhagen, Valby, Denmark J .E. SCOTT, Department of Medical Biochemistry, University of Manchester, Manchester M l 3 YPT, England E.C. SLATER, Laboratmy of BiochemistT, B.C.P. Jansen Institute University of Amsterdam, Amsterdam-C., The Netherhnds B.L. VALLEE, Centerfor Biochemical and Biophysical Sciences and Medicine, Harvard University, Boston, M A 02115 P. VENETIANER, Institute of Biochemistry, Hungarian Academy of Sciences, Szeged 6701, Hungary M. WIKSTROM, Department ofhledical Chemistv, University of Helsinki, SF 001 70 Helsinki 17, Finland K. YAGI, Imtitute of Applied Biochemistry, Y a p Memorial Park, Mitab, Gifu 505-01, Japan

METHODS OF BIOCHEMICAL ANALYSIS

Edited by DAVID GLICK Cancer Biology Research Laboratory Stanford University Medical Center Stanford, California

VOLUME 30

An Intersciences Publication JOHN WILEY & SONS NewYork

Chichester

Brisbane

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An Interscience@Publication Copyright 0 1984 by John Wiley & Sons, Inc. All rights reserved. Published simultaneously in Canada. Reproduction or translation of any part of this work beyond that permitted by Section 107 or 108 of the 1976 United States Copyright Act without the permission of the copyright owner is unlawful. Requests for permission or further information should be addressed to the Permissions Department, John Wiley & Sons, Inc. Library of Congress Catalog Card Number: 54-7232 ISBN 0-471-80276-X Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

METHODS OF BIOCHEMICAL ANALYSIS

VOLUME 30

PREFACE

Annual review volumes dealing with many different fields of science have proved their value repeatedly and are now widely used and well established. These reviews have been concerned, not only with the results in the developing fields, but also with the techniques and methods employed, and they have served to keep the ever-expanding scene within the view of the investigator, applier, the teacher, and the student. It is particularly important that review services of this nature should have included the area of methods and techniques, because it is becoming increasingly difficult to keep abreast of the manifold experimental innovations and improvements which constitute the limiting factor in many cases for the growth of the experimental sciences. Concepts and vision of creative scientists far outrun that which can actually be attained in present practice. Therefore, an emphasis on methodology and instrumentation is a fundamental need in order for material achievement to keep in sight of the advance of useful ideas. The volumes in this series are designed to try to meet the need in the field of biochemical analysis. The topics to be included are chemical, physical, microbiological, and if necessary, animal assays, as well as basic techniques and instrumentation for the determination of enzymes, vitamins, hormones, lipids, carboydrates, proteins and their products, minerals, antimetabolites, etc. Certain chapters will deal with well-established methods or techniques which have undergone sufficient improvement to merit recapitulation, reappraisal, and new recommendations. Other chapters will be concerned with essentially new approaches which bear promise of great usefulness. Relatively few subjects can be included in any single volume, but as they accumulate, these volumes should comprise a self-modernizing encyclopedia of methods of biochemical analysis. By judicious selection of topics it is planned that most subjects of current importance will receive treatment in these volumes. The general plan followed in the organization of the individual chapters is a discussion of the background and previous work, a critical evaluaV

vi

PREFACE

tion of the various approaches, and a presentation of the procedural details of the method or methods recommended by the author. T h e presentation of the experimental details is to be given in a manner that will furnish the laboratory worker with the complete information required to carry out the analysis. Within this comprehensive scheme the reader may note that the treatments vary widely with respect to taste, and point of view. It is the Editor’s policy to encourage individual expression in these presentations because it is stifling to originality and justifiably annoying to many authors to submerge themselves in a standard mold. Scientific writing need not be as dull and uniform as it too often is. In certain technical details, a consistent pattern is followed for the sake of convenience, as in the form used for reference citations and indexing. The success of the treatment of any topic will depend primarily on the experience, critical ability, and capacity to communicate of the author. Those invited to prepare the respective chapters are scientists who either have originated the methods they discuss or have had intimate personal experience with them. It is the wish of the Advisory Board and the Editor to make this series of volumes as useful as possible and to this end suggestions will be always welcome.

DAVIDGLICK

METHODS OF BIOCHEMICAL ANALYSIS

VOLUME 30

CONTENTS

The pH Jump: Probing of Macromolecules and Solutions by a Laser-Induced, Ultrashort Proton PulseTheory and Applications in Biochemistry. By Menachem Gutmun .............................................

1

Laser Photolysis in Biochemistry. By Shirley S. Chan and Robert H . Austin .....................................................

105

Density Gradient Electrophoresis of Mammalian Cells. By Abraham Tulp ......................................................... 141 Quantitation of Lipid Transfer Activity. ByJohn R. Wetterau and Donald B. Zalversmit ......................................... 199 Measurement of Oxygen Consumption by the Spectrophotometric Oxyhemoglobin Method. By Octavian Brirzu ...................................................................... 227 Historical Development and Newer Means of Temperature. Measurement in Biochemistry. By Robert L. Berger, Thomas R. Clem, Sr., Victoria A. Harden, and B. W .Mangum ....................................................... 269 Author Index

..................................................................

333

Subject Index

...................................................................

345

Cumulative Author Index, Volumes 1-30 and Supplemental Volume ...................................................... 353 Cumulative Subject Index, Volumes 1-30 and Supplemental Volume ..................................................... 965; vii

Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc. METHODS OF BIOCHEMICAL ANALYSIS

VOLUME 30

The pH Jump: Probing of Macromolecules and Solutions by a Laser-Induced, Ultrashort Proton Pulse-Theory and Applications in Biochemistry MENACHEMGUTMAN BiochemGtly, Tel Auiu University, Tel Auiu, Israel

I. Introduction .................................................................................................. 11. Methodology and Instrumentation 1. Dynamics of Proton Dissociation from Excited Molecules ............... 2. Dynamics of Protonation mpounds .................. ............................................. A. Excitation Pulse ............. B. Monitoring Light ................................. ... C. Measuring Equipment ......,............................. D. Geometry of Excit earns ......................... .,,.....,,.,,....................... 111. Kinetics of Proton Dissociation 1. Determination of the Rate of Proton Dissociation .............. 2. The Effect of pK on Rate of Dissociation ......................................... 3. The Effect of the Solvent on the Rate of Proton Dissociation ......... 4. Proton Dissociation in Concentrated Salt Solution ........................... 5. Conclusion ................................................... ....,...,....,.. IV. Detection of the Proton by Its Reaction with the Proton Emitter ....................................... 1. Reactions in a Small, Open, Hydrating Microcavity ......................... ......................................... A. Steady-State Fluorescence B. Time-Resolved Fluorescence ............................... 2. Proton Dissociation in a Poorly Hydrating Site ................................ 3. Proton Dissociation-Recombination in the Inner Space ........................ .............................. ... of a Liposome ........... s ............................................ 4. Discussion and Conch V. The Reaction of the Proton with a Molecular Proton Detector ................. 1. The Detection of Protons by Their Reaction with the Ground... State Anion of the Proton Emitter ......................

1

1 3 4 4 5 5 5 6 6 7 10 10

15 21

22 24 24 27 .33 35 38 43 45

2

MENACHEM GUTMAN

2. The Reaction of Proton with Indicator ............................................. A. Dynamics of Proton Cycle in the Absence of Direct Proton Exchange ...................................................................... B. The Effect of Initial Conditions on the Macroscopic .................... ,............... Parameters ............................... sand Its Effect on the 3. The Direct Proton Exchange between Dynamics of the Proton Cycle ........................................................... 4. Alkalinization Pulse by the Conjugate Base of the Proton Emitter 5. Limitations and Inaccuracies ............................................... A. Reactants Concentration ...................... ................................... B. Accuracy of the Macroscopic Parameters .............................. VI. Kinetics of Protonation of High-Molecular-Weight Structures .................. 1. Protonation of Uncharged Target Adsorbed on Uncharged Carrier 2. T h e Effect of Charge on Rate of Protonation .................................. 3. The Effect of Postprotonation Reaction on the Dynamics ............... A. Simulation of Protonation of Adsorbed Bromo Cresol Green B. Classification of Postprotonation Reactions ........................... VII. Proton Transfer on the Surface of Macromolecular Structure .................. VIII. The Effect of Buffer on the Dynamics of the Proton Cycle ...................... 1. Two-Component Systems: Buffer and Proton Emitter ................... 2. Three-Component System: Emitter, Detector, and Buffer ............. A. Simulative Solution ............................... B. Effect of Initial Conditions .... .,...............,.....,...............

........,...........................

46 47 51

57 62 63 63 65 66 68 69 73 75 78 84

90 91 94 94 96 96 98 99 100 100 101

I. INTRODUCTION The chemiosmotic hypothesis, alias the Mitchell theory, was accepted in biochemistry with a whole glossary of new terms: proton-motive force, proteicity, proton well, local pH, proton-driven reaction, protogenic site, proton symport, etc. All these terms were intended to describe a specific thermodynamic parameter, or chemical mechanism, with the assumption that their meaning is well defined in the Biophysical-BiochemzcacalDictionary. Although these terms are made up of familiar explicit words, the interpretation of some composite terms is only vaguely implicit. Presently, it is generally accepted that the free energy released by proton transfer between phases of different electrochemical proton potential is converted into other forms of chemical energy (ATP synthesis, active transport, redox reaction). Still, the identity of these phases is not agreed upon. The phases are identified with the whole aqueous bulk, a thin nearly

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

3

unimolecular layer on the surface of a membrane, a single proton trapped in an active site, or even an anhydrous hydrochloric acid in the dry lipid interior of a membrane. The various models describing proton transfer through a transmembranal protein consider an array of hydrophilic semirigid carriers spanning the protein, a water molecule channel, an ice-like microthread, or even approximate the proton channel by Al(0H)s crystal at 300°C. Proton transfer in biochemical systems is measured, in most cases, as an outcome of external force (ATP, redox potential, etc.) mediated by an enzyme. Enzymic turnover is a million to a billion times slower than the basic events of proton transfer. Because of this huge difference in time scale, enzyme-driven proton transfer is blurred by the noncoherent catalysis. By the time the first turnover is completed, the proton had ample time to equilibrate with the whole bulk of the solution. Because of these reasons, my colleagues and I initiated a few years ago a detailed study of proton transfer in an aqueous system, where the event is synchronized by a laser pulse. This technique, using signal averaging, retains the temporal parameters of the event and allows the evaluation of the probabilities of finding a proton in putative environments assigned for it by the different bioenergetic models. During these studies, it became apparent that proton transfer is an extremely sharp instrument for gauging the water in the immediate environment surrounding the site of dissociation. It turned out that the general biological solvent, the water, acquires different properties at the site where biochemical reaction takes place-the surface of the enzyme. These local properties of the water can be measured through the technique of the laser-induced proton pulse, free of perturbation caused by the huge mass of the bulk water. In this chapter, I shall describe the basic methodology of the laserinduced proton pulse. Starting with the initial event of a synchronous proton dissociation, going through the reaction of a proton with other solutes in a true solution, and ending with the complex multiphasic system of protons, macromolecules, and interfaces associated with the real life of biochemical reaction. In each level of complexity, I shall point out the pertinent information available for interpretation and the mode of mathematical and physical analysis. In some cases, I shall also reflect the conclusions on current hypotheses of biochemical proton transfer. 11. METHODOLOGY AND INSTRUMENTATION

The experiments described in this chapter can be carried out in any laser laboratory equipped for monitoring fast photochemical reactions.

4

MENACHEM GUTMAN

1. Dynamics of Proton Dissociation from Excited Molecules This reaction is observed through time-resolved fluorescence measurements. T h e sample is excited by a short laser pulse and the fluorescence intensity at the proper wavelength is followed with time. Thelifetimeofthemeasuredeventsvariesbetween l00psecto -20nsec. The time constant of the measured reaction limits the duration of the excitation pulse. Unless the pulse is shorter than 10%of the lifetime of the measured reaction, the observed signal must be deconvoluted to correct for the time profile of the perturbing event. T h e intensity of the excitation pulse is not critical, yet it is advisable to use low-energy density. High-energy flux enhances the probability of undesired two-photon effects. T h e light source for time-resolved fluorescence can be a nanosecond pulse of Blum-line nitrogen laser, triple harmonics of yttrium-aluminumgarnet (YAG) laser, second harmonics of mode-locked dye, or gas laser. T h e fluorescence decay can be measured with a streak camera, a very fast photomultiplier tube-like Hamamatsu 1294U attached to Tektronix transient digitizer, a box car integrator, or photon counting.

2. Dynamics of Protonation of Ground-State Compounds The reaction is followed through transient absorbance measurements. The sample is excited by intensive laser pulse (0.2-2 MW/cm2) and absorbance changes in the irradiated volume (usually 0.05-0.1 ml) are monitored by a probing light beam at the proper wavelength (see Figure 1).

Figure 1. Optical arrangement and elements needed for transient absorbance measurements. C, observation cell; PL, pulse laser; CWL, C W laser; Mr, mirror; F1, filter; MC, monochromator; PM photo multiplier; Trig, triggering photo diode; T R , transient recorder; AV, signal averager; COM, computer; X-Y, XY recorder.

THE pH JUMP PROBING OF MACROMOLECULES AND SOLUTIONS

5

A. EXCITATION PULSE

The excitation pulse should be less than 10%of the fastest time constant that is measured. A pulse of suitable duration (1- 10 nsec) and energy (0.5- 10 mJ) can be obtained by many commercially available nitrogen or excimer lasers. High-energy input into the solution (more than 10 mJ) can lead to rapid accumulation of undesired photoproducts. Thus, it is better to use a short (1-2 nsec), less intensive pulse (0.5-2 mJ) than massive (5-50 mJ) longer ones (5-20 nsec). The length of the proton cycle is 10-300 pec. Thus, even at high repetition rate (100-400 Hz) available with some gas lasers, the system will relax to its prepulse state before the next pulse. Thus, high repetition rate cannot compensate for low peak power of the laser. I found it practically impossible to use a peak power of less than 50 KW. B. MONITORING LIGHT

The monitoring light should fulfill the following requirement: Its energy-density modulation at the entrance slit of the monochromator should be higher than the energy of the fluorescence emitted from the observation cell. About 1- 10%of the MW excitation pulse is emitted as fluorescence over a wide spectral range. Even if 0.1% of the fluorescence falls at the wavelength of the monitoring beam, it amounts to 10- 100 W of light energy. To prevent it from saturating the photomultiplier, it must be damped below the energy of the oncoming signal. The simplest way to reduce the fluorescent light is to keep the monochromator far from the reaction cell, 1-3 m. The monitoring beam should probe only the irradiated volume, that is, not more than 1- 1.5 mm in diameter, and all of its energy should reach the entrance slit of the monochromator-without the assistance of a lens. A lens will focus both monitoring and fluorescent light and no advantage is gained. The fluorescence spans a wide spectral range, thus the narrower is the wavelength of the probing source, the lesser will be the incremental energy of the fluorescence. Thus the monitoring light should be highly monochromatic collimated intensive beam, that is, the output of a cw laser. C. MEASURING EQUIPMENT

The measuring equipment needed to follow transient absorbance can be as simple as a fast oscilloscope and a Polaroid camera. But a transient recorder coupled to signal averager, q recorder, and computer have a certain advantage.

6

MENACHEM GUTMAN

The measured signals are small-in most cases less than 2-3 mV deviation from the constant voltage of the photomultiplier (-50 mV at 50 R entrance impedance) which corresponds in the above case to AA = log (50 + 2)/50 = 0.017. Many events (50-5000) should be recorded to obtain a clear signal. A few thousand pulses of a megawatt photon flux can bleach even photostable compounds. To avoid this outcome, it is highly recommended to mix the content of the cell. As the irradiated volume is rather small (0.05-0.1 ml), moderate stirring can replenish the irradiated volume with fresh reactants. Under such conditions, 10,000-50,000 events can be recorded without decrease of intensity of signal. Stirring cannot remove transient photoproducts with lifetime of few nanoseconds to -300 Fsec. These photoproducts may appear in varying quantities, depending on the proton emitter, energy density, pH, impurities, etc. Most of these photoproducts, solvated electrons, stable free radicals, and triplets are formed by two-photon reaction and their yield is higher with ground-state ionized emitters (+O-) than with +OH. Thus, it is recommended to keep the pH of the solution below the pK of the proton emitter and to lower the excitation energy density as long as it does not reduce the quality of the measurement. Sometimes these precautions are insufficient. Under these conditions a two-channel signal averager can be very useful for subtracting the imprint of the transient photoproducts from the measured event. D. GEOMETRY OF EXCITATION A N D PROBING BEAMS

The irradiated volume should not be bigger than needed to obtain a good overlap with the probing light. The perpendicular-crossing beams are the geometry of choice (Figure 1), because they allow the use of a constantlength optical path and probing the space very close to the front surface of the cell, where the excitation pulse is at its maximumLThis beam geometry spreads the irradiating beam and lowers the energy density. Lasers with low output should be focused to a small spot that necessitates the colinear alignment of the probing beatn (Figure 1). In such an arrangement, care should be taken that the sample is optically thin. High absorbance of the excitation pulse will render most of the probed space inactive, with concommitant reduction of the measured signal. 111. KINETICS OF PROTON DISSOCIATION

In the classical chemistry of aqueous solutions, proton dissociation is treated as a simple first-order reaction. Actually it is a very complex

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

7

reaction involving more than one step (Huppert et al., 1982).The initial event is the charge separations:

AH,, G A;,

- - - H+

where the dissociating bond of the proton donor stretches to a distance where charge separation takes place and an ion pair is formed. The lifetime of this transient state is very short-comparable with the vibration time, for an OH bond it is about 30 fsec. Compounds with low pK will reach the A- - - . H + state with high frequency whereas those with high pK will have to go through many vibrations until the energy of stretching will overcome the threshold to form the ion pair. The A- - - H+ state is very unstable, being subjected to enormous electrostatic attraction. Thus, no dissociation will take place without the assistance of the solvent. The positively charged proton interacts with the dipole of the surrounding water molecules, and a hydration shell is formed within a time frame of 20-50 fsec (Rao and Berne, 1981), (Warshel, 1982). The stabilized proton can now diffuse away from its conjugated base, but this diffusion, still within the radius of the Coulomb cage, is subjected to electrostatic forces. The rate of the escape of the proton out of the Coulomb cage (where electrostatic force is higher than the thermal energy) is given by Equation (1) (Hauser et al., 1977; Eigen et al., 1964; Eigen, 1964.)

k,

32D

=-

72

6 1 - e-'

where XD is the sum of the diffusion coefficient of the proton and the conju ate base, T is the radius of encounter, and 6 is given by 6 = Z I Z p e lwkT. For r = 5A and residual charge of the conjugate base of - 1, -2, -3, the rate of proton escape out of the Coulomb cage will be 5 x lo", 2 X lo", 0.7 X lo", sec-l, respectively (Eigen et al., 1964). As the rates of proton hydration and escape are comparable for all acids, the big difference between kdiss (or pK) of acids stems from the probability that the dissociating bond will stretch to its A- - - - H+ state. This correlation between the pK and the rate of dissociation is described by the empirical valance bond formalism of Warshel (1982).

B

1. Determination of the Rate of Proton Dissociation

In their first electronic singlet state, hydroxy aromatic compounds are much stronger acids than in their ground state (Weller, 1961; Gutman et al., 1981; Schullman, 1977). The pK shift ApK = pK* - pKo can be

8

MENACHEM CUTMAN

estimated from the wavelength of emission of the neutral and the ionized excited states, using the Forster cycle calculation (Forster, 1950).

where u(+~)-,and u ( + ~are ~ the ) frequency of maximal emission of 40and +OH, respectively,(For compilation of pK* of many compounds, see Ireland and Wyatt, 1976). The rate of proton dissociation can be obtained, either by steady-state or time-resolved measurements. The reaction describing the proton dissociation from the excited molecule is summarized in Scheme I

+OH

k

& $0-*+ H + k- 1

Scheme I The excited molecule can decay, by irradiative ( k ( f ) ) and nonirradiative (Iqn,.))processes into its ground state with a time constant 70 = (k(f) + k(nr))-i. Alternately, it may first dissociate and then decay as an excited anion by the same processes (7'0 = (k'(f) + k'("&*). In a case where dissociation is faster than the decay of +OH*, (Itl>> k ( f ) k,,,) the emission of +OH* will decline very rapidly, due to the depletion of population by the dissociation reaction. The steady-state reflection of this rapid dissociation will be a very weak emission of the +OH* species. Slow dissociation of +OH*, or rapid recombination of the excited anion with the proton (H+-kL1>k1)will enhance the emission of +OH* (observed by steady-state fluorescence)and prolong the decay time of +OH* as observed by time-resolved measurements. The interrelation between the kinetic constants and the steady-state fluorescence of the two species is given by the following equations (Weller, 1961).

+

where is the ratio between the quantum yield measured under the experimental condition (4) and that measured under conditions where

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

9

dissociation is totally supressed (40).This ratio can be approximated by the intensities of the +H* emission measured under experimental conditions and when +OH is dissolved in strong acid or in organic solvent where no dissociation takes place. +'/+A is the ratio of $0-*emission under the experimental conditions (+') and under conditions where no recombination takes place (+A), that is, at pH>>pK*. T~ and TA are the decay lifetime of +OH* and +O-*, respectively. T~ can be approximated by the decay time of +OH* measured at pH >pK*. By dividing Equations (3) and (4), we obtain

Drawing the ratio of the quantum yields (or the relative intensities) vs. H+ can yield the rate constants, given that the lifetimes of the two states are known. Because of the simplicity of the measurement and the availability of spectrofluorimeters, this method is readily applicable. Still, the lifetime of +OH* and $0-*should be measured and verified for each experimental system. The other method for calculation of the rate of proton dissociation calls for time-resolved measurements. The differential rate equations for OH* and ~$0-*

+

can be integrated to give the time dependence of these two species.

The apparent, macroscopic time constants y1 and y2 are related with the partial rate constant

10

MENACHEM GUTMAN

According to these expressions, the intensity of the +OH*emission will decay as a biexponent, the rapid initial phase y2 represents the reaction as it proceeds until the velocity of dissociation and recombination become equal. The slower phase y1 represents the decay when the two populations (+OH* and +O-*) are in equilibrium with each other. The relative amplitudes of the two phases A r = (a2, - y1)/(y2- yl) and the macroscopic rate constants (y1,y2) allow one to calculate the rate of all partial reactions. The agreement between rate constants calculated by time-resolved measurements and steady-state kinetics is usually'good. In a limiting case, where the rate of recombination is much slower than dissociation pKo > pH >> pK*, the amplitude of the slow phase representing recombination will diminish to zero and the emission of the +OH* state will decay in a single exponent curve with a macroscopic rate constant y2 = k l + k(f,,,) = k l .

2. The Effect of pK on Rate of Dissociation The rate of proton dissociation is controlled by three parameters: the frequency of ion pair formation, the rate of stabilization of the proton by hydration, and the rate of escape out of the Coulomb cage. Measurements carried out in dilute salt solutions, that is, 10- l O O m M , will not be influenced by the two later steps. The activity of the water is invariable whereas the ionic atmosphere will screen the electrostatic attraction. Under such conditions, the rate of dissociation should be a direct function of the probability that the stretching covalent bond will reach the dissociation distance. As demonstrated in Figure 2, this expected correlation is observed over a wide range of pKs. Under these conditions, a reversible dissociation will comply with the relationship Kdiss = k l / k - l . As the recombination reaction for all acids is a diffusion-controlled reaction, we = 10'' * Kdi,,(sec-'). can approximate kl = k-1

-

3. The Effect of the Solvent on the Rate of Proton Dissociation The solvent can affect the rate of dissociation by three independent mechanisms: 1. The rate of proton hydration.

2. The rate of proton diffusion. 3. The dielectric constant of the medium. The first mechanism has already been discussed. The latter two both affect the rate at which the proton escapes out of the Coulomb cage. Diffusion of proton is mediated through rapid exchange of hydrogen bond-covalent bond through the quasistatic continuous network of hy-

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

11

10

Q A

2 8

3

* B

7 6

5

4 3 2 1

1

2

3

4

6

PK

6

7

8

0

Figure 2. Correlation between rate constant of proton dissociation and pK of acids. (M) 8-hydroxypyrene- 1,3,6-trisulfonate, excited state; (U)2-naphthol-3,6-disulfonate,excited state; (A) 2-naphthol-6-sulfonate, excited state; (V) 2-naphthol, excited state; (0)Bromo Cresol Green; (0)Bromo Cresol Purple; (V)Bromo Fymole Blue; (A) 8-Hydroxypyrene 1,3,6 trisulfonate, ground state; (8)2-naphthol 3,6 disulfonate, ground state; ( 0 )2naphthol, ground state.

drogen bonds (Belch et al., 1981). Any rupture of the network will shorten the distance over which the proton can migrate at a rate faster than the relaxation time of the network. Thus, organic solvents, may shorten the fast passage stretch, and slow the proton on its way out of the Coulomb cage. The low dielectric constant of organic solvent will expand the size of the Coulomb cage, which may also lower the probability of successful escape. T o evaluate the relative contribution of each mechanism, Huppert et al., (1981) and Huppert and Kolodney (1981) measured the rate of proton dissociation in organic solvent-water mixtures. The effect of organic solvent on proton dissociation is easily demonstrated through the emission spectrum of HPTS (Figure 3). In water, the rate of dissociation is so fast that 95% of the emission is at the wavelength

12

MENACHEM GUTMAN

450

500

nm

555

Figure 3 . Fluorescence emission spectra of B-hydroxypyrene-l,3,6-trisulfonate in water (---) and in 40% vollvol ethanol water.

of the anion (~$0~"). Increasing the mole fraction of ethanol in the mixture enhances the emission of the neutral species (@OH*)at the expense of that of @O-*,that is, the rate of dissociation is slowed to the extent that the radiative (plus nonradiative) decay of +OH* can successfully compete with the dissociation. T h e kinetic reflection of the enhanced emission of the neutral form is demonstrated in Figure 4. In pure water, @OH*emission decays rapidly (7 = 100 psec) due to dissociation (Figure 4A), but in 50% (vol/vol) of ethanol in water, it is already 25 times longer (Figure 4B) and so is the rise time of 40-* (Figure 4C). T h e dependence of the dissociation rate on the mole fraction of the organic solvent is depicted in Figure 5. As seen in Figure 5 , the rate of dissociation decreases exponentially with the mole fraction of the ethanol. This decrease in rate of dissociation cannot be attributed to the effect of the solvent on the dielectric constant of the solution. At Xethanol= 0.2, the dielectric constant of the mixture is 66.2 (vs. 77.5 of water), but the rate of dissociation is slowed by an order of magnitude. T h e proton conductivity of the water-ethanol mixture decreases with the mole fraction of the solvent, but this decrease is not steep enough to account for the measured effect on the rate of dissociation (see Figure 5). This reasoning

" O S

cn-

8 hydroxy p m n e 2.3,6 T r i sulfonate

Time (psec) (A)

HPS IN E T H A N O L WATER MIXTURE

5 0 VOL.% E T H A N O L O B S E R V E D A T 54.7"

L0

2000 T I M E ( psec) ( B)

Figure 4. Time-resolved fluorescence of 8-hydroxypyrene-1,3,6-trisulfonate in waterethanol mixture. The samples were excited by a 6-psec laser pulse (352 nm) and the emission was recorded by Hammamatsu C939 streak camera combined with optical multichannel analyzer (PAR 1205 D): (A) the emission of the undissociated state, measured in pure water at the spectral range 400-470 nm; (B) the emission of the undissociated state (400-470 nm) measured in 50% voVvol ethanol-water mixture; (C) fluorescence rise time (540 nm) of the dissociated excited form, 45% voVvol ethanol- water mixture.

13

I

HPS IN 'ETHANOl! WATER 'MIXTURi 4 5 VOL.% ETHANOL

fl

ANION FLUORESCENC RISE

J

I

2 3 TIME (nsec)

4

5

(C)

Figure 4. (Continued)

30

0

3. I MOL X E t O H

Figure 5. The dependence of the rate of proton dissociation from excited S-hydroxypyrene- 1,3,6-trisulfonate on the mole fraction of ethanol in water, and the respective proton conductivity of the mixtures. The rate of proton dissociation was measured by time resolved (0)or steady-state (D) fluorescence. The proton conductivity of the solutions (A)is normalized for pure water conductivity. Data taken from Erdey-Grutz and Lengyel (1977).

14

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

15

suggests that the main effect of the organic solvent is to delay the hydration of the proton, increasing the probability of abortive dissociation attempts. Still the present information is insufficient to state whether the slow hydration is the only mechanism that slows dissociation, or whether the expansion of the Coulomb cage and reduced prototropic mobility contribute also to the observed phenomena. 4.

Proton Dissociation in Concentrated Salt Solution

Figure 2 demonstrated the free-energy relationship for proton transfer from various donors to the same acceptor, H20. In the present section, we shall extend these studies by relating the rate to the chemical potential of the acceptor, the water. The water-alcohol mixtures described above are not convenient for measuring the role of water in the proton dissociation reaction. The large Coulomb cage (3081 for 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) (Hauser et al., 1977) and its expansion upon lowering of the dielectric constant, introduces a nontrivial contribution of the ion pair recombination to the observed reaction. Concentrated solutions of strong electrolytes are a much better system. At concentrations above 1M of strong electrolyte, the electrostatic screening shrinks the Coulomb cage to be about the molecular diameter of HPTS. This effective electrostatic screening practically eliminates the role of the Coulomb cage in the recombination. The dissociative step itself will already place the proton out of the range of the electrostatic attraction. Under these conditions, we can investigatethe primary event of proton hydration. What is more, as hydration is a femtoseconds event (Rao and Berne, 1981; Warshel, 1982), only those water molecules that are in the range of the hydration shell can stabilize the dissociating proton. Consequently such studies may be used to gauge the properties of water in microenvironments, such as the active site of an enzyme. The effect of salt on the rate of proton dissociation from excited hydroxypyrene trisulfonate is demonstrated in Figure 6. The effect on the steady-state fluorescence is similar to that shown in Figure 3. The emission of the neutral form is intensified while that of the anion decreases. Figure 7 relates the rates of proton dissociation, as measured by timeresolved fluorescence and steady-state fluorescence with the molar concentrations of LiC104 and MgC12. As in the case of organic solvents (Figure 5),the decreased proton conductivity is insufficient to account for the decrease in the rate of dissociation. The advantage of the strong electrolyte solution, with respect to organic solvent, is their adherence to Rault’s law. Thus the activity coefficient of the water can be easily obtained from the vapor pressure data (Grollman, 1928; Kracek, 1928).

I

I

I

I \ 500

1000

I

I

rF = 180 psec

1500 2000 2500 3 )OO

TIME ( p s e c )

Figure 6. Time-resolved fluorescence of the neutral form of 8-hydroxypyrene- 1,3,6-trisulfonate in concentrated LiC104 solution. Measurements were carried out as in Figure 4. Line (a) 1M LiCIO,; (b) 2.5 M LiCIO,.

Figure 7. T h e variation of the rate of proton dissociation from excited hydroxypyrene trisulfonate on the molar concentration of the salt: (0,0 ) time-resolved fluorescence measurements; (0,U) steady-state fluorescence measurements; (A)proton diffusion coefficient, normalized for pure water (data from Glietenberg et al. 1968). Open symbols, MgCI,; closed symbols, LiCIO,.

16

THE PH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

17

Figure 8 demonstrates the linear log-log correlation between the rate of proton dissociation and the activity coefficient of the water in concentrated solutions of NaCI, LiBr, and MgCI2. The same correlation has been measured for five more compounds whose kdiss values (measured in pure water) differ by five orders of magnithdes (Figure 9). T h e slope of the lines vary with the structure of the compound. Compounds which have a hydrophylic moiety (SO;) or polarizable substituent (Br) in ortho position with the dissociating proton (2-naphthol-3,6-disulfonate and Bromocresol Green) are somewhat less affected by water activity than compounds where a hydrophylic moiety is in a more remote position (2-naphthol-6-sulfonate) or no hydrophylic substitution at all @-naphthol). The realiability of this fluorometric technique for determination of u ( ~ , Ois ) demonstrated in Figures 10 and 1 1. Figure 10 relates the activity of water with the molar concentration of NaC1, as determined by the rate

1

I

u

I

I

46

I

1

0.55

acH,O)

Figure 8. The free-energy relationship between rate of proton dissociation from excited 8-hydroxypyrene-l,3,6-trisulfonate and the activity coefficient of the water. Water activity coefficient was varied by concentrated solution of NaCl (El),LiBr(A), and M g C12 (0,O).

18

MENACHEM GUTMAN

t

1

log

aH20

Figure 9. The dependence of the proton transfer rate on water activity for various excited hydroxy aromatic compounds. (0) 2-naphthol-3,6-disulfonate; (A) 2-naphthol-6,8-disulfonate;(A)8-hydroxypyrene-1,3,6-trisulfonate;(+) 2-naphthol-6-sulfonate;( 0 )2-naphthol; (m)Bromo Cresol Green (ground state). Note the discontinuity of the ordinate.

of proton dissociation from two proton emitters or as measured by the colligative properties of the solution. Figure 1 1 demonstrates the equivalence of u(H?O)as estimated for the same salt solutions by the rate of proton dissociation from two proton emitters. Thus, the kinetic method for determination of u ( H , O ) can be regarded as a reliably accurate technique. The most trivial explanation for the effect of electrolytes on rate of proton dissociation is to consider the effect of salts on the dielectric constant of the solution (see also Equation 1). In concentrated salt solutions, a considerable fraction of the water moiecules are oriented in an hydration shell around the ions; thus, their dielectric constant is smaller than in pure water (Hasted et al., 1948). A decreased dielectric constant will accelerate ion-pair recombination and slow down ion-pair separation.

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

1

2

3

NaCl (M)

4

19

5

Figure 10. The reduction of water activity by high concentration of NaCI. Water activity was measured by the rate of proton dissociation fromexcited hydroxypyrene trisulfonate (A), excited 2-naphthol-6-sulfonate (O),or using the published vapor pressure (0)(Grollrnan, 1928; Kracek, 1928).

The combination of these two effects will lower the probability of proton dissociation in accord with our observation. Yet, this explanation is not applicable for our case. An appreciable decrease of the solution’s dielectric constant occurs above 1 molar of electrolyte. At such concentration (1M) the ionic atmosphere will effectively screen the proton from the electric charge of the conjugated base at a distance of 3 A. Under such effective screening, the contribution of the 10%decrease of the dielectric constant (at 1M NaCl) will have a trivial contribution. Apparently neither electrostatic interactions nor reduced diffusibility of protons is the major cause for the decrease in the proton transfer rate. As these effects are dominating in ion-pair recombination and ion-pair separation, we have to focus our attention to the primary event in proton dissociation: ion-pair formation. In this reaction, the hydrogen of the OH bond of the excited parent molecule forms a hydrogen bond with the nearest H 2 0 molecule, which itself is hydrogen bonded to other water

20

MENACHEM GUTMAN

7

aH20

1

(Pyrene)

Figure 1 1. Correlation between water activity coefficient o f MgC1, and NaC104 solutions as estimated from the rate of proton dissociation from two proton emitters, 2-naphthol6-sulfonate (ordinate) and hydroxypyrene trisulfonate (abcissa). (0)MgCI,; (m)NaC104.

molecules nearby. If the proton moves by 0.5 A, along the lineconnecting it to the nearest H20, the OH bond breaks and H 3 0 + is formed. T h e enthalpy of proton hydration is 270 kcal/mol, whereas the enthalpy of formation of H 3 0 + is estimated to be 170 kcal/mol (Conway, 1964). T h e energy difference of 100 kcal is attributed to further solvation of H 3 0 + by additional water molecules. Within the timeframe of proton dissociation, a stable hydronium ion must be formed: otherwise, the proton will revert to the parent molecule. Molecular dynamic simulations indicate that the formation of a hydration shell around the central ion is completed within 0.05 psec (Rao and Berne, 198 1). Just because the stabilization of proton in the hydration complex is comparable with the OH vibration time (0.03 psec), any perturbation at this step might be crucial for the rest of the reaction to occur. T h e dynamics of proton hydration is too fast to be directly measured, but the thermodynamics of stepwise hydration of proton was measured as clustering of water molecules around free protons in a gas phase (Searcy and Fenn 1974; Kebarle, 1975). Clusters with varying size were observed

T H E p H JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

21

and the respective enthalpy of formation was calculated. The difference in enthalpy of hydration of a proton vs. the cluster number n,designated as -AHo,,,+ shows a remarkable decrease while increasing the cluster number n. The hydration enthalpy difference between a monomer H 3 0 + and a dimer is AHl,z = -32 kcal/mol, while AHo2,3= -22 kcal/mol. These values are comparable with the results obtained by quantum mechanical calculations (Kraemer and Diercksen, 1970;Newton and Ehrenson, 1971). The hydration enthalpy AHo,,,,+l is reaching a limiting value of about - 10 kcal/mol when the cluster number is about 10. Analogous to these results, the enthalpy of proton hydration in solution will also increase with the size of the hydration complex. Yet, in liquid water, one exception should be made: to increase the size of the complex by one water molecule, a water molecule must first be removed from the bulk, with energy investment of 10 kcal/mol (heat of evaporation of water). Therefore, the hydration complex of a proton will not exceed the state where the energy gain of further hydration will be comparable with the heat of evaporation. Using the results of Kebarle (1975) and Searcy and Fenn (1974), we estimate that the hydrating complex in dilute electrolyte solution (u(H,*) = 1) will be of 10 water molecules, or less. In concentrated salt solutions, the vapor pressure is lower than that of pure water, and hence it exhibits reduced water activity. This phenomenon is explained by the fact that a considerable fraction of the water molecules are associated with the hydration of the salt ions. The binding energy of these water molecules (which forms the first and the second hydration shells) to the center ion is larger than 10 kcal/mol; therefore, they are less likely to participate in the hydration of the newly formed proton. To observe successful proton dissociation, the thermodynamic stable complex must be formed within the ion-pair lifetime. The depletion of the solution from water molecules available for this reaction will lower the probability of the successful dissociation. As demonstrated in Figure 9, this function decreases with the activity of the water in the solution. 5. Conclusion

The rate constant of proton dissociation is extremely sensitive to its environment. Except for very strong acids (pK < 0), no dissociation will take place unless water molecules are in the immediate vicinity to act as proton acceptors. What is more, these water molecules must be free to react with the dissociating proton at a time scale comparable with the vibration time (30fsec). During such a short period, the water molecules are practically fixed in space and only those molecules that are at the

22

MENACHEM GUTMAN

distance of the first and second hydration shell will participate in the reaction. The molecular dynamic simulations (Rao and Berne, 1981) demonstrated that the total hydration process of a positive charge ion take place within a shell of less that 4 A. As long as the activity coefficient of the water is measured in homogeneous solution, the kinetics of proton dissociation is just another technique for measuring and probably not the most accurate one. On the other hand, if we can introduce the dissociable proton into a defined microspace, the kinetics of its hydration can be used as a specific method for measuring 0 at the microspace. 2 ) In the next section, we shall describe how the dissociation dynamics are applied for measuring the properties of the space bounded by an active site of a protein.

IV. DETECTION OF THE PROTON BY ITS REACTION WITH THE EXCITED ANION OF THE PROTON EMITTER Immediately after its formation, the hydrated proton reacts in a diffusion controlled reaction with any component in the solution. Reaction of the proton with stable compounds may be followed at any time scale, but the reaction with the excited anion is measurable only during the few nanoseconds of 40-* lifetime. This short observation window through which the reaction is monitored focuses our measurement only to those protons that are at a diffusion distance (within the limited time frame) from +O-*. In a homogenous solution the reaction volume, where $0-* probed for proton, is defined by a radius of r = = ( D L is the diffusion coefficient of H+ and T’ is the lifetime of +O-*). For excited anion with T ’ -6 nsec, r -70 A. If the proton emitter is placed in a microcavity, such as active site of enzyme, the resolution of the observation increases as the probed volume is smaller than the reaction volume. Consequently, the recombination reaction will be faster, reflecting the high formal concentration of protons in the site. If the microcavity is open to the bulk, some of the sites will lose their protons within the +O-* lifetime. A proton that emerges into the bulk is diluted to the bulk pH. In neutral solutions, no proton reentry will take place during the observation window. Thus, those sites that lost their proton will be clearly distinguished from the rest, they are equated with the $0-*molecules that cannot be reprotonated. The detailed analysis of the results furnishes the formalism for probing the interior of a protein (or vesicle) with hydrated protons and concludes about the physical properties of the confined space. It must be stressed that this type of measurements is a close

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

23

approximation to a proton in a box. Each cavity contains a single $0-* and an extra free proton, and within the observation time there is no exchange of matter between the “boxes.” The first reaction taking place in the microscope is the reversible dissociation of the excited state

where the brackets represent events taking place in the cavity. The exchange of the proton between the cavity and the bulk is given by a second reaction {+O-*(aq) + H+(aq))

k23

k32

{+0-*j + H+(aq)

(12)

This rather simple kinetic model is getting obscured by the fact that we cannot measure the protons but the fluorescence of +OH* and +O-*, each decaying to its ground state with a characteristic lifetime (T and T’, respectively). It should also be remembered that the lifetime is affected by radiative and nonradiative transition and the rate of the nonradiative transition may vary with the conditions prevailing in the microspace (Kosower et al., 1975 and 1978; Kosower and Dodiuk, 1978; Dodiuk et al., 1979). The combination of the chemical reactions and the decay of reactants is described in Scheme I1 as a sum of three populations N 1 , N 2 , N 3 corresponding with the three states of the proton and the emitter in the cavity.

24

MENACHEM GUTMAN

and for simplicity we shall define

These three coupled differential equations were solved by the Laplace transform method using the initial conditions Nl(,=o, = 1; N Z ( ~ ==~ ) N3(t=o) = O.* ?'he three populations can be monitored by their emission at the respective wavelength. The emission at hv2 corresponds with N2 and N3 populations and the dynamics are inherently complex. Thus, we shall rather follow the events at hul emission, where only the single population N 1 is observed. The integrated rate equation for N is given in Equation (18). Nl(t) = (k2 - Yl)e-Y't

+ (y2 - k2)e-Y2t

(18)

The function is a sum of t w o terms, one is characterized by a fast (y2)and the other by slow (y1) macroscopic rate constants. The macroscopic rate constants y1 and y2 are the roots of the Laplace polynomial and are given by Equation (19),which relates the macroscopic with the microscopic rate constants Y1.2

=

(kl

+ k 2 ) * V(k1 + k 2 Y 2

- 4 (klk2 - k 1 2 M

(19)

We shall use the expression for analyzing the results of three experimental systems, each representing a typical case of emitter in a microcavity. 1. Reactions in a Small, Open, Hydrating Microcavity A. STEADY-STATE FLUORESCENCE

8-hydroxypyrene- 1,3,6-trisulfonate is a very convenient proton emitter. Its ground state pKo = 7.7 is sufficiently high to work under conditions

'These initial conditions apply only when the ground state of the proton emittor is mostly in its protonated state, i.e., pHGpK,, - 2. If the experiment is carried out under conditions where [+OH] = [+O-],the analyst must excite +OH at a wavelength were 40- has a low extinction coefficient or alternatively modify the initial condition terms during the integration.

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

25

where more than 98% is in the +OH state, and its low pK* (0.5)ensures a very rapid dissociation (Smith et al., 1979; Gutman et al., 1981). The molecule is highly soluble in water, but due to its big hydrophobic surface (Bondi, 1964; Valvani, 1976), it may participate in strong hydrophobic interactions (Melander and Horwath, 1977; Horwath et al., 1976). Thus, in spite of the three sulfono groups, it binds with high affinity to bovine serum albumin (Gutman et al., 1982) and to the heme-binding site of apomyoglobin (Gutman et al., 1982a) (Ka = 7 X 106M-' and 9 x ~ o ~ M -respectively.) ', Figure 12 (line A) depicts the emission spectrum of hydroxypyrene trisulfonate dissolved in diluted buffer (pH 5.0). At this pH, the ground state is fully protonated (pKo = 7.7), but not so the first excited singlet state (pK* = 0.5). The excited molecules dissociate and 95% of the emission is at the wavelength of the excited anion (515 nm). The dissociation can be prevented if the compound is dissolved in acid solution, pH < pK*, such as 2MHCl (line B). Under such conditions, we observe the emission of the neutral form with maximum at 445 nm. Upon ligation to apomyoglobin, the fluorescence of hydroxypyrene trisulfonate consists of two

excitation 400 nm

.-.

450

QOH'HCI

500

nrn

550

Figure 12. Steady-state fluorescence emission of hydroxypyrene trisuIfonate. Fluorescence of 20pM hydroxypyrene trisulfonate. (A) at pH 5.0, (B) in 2M HCI, and (C) in 30@ apomyoglobin pH 5.0. Excitation at 400 nm. Fluorescence measured in arbitrary units at identical instrumental set up.

26

MENACHEM GUTMAN

bands (line C) of equal intensity, typical for the anionic state (515 nm) and the neutral form, slightly shifted to shortened wavelength (435nm). Such shifts are common in organic solvents (Kosower et al., 1975;Kosower, 1968).The total light emission of the two bands of bound hydroxoypyrene trisulfonate is 84% of the emission of the free ligand. The same effects are observed with hydroxypyrene adsorbed to bovine serum albumin (Figure 13).The emission of the neutral form increases to 30% with a distinct blue shift observed as a broad peak between 425 nm and 445 nm. T h e overall emission intensity of bound hydroxypyrene (measured as the area below the emission curves) is 64% of that of the free molecule, probably reflecting the effect of the local dielectric constant. The enhanced emission of the neutral form recorded in these two examples may originate either from rapid recombination of Hf with $0-*(see Scheme 11) or from slow dissociation of +OH* due to reduced activity of water in the cavity (see Figure 9).The contribution of each mechanism to the overall observation cannot be deduced from steadystate fluorescence measurements and necessitate kinetic analysis according to Equation (18).

7-

6S-

Figure 13. Fluorescence emission spectra of free and protein-bound hydroxypyrene trisulfonate. I . I S M hydroxypyrene trisulfonate in water (A) or 1% bovine serum albumin (B) adjusted to pH 6.0. The samples were excited at 400 nm and the emission spectra were measured under identical instrumental set up.

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

27

B. TIME-RESOLVED FLUORESCENCE

Figure 14 depicts the shape of the laser excitation pulse and the most initial emission of hydroxypyrene bound at the heme-binding site of apomyoglobin. A rapid decay of [+OH*] population is observed, but it slows down after 100 psec (about the middle of the frame). Figure 15 was recorded at a slower streak speed and resolves the phases. The emission of the neutral form decays with rapid kinetics during the first 0.3 nsec (Table I), actompanied by coevolution of the 40-* population (Figure 15). Between 0.3-1.5 nsec, the +OH* population decays while $0-*is in a steady state. Depletion of the +O-* population by radiative plus nonradiative decay is compensated by further dissociation of +OH-*. Only at t > 3 nsec, where the +OH* population is nearly exhausted, does $0-*decay with monotonic first-order kinetics (Figure 16) (7 = 5.9 nsec). The fluorescence dynamics of the same ligand bound to bovine serum albumin is presented in Figure 17. There is a rapid phase of +OH* decay with coevolution of +O-*, followed by a slower +OH* decay ( ~ =1 3.3 nsec). During the initial 1-2 nsec of the second phase the emission intensity of $0-*is practically constant, indicating that +O-* (N2 + N3 population) is replenished by further

i

32ps

c--*

Figure 14. Fluorescence decay time of hydroxypyrene trisulfonate bound to apomyoglobin: (A) 60#4 apomyoglobin, 50#4 hydroxypyrene trisulfonate in lOmM Mes buffer pH 5.0. The emission was measured (in arbitrary units) at a streak speed of 15 mmlnsec at the wavelengths 400-450 nm using BG-3 (3 mm) and GG 400 Schott glass filters. (B) The excitation laser pulse measured under identical conditions as seen by reflection from the front and back surfaces of an empty 0.5-cm cuvette.

...

I

0

1

1

.

1

2

,

3

I 4

B

1

5

6

ins)

Figure 15. Time-resolved fluorescence of neutral and anionic hydroxypyrene trisulfonate bound to apomyoglobin: (A) the emission of the neutral form as measured through BG3, 3 mm, and GG 400 glass filters; (B) the emission of the anionic form measured through a KV 550 glass filter. (A) and (B) were measured at a streak speed of 1.5 mm/nsec with 60@ apomyoglobin, 50@ hydroxypyrene trisulfonate, lOmM Mes buffer (pH 5.0).

I Figure 16. The lifetime of the excited anion of hydroxypyrene trisulfonate bound to apomyoglobin. T h e sample was excited by a nitrogen laser (337 nm, 1 nsec full width at half maximum) and the emission of the anionic form was monitored (in arbitrary units) through a KV 550 filter by a photomultiplier attached to a Tektronix 7912 AD transient digitizer equipped with a 7A19 vertical amplifier.

28

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

M

‘

29

I

Figure 17. Time-resolved fluorescence of 8-hydroxypyrene-l,3,6-trisulfonatebound to bovine serum albumin. Sample solution: 250p.M hydroxypyrene in 700p.M bovine serum albumin solution pH 5.96. (A) Emission of neutral form measured through BG-3 2-mm filter; (B) emission of anionic form measured through an orange-green 550-nm filter. The time constants of the fast decay of (A) and the rise of (B) are 350 and 290 psec, respectively.

dissociation of +OH* (NI). The decay time of [+O-*] is identical with that measured for the free emitter (Table I). Figure 18A and B are the semilogarithmic plots of [+OH*] decay, as measured for hydroxypyrene trisulfonate at the myoglobin’s heme binding site and the bovine serum albumin site, respectively. Both kinetics follow a biexponential decay: one is very fast with a time constant of less than 1 nsec; the other is slower, yet its time constant is faster than T , indicating that the +OH* population is consumed by more than simple fluorescence decay. The decay of N , is a biexponential reaction, characterized by a fast (y2) and slow (yl) time constant. The amplitude of the slow reaction is represented by the normalized value A,. These three parameters are sufficient for calculating the rate constants of the partial reactions, as described below. The value A, as derived from Equation (18) is A,. = ( k 2 - y1)/(y2 - 71). As both y1 and y2 are measured, k2 can be obtained with no difficulties. The value k2 is the sum of [ k 2 1 * H+], k23, and k’f,n,+,und). The latter is directly measurable. This is the decay constant of +O-* measured at a time where +OH* signal has completely vanished (see Figure 16 and Table I). The rate constant of proton escape from the cavity ( k 2 3 ) can be obtained for y1. This macroscopic rate constant is due to the contribution

02 I 1

I

,

I

2

' I

I

3

I

*ns

0

3

I

4

(B)

Figure 18. Kinetic analysis of the fluorescence decay of neutral hydroxypyrene trisulfonate hound to apomyoglohin (A) or bovine serum albumin (B). The results are taken from experiments carried out as detailed in Figures 15 and 17, using various streak speeds. The values of y,, y2 and AR are listed in Table 11.

30

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

31

TABLE I Macroscopic Rate Constants of 8-hydroxypyrene-1,3,6-trisulfonateFluorescence in Water and in Protein Complexes Decay time (nsec)

Fluorescenting species

Rise time (nsec)

Free +OH, pH = 5 Free +OH, 2M HCl Free $0-,pH = 5 Apomyoglobin4OH Apomyoglobin~OBSA-+OH

-

0.1 1 f 0.01 5.9 ? 0.1

0.11 2 0.01

5.9 2 0.1

BSA-+O-

-

0.52 t 0.2

-

0.29 t 0.02

Y1

0.545 t 0.05 6.1 2 0.1 0.352 t 0.01 0.252 2 0.01 5.76 t 0.1

Y2

-

-

3.2 t 0.1

-

3.3 2 0.1 3.3 -t 0.1

of the two irreversible reactions that consume the bound +OH* population. One reaction is the fluorescence decay and the other is the escape of H+ from the cavity. As long as the proton is in the cavity, +OH* can be reformed by recombination, but once it escapes, the probability of recombination within the lifetime of the excited singlet is practically nil. Insertion of Equations (16) and (17) into Equation (19) leads, in the range pK* < pH C pKo, to a solution y1 = k23 + k,,n,. As the decay times of the free hydroxypyrene in its undissociated state and in its ionized state are the same and equal to that of bound +O-* (see Table I), we can approximate kf,nl-(bund)= k’f,nr(bund). (It is very unlikely that only one species, +OH* or +O-*, will be subjected in the site to selective rapid nonradiative decay.) Using the approximation of k,,, and the calculated values of k23 and k2, the rate of recombination (k21*H+) can be obtained. Finally, using the following expression

y1 + 7 2 = k12 + k21[H+1 + k23

+ kf,m + k’f.7~

(20)

the rate of dissociation in the cavity k I 2 is obtained. The respective values for the dissociation, recombination reaction in the apomyoglobin and the bovine serum albumin are listed in Table 11. There are some similarities between these two sites: the lifetime of +O-*, the rate of proton escape (k23), and even the apparent rate of The implication of these values will be proton recombination (kZ1-[H+]). discussed below. What markedly differentiates the two sites is the rate of proton dissociation (k12). In both sites, the rate of proton dissociation is significantly slower than in water, implying that in these sites the water molecules are at a state that is not suitable for rapid (sub-picosecond) hydration of the discharged proton. The equivalent water activity coefficients, as estimated from the kinetic method described in Section 111. are

-

3 x 10' 3 x 108

"Forster and Volkers (1975).

HPTSr,,, Apomyoglobin Bovine serum albumin

-

1.8 x 109 3.3 x 109

-

0.63 0.31

10'0

6.9 x 10' 2.2 x 109

5.10'" X [H']" 9.3 x 108 9.3 x 108

-

1.38 x 10' 1.3 x 10'

1.66 x 10' 1.6 x 10' 1.7 x 108

The Macroscopic Rate Constant and the Partial Rate Constants of Proton Dissociation, Recombination, and Escape Out of the Apomyoglobin Heme-Binding Site and Hydroxypyrene Binding of Bovine Serum Albumin

TABLE I1

1.oo

0.68 0.8

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

33

0.68 and 0.8 for the myoglobin and bovine serum albumin, respectively. These values indicate that the water molecules in these two cavities are well below the values of the bulk. The implication of this finding will be discussed below.

2. Proton Dissociation in a Poorly Hydrating Site 2-naphthol-3,6-disulfonate is a very efficient proton emitter (pKo = 9.3; pK* = 0.3).In water, the proton dissociates in less than 100 psec. Binding of 2-naphthol disulfonate to bovine serum albumin increases the emission of +OH* with concommitant decrease of +O-* fluorescence (Figure 19). This effect is concentration dependent (Figure 20), following a typical saturation curve. A Scatchard plot of these results indicate a 1:1 stoichiometry. Each protein molecule carries one binding site ( K , = 2. 106M- ') where the lifetime of the +OH* state is significantly prolonged with respect to that measured in water (Gutman et al., 1982). It should be stressed that in this type of analysis other sites that do not change the emissions intensity ratio with respect to water are not observed. T h e kinetic study of the dissociation of 2-naphthol disulfonate is hampered by some technical limitations. The emission of the +OH* is at a short wavelength, which is already absorbed by the optical components of I

I

I

1

nm

Figure 19. Fluorescence emission spectra of free and protein-bound 2-naphthol-3,6disulfonate. The spectra were recorded at pH 6 with lOOpA4 of the free ligand (a) or in the presence of 140pA4 bovine serum albumin (b). Excitation at 330 nm.

34

MENACHEM GUTMAN

0.5

1.o

Figure 20. Titration of bovine serum albumin by 2-naphthol-3,6-disulfonate. Titration was carried out with 14.7p.M protein (pH = 6.0) and increasing ligand concentration. The fluorescence emission ratio of the dissociatedvs. undissociatedemitter is drawn with respect to the molar ratio of protein to ligand. The emission ratio of free ligand is indicated. Note discontinuity of abcissa and ordinate. (Insert). Sketchard plot of the titration.

our streak camera. Consequently, we miss both the high time resolution and the simple analysis according to Equation (18).Still, the difficulty can be overcome by utilizing another fluorescence property of this proton emitter: the difference in the fluorescence decay time between the neutral and dissociated species. The decay of the neutral state of 2-naphthol3,6-disulfonate is twice as fast as that of the excited anion (Table 111).This difference can be reduced at pH = pK*. Each collision between +O-* and H+ will enable a faster decay pathway for the excited state, through the faster route of +OH*. Indeed, at pH = 1, the lifetime of +O-* is shortened and approaches that of +OH* (compare lines 1,3,and 4, Table 111). As we observe no accelerated decay of 40-* in the protein-emitter complex (lines 6 and 3, Table III), we conclude that the site where $0-*is located cannot confine the free ejected proton for any appreciable time. emission originates must be well exposed to the The site where $0-* bulk, or contain a basic group that can trap the proton and retain it for a time with affinity that will prevent its retransfer to the excited anion. The above conclusion implies that the enhanced +OH* emission represents a reaction in a different site where proton dissociation is suppressed by very low local activity of the water.

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

35

TABLE 111 Fluorescence Lifetime of Free and Bound 2-Naphthol-3,6-Disulfonate

1.

2. 3. 4. 5. 6.

Observed parameter

Experimental condition

Fluorescence lifetime (nsec)

+OH* free +OH* free 40-* free +O-* free +OH*-BSA +O-*-BSA

2M HC1 pH = 7 pH = 7 pH = 1 pH = 5.96 pH = 5.96

13.4 0.08" 22.2 12. 11.1 23.8

"Measured by streak camera as rise time of +O-*.

The slow decay of +OH* would be accompanied by a comparable slow rise of 40-*which is not the case. The rise time of $0-*emission of 2-naphthol disulfonate- bovine serum albumin complex was as fast as measured for the free ligand (trise re h e equilibrium concentrations of these reactants. As k2@H = kI+O- - H + . The equation is reduced to

dx - = -(k2 dt

+ kI(@- + H + ) ) X - klX2

In the classical treatment of chemical relaxation, the nonlinear terms (x2) are neglected with the assumption that the increment X is much smaller than the equilibrium concentration of the reactants. If this assumption is made, the decay of X is characterized by a single exponential decay with time constant: 7-1

=

[k2 + k,(@-

+ H+)]

(28)

Despite this convenience, such approximation is seldom permitted by the initial conditions of the experiment. Suppose that a dilute neutral solution (loo@, pH = 7) of hydroxypyrene trisulfonate (pK = 7.7) is pulsed

46

MENACHEM GUTMAN

with a medium-energy density (0.4MW/cm2) and lOpM of +OH dissociate. In this case, the term X2 = (lo-'') is not negligible with respect to (@- + H + ) * X = (1.6 X lo-"). Thus, the actual decay will not follow a single exponential decay (Pines 1981).Suitable conditions for the single exponential decay call for pH > pK so that &6->> X, or pHX. The employment of alkaline initial conditions reduces the fractional increment of the signal (Xlw-) that reduces the signal-tonoise ratio. What is more, it may introduce OH- as a possible proton acceptor in the system. The acidic conditions lead to very rapid recombination, (T < 1psec) which calls for high time-resolution instrumentation. Because of these reasons, it is better not to manipulate the experimental conditions for the sake of simpler kinetic analysis. The numerical analysis is applicable for any initial conditions and allows one to select the conditions according to the biochemical system under investigation. The intrinsic simplicityof the single-component system renders it ideal for measurements of basic reaction in chemistry like the kinetics of recombination in Coulomb cage (Forster and Volkers, 1975), the diffusion coefficient of a proton in water (Gutman et al., 1981) or solids like ice or urea (Pines, 1981). Due to the indirect applicability of these subjects to biochemistry, these subjects will not be further discussed.

2. The Reaction of Proton with Indicator The pH indicators are a large group of compounds with a huge selection of pKs, solubilities, structural properties, and adsorption bands to fit any special demand. As a general rule for monitoring rapid reactions, color changes associated with protonation-dissociation of carbon atoms (like crystal violet or malachite green) should be avoided-as these reactions are not diffusion controlled (Eigen, 1964; Duynstee and Grunwald, 1959a,b).The reaction of amines, azoaraomatic rings, phenols, or carboxylates are fast diffusion-controlled reactions (Eigen, 1964).The time scale of the observations depends on the indicator. In all cases, the protonation is a fast reaction, reaching its maximum in the microsecond time scale. The relaxation of the signal, that is, deprotonation of the indicator, varies with the pK of the indicator. The time constant of the decay can be roughly approximated by T = (5 x 10" - K d i s S ) - l . Thus, indicators with pK = 5 will dissociate in the microsecond time scale whereas those with a pK of 7-8 may linger in their acidic state for approximately 1 msec after the pulse. The reactions that take place in two-component systems, proton emitter, and pH indicator dissolved in water, are given below:

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

47

plus the following reactions: 1.4 x 10" H+ + O H - . ' H20 +OH + OH- e40- + H 2 0 (hydrolysis) (33) HIn + OH- eIn- + H 2 0 (hydrolysis) (34) The time constant for hydrolysis is approximated by Thydrolysis = K , X 1O5 (sec). Thus, for proton emitters with pKo < 10 this reaction will be too slow to affect of overall dynamics. The reaction of the proton with OH-, should always be considered, due to the high rate constant of this reaction, k = 1.4 X 10"M-' sec-'. In order to avoid efficient competition between In- and OH- for H+, [OH-] in the solution should be at least 100-fold smaller than [In-], that is, better keep the pH of the solution below pH = 8.5. Abiding with the above precautions, we can limit our consideration only to reactions (29) to (31). Reaction (31) is strongly modulated by electrostatic interactions. In a case where both 40- and HIn are negatively charged, the rate of the direct reaction is negligible with respect to reactions (29) and (30). We shall first deal with such a case, and then demonstrate the effect of reaction (31) on the dynamics. A. DYNAMICS OF PROTON CYCLE IN T H E ABSENCE OF DIRECT PROTON EXCHANGE

-

-

The experimental system consists of double charged indicator, Bromo Cresol Green BCG-- + H+ BCGH- (pK 4.7) and triple-charged proton emitter hydroxypyrene trisulfonate: +OH-3 40-4 + H+ (pK = 7.7). Because of the large charge product, Z1Z2 = +4, the contribution of reaction (31)to the relaxing system is rather small, less than 1%, and we can limit our analysis only to reactions (29) and (30). The laser pulse dissociates X molecules of +OH, and the discharged protons react with Y molecules of In-. Thus, the concentration of the reactants at time t is given by

w-

(+O-)t= - + X; (In-)t = In- - Y; (H'), = E++ X - Y

(+OH), = +OH - X (HIn), = m n + Y

48

MENACHEM GUTMAN

At t = 0, X ( 0 ) = XO and Y ( 0 ) = 0. The differential rate equations which describe the time dependence of X and Y are:

d[+O-I dt

--

-

dx

- = (--k'([H+]+ [qz-]) - k*)X dt + k l [ w - ] Y - k l X 2 + klXY

d[In-] dY ----= dt dl

(35)

k3rG-W - (k3([F+]+[ln-]) - k*)Y - k3XY

+ k3Y2

(36)

Three independent parameters control the numerical solution: kl k3, and X,. The other two rate constants (k2 and k4) are already set by the equilibrium constants: K43 = K4/k3; KZI = k 2 / k l . To find the best fit, the two diffusion-controlled rate constants of protonation, k 1 and kJ7 are varied in the range of lo9 to 20 x 10'OM-I sec-' (which is the range of diffusion-controlled reaction between small molecules and H+). The other independent parameter, XO,is estimated from the initial rate of HIn formation. It is assumed that the initial velocity of HIn formation is given by Vinit = k3 [In-][H+]. Usin? the known value of [In-] and an estimation k3 = 5 X 10'oM-l sec- , [H+Io can be estimated with about 30% accuracy. Within this range of estimated parameters, simulations are systematically iterated until a set of parameters (Al, k 3 , X o ) is found which yields the best curve fitted to the experimental one. Figure 23 depicts such a typical simulation. The initial velocity of the reaction led to an estimate forXoof 4-5.5p.M, and within this range ofXo we varied systematically both k l and k 3 . Figure 23A depicts simulations withkl = 1.8 X lO"M-'sec-' andthreevaluesofkg(3,4.2,5X 10'oM-' set-I). In Figure 23B,k3 was kept constant (4.2X 101oM-' sec-') and k l was varied (1.6, 1.8, 2.2 X 10"M-' sec-I). In both figures, Xo = Figure 23. Experimental results and simulated curves of the proton cycle. The experiment was carried out in the presence of 4 0 m Bromo Cresol Green, 1OOphf 8-hydroxypyrene1,3,6-trisuIfonate, pH 5.88, room temperature. Fifty readings of the experimental curve, in the range 0-3.3 psec, at 68-nsec intervals, and 50 readings in the range of 1.0- 17.7 psec at 340-nsec intervals are drawn, together with the simulated curves. (A) The simulated reactions were computed for the following parameters: k l = 18 X lO'"M-' sec-'; X, = 4.25pN; k3 = 3.2 x 10'" (---), 4.2 x 10'" (-), 5.2 x 10'" (--), M-' sec-I. After 5 psec the lines for 4.2 and 5.2 x 10'" M-' sec- practically overlap. (B) T h e simulated reactions were computed for the following parameters: k3 = 4.2 x 10'" M-' sec-'; X, = 4.25pM; k l = 16 X 10'" (---), 18 X 10'" (-), 22 X 10'" (-.-), M - ' sec-'. (C) Simulation of the variation with time of (40-1, (---), (Hln), experimental and simulation (-), and free proton concentration (-.). k l = 18 X 10'" M-' sec-'; X, = 4.25pN; k3 = 4.2 X 10" M - ' sec-'.

'

49

50

MENACHEM GUTMAN

4.0

z

Q

4

I

I

I

-

-

\

-

3.0\,\

';\

-

2.0 j:, \,

-

-

- I

I

I

TABLE I V Association and Dissociation Rate Constants of Protons from the Ground State of Proton Emitters and'hdicators Compound 8-Hydroxypyrene-l,3,6trisulfonate 2-naphthol-3,6disulfonate 2-naphthol-6-sulfonate p-naph tho1 Bromo Cresol Green

has," ( M - ' sec-')

(sec-')

PK

1

18 2 1.5 X 10"

3600

7.7

0.0006

45

9.2

0.004

9.2 9.3 4.95

0.002 0.001 0.0006

7

2

1.5 X 10"

7.6 2 0.4 X 10" 1 2 0.1 x 10'0 4.2 2 0.1 X 10"

hiss

48 5 4 . 7 . 105

"The values given are the average of five to seven independent experiments.

T H E p H J U M P PROBING OF MACROMOLECULES AND SOLUTIONS

5I

et al., 1983) on the experimental one is a very critical evaluation of the accuracy and fit of the simulation, yet because of its unqualitative nature it is inconvenient for comparison between experiments. Thus, we prefer to describe the dynamics of the proton cycle by some measurable parameters-called macroscopic parameters. These are T,,, and Y,,,, which are the time and amplitude coordinates of the maximum of the curve, and two macroscopic rate constants y1 and y2 corresponding with signal build-up and decay. It should be stressed that neither the experimental nor the simulated curves behave as a sum of two exponents. Both the build-up and the decay are nonexponential curves. Thus, we characterize them by approximation. About 60% of the signal is built up and the initial 60% of signal decay are fairly approximated by a mono exponential function. Within these limits, y1 and y2 were calculated and employed as convenient parameters for quantitative characterization of the curve. The usage of this macroscopic parameter for calculation of the partial rate constants is demonstrated in Figure 24. The computer was programmed to produce the value of the macroscopic parameters as a function of k I and k3. Figure 24A depicts the dependence of y l and y2 on ks with a constant value of k l , whereas Figure 24B relates T,, and Y,, with the same parameters. The macroscopic parameters calculated from the experimental curve fit the theoretical curves for k3 = 4 k 0.2 X 101OM-'sec-'. Figure 25, A-D, represents the variation of the macroscopic parameter as a function of kI-for three values of k 3 , 3.2, 4.2, and 6.2 X 101OM-'sec-'. The accurate values of k 1 and k3 are those that will generate simultaneously the correct value of four macroscopic parameters. As seen in Figure 25, that requisite is met by the combination k1 = 1.6 rfr 0.2 X 10"M-'sec-' and k3 = 4.2 x 10'oM-'sec-'. B. T H E EFFECT OF INITIAL CONDITIONS O N T H E MACROSCOPIC PARAMETERS

Figures 23- 25 demonstrate that the simulation technique is suitable for reproducing the outcome of the experiment, thus these calculations may be convenient for understanding the complex relationship between the various parameters appearing in the differential rate equations and the observed macroscopic parameters. The most dominating process during the proton cycle is the continuous competition between +O_Land In- for the protons. In a case of a large perturbation (Xo >> +O-; K-), the initial conditions will have minor effect on the outcome of the proton cycle, but otherwise the dynamics are very much influenced by the prepulse concentration. The

Figure 24. The dependence of the macroscopic parameters on the rate constants of the reaction of the indicator with the proton. The macroscopic parameters were calculated for simulations describing the experimental conditions defined in Figure 23. T h e rate constant k l = 1.8 x 10" M - ' sec-l and X , = 4.25fl were kept constant and k3 was varied from 10'' to 10" M - ' sec-I. The macroscopic parameters with their error bars, which were measured from the experimental results, are indicated. (A) y1 and y2 vs. k,; (B) T,,, and Y,,, vs. k?. T h e vertical lines marked the accuracy of k3 determination.

52

0

I 5

I

I

10

k,

I

15

I

1 20

I

25

10-'0

(A)

0.E

p

+)

0.4

N

P

0.2

0

k,=

10-10

(6 )

Figure 25. The dependence of the macroscopic parameters on the rate constant of proton recombination with the proton emitter anion. The macroscopic parameters were calculated for simulations describing the experimental conditions defined in Figure 23. The frames represent y1 (A), y2 (B), Tmax(C), and Y,, (D) as a function of the rate of protonation of $0-.In each figure, there are three curves calculated for k3 with the values of 3.2 x 10" M-' sec-* (-), 4.2 X 10'' M-' sec-* (-), and 6.2 x 10" M-' sec-' (---). The experimentally determined macroscopic parameters are indicated as parallel horizontal lines. The vertical lines denote the range of k l values that will yield macroscopic parameters compatible with the measured ones.

53

-

a

v)

I

I

I

I

’

I

1

I

0

5

10

k,

a

I I

I 20

15

10“~

(C)

10

-

I

I

I

54

I

I

I

i

I 1

1

I

5

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

55

effect of the prepulse conditions, pH, reactants concentration, and pulse size are represented below. Figure 26 relates the effect of the prepulse pH on the macroscopic parameters ( X , = 4.25pM). Within the range where the experimental results are conveniently measured, the computed macroscopic parameters and the measured ones are essentially identical. It is of interest to point out that at low pH both y1 and y2 increase but at the upper limit y2 does not vary much. This is the range where the concentration increases to such an extent that it competes effectively for any H+ dissociating from HIn, thus y2 approaches the value of It4. The signal size has a clear maximum, determined mostly by the pK values of the proton emitter and the proton detector. At low pH, the depletion of G- limits the formation of HIn, whereas at high pH the absence of undissociated +OH reduces the size of the pulse and increases the competitivity of $0for the protons.

w-

Figure 26. The effect of initial pH on the macroscopic parameters characterizing the Bromo Cresol Green-hydroxypyrene trisulfonate system. The macroscopic parameters were calculated for the experimental conditions described in Figure 23 and the rate constant listed in Table IV. X o = 4.25pW. The experimental values are drawn with their error bars. (A) 71 VS.pH; (B) 72 VS. pH; (C) T,,, VS. pH.

2.

I

I

Figure 26. (Continued)

56

I

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

57

T h e effect of $0- concentration, at a constant pH, on y1 and y2 is given in Figure 27. Increasing the competitiveness of $0- for H + enlarges both y1 and y2. T,,, is shortened and the amount of HIn formed diminishes. It is of interest to note that above certain concentrations of $0-,y2 remains constant. This is the limiting case where $0- competes successfully for any proton dissociating from HIn, consequently the ratelimiting step of the reaction becomes k4. If the proton cycle can be measured under conditions where [$O-] > [In-], then y2 can be accurately equated with kq. This advantage has its drawbacks, for at high excess of $0-,the signal diminishes in size and reduces the accuracy of the measurement. T h e effect of [In-] is given in Figure 28. As expected, there &hardly anyeffect on 439, whereas y1 increases nearly linearly. Once [In-] >> ([$O-] + X), the formation of HIn becomes effectively a pseudo firstorder reaction and y1 will become a linear function of [In-] with a slope of kS. Xo,the size of the proton pulse, depends linearly on the energy density of the excitation beam (Gutman et al., 1981) and the emitter concentrations. As a result, the pulse size may vary between successive experiments. T o estimate the effect of this variance on the macroscopic parameters, we simulated a set of experiments where the pulse size was varied from X o = 1 to 401J.M(Figure 29). During the pulse, the protons are produced together with their conjugate bases and the prepulse pH is regained very rapidly (Figure 23C). Consequently, the effect of the pulse size differs from - the effect on the prepulse concentration of the other reactants, [$O-] o r Y,,, increases steeply with X o , but at higher values the line curves, reflecting the higher probability of an encounter between H + and $0-. T,,, is less sensitive to X o , while the two rate constants, y1 and y2, are nearly independent of the pulse size.

[z-].

3. The Direct Proton Exchange between Reactants and Its Effect on the Dynamics of the Proton Cycle In the above examples, the contribution of a direct proton exchange has been disregarded. Considering the fact that biochemical reactions and physiological events take place in well-buffered solution where the free proton concentration is low (pH > 6) and the buffer concentration is high, the direct proton exchange cannot be neglected. As a matter of fact, the protonation of surface components on a membrane across the unstirred layer is very likely mediated by protonated buffer molecules (McLaughlin and Dilger, 1980; Benz and McLaughlin, 1983). Direct proton exchange can be easily included in our formalism and, under certain experimental conditions, can be measured.

[OO-] (pM1 ( 5)

Figure 27. The dependence of the macroscopic parameters on the $0- concentration. The experimental conditions, except for emitter concentrations, are defined in Figure 23. The rate constants for 8-hydroxypyrene-1,3,6-trisulfonateand Bromo Cresol Green are listed in Table IV. X, = 4.25M. (A) y1 and y2; (B) T,,, and Y,,,.

58

Figure 28. The dependence of the macroscopic parameters on the indicator concentration. See legend to Figure 27.

59

X(o)

(pM) tB)

Figure 29. The dependence of the macroscopic parameters on the pulse size (XJ.The experimental conditions are defined in Figure 23. The rate constants are listed in Table IV. The pulse size, X,, was varied between 1 to 4 0 N . (A) y I and y2; (3)T,,, and Y,,,.

60

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

61

T h e intensive electrostatic repulsion between the pyrenate trisulfonate anion (2 = -4) and the protonated indicator (BCGH-) lowers the probabiIity of their encounter by a hundredfold (Eigen et al., 1964). Thus, a direct proton exchange between these two reactants is slowed both by slow diffusion (D= 2-3 X cm2 sec-') and electric repulsion. To evaluate whether our experimental system can detect the contribution of a direct reaction, we substituted the highly charged emitter with the neutral one, @-naphthol. T h e protonation cycle of Bromo Cresol Green, in the presence of P-naphthol, is depicted in Figure 30. Calculations that did not account for the contribution of the direct proton exchange (reaction 3 1) gave unsatisfactory simulations. However, including the rate constants of reaction (30) in the differential rate equations dX

(Hf)]+ k 2 + k&H)

= -{k,[(@-) dt

- k(j(h-)}X

+[kl(+O-) - k 5 ( + 0 - ) - k6(+OH )1y -k*X2 + [k* - k5 + KGIXY

dY

- ~,(HI~)W+ (H+)] + k4 + k6(+OH ) + k 5 ( + 5 - ) } Y

- -- [ k 3 ( G 1 - k6(E-) dt

(37)

-{ks[(In-) +k3Y2 + [-k3

_ .

k6 - k5lXY

(38)

led to the simulation given in Figure 30. The rate constant of the spontaneous direction of the proton exchange k5 = 5 x 109M-' sec-' is compatible with the value estimated according to Debye and the viscosity of the solution, but the rate of @-naphtholate protonation ( k , ) also derived from the simulation is only k 1 = 1.5 x 10'oM-'sec-'. This value is only 30% of the value estimated by Weller (1958) for this reaction, or as expected from Debye's equation for diffusion-controlled reaction. The ratio between the measured rate constant of a diffusion-controlled reaction and the value predicted according to Debye's equation is an estimation of the steric factor u. Thus, for protonation of P-naphtholate, u = 0.2-0.3, whereas for proton exchange between protonated indicator and P-naphtholate u 1.0. T h e steric factor u appearing in Debye's equation is a complex function representing not only the geometry of the reacting molecules, that is, the fraction of the reacting surface and axis ratio for nonspheric molecules, but also the rate of rotation of the reactants (Richter and Eigen, 1974; Solc and Stockmayer, 1973; Shoup et al., 1981). A fast rotation of the reactants increases the probability of reaction, but this event must take place before the encounter complex separates. Thus, while fast rotational diffusion increases u, a fast translational diffusion lowers it (Shoup et al., 1981). Consequently, the proton exchange between the rapidly rotating, slowdiffusing molecules P-naphtholate and protonated Bromo-Cresol Green

-

62

MENACHEM GUTMAN 2D

I

I

I

psec

Figure 30. Experimental results and simulated curve for the proton cycle measured with B-naphthol (I&), and Bromo Cresol Green (40 pM) (pH = 7.3). The rate constants for Bromo Cresol Green are taken from Table IV.The simulation curve given in the figure (---) corresponds with k, = 1.0 x 10" M--' sec-I, k5 = 5 x lo9 M - ' sec-I, X o = 2.7p.M.

has a higher steric factor than the reaction between P-naptholate and a free proton. T h e steric factor estimated for the latter reaction (+ -0.3 is comparable with the surface fraction of the hydroxyl on the @-naphthol (Bondi, 1964), indicating that in this reaction the rotational diffusion is not fast enough to affect the outcome of the collision.

4. Alkalinization Pulse by the Conjugate Base of the Proton Emitter In the above sections, we referred only to one product of the photodissociation-the proton. Still, it is produced together with a strong base, the ground-state anion of the proton emitter. T h e reason that the effect of $0-was not observed is due to the experimental conditions that were set to mimimize it. T h e diffusion of the conjugate base is 10 times slower than that of the proton. Thus, the reactions of the proton with other solutes is the dominating event. Furthermore, the electrostatic repulsion between the emitter and the detector reduces the contribution of $0-to the measured dynamics. Once these factors are understood, it is possible to predict and set experimental conditions where the reaction of $0with the indicator will dominate the observed reaction. By setting the experimental pH to be lower than the pK of the proton detector (pH
-,

we can approximate this by:

At the pH below pH’, the reaction with the conjugated base dominates. pH’ differs from the pK by log (k3/k5),the ratio of the rates of Inprotonatiorr(k3) and HIn reaction with a conjugated base (k5). The effect of the initial pH on the direction of HIn response is demonstrated in Figure 31. At pH 7.1, the Bromo Cresol Purple (pK = 6.3) is protonated; at a lower pH (5.12), it gets deprotonated by the reaction with the P-naphtholate formed by the pulse (Gutman et al., 1983).

5. Limitations and Inaccuracies A.

REACTANTS CONCENTRATION

In the present section, I shall point out the sources of inaccuracies and how to minimize them by proper selection of the experimental conditions.

64 A

MENACHEM GUTMAN

I B

Figure 3 1. The effect of pH on the response of Brorno Cresol Purple during the proton pulse. The reaction mixture contained Bromo Cresol Purple (100pM) and P-naphthol (1mM) at pH 7.05 (A) and 5.12 (B). Each tracing is an average of 4096 events. Downward deflection corresponds with protonation of indicator.

We found it convenient to limit the indicator concentration to absorbance range of 0.2-3A. .4t lower concentrations of indicator, the dilution flattens the signal and makes it poorly resolved. T h e rise time becomes shorter, the decay is faster, and the signal diminishes in size (Figure 28). T h e upper limit of indicator concentration is limited by the fluorescence of the proton emitter. It should be remembered that of the 1 MW of excitation energy 10- 100 k W are irradiated as fluorescence. Unless this emission is reduced at the entrance slit of the photomultiplier below the saturation energy of the photomultiplier, nonlinear responses are expected. On the other hand, the transient intensity of the probing beam must be big enough to be recorded. Thus, a combination of intensive monitoring beam and selective damping of the fluorescence are always needed. Once the absorbance of the monitoring beam increases, more light should be introduced to the monochromator with consequent admission of more fluorescence. The concentration of the emitter can vary, in practice, only within a certain range. At low concentration, the perturbation is small whereas at high concentration all of the excitation energy may be absorbed at the first few millimeters of the solution. Under the latter conditions, the size of the perturbation is highly dependent on the distance of the probed space

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

65

from the front surface of the cuvette. As pointed out in Figure 29, the magnitude of X o has a marked effect on all macroscopic parameters. This may introduce an apparent irreproducibility with every minor change of the monitoring beam’s location. Finally, a possible source of error is the accumulation of photoproducts (Klaning et al., 1973; Lachish et al., 1977). At an energy density of less than 1 MW/cm2, no deviations due to photoproducts were observed (Gutman et al., 1981). Still, whenever many events are accumulated over a long time period, 3-30 ml of irradiated solution and proper mixing are recommended. B. ACCURACY OF THE MACROSCOPIC PARAMETERS

T h e dynamics most accurately measured are the decay of the signal, y2. T h e relaxation is slow enough to be conveniently measured by currently available instruments. The signal relaxation is a complex function; its initial phase is described by a single exponent, but as it proceeds the relaxation slows into a long tail. [Some of the tail might even be attributed to stable photo products (Klaning et al., 1973; Lachish et al., 1977)l. Most of the data, characterized by high signal-to-noise ratio, is clustered in the early exponential decay curve represented by y2. In most cases, y2 can be measured with + l o % error. T h e rate constant of signal rise, yl, is less accurately determined. This signal is a fast event, reaching its maximum in a few microseconds or less. Thus, the ascending section of the curve is limited in resolution; its initial part has an exponential rise but a poor signal-to-noise ratio due to the small signal size. Once the measured values are bigger, the curve begins to deviate from the exponential function and the data become unsuitable for calculating 71. For these reasons, the value for y1 is less accurate than that of y2. In some experiments, the error might be as high as 30%. T,,,, the time when the signal reaches its maximal height, has its own uncertainty due to the flatness of the curve at its maximum and the electronic noise. In the case of a sharply rising, steeply decaying signal, T,,, can be estimated with an error of 270 nsec. On the other hand, slowly decaying signals have undefined maxima and inaccuracy may be as high as 2 1 psec. T h e value of Y,,, for a given experiment can be measured very accurately, with an error of less than 5%. Thus, for the purpose of simulation, the amplitude is a very informative parameter. However, as demonstrated in Figure 29, Y,,, is highly dependent on X o , which is a function of the excitation laser output, pulse-beam dimensions, and observation cell and monitoring-beam geometries. The other parameters, T,,,, yl, and y2 are much less affected by X,. Thus, the amplitude is a

66

MENACHEM GUTMAN

parameter that may vary quite independently between experiments. Therefore, despite the fact that Y,,, can be an informative parameter for a given experiment, it is unsuited for comparison between different experiments. For these reasons, we did not compare the Y,,, values among the experiments. Even though each of the experimental parameters bears its own inaccuracy, the combination of the four leads to unambiguous determination of the reaction rate constants (Figure 25). Table IV lists the means of five to seven independent measurements, carried out under different initial conditions. T h e results are all reproducible within 20%.

VI. KINETICS OF PROTONATION OF HIGH-MOLECULAR-WEIGHT STRUCTURE T h e charge of a macromolecule modifies the ionic atmosphere in its immediate vicinity, as described by the Gouy-Chapman diffused double layer (Adamson, 1960). At the surface of the protein, the concentration of counter ions will exceed that of theothers. As a result, the measured pK of surface groups will be shifted, reflecting the difference between the surface and bulk concentration of the protons (Goldstein, 1972). This surface phenomenon reflects the total charge of the molecule, even of charges located far from the site of protonation. Because of the low dielectric constant of the interior of the protein, the electric field of buried charges propagate to the surface without any attenuation by the ionic atmosphere (Matthew et al., 1979a,b; Matthew and Richards, 1982; Russu et al., 1982). T h e electric charge of the protein (or any other macromolecular structure) affects the rate of protonation of any surface group, as given by the electrostatic terms of the Debye Shmulchowski equation for diffusioncontrolled reactions (Eigen et al., 1964). Besides this charge effect, the probability of protonation of ;I surface group on a protein is modulated by the following factors: 1. T h e rate of encounter between a proton and a specific target on a macromolecular weight structure is a function of the ratio of the target (rt) and macromolecule (r,) radii. 2. T h e surface of a macromolecule forms a two-dimensional diffusion plane. As pointed out by Adam and Delbruck (1968), the probability of encounter between mobile ligand and a target on surface can be higher than encounter in three-dimensional space. 3. T h e matrix through which the proton diffuses, water, changes its properties in the immediate vicinity of the interface. T h e interface is

THE PH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

67

covered by a hydration layer (Le Neveu et al., 1977);(Rand et al., 1980); Parsegian et al., 1979; Lis et al., 1982) characterized by lowered activity coefficient. In this space, the rate of proton transfer will differ from those taking place in the bulk (reactions 16 and 13). What is more, this hydration layer will act as a reflecting surface for an incoming proton. The penetration of a proton into the hydration layer calls for reorientation of the water molecules by the proton (reaction 13).Thus, the hydration layer is a thermodynamically unfavorable space for a free proton, modulating the rate of protonation by a bulk proton. The considerations listed above are concerned with the direct interaction of the proton with its target, yet we also have to consider events that will affect the apparent properties of the measured system. In this category, I include the postprotonation reaction, which can be detected by kinetic analysis but is obscured in equilibrium measurements. The kinetic analysis of protonation (as in reaction 27) is much facilitated by having the equilibrium constants of the reactions. The pK of a surface group depends not only on the total charge of the protein (Tanford, 1955, 1976; Tanford et al., 1955; Tanford and Kirkwood, 1957) but also on the solvent accessibility (Matthew et al., 1979a,b; Matthew and Richards, 1982; Russa et al., 1982). Some of the solvent accessibility abberrations are accounted for by the three mechanisms described above. But solvent accessibility, which reflects the precise configuration of the binding site, points to another factor that must be considered-the selective interactions of the immediate environment with the two forms of the proton acceptor. Highly polarizable microenvironment will favor the charged state of the proton acceptor, whereas local charges will selectively stabilize the counter-charged form of the acceptor. If the acceptor protein can assume two configurations (Tong and Glesmann, 1957; Gutman et al., 1983a) reflecting the selective stabilization energy, we shall observe a postprotonation reaction that will modify the measured equilibrium parameters of the measured system. In such a case, the reaction is not a straightforward reversible-binding-dissociationbut a successive two-step reaction. For such a case, we do not have the advantage of relating the partial rate constant of proton association-dissociation with the observed equilibrium constant, that is, K,# (k4Ik.3). Consequently, the analysis of protonation of macromolecule calls for differentiation between the observed pK (Kobs) and the surface one, the one that reflects the actual step of the fast, reversible protonation. In the following section, we shall define K , = k4/k3. Due to their heterogeneous surface, proteins are too complex to serve as a model system. The one we selected is the indicator-micelle system (Gutman et al., 1981a; Gutman et al., 1983a). Once the formalism is

68

MENACHEM GUTMAN

defined and its applicability is proven, it can be employed in more complex systems like protein or membrane surface. 1. Protonation of Uncharged Target Adsorbed on Uncharged Carrier The simplest model for protonation of a surface group is the reaction between the uncharged indicator Neutral Red (Scheme 111) adsorbed on uncharged micelle of Brij 58. Neutral Red is uncharged in its alkaline state. Its water solubility is extremely low, but it is freely soluble in inorganic solvents. Protonation increases its water solubility. Both the alkaline or acidic state are adsorbed to micelles. T h e distribution ratio between the aqueous and the micellar phases, for the acidic (a)and alkaline (p) states of the indicator, are a = 130 ? 10, p = 1400 100 (Gutman et al., 1983a). T h e apparent pK of the adsorbed indicator is practically independent of ionic strength (Table V), indicating that the surface charge of the micelles is very close to zero (Tanford, 1976). T h e kinetic experiments (Figure 32) were carried out with 501J-M Neutral Red and 40 mg/ml Brij 58 (equivalent to -500@4 of micellar concentration). At this concentration of detergent, 98%of the indicator is adsorbed. T h e initial pH of the experiment (7-7.5) ensured that before perturbation the Neutral Red was mostly deprotonated, whereas the proton emitter (2-naphthol, 3,6-disulfonate, pKo = 9.3) was undissociated. Perturbation of the equilibrium by a laser pulse, dissociates X o molecules of +OH, and the relaxation of the system is described by equations (37) and (38). (Direct proton exchange between +O- and bound indicator can be ignored in this case.) T h e experimental curve and the simulated function are given in Figure 32. T h e rate constants of the reaction are listed in Table V.

*

TABLE V Thermodynamic and Kinetic Parameters for Protonation of Neutral Red Adsorbed on Urij 58 Micelles"

KCI (mM)

p%bs

0 40 100

5.5 ? 0.05 5.55 t 0.05 5.55 t 0.05

-log

k4

k3

5.53 5.60 5.61

kl

kS

(M-' sec-I)

( M - sec- I )

I

7 4 I x 10'' 5.5 4 0.9 X 10" 4.5 4 0.7 x 10"

0.9 2 0.3 x lo'* 1.3 k 0.3 X 10'' 0.9 k 0.3 X 10''

0.006 0.046 0.106

"The experiments were carried out in the presence of 50p.M Neutral Red, 40 mg Brij 58/ml, corresponding with 5OOp.M of micellar concentration), 1 mM of 2-naphthol-3,6disulfonate, and KCI as indicated. T h e k l values appearing in the table were computed simultaneously with k3.

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

69

Scheme I11

;

time (psec) Figure 32. Experimental results and simulation curve for the protonation cycle of Neutral Red adsorbed on Brij 58 micelles. The experiment was carried out in the presence of 1mM 2-naphthol-3,6-disulfonate, 40pM Neutral Red, 40 mg/ml Brij 58 (pH 7.3). The rate constants used for the simulation were k , = 7.0 X lo-'" M - ' sec-'; ks = 0.85 x 10" M-' sec-'; pK, = pK43 = 5.53 X , = 1 O p M . (-) Experimental; (---) simulation.

2. The Effect of Charge on Rate of Protonation The time-independent diffusion-controlled rate constant for reaction between charged reactant is given in Equation (42) (Eigen et al., 1964).

T h e first term, ken, is the rate of encounter between the reactants in absence of electrostatic interaction; the second term represents the contribution of the electrostatic interactions at I = 0, while the last one is a correction for ionic screening at I > 0; x is the radius of the ionic atmosphere [ x = 3.27 x lo7 fi(cm-')I; and rg is the radius of the encounter between the reactants. T h e rate of encounter of small molecules is given by 4?r

ken = -N C D r q a 1000

(43)

70

MENACHEM GUTMAN

where ED is the sum of the diffusion coefficients of the reactants and u is the steric factor (Eigen et al., 1964). The two electrostatic terms are functions of 6,which is the ratio between the Debye radius of the molecule RD = Z1Z2eglEkT (the distance at which the electrostatic force equals to the thermal energy k T ) and rq.

1 At room temperature, we can approximate 6 = 7 * Z l Z p - (given in A

rrj units). The replacement of one reactant by a large molecule bearing a specific site for the small ligand modifies all terms of the equation. The rate of encounter has to account for the fact that the radius of the site (rt)is smaller than the radius of the macromolecule (r,). Furthermore, whereas the slow translational diffusion of the macromolecule can be ignored, the rotational diffusion of the macromolecule becomes crucial. A fast rotation of the macromolecule can compensate for the small radius of the site. In a limiting case of slow rotating macromolecule and a fast translational diffusion of the mobile reactant (a proton in our case) (for details, see Shoup et al., 1981), we can approximate

3 ken = - .ir2NDH+ r(,) 8

-

(44)

The electrostatic interaction terms should also be modified to account for the unequal size of the reactants. The terms appearing in Equation (42) represent the electrostatic potential at the encounter distance rv; if one of the reactants is a macromolecule, we have to calculate the electrostatic potential on its surface and thus r, will replace rq in the two last terms of Equation (42), that is, RD rm

and

~

rmx 1 + r,x

The effect of charge on the rate of protonation of specific site adsorbed on high-molecular-weight structure was measured with the Neutral RedBrij system described above (Equation 42)using the negatively charged detergent, SDS, to modify the micellar charge (Gutman et al., 198la). The charge of these mixed micelles (Fromherz, 1973) was calculated according to Tanford using the pK shift of the bound indicator (Figure 33).The pK of a protonable group carried on the surface of an impenetrable sphere without a radius of exclusion (both solvent and solutes can freely approach the surface of the impenetrable sphere) varies with the charge of the sphere and with the ionic strength of the solution (Tanford, 1976).

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

71

SDS/rnicelle Figure 33. The dependence of the pK of adsorbed Neutral Red on the average sodium dodecyl sulfate content of mixed micelles.

pWAZ = 0.867W where AZ is the charge increment of the sphere and W, the electrostatic free energy on the sphere surface, given by W=

e3

1

2rmE kT 1 + r,x

(45)

At I + 0, the second term approaches unity. Under such conditions, the value of W for a micelle with r, = 17 A (Tanford, 1973) will be W = 0.205. However, the experimentally measured value (W = 0.0674) is much smaller, indicating that only 33% of the adsorbed SDS molecules are not neutralized by counterions (the extent of neutralization by counterion in pure SDS micelle is 85% (Finstein and Rosano, 1967)). The fractional charge of SDS on micelle and the micellar composition allows calculation of the average charge of the micelles. The micellar charge was also corroborated by the Guoy-Chapman diffused, double-layer model. At equilibrium, the surface charge of the micelle alters the ionic composition of the interface with respect to the bulk concentration. The difference between the actual proton concentration on the interface and the one measured at the bulk by pH electrode is observed as a pK shift of the indicator and is related with the GouyChapman potential (Goldstein, 1972). '4'~ = 60A pK

72

MENACHEM GUTMAN

of which the charge density (or charges per micelle) can be obtained (Adamson, 1960).As seen in Figure 34, the estimates of surface charge by the two methods are compatible. The rate constants of protonation of Neutral Red adsorbed on these mixed micelles was measured, the rates were extrapolated to I = 0. The dependence of the extrapolated values on the micellar charge is given in Figure 35. Similar studies were carried out using photoionization of benzidine derivatives (Narayana et al., 1982),phenothiazine (Alkaitiset al., 1975),or pyrene (Gratzel and Thomas, 1974) dissolved in the hydrophobic core of charged micelles. A laser pulse ejects an electron which is rapidly hydrated and then decays into hydroxy radicals, or reacts with the excited cation. The charge of the micelle has a dramatic effect on the rate of the latter reaction but the presence of the former reaction forbade a precise kinetic analysis of the overall reaction. The adherence of our experimental results to the predicted curve (Figure 35)bears a straightforward implication. Any increment in macro-

SDS yh4

Figure 34. Determination of micellar charge from equilibrium and kinetic measurements. The decrement of micellar charge as a function of sodium dodecyl sulfate added to Brij 58 micelles was calculated from the pK shift according to Gouy-Chapman equation (I = lo&) (A) or from the second-order rate constant of protonation using Debye’s equation (Eigen et al., 1964)for rates measured in the presence of ionic screening (0)at I = 1O M , or from rates extrapolated to I = 0 (0)(Gutman et al., 1981a).

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

73

15

10

5

Figure 35. The correlation between the second-order diffusion-controlled rateconstant of protonation of adsorbed Neutral Red and the micellar charge. The micellar charge was calculated as described in the text. The continuous line was calculated according to Debye’s equation (Eigen et al., 1964).

molecular charge will be reflected by an immediate pK shift of all protonable group on its surface, and these groups will react with the protons in the solution in a diffusion-controlled reaction; a pH indicator adsorbed on a macromolecule (or membrane) will adjust its state of protonation with the momentary charge of the protein. The use of pH indicator for measurement of rapid changes in charge density of protein has been employed by Carmeli and Gutman (1982). Bromo Cresol Green was adsorbed on bacterial rhodopsin membrane sheets suspended in well-buffered solution ( l O O m M Mes buffer, pH = 5 ) and the photocycle of the bacteriorhodopsin was initiated by 10-nseclaser pulses. The protonation state of the indicator was probed by an He-Ne CW laser and the transient absorption was averaged. In spite of the high buffer capacity of the medium, a transient acidification of the adsorbed indicator was measured-reflecting the charge modulation of the protein and the subsequent redistribution of protons in the Gouy-Chapman diffused double layer.

3. The Effect of Postprotonation Reaction on the Dynamics The pK of a solvent-accessiblegroup on a protein may deviate markedly from the pK of the free species (Russu et al., 1982).This deviation might be a consequence of pK shift by electrostaticinteraction with other charges on the proteih, or result from selective stabilization of one form of the proton acceptor by the immediate vicinity of the protein. For example,

74

MENACHEM GUTMAN

the pK of His $3146of carbomonoxyhemoglobin A, as measured in high ionic salt solution, is pK = 7.85. Such high value cannot be attributed to electrostatic interaction in the solution (Z = 0.lM) but rather to the salt bridge with the nearby carboxyl of Asp p94. Another type of in situ stabilization was noted by Klotz and Fiess (1960). The pK of the dimethylamino naphthalen sulfonyl adduct to bovine serum albumin was measured and found to be lower than that of the free ligand and independent of the ionic strength. The pK shift is attributed to selective stabilization of the uncharged form of the probe by the hydrophobic pocket where it is bound to. To protonate the ligand in the site (or to extract it to a more aqueous phase where the acidic state is stable), an incremental free energy is needed. This free energy is supplied by the 100-fold increase of H + needed to protonate the amine in its hydrophobic pocket. Apparently, the pK shift of a surface group can be qither due to variation of the rate constants of the reaction or reflect some postprotonation reaction. Thus, we must be capable to discriminate in our analysis between the two mechanisms. The model used for this purpose was Bromo Cresol Green (Scheme 1V) adsorbed on Brij 58 micelles (Gutman et al., 1983a). Bromo Cresol Green, due to the solfono group, is water soluble both in the acidic and the alkaline states. This group also prevents it from diffusing through phospholipid bilayers. Due to the large hydrophobic surface of the three aromatic rings, it is well adsorbed to micelles and liposomes both in alkaline and protonated states. In its alkaline state, the phenolate and the quinoid rings are in resonance and the charge is delocalized. This form is fairly soluble in the lipid core of the micelle. A much higher lipophylicity is achieved in the acidic state. The disruption of the resonance by the protonation yields two distinguished nonresonant structures (phenolic and quinoid rings) which are more lipid soluble than the resonant form. Thus, protonation of the indicator on the interface will reorient the molecule in such a way that both phenolic and quinoid rings will have a better contact with the lipid region of the interface. Such reorientation will insert the dissociable proton into an environment where it cannot dissociate (Huppert et al., 1982). Consequently, the dissociation of the

Scheme IV

75

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

proton will be delayed until the phenolic ring reaches the aqueous phase of the interface. Bromo Cresol Green adsorbed on Brij 58 micelles shifts its pK from pK = 4.96 (I+ 0) in water to pK,b, = 6.5 0.05. The distribution ratio of the acidic (a)and alkaline (0) states are a = 30,000 k 1000 and 0 = 1100 2 100. Thus, protonation increases the lipophilicity of the indicator by 30-fold. The pK of the adsorbed indicator is independent of the ionic strength (Table VI), suggesting that the micellar charge is close to zero. T h e simulation of the dynamics of protonation of bound Bromo Cresol Green, using Equations (37) and (38), failed to reproduce the observed dynamics as long as we assumed that the measured pK is a direct function of the forward and backward rates of proton transfer. This inadequacy led to the conclusion that for this case K o b s f k 4 / k 3 and provision must be made for a mechanism where the postprotonation reaction modulates the observed dynamic and equilibrium.

*

A. SIMULATION OF PROTONATION OF ADSORBED BROMO CRESOL GREEN

Protonation of adsorbed Bromo Cresol Green is a fast, diffusion controlled reaction (Gutman et al., 1983a, Gutman et al., 1981a) (see Table VI), implying that the encounter between the proton and the phenolate ring of the alkaline state is the rate-limiting step in HIn formation; that is, the alkaline form of the adsorbed indicator does not insert both resonating rings into an environment where protons cannot diffuse. T h e acidic state of the indicator is 30-fold more lipid soluble than the alkaline state. T h e mechanistic equivalent of this enhanced stability is an insertion of the neutral, proton-bearing hydroxyl into the hydrophobic environment of the micellar surface (Mukerjee and Cardinal, 1978; Dill and Flory, 1980; Narayana et al., 1982). Thus, we have to account for three populations of TABLE VI Thermodynamic and Kinetic Parameters for Protonation of Bromo Cresol Green Adsorbed on Brij 58 Micelles" kin

-RTln -

Salt

(d) pKob, 0 100, KC1 100, KCNS

6.5 2 0.05 '6.5 k 0.05 6.5 k 0.05

PKS 5.35 ? 0.05 5.5 f 0.05 5.3 ? 0.05

k3

kout

(M-' sec-') 0.65 2 0.05 1.00 k 0.05 0.55 ? 0.05

X X X

10'" 10'" 10'"

kou,

(kcal/mol)

(sec-')

-1.56 -1.3 -1.60

150 ? 50 150 50 100 2 50

*

"The experiment was carried out in the presence of 50pW indicator, 40 mg BrlJ 58/ml, and I d of Z-naphthol-3,6-disulfonate.

76

MEkACHEM GUTMAN

the indicator In,,, HIn(,,, and HIn(,) which are the surface-bound form of In-, the surface-bound form of HIn, and the more hydrophobic location of the proton-bearing hydroxyl [HIn(,,]. (The free forms of the indicator are ignored, as they consist of less than 2% of the total indicator.) The equilibrium between the reacting components is given by reactions (46), (47), and (48).

The correlations between the observed equilibrium constant and the various rate constants are k4 k3 K, Kobs =kin I+K,

-

(49)

kOUt

The corresponding differential rate equations are written with three time-dependent variables: X and Y, as defined before, plus a third one, Z, the increment of HIn(,,. HIn,,, is now given by HIn,,) = Y

-

2

The differential rate equations are:

dX

-=

dt dIndt =

[-k,([Rs-] -

+ [Hf]) -k2

dY dt

k3[Gv(

-

[k3([E+] +

]X

+ kl Y - kl X 2 + kl X Y

[In-l)+ k4]Y - k&Y + k3Y2 + k4Z

(51)

(52)

For simulating the reactions summarized by Equations (51-53), we have to negotiate with three independent unknown variables k3, K,, and

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

77

k,,, (or kin). T o avoid a laborious computer search over a three-dimensional matrix, we tried to estimate the magnitude of k3, the diffusioncontrolled rate constant of protonation of bound Bromo Cresol Green. As discussed by Shoup et al. (198l), the rate constant of a diffusion-controlled reaction of a small ligand with specific site on a macromolecule (ignoring the charge effect) is a function of the site's size. As Neutral Red is comparable in size and adsorbed on the same type of micelle, we can estimate that k3 for Bromo Cresol Green will be similar to that measured for Neutral Red, that is, k3 = 1 x 10'oM-' sec-I. With one variable temporarily determined, we systematically varied the other two. We let the pK, of adsorbed Bromo Cresol Green vary, from the pK measured in water (4.96) up to pKobs, with 0.1 pK unit increments. At each pK value (which sets a certain ratio of kin/kout, Equation 50) we varied k,,, over a wide range (10- lo6) and looked for values that superimposed the simulated curve over the experimental one. Once the simulated curves approximated the experimental one, we varied k3 to ascertain the accuracy of our estimated value; we repeated the search over the pK, and KO,, matrix until superpositioned curves were obtained. T h e result is shown in Figure 36. T h e surface pK for the bound indicator is pK = 5.5, corre-

o 20

40

time

so

80

(psec)

Figure 36. , Experimental results and simulated curve for the protonation cycle of Bromo Cresol Green adsorbed on Brij 58 micelles. The experiment was carried out in the presence of 1mM 2-naphthol-3,6-disulfonate,40 mg/ml Brij 58,57@4 Bromo Cresol Green, l O O m M KCI (pH 7.07). The rate constants used for simulation were k, = 5.0 X 10" M-' sec-'; k3 = 1.0 X M-' sec-'; k,,, = 160 sec-'; X , = 7.75M pK, = 5.51 and pKOb,= 6.5. (-) Experimental; (---) simulation.

78

MENACHEM GUTMAN

sponding to kinkout = 9. The best fit is obtained with k3 = 1 X 10'oA4-' sec-', and k,,, = 150 50 sec-'. The rate constants for this experiment and of those carried out in absence of added salt, or in the presence of lOOmM KCNS are listed in Table VI.

*

B. CLASSIFICATION OF POSTPROTONATION REACTIONS

For the purpose of the coming discussion, we shall assign all systems that undergo a postprotonation reaction into two classes. In class I we include those where the microenvironment stabilizes the nonprotonated configuration, such as the hydrophobic site of dimethyl amino naphthalene in bovine serum albumin (Klotz and Fiess, 1960) or the Neutral Red-Brij micellar system (Gutman et al., 1983a). Class I1 will include those cases where the protonated state is the favored one. In this class, we include the Bromo Cresol Green-Brij micelle, or the 146phistidine of carbomonoxyhemoglobin (Russu et al., 1982). The effect of the stabilization of class-I1 compounds in their protonated state will be analyzed using the Bromo Cresol Green. Protonation of this indicator increases its binding energy from AG = -4.1 kcal/mol, (corresponding with p = 1100) to -6.1 kcal/ mol (a= 30,000). This 2 kcaymol increment is mostly due to the transition of the protonated indicator from the (s) to (c) position (-RT In (kin/ kOuJ = - 1.5kcaymol;Table VI). The remainder ofthe increment (--0.5 kcal/mol) can be attributed to the preferential stabilization of HIn,,, with respect to In-,,). Indeed, the pK, of Bromo Cresol Green is -0.35 pK units (--0.5 kcal/mol) higher than that measured in water (Table Vl). The postprotonation reaction, that is, the translocation of the proton-bearing hydroxyl into a more hydrophobic environment, slows the dissociation of the proton from the adsorbed indicator. We have already demonstrated that proton dissociation necessitates the presence of water molecules which act as proton acceptors (Huppert et al., 1982).Thus, whenever the proton-bearing hydroxyl is inserted into the (c) phase, dissociation is delayed until the indicator resumes its (s)-phase orientation. Consequently, the HIn,,) and HIn,,) have different decay time constants and the ratio of these populations is a function of time and of k;,/k4. Figure 37 demonstrates the effect of the postprotonation event on the following parameters of the observed dynamics: HIn(,), HIn,,), and y2. These parameters were computed for a class-I1compound with the same kinlkOut, pK, and k3 which were measured for Bromo Cresol Green, only that Kin was varied from 10 to lo6 sec-'. In systems were ki,,/k4 > kq,a direct relationship between the microscopicrate constant and the apparent ones can be obtained. The fast

3

3 I

0

=4 L 3-2

-

1

3

20

0

60

40

%* I /

/

/

/

/

/

0

1

3

a*

5

0

1

1

20

1

1

40

1

1

60

1

1

I

I

I

I

C

1

20

I

1

60 time ( p s e c ) 40

I

80

Figure 38. Simulated dynamics of protonation of class-I1 components at varying rations of kin/k4. The simulations were run for the rate constants pK,, pkb,, pH, and concentration listed for the Bromo Cresol Green, Brij 58 system in Figure 36. (A) ki,/kr = 0.1. (B) ki,lk4 = 1.0. (C) kin/kr = 10. (-) HIn total; (---) HIn,,,. The inserts are the semilogarithmic plots of HIn(cota,trelaxation.

80

T H E pH JUMP: PROBING OF MACROMOLECULES AND S O L U T I O N S

81

exchange of the HIn between its two locations allows us to regard HIn,,) as a steady-state intermediate dHIn,,, - d(Y - 2 ) -=o dt dt This steady-state equation leads to the expressions that relate the apparent pseudo first-order rate constants kS(app) and k4(app) with the kinetic rate constants

and their ratio:

kapp) = kapp)

k4/k3

1 + kin/kout

= Kobs

(see Equation 50)

In the case where both kin and kout are fast, the ratio of the apparent forward and backward rate constants is identical with the dissociation constant determined by equilibrium methods. A different situation prevails in the range where the ratio kink4 is very small. Under such a regime, the brief protonation-dissociation cycle will not propagate into the core, and the dynamics will be controlled only by the rate of the surface reactions. For such specific cases there will be a marked difference between the pK,, derived from the kinetic experiment, and pK,bs obtained by equilibrium measurements. Consequently, it is only the accurate kinetic analysis that can furnish the precise description of the system and yields the infcrmation about the events that follow the protonation. The interrelationship between k&s, K,, and the ratio kin/kOut for class-I compounds is derived by replacing reaction (48)by reaction (56). I"(,)

kin

kou,

,

I"@)

(56)

The replacement leads to Equation (57) which substitutes Equation 50 for class-I compounds.

The numerical analysis of the kinetics of adsorbed Neutral Red (Figure 32) yields a value for K , which is identical with the Kobsmeasured under equilibrium conditions. Thus, for this indicator, adsorbed on Brij 58 micelles (kin/kout) 435 nm has a fast rise time, much faster than that measured for P-naphthol in water, and a decay time characteristic for the

Figure 41. Fluorescence emission spectra of P-naphthol (lo@) in water (A), or adsorbed

to Brij 58 micelles (4 rng/ml) excitation at 300 nm (B).

86

MENACHEMGUTMAN

+O-* species. This fluorescence dynamics indicates that, when adsorbed on the micelle, a small fraction of P-naphthol molecules are oriented in such configuration that their hydroxyl protons are in contact with the aqueous phase. The protonation of adsorbed indicator can proceed by two pathways. The proton may come from the same micelle or from an adjacent one. We shall define the products of each pathway by HInl and HIn2 respectively. The magnitude of HIn, is a function of micelles carrying both emitter and detector HInl=fliigN.nBcG.[Micelle] where iip~=[PN]/[MiCelk] and nBcG=[BCG]/[Micelle).f l is an empirical term reflecting, besides controllable conditions (light intensity, pH, ionic strength), the proton quantum yield and the various rate constants involved in the proton cycle (sections V and VI). The magnitude of HIn2 is related with the concentration of the reactants HIn2 = fyiiPN. [Micelle].iiBc~-[Micelle].where f2 is a proportional factor (measured in M-' units) which reflects the same parameters affecting f , plus those which modulate proton diffusion in the bulk. By themselves f l and f 2 are too complex to be interpreted, but their relative magnitudes characterize the nature of the proton transfer in a given solution. The total product of the two pathways is given by HInT = [BCG] [PN]. (fl [Micellel-' + f2). This relationship is given in Figure 42, where the slope of the line equals (fl [Micellel-' + f2). By

Figure 42. The correlation between the occupancy of a micelle by both emitter and detector, with the maximal amount of protonated detector measured after the excitation pulse. The results are drawn according to equation the HIn, = [BCG] [PN] (fl[MiC]-'+f2). The reactants concentrations in the experiment were 40 mg/ml Brij -58 and p naphthol 100-1000 (500 +M, rnicellar concentration). Bromocresol green 50 a.pH 7.5.

m

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

87

repeating the measurements at different micellar concentrations (not shown) f l and f2 were measured as f l = (2.9 0.02) lo-’ and f2 = 1.58 f 0.05 M-I. As long as the product f 2 - [PN] is smaller than fl the dominating pathway is the proton transfer between reactants adsorbed on the same micelle. Each micelle is an isolated system whereas the macroscopic observation is a sum of many singular stochastic events. To exclude the possibility that the proton transfer takes place in the core of the micelle, it was tested whether a soluble, highly hydrophylic acceptor phosphate anion can compete with the indicator for the proton. As demonstrated in Figure 43, phosphate competes with the indicator whether the emitter and the indicator are adsorbed on the micelle or are free in solution. Thus, the protons are formed on the interface of the micelle and are not confined in any kind of microenvironment that secludes them from the bulk. The analysis of these kinetic measurements should employ a stochastic approach. Each micelle can have a discrete integer number of emitter and detector molecules, as given by the Poisson distribution (Miller, 1978; McQuarri, 1963; Miller et al., 1980). Still, as was argued by Vass (1980), the stochastic dynamics will differ from classical dynamics by not more than 15%.Thus, being aware of this Iimitation, we simulated the experimental results using the classical kinetic equations instead of the stochastic formalism. In these simulations,we accounted for the (S) + (C)transition of the protonated indicator, the K , value and kOu, as derived in Equation (50) (see Table VI) and varied the values of the following parameters: kl, k S , Xo, and [In-] until a satisfactory simulation was obtained (Figure 44). The results of a series of such measurements are listed in Table VII. Because of the discrete nature of the measured events, and the averaged

*

-

a1 a2 c23 0.4 0.5 KPI WM

Figure 43. The competition between phosphate buffer and indicator for the pulse-emitted protons. 40pM Bromo Cresol Green, 200p.M p-naphthol. The reaction was measured in water (0)or in micellar system (250pMmicellarconcentration) (Cl).The resultsare normalized for HInmaxmeasured in the absence of buffer.

TABLE VII Amplitudes and Dynamic Parameters of Proton Transfer between @-Naphtholand Bromo Cresol Green Adsorbed on Brij 58 Micelles"

fi,,

micelle

0.2 0.4 0.5 0.6 0.8 1.0 1.5

2.0 3.0 4.0 6.0

[HInI,,, measured

Effective pulse size

Effective indicator concentration

(W)

k l X lo-'' (M-' sec-I)

k3 x lo-'" ( M - ' sec-')

0.05 0.082 0.100 0.1 12 0.171 0.182 0.267 0.366 0.455 0.513 0.652

0.47 2 0.03 0.60 2 0.03 0.60 2 0.03 0.72 2 0.03 1.00 2 0.05 1.25 2 0.10 1.75 2 0.10 3.00 2 0.10 4.50 2 0.20 6.00 2 0.20 9.00 2 0.20

0.50 2 0.02 0.70 2 0.02 0.75 2 0.02 0.80 5 0.02 0.90 2 0.02 1.05 2 0.02 1.05 2 0.02 1.00 2 0.02 1.00 2 0.02 1.00 2 0.02 1.05 2 0.02

2.50 2 0.25 2.75 2 0.25 1.25 2 0.25 1.60 2 0.15 1.50 2 0.10 1.50 2 0.10 1.50 2 0.10 0.90 5 0.05 0.85 2 0.05 0.62 2 0.03 0.57 0.03

20 2 5 20 5 5 17 ? 5 17 2 5 15 k 5 10 ? 1 9.5 2 0.5 7.0 2 0.5 5.5 5 0.5 5.0 2 0.5 4.5 2 0.5

(W)

(W)

*

"Bromo Cresol Green, 50pM; @-naphthol,0.1 -3.OmM (pH 7.4-7.5); Brij 58, 40 mg/ml (500p.M in micellar concentrations).

Figure 44. Experimental results and simulated dynamics of proton transfer between emitter and detector bound to the same micelle. k l = 2.5 x 10" M-' sec-' and 3.0 x 10" M-' sec-' for upper and lower simulated curves; ks = 2.5 X 10" M-' sec-'; h,,, = 150 pK, = 5.35.40p.M Bromo Cresol Green, 200p.M f3-naphthol, 500 p M Brij 58 (micellar concentration).

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

89

output of the calculations, the values listed in Table VII call for further anaIysis to derive the physical interpretation of these figures. The amount of protonated indicator detected after the pulse increases linearly with the average content of emitter (Figure 45).The slope of this line corresponds with a yield of 0.003 H+/P-naphthol (mol/mol) which was successfully transferred to the indicator. As the indicator occupies only 10% of the micelles, the dissociation is undetected in 90% of the events. Thus the true yield of protons is 3% of the P-naphthol present. Only 1 of -30 adsorbed P-naphthol molecules is located in such configuration with respect to the micellar surface that the proton can dissociate, in accordance with the negligible emission of adsorbed +O-*. Considering the low efficiency of proton dissociation, the probability of two protons ejected on the surface of the same micelle is very low. Still, the linear correlation between iipN and probability of proton dissociation affects the apparent indicator concentration. A micelle carrying one indicator and few emitters will always record a single proton transfer, but the probability of such an event increases with the number of emitters sharing the same micelle. This increased probability is translated by the classical kinetic formalism as a higher effective concentration of the indicator (Table VII). As long as the physical properties of the micelle are invariable, a linear dependence of the effective indicator concentration on iipN is expected. The deviation from linearity at iipN > 1 may imply that the micelle vary some of its properties. This consideration becomes

1

2

3

-%em

4

5

6

Figure 45. The dependence of the pulse size on the average occupancy number of @-naphthol.5 0 w Bromo Cresol Green; 5 0 0 M micellar concentration and varying pnaphthol. Data taken from Table VII.

90

MENACHEM GUTMAN

evident once we consider the actual distribution of the discrete number of molecules per micelle given by the Poisson distribution

At ii < 0.4, some 80% of the population consists of micelle with only one P-naphthol molecule. At ii = 1.0, 40% of the population contains more than one emitter and at ii = 3 it is -5%. Thus, the higher is the occupation number the less homogeneous is the observed population. As long as we cannot exclude experimentally any secondary interactions between the adsorbed reactants, or modification of the physical properties of the measured system, it is better to limit the interpretation to low occupancy numbers where only one emitter-detector pair share the micelle. The rate constants we measured under these conditions, both for protonation of the indicator and the P-naphtholate are significantly faster than those measured for the free reactants. The rate of protonation of free P-naphtholate is (1 -C 0.1) X 10'oM-' sec-' and free Bromo Cresol Green reacts with the proton at a rate constant of (4.2 0.1) IO'OM-' sec-' (Gutman et al., 1983; Table IV.). The rate of Bromo Cresol Green protonation by a proton produced on the same micelle is 20 times faster than protonation of bound indicator by a proton coming from the bulk 20 X 10" and 1 X 10'oM-' sec-', respectively (Table VII). There may be many reasons for this fast reaction. The most likely one is the nonrandom distribution of distance between the emitter and the detector; it cannot exceed the halfcircumferenceof the micelle (-60 A).Therole of other factors, such as high proton conductivity of the hydration layer, and the two dimensionality of the trajectory (Adam and Delbruk, 1968)is a subject for further research.

*

VIII. THE EFFECT OF BUFFER ON THE DYNAMICS OF THE PROTON CYCLE The experimental systems described above consisted of the minimal number of components: proton emitter and detector. If one wishes to probe a specific site on a protein, he should consider the presence of a third component-the other proton-binding site on the protein. Any species with which the proton may react, referred to here as buffer, will modify the progress of the reaction and perturb the shape of the observed parameter. As the general strategy is to extract the partial rate constant out of the macroscopic ones, we have to retrace the effect of the partial

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

91

reactions of the buffer and evaluate their effect on the macroscopic parameters. T h e reactions which should be considered are (58)-(63). k2

@OH-40kl

HIn

+ H+

k

&In- + H'

(59)

k3

BH B-'

k6 k5

+ H+

plus direct proton exchange between the reactants. k7

H I n + B--BH ks BH

ho + $0- -$OH

+ B-

k12 + $0- /$OH

+ In-

k9

HIn

+ In-

k11

(63)

The corresponding differential rate equations for X,Y , and Z (the increment of buffer protonation) are given in the Appendix. It is obvious that so many variables make a simulative solution a heroic endeavor unless some of them can be obtained by independent experiments. Considering the fact that any application of the proton pulse in an enzymic system must deal with appreciable buffer capacity of the protein or substrates, I shall demonstrate the general procedure of a simulative solution for a buffered solution.

1. Two-Component Systems: Buffer and Proton Emitter T h e three-component system is first dismembered into two-component reaction systems: +OH + indicator (see Section V) and +OH + buffer. Mathematically, these two are identical and can be simulated as detailed above. Yet, practically, there are basic differences between them. T h e monitoring of the indicator-emitter system is through the increment of HIn(Y), while the buffer-emitter pair must be monitored through the transient absorbance of +O-(X) (see Section V.1). In practice this may limit the study to proton emitters that have an absorption band at a wavelength suitable for monitoring.

92

MENACHEM GUTMAN

The second difference concerns the concentration of the reactants. Although indicator concentration can be reduced to the limit of detection, the buffer concentration cannot be easily manipulated. The buffer capacity is generally imposed on the experimental system. Thus, measurements with the buffer-emitter system are carried out in well-buffered solution. Furthermore, while indicator is a noninformative detector at pH < pK, the behavior of the buffer must be considered both above and below its

PK-

The rate constants associated with the buffer system consist of its acid dissociation (k5 and k6) and its direct proton exchange reaction with the emitter (kg and klo). The latter two can be approximated by measuring the effect of the buffer on the dynamics of $0-at pH 7. The proton transfer between $OH and B- can be mediated through proton diffusion (DH+= 9.3 x lop5 cm2/sec) (reactions 58-60) or through the diffusion collision of the reactants (D = 1 X cm2/sec)(reaction 62). In a well-buffered solution near the pK of the reactant ([B- + BH] >> X O ; pH = pK65 = pK21), the increment of Hf after the pulse will be so small that most or all of the relaxation reaction will proceed through the direct proton exchange pathway. Under these specific conditions, the linear approximations of Eigen (1964)are applicable and lead to a simple expression:

-

kobs =

+ BH) kg($GH +B-) + K H ) vs. (WH +K-)/(@+BH)

klo(@-

(64)

A drawing of hobs/(@should yield a straight line with an intercept k l o and slope of k g [Figure 46). It must be stressed that this is an approximative solution that ignores the contribution of the proton diffusion pathway. This omission leads to a systematic error: The equilibrium constant K l o . gas derived by equilib= 5, whereas the value derived rium measurement is K ~ O=.K21/K65 ~ from the rate constant (Figure 46)is kl,,/kg = 23. The approximative values for k g and k l o can be incorporated into a in order to obtain k5 full simulative solution of the relaxation of $0-, (and k6) and refine the values of k l o and kg. The presence of high buffer concentration has a mixed effect on the relaxation of $0-. At pH > pK,j5, B- will compete successfully with $0- for the ejected protons and will slow the relaxation to its prepulse level. At pH < pK, where most of the buffer will be protonated, it will be a poor proton acceptor and efficient proton donor (by direct proton exchange). At this pH range, the buffer will accelerate the decay of $0-to its prepulse level. This effect is demonstrated in Figure 47 which relates the rate of $0-relaxation with the initial pH.

18x10.

10X10*

I

I

I

I

I

lo

1

1

I

-

I 20

C+PhOH BH+PhO'

Figure 46. The effect of buffer on the rate of 40- relaxation after a laser pulse. 100pM hydroxypyrene trisulfonate, 0.1- lOmM of imidazol. The pH of the experiment was varied between 6.5 to 8.5. Intercept = 1.8 X lo9 M-' sec-'. , slope = 7.7 X 10' M-' sec-'. The reaction was monitored at 445 nm using CW HeCd laser as a probing light.

>

6.0

7.0

PH

8.0

Figure 47. The effect of pH on the rate of +O- reprotonation in an emitter-buffer system. loo@ hydroxypyrene trisulfonate, 2mM imidazol buffer. The reaction was followed at 440 nm using CW dye laser. The continuous line is computed for the emitterbuffer system using the rate constants R1 = 1.6 X 10" M-1 sec-'; k5 = 3.5 x 10" M-' sec-'; klo = 2.1 X lo9 M-' sec-I. Measurements in the absence of buffer (0)or in the presence of 2mM imidazol (m).

93

94

MENACHEM GUTMAN

2. Three-Component System: Emitter, Detector, and Buffer A. SIMULATIVE SOLUTION

A simulative solution for the rate equations (detailed in Appendix) describing a three-component system is possible only if the rate constants are previously measured through binary systems (except those given by Equation 61). But once at hand, simulation is feasible as demonstrated in Figure 48. This figure simulates the transient protonation of Bromo Cresol Green by pulse dissociation of 2-naphthol-3,6-disulfonate in presence of 0.25mM imidazol (pK = 7.0). T h e same simulative procedure was used for describing the reaction in a hydroxy pyrene-Bromo Cresol Green - imidazol system and the characteristic rate constants, needed for superimposing of the simulated curve on the measured one, are listed in Table VIII. As evident from the table, the parameters that really govern the dynamics are those that were determined by high degree of accuracy-the reaction of each component ( I t , , k 3 , and It5) with the free proton. These parameters determine the shape of the simulated dynamics and are critical for superimposing the simulation on the experimental curve.

-.2

0

I

I

1

,766

1

I

I

1

2.297 MICRO SECONDS

I

1.532

a

a

1

L

I

3.063 I

1

I

1

3829

Figure 48. Experimental results and simulated curves for a three component reaction. T h e experimental system consisted of 1mM 2-naphthol-3,6-disulfonate, 20pM Bromo Cresol Green, and 0.25mM imidazol (pH 7.0). T h e simulations demonstrate the effect of increasing the buffer concentration; 0.25mM (upper simulation) 0.475; 0.738; 1; 1.3; 1.6 and 2mM (lower curve).

0.25-4

2.0 4.0

1.o

0.5

7.0

5.73 6.1 6.75 5.65 5.9 6.05 5.54

PH

4

0.7 t 0.05 f 0.2

4 t 0.2 4 f 0.2

1.6 t 0.1 1.6 -+ 0.1

f 0.2

f 0.2

4

4

k3 X lo-'' (M-' sec-')

1.6 f 0.1

1.6 f 0.1

(M-' sec-')

k ] x 10-11

2.0

f 0.1

3.25 t 0.25 2.75 f 0.25 2.75 f 0.25 2.75 f 0.25 3.25 t 0.25 2.75 t 0.25 2.75 f 0.5

k5 x lo-'' (M-* sec-')

OIOOpM8-hydroxypyrene-I ,3,6-trisulfonate, 20M Bromo Cresol Green, and imidazole. * I mM 2-naphthol-3,6-disulfonate, 20@ Bromo Cresol Green and imidazole.

Bb

A"

(M)

Imidazol

1

322 3?1

322

322

k7 X lo-' (M-' sec-')

*

f

1

5 2 2

2.5 1 2.5 t 1

2.5

2.5 t 1

k l o x lo-' (M-' sec-')

The Kinetic Parameters Characterizing the Dynamics of a Three-Component System: Emitter, Detector, and Buffer

TABLE VIII

10

1 cm) is composed of erythrocytes predominantly. Separation of nucleated cells took place within 0.5 cm, underlining the excellent hydrodynamic properties of the device. For comparison, the separation of CBA spleen cells with the use of free-flow electrophoresis is given in the inset of Figure 15 (Zeiller and Pascher, 1973). These authors demonstrated that the highest peak in

172

ABRAHAM TULP

0

0.5

1.0

M i g r a t i o n Distance(cm 1 Figure 15. Electrophoresis of murine spleen cells. Murine (CBA strain) spleen cells (4 x 10’) suspended in 5 ml 1.5% Ficoll are layered on a 3-13% Ficoll density gradient followed by an overlay of 5 ml. Electrophoresis proceeds for 15 min at 250 mA and 4°C. Fractions of 1-2 rnl are taken. For comparison, in the inset the separation of CBA mouse spleen cells by free-flow electrophoresis is given. [Redrawn from Zeiller and Pascher (1973) with kind permission of Dr. K. Hannig and Verlag Chemie.]

the migration profile is composed of B cells whereas the more rapidly migrating small peak is composed of T cells. In view of the almost identical migration profiles obtained by FFE and by the present device, w e may conclude that the resolving power is about equal. Because only 15 min is necessary for separation, the method is rapid. Since the density gradient extends from p = 1.02- 1.05, cells are relatively close to their buoying density so that rather large numbers of cells can be layered without a trace of streaming. In Figure 16, 10’ spleen cells from a GR strain mouse are layered onto the density gradient and electrophoresed €or 25 min at 250 mA. Again, two migrating bands can be seen when the chamber is viewed from the side but there is considerable substructure in the migration profile. In-

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS 173

0

1.o

0.5

Migration Distance ( c m )

Figure 16. Electrophoretic separation of murine (GR strain) lymphoid cells. Unless otherwise stated, cells suspended in 5 ml 1.5% Ficoll were electrophoresed for 25 min at 250 mA at 4°C. Hatched area represents initial cell band width. (*---*) 3.2 x lo' thymocytes; (0-0) 1 x 10' spleen cells are separated by unit gravity sedimentation alone for 25 min; (0-0) 1 x 10' spleen cells are separated by unit gravity sedimentation plus electrophoresis.

deed, different mouse strains show totally different migration profiles Whereas electrophoresis (see Platscoucas and others in Section V. 1C). separates the migrating spleen cells into high-, intermediate-, and lowmobility populations, it is worth mentioning that the cell band under influence of gravity alone remains very thin (Figure 16). At the initial interface of sample layer and density gradient, a jump in viscosity and density occurs using 1.5 to 3% Ficoll70 with the effect that the migrating cell band (by gravity force alone) is compressed by a factor,

rl PP - Po -rlo

PP

- PM

where qo = viscosity of sample layer; q = viscosity of top of the density gradient; po = density of sample medium; and pM = density of top of gradient.

174

ABRAHAM TULP

Whereas the theoretical half-width of the sample layer as loaded is 1 mm, after 25 min sedimentation at unit gravity in the absence of an electrical field it is only 0.36 mm due to the compressing effect of medium viscosity and density (Figure 16). It is this band-width that determines resolving power (Section 111.3). For adequate electrophoretic separations, it is necessary that the unit gravity sedimentation profile be different from that obtained after combined sedimentation and electrophoresis (see our criticism in Section V.1.F). Figure 16 shows the electrophoretic migration of CBA mouse thymocytes. The profile is more or less similar to that obtained by FFE. These T cells have a lower mobility than the B cells from spleen. Low-mobility thymocytes possess the highest 8 antigen content that disappears at the same rate as the negative surface charge increases (Zeiller et al., 1974). These authors suggested the following sequence: thymocytes of low mobility + thymocytes of high mobility + peripheral T cells of high mobility.

3. Analytical Electrophoresis of Cells in Density Gradient Two of the previously described preparative columns can also be used analytically (i.e., Section IV.l.C, Section IV. l.F). A. TRANSANALYZER

An instrument for electrophoresis with continuous optical scanning was developed by Catsimpoolas et al. (1975) and Catsimpoolas and Griffith (1977b). It consists of a cooled electrophoresis cell cassette to accomodate two qLartz columns (0.6-cm i.d., 14.2-cm length). One column carries the sample for analysisand the density gradient. The other is used as reference background. The bottom of the quartz tube is closed by a semipermeable membrane or polyacrylamide gel plug. The quartz tube is coated by methyl cellulose (Dow Chemical Company) to prevent electroosmosis. Buffers are circulated through the upper and lower electrode reservoirs to remove products of electrolysis. The quartz column is moved vertically up and down by a synchronous stepping motor (Catsimpoolas, 1973). Absorbance of light is detected by a photomultiplier as subsequent layers of the column pass the photocell. During electrophoresis, the column is scanned sequentially several times so that a transient electrophoretic analysis of the migrating cells is possible. The device was modified later (Catsimpoolasand Griffith, 1978): the “Transanalyzer” (Bascom-Turner Instruments, Newton, Mass.), contained six removable columns. Five cell samples can be analyzed simultaneously because the columns move side-

DENSITY GRADIENT ELECTROPHORESIS O F MAMMALIAN CELLS

175

ways while a vertical moving light beam scans the columns. Blueprints of these rather complicated devices may be obtained from Catsimpoolas. B. COLUMN W I T H LASER BEAM

A glass tube, 0.5-cm i.d., 11-cm length, treated with Siliclad, was scanned by a laser beam in the device of Lim et al. (1977)by vertical movement of the density gradient column. The electrical field was repeatedly applied for 60 sec (8 Vkm, 10 mA) and then turned off for 80 sec to allow heat to dissipate. C. ELECTROPHORESIS ALONG A DISCONTINUOUS DENSITY INTERFACE

A discontinuous Ficoll gradient was used by Pradac et al. (1978) to transport a front of erythrocytes horizontally along the Ficollbuffer interface in a cuvette of 2 x 0.2 x 4.5 cm3 at 5.5 V/cm. 4. Experimental Conditions A. DENSITY SOLUTE

The solute contributing to the density increment must be: highly soluble in electrophoresis buffer, of a high density increment per gram, electrically neutral, nontoxic to cells, of a low intrinsic viscosity, not, or only slightly, contributing to osmolarity, and easy to remove after electrophoretic separation. Sucrose has been used in a few cases as a solute for zonal electrophoresis of cells (Carty et al., 1975;Gear, 1977)or for isoelectricfocusingof cells (vide infra),but, it is not desirable due to its osmotic effect. A mixed type isoosmotic gradient of sucrose and Metrizamide (Nyegaard, Oslo, Norway) seems more promising. Ficoll M 400,000 and M 70,000 have been the solutes of choice. Catsimpoolas advised dialysis of Ficoll400,OOO prior to its use since it “dramatical1y”reduced cell clumping. No such clumping effects were found with Ficoll70,OOO. Ficoll gradients are transparent and visual inspection of migrating zones often yield as much information as electronic scanning of the column. Physical properties of Ficoll400,OOO are given by Boltz et al. (1973) and those of Ficoll 70,000 by Tulp et al. (1982a). According to Pretlow et al. (1975), prolonged autoclaving of Ficoll solutions (>16 min) promotes depolymerization and gives rise to osmotic pressure effects. According to Loos and Roos (1974),a 10% Ficoll solution has an osmolarity of about 7.5 mOsm. Apparently due to the autoclaving procedure, it was necessary for Boltz et al. (1973)and Griffith et al. (1975) to vary both sucrose and Ficoll concentrations to obtain an isoosmotic

176

ABRAHAM T U L P

density gradient (303 mOsm, from 10% Ficoll plus 5.1% sucrose to 2.5% Ficoll plus 6.35% sucrose in electrophoresis buffer). B. IONIC COMPOSITION

In view of the higher mobilities of cells, buffers of low ionic strength should be used. Buffer capacity should be as high as dilution permits with respect to the stability of cells. Preferentially, ions with low electrophoretic mobilities should be used. Ionic composition as well as the nature of the solute determining osmolarity can be found in a Table of Hannig (1971)depending upon cell type, or in Hannig and Heidrich (1977). Boltz et al. (1973) recommend a buffer of the following composition: 0.2 g KCI, 1.15 g Na2HP04,0.20 g KH2PO4, 0.10 g MgCI2 6H20, 10 g glucose per liter (pH 7.2), specific conductivity 1 mmhokm. This buffer was modified by Griffith et al. (1975) by omitting MgC12 and adding 0.12 g sodium acetate. A buffer, slightly adapted from Hannig and Zeiller ( 1969),containing 18g glycine, 2.24 g triethanolamine, 0.3 g K-acetate, and 7.2 g glucose per liter, pH 7.2 specific conductivity 0.9 mmhokm, is very satisfactory for the separation of lymphoid cells, Tulp (this volume). C. ELECTRODES

The electrodes conventionally used for electrophoresis are noble metal and metal/insoluble metal salt (e.g., Ag/AgCI/Cl-). This electrode requires the installment of barriers to prevent metal ions from entering the separation compartment. Also platinum electrodes, generally used in cell separation studies, must be isolated hydrodynamically from the separation density column to prevent mixing and serious distortion due to electrolytically developed H2 and O2bubbles. In some separation devices (but not yet applied) the use of a less expensive palladium electrode as a cathode might be satisfactory. Palladium dissolves considerable amounts of hydrogen so that no gas is evolved at the cathode and a H2-saturated palladium anode might stop the evolution of oxygen (Neihof and Schuldiner, 1960). D. POWER SUPPLY

Several commercial power supplies for constant current exist. We found a low-cost, regulated power supply (Delta Elektronika BV, Zierikzee, the Netherlands) Type E 060-0.6 for 0-60 V or 0-600 mA entirely satisfactory.

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

177

E. ELECTROENDOOSMOSIS

Due to the surface charge of the separation vessel, waterflow is induced by the electrical force. In the relatively large (cross section) separation columns used for preparative (but not analytical) electrophoresis, electroendoosmosis is very small. Although Rilbe (1977)originally thought that electroosmosis is damped in density gradients, it appeared that at high field strength everywhere at the wall small circulatory liquid movements over very small density intervals occur. These microwhirlpools tend to deteriorate zonal layers and they, rather than heat convection, are the limiting factors for the voltage that can be applied. In cell separation studies, the electrical field strength is only a few V/cm so this kind of disturbance is not operative. Moreover for several types of glass surfaces, coatings have been devised to reduce surface charge and thus electroendoosmosis(Van der Hoff et al., 1977,1978;Van der Hoff and Van Oss, 1979; Van der Hoff and Micale, 1979. Even Perspex (polymethyl methacrylate) may be treated by hydrolysis of the surface in sodium hydroxide followed by neutralization and reaction with Methocel (Van der Hoff et al., 1977).Rilbe (1977)remarks that “even if it were possible to prepare a completely uncharged solid phase, there would be a permanent risk that this complete neutrality could get lost by absorption of solutes with ionogenic groups.”

V. APPLICATIONS

At neutral pH, mammalian cells possess a negative surface charge mainly originating from the carboxylate group of N-acetylneuramic acid residues coupled to glycoproteins. Therefore, cells migrate to the anode.

1. Lymphoid, Blood, and Hemopoietic Cells A. PLATELETS

Platelets of man and rat have been resolved in age groups by Carty et al. (1975). B. ERYTHROCYTES

As prototypic mammalian cells, erythrocytes have been used almost solely to test the resolving power of density gradient electrophoresis columns. To perform a large number of tests, erythrocytes may be fixed by dilute aldehyde solutions (Vassar et al., 1972)to obtain particles that are chemi-

178

ABRAHAM TULP

cally stable and have stable surface charge for several months of storage in physiological saline. In particular, the separation of an artificial mixture of rat, chicken, and rabbit erythrocytes (Boltz et al., 1976; Todd et al., 1979)is a good test of resolving power. Table 111shows the separation of artificial mixtures of various erythrocyte species either on a analytical or preparative scale. The only separation into functional entities has been described by Gear (1977), who obtained a separation of old v. young erythrocytes. Questionable in the experiments is the high ambient osmotic pressure (>889 mOsm) in the density gradient column. C. SPLEEN CELLS

Separation of spleen cells has been studied extensively by Catsimpoolas and co-workers (Catsimpoolas and Griffith, 1975, 1977a,b,c 1978; Catsimpoolas et al., 1980; Griffith et al., 1975, 1976; Platsoucas and Catsimpoolas 1978, 1979, 1980; Platsoucas et al., 1976, 1980). Electrophoretic separation of mouse spleen cells into two visible bands was first reported by Griffith et al. (1975). Athymic nude mouse spleen cells showed only one migration band during electrophoresis (Griffith et al., 1976). Resolution of mouse spleen cells, using analytical quartz and small columns, in at least three distributions after 6 hr migration, was reported by Catsimpoolas and Griffith (1975, 1977a,b, 1978). Evidently resolution TABLE 111 Electrophoretic Separation of Erythrocytes Mixture of erythrocytes

Electrophoresis Analytical Preparative

+ + + Ra,C,R H;R H,Ra(f)lM,Ra H,ghosts R H,C H,H(n) HNf)

+ +

+ + + + + +

Explicitly resolved in

References

R;C R;C;Ra Ra;C;E H;Ra

Boltz and Todd (1979) Boltz et al. (1976) Bronson and Van Oss (1979) Catsimpoolas and Griffith (1975, 1977b) Ra;C;R Gaines et al. (1974) o1d;young eryth. Gear (1977) H ;R/M ;Ra Griffth et al. (1975) Lim et al. (1977) Me1 et al. (1973) Michalik et al. (1980) Tulp et al. (1982a, 1983) Van Oss and Bronson (1979)

Abbreviations: C = chicken; E = equine; H = human; M = mouse; Ra = rabbit; R = rat; (f) = fixed; (n) = neuraminidase treated.

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

179

could have been increased if the sample layers applied to the analytical column were not 2.5 mm high but 1 mm instead. Figure 17 shows that almost complete separation of murine (CBA/ H/TGJ) B and T cells was obtained (Catsimpoolasand Griffith, 1977c)as is evident from the separation of 8-antigen-positive(T)and Ig-positive cells (B), respectively. The high-mobility &positive cells responded in vitro to stimulation by phytohemagglutinin, whereas the low-mobility Ig-positive cells were activated by lipopolysaccharidefrom E. coli. In the intermediate fractions of Figure 17, double negative (for 8 and Ig) was present in a relatively high percentage as can be calculated from Figure 17. These

-s m 0

f

B

60

) .

=

8

40

20

30

40

50

60

70 80 9( fraction number

Figure i7. Electrophoretic separation of murine spleen B and T cells. Spleen cells from a CBA mouse were electrophoresed in Buchler Polyprep apparatus for 4.5 hr at 20 mA and 4°C. [Redrawn from Catsimpoolas and Griffith (1977~)with kind permission of the authors and Plenum Press.]

180

ABRAHAM T U L P

results are entirely comparable with those obtained by FFE (Zeiller and Pascher, 1973; Zeiller et al., 1974; Boehmer et al., 1974). Moreover, Platsoucas et al. (1980) found that BALB/c lymphocytes, responding by proliferation to allogenic (CBA/H/TGj)spleen cells in a mixed lymphocyte culture, were exclusively found in the high-mobility fractions. Whereas two peaks with moderate skewness and little substructure are shown, Figure 17, a variable and large number of peaks were detected in the electrophoretic migration profile of spleen cells derived from C57B1/ 6J mice (Platsoucas et al., 1976). These authors ascribe the variability of peak height and peak number to a removal of certain subpopulations in a nonreproducible manner during cell preparation prior to electrophoresis. Their alternate explanation that the variable subpopulations represent dynamic changes of lymphocytes in individual animals due to exposure to different environmental antigens remains open. Not only a putative variability in individual animals was reported but also a straindependency of the electrophoretic profiles was described by Platsoucas and Catsimpoolas (1978, 1979). They suggest that the surface charge of the cells is genetically determined, although its dependency on the H-2 locus is in doubt. For BALB/c mouse spleen cells as well as for other strains (Platsoucas and Catsimpoolas, 1980),an age-related difference in electrophoretic migration profiles was detected. Regardless of age, a separation of T and B cells was always obtained. Thymectomy had profound effects on the age dependency of the electrophoretic profile. Rat spleen cells could also be separated into two populations (B and T cells, Platsoucas and Catsimpoolas, 1979). Platsoucas et al. (1981b) subjected human spleen cells from patients with Hodgkin’s disease to electrophoretic separation. Of the five (pooled) fractions obtained, B lymphocytes were highly enriched in the low-mobility fractions. At high mobility, enrichment of T p cells was measured with minimal contamination of T y cells. In low-mobility fractions, T y cells were significantly enriched, and lymphocytes of intermediate mobility were considerably enriched as a population of rosette-forming cells with Ripley’s serumcoated human red blood cells. In another experiment, these authors separated human splenic T lymphocytes, after rosetting with neuraminidase-treated sheep erythrocytes, into Tp (high mobility) and T y (low mobility) cells, resolution being comparable to that achieved with cells from peripheral blood. D. PERIPHERAL BLOOD CELLS

In 1976 Catsimpoolas et al. suggested that the very clear-cut bimodal electrophoretic migration profile of human blood cells (derived from a

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

181

Lymphoprep interface) was due to the presence of B and T cells, respectively. Depending on the donor, the bimodality was more or less apparent. Lymphocytes, preincubated for 2 hr in autologous plasma, showed a monomodal distribution skewed positively, and ascribed by these authors to “shedding” of charged membrane components. Ault et al. (1976) made a study of the cellular constituents in these electrophoretic profiles. It appeared that monocytes were skewed toward the faster moving fractions; T cells (Ig-negativecells)were distributed fairly uniformly throughout the electrophoretic column. B cells were 65% pure in the slower moving fractions. The overall recovery ranged from 30 to 50% and since most of the cell loss occurred at the time of dilution in electrophoresis buffer, some care is recommended in the use of this low-conductivity buffer (see also criticism of Pretlow and Pretlow, 1979).Bronson and Van Oss (1979) reported the separation of 96% pure human T cells and of about 57% pure cells in the slowest moving fraction. Platsoucas et al. (1979a,b) made a further subdivision and were able to show that Tp (possessing receptors for IgM), Ty (which have receptors for IgG), and T+ (lacking both receptors) could be enriched electrophoretically from T-cell preparations that were first depleted from monocytes and next rosetted with sheep red blood cells. Of the cells applied, that is not counting the loss due to dilution in electrophoresis buffer, 64% were recovered with a viability greater than 95%. The high-mobility fractions contained 85% pure Tp cells, the low-mobility fraction 45% T y cells, and the intermediate mobility fractions were mostly (70%)T+ cells. This particular separation procedure yields cellular preparations that are not modulated with respect to the Fc receptors unlike rosetting techniques with the appropriate rabbit IgM of IgG anti-ox red blood cell antibodies. Platsoucas ( 1983)applied density gradient electrophoresis to the separation of cells from untreated patients with chronic lymphocyticleukemia (B-CLL).The heterogeneity of separated cells could be subdivided with the use of a panel of surface markers. Lymphocytes able to differentiate into plasma cells were highly enriched in the low-mobility fractions and contained the light chain (K-chain) that was already present on the cell surface before stimulation. Using fluorescence-activated cell sorting, it was further demonstrated that immunoglobulin-positivecells had significant increase of cell-surface immunoglobulin expression in the lowmobility fractions. It thus shows that subpopulations of leukemic B cells in different stages of differentiation and maturation can be obtained electrophoretically. Platsoucas and Catsimpoolas (1978) made the observation that peripheral rat blood cells can be separated by electrophoresis in 80% pure B

182

ABRAHAM T U L P

(Ig+)cells and in almost 98% pure PHA responsive cells, overlapping of B and T cells in the migration profile was minimal. E. TONSILLAR CELLS

Platsoucas et al. (1980) subjected human faucal tonsil cells to density gradient electrophoresis. Low-mobility fractions were highly enriched with B cells and were contaminated only with 10-3076 T cells. T h e high-mobility fractions were comprised of T cells with minimal contamination of B cells. Intermediate fractions contained approximately equal amounts of T and B lymphocytes. Human tonsillar lymphocytes, preenriched by E rosetting, showed a much more narrow migration profile but purification of Tp, T y , and Ta (IgA receptor) cells was not impressive. if achieved at all. F. BONE MARROW CELLS

Platsoucas et al. (198 la,c) showed that electrophoretic separation of human bone marrow cells resulted in significantly enriched colony (in vitro) forming cells in the high- and intermediate-mobility fractions. T y cells were enriched in the low-mobility fractions and Tk cells in the intermediate ones. These authors explained circumstantially that the contribution of unit gravity sedimentation of cells to the final electrophoretic profile of bone marrow cells is minimal. Although they suggested that unit gravity sedimentation contributed only 15%to the migration profile, we interpret their results quite differently. The sedimentation profile (in the absence of electrophoresis) was nearly similar in shape and band width to the electrophoretic profile (their Figure 3). It would appear, then, that the unit gravity profile is only displaced and that, unless fortituitous combinations of sedimentation velocity and surface charge exist that give rise to an almost similar migration profile, one must conclude that cells all having about the same surface charge were not separated better by the electrical force than they already were by the gravity force. Van Beek et al. (1982) showed that, with unit gravity sedimentation of human bone marrow cells, the relatively fast-sedimenting, large cells are highly enriched myeloid precursor cells, the slowest-sedimenting cells are monocytes (unpublished results), and that cells with intermediate velocities are lymphocytic cells. Also Wells et al. (1977) demonstrated that cells of intermediate and high sedimentation velocity are colony-forming cells. These results are in line with those of Platsoucas et al. (1981a) who found that colony-forming cells are in the intermediate and'high-mobility fractions and that the (large) immature myeloid cells dominated the high-

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

183

mobility fraction whereas monocytes are in the low-mobility fractions. Platsoucas et al. (1981~) furthermore showed that in vitro treatment of human bone marrow cells with ubiquitin, but not with thymopoietin, resulted in increased proportions of IgM-bearing cells in the fractions of intermediate electrophoretic mobility. G. THYMOCYTES

Thymocytes from mouse migrated as a single band (Griffith et al., 1975, 1976) and no functional differentiation was recorded.

2. Miscellaneous For demonstration purposes only, Boltz et al. (1973)showed the different migration profile of Chinese hamster cells due to gravity force alone and to the electrical plus gravity force, respectively. Boltz et al. (1976) demonstrated that rat embryonic cells resolved in several electrophoretic subpopulations. The shift in mobility distribution following infection with Herpes simplex virus was ascribed to a depletion of cells from the most rapidly migrating fractions. These authors also showed that human cervical biopsy cells were resolved from erythrocytes and (among others) keratinized squamous cells, a result that can also be obtained by velocity sedimentation at unit gravity. Furcinitti and Hunter (1978) reported on preliminary experiments concerning the electrophoresis of quail femoral medullary cavity cells. Cells from nonestrogenized birds electrophoresed in at least three broad bands, including red blood cells. The highest thymidine incorporation 27 hr after estrogen injection coincided with the peak of large volume cells. BZrjesson et al. (1981) subjected cells from human breast cancer first to velocity sedimentation in a density gradient, and then transferred the whole gradient to an electrophoresis tube. After 6 hr electrophoresis, a correlation between large cancer cells and estradiol uptake was observed. Todd et al. (1981) separated by upward electrophoresis dividing cells of the epithelial cell line T 1;low-mobilitycells were enriched for Gn-phase cells of the cell cycle but this was probably due to the action of the gravity force that pulls larger cells down into the electrophoresis column. Generally, it is more feasible to separate dividing GI-, S- and G2-phase cells by unit gravity alone (e.g., McDonald and Miller, 1970; Tulp et al., 1982~). Human embryonic kidney cells were electrophoresed into a highmobility fraction, possessing the highest level of urokinase and a plasminogen activator, as compared with other fractions (Todd et al., 1981). Todd et al. (1981) deduced from their experiments that a small population of somatropin-secreting cells of low mobility is responsible for the

184

ABRAHAM TULP

production of relatively large quantities of growth hormone in the rat anterior pituitary.

VI. FUTURE PROSPECTS It is hoped that the present review will encourage those active in the field of cell separation to apply low-cost electrophoresis columns to cell-separation problems. In our opinion, much can be expected from the small height and large surface area chamber for rapid electrophoretic separation of cells and cell organelles. Although the device (Section IV.2.1.b) is certainly subject to technical improvements to facilitate operation, the method can open new areas because monoclonal antibodies, reacting with lymphocyte subpopulations under noncapping conditions, may give differential surface charges to various cell classes. These antibody-coated cells may then be separated electrophoretically as in the ASECS- FFE method of Hansen and Hannig (1982). Alternatively, cell surface charge may also be modulated by adhering microspheres coated with specific antibodies (immunospheres) to the cells thus enhancing mobility differences of cells that otherwise have almost equal mobilities (Smolka et al., 1979).

VII. ISOELECTRIC FOCUSING OF CELLS IN DENSITY GRADIENTS 1.

Introduction

In isoelectric focusing (IEF), a “natural” pH gradient is generated by the electrical field increasing from anode to cathode and stabilized by a density gradient (Haglund, 1971).An ampholyte loaded on such a gradient migrates toward a position where its net charge is zero and stays there. The method is essentially an equilibrium method. Whereas ratezonal electrophoresis allows probing of the cell surface to a depth of 1.4 nm beneath the surface of shear, IEF allows probing to a depth of 6-7 nm (Sherbet, 1978). A priori, one may conceive several objections, even leading to scepticism, to the possibility of applying IEF as a method that maintains cellular viability. Just and Werner (1979) and Hannig (1978) in particular have been sceptical. The latter author remarks: “the isoelectric point of cells is not a physical constant as with proteins . . . the importance of cell isoelectric focusing becomes doubtful . . . and cell viability is usually completely

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

185

lost.” And even Sherbet (1978) states that “isoelectric focusing does not appear to be suitable for separation of cells, as we had hoped, due to drawbacks inherent in the technique.” These drawbacks can be listed as follows: 1. Under the ambient conditions of IEF (176 ampholine, LKB Produkter, Bromma, Sweden)extremely low specific conductivity is reached: 0.2 mmhokm (Haglund, 1971)or even 0.1 mmhokm (Just and Werner, 1979) corresponding with a very low “ionic strength” of 0.5- 1 mg ion/ liter (Righetti, 1980), affecting the stability of the cellular plasma membrane. 2. Low pH values per se in the separation column must have a deleterious effect on cells. Erythrocytes (Heard and Seaman, 1960; McGuire et al., 1980) and lymphocytes (Mehrishi and Thomson, 1968) are electrokinetically and irreversibly unstable at low pH. Murine lymphoid cells and Chinese hamster fibroblasts show reversible electrokinetic properties after a brief exposure (15 min) to low pH (Greig et al., 1976).Heard and Seaman (1960) showed that the stability of the erythrocyte membrane at low ionic strength and low pH is so impaired that lysis ensues. McGuire et al. (1980) showed that a combination of low pH and low osmolarity favors lysis of cells. These two factors, low “ionic strength” and low pH, weigh more heavily since IEF of cells requires several hours up to 24-48 hr (Leise and Lesane, 1974). Catsimpoolas and Griffith (1977) observed that cells remain focused after about 1 hr but start to defocus after 2 hr, followed by lysis. 3. It is essential that the ampholyte building up the pH gradient does not form complexeswith cellular membrane components, but actually the LKB ampholine does, among others with sulfated and carboxylated polysaccharides (Righetti et al., 1978; Righetti and Gianazza, 1978, 1980; Gianazza and Righetti, 1978).A model for this binding has been proposed by McGuire et al. (1980). The protonated nitrogens, separated by two methylene groups, in the backbone of the carrier ampholyte are thought to bind to sialic acids or sulfate groups attached to the carbohydrate chains of membrane glycopeptides. Moreover, according to Galante et al. (1975), carrier ampholytes form chelates with M2+ metal ions. 4. Insertion of cells at low pH may result in the extraction of cellular proteins. For IEF of HeLa cell metaphase chromosomes, it was found that stripping of basic proteins occurred in the pH gradient (Landel et al., 1972). In this respect, Boltz et al. (1977) and Hammerstedt et al. (1979a) noted that the focusing pH value of sperm cells was related to the pH of sample injection, certainly due to the extraction of basic proteins at low pH. Also sperm became immotile after suspension in electrofocusing

186

ABRAHAM TULP

media (Hammersted et al. 1979a). Despite the fact that PIS of cells are reproducible, they must be considered with caution. 2. The Lack of Theory A reliable theory that predicts IEP of cells based on the pK of charged membrane groups and on their stoichiometry is seriously lacking. When the PI of a cell is not a physical constant, it is even not worthwhile to develop such a theory. Phenomological studies would at least indicate the factors that are involved (Heard and Seaman, 1960).The cell (as a macropolyanion) can be considered as an analog to an amphoteric amino acid. The PI of a cell may be defined as the pH where the number of protonated basic groups is equal to the number of protons dissociated from the acid groups, thus leading to zero surface charge and electrophoretic immobility. Based on very simplified assumptions (Thompson et al., 1978) that the negative surface charge depends on a glycosidic carboxyl group GCH (pK = 2.6) and the positive charge on amino groups NH: (7.6 S pK S 12.5), the following relations hold: GCH NH,f

F=

GC- + H+ NHn-l + H+

in which

The ratio P = total glycosidic carboxyl residues over total N residues can be expressed as (20)

at pH = PI, [GC-] = [NH,f] and solving Equation (20) for [H']

The PI ofE. coli is given as 5.6 (Sherbet, 1978),nearly the same as the PI of an erythrocyte (Just and Werner, 1979). In E. coli the ratio of negative to positive charges is 2 (Sherbet, 1978), whereas in erythrocytes this ratio is as high as 25. Whatever the pKs of membrane groups for the two cell types may be, Equation (20a) will not yield high values of pI for these cell types, as close inspection of Figure 18 shows. Theory and experimental values at present

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS 4-

I

t

-a

187

I

I 1

I

I

I

2

I

3

’

I

- ’ 1

4

25

P

Figure 18. Theoretical dependency of isoelectric focusing pH (PI) on the ratio of glycosidic carboxyl residuedtotal ‘“”-residues (P). Equation (20a) was applied.

clearly exclude one another. It may well be that at low ionic strength (ambient conditions during IEF) charged groups become “visible” that are not detectable at high ionic strength and low pH. Treatment of the cell-surface charge in the manner of the “smeared site” model for a rigid impenetrable sphere at finite ionic strength (Linderstrflm-Lang and Nielsen, 1959) might bring insight into this problem. Sherbet (1978) derived a formula that connects electrophoretic mobility of a cell at pH 7 with its IP, and for several cell types a good fit was observed. The human erythrocyte however falls completely out of this particular relationship. Righetti et al. (1980) consider this lack of good fit makes the theory suspicious. 3. Applications Being an equilibrium method, cells can be added to the separation column as a zonal layer or may be mixed throughout the gradient at the time the density gradient is introduced into the column. In the latter case, focusing times are extremely long because building up of the pH gradient is a slow process. On banding of the cells at their PI, the banding capacity of the rather weak density gradient can be exceeded according to Mason’s equation (Equation 4 in Section 111.1) and streaming phenomena may occur. Floating of individual cells (at a gradient density > density of the cell) on the other hand is unlikely to occur since this buoying movement at unit gravity is exceedingly slow, as calculation shows.

W

-

3T3 SV-3T3 SV-BHK Py-3T3 3T3+formaldehyd 3T3+ethyleneimine SV-STS+forrnaldehyd SV-3T3+ethyleneimine Butter yellow-induced rat hepatoma

b

Yoshida ascites Ehrlich ascites HeLa y-Globulin-treated Hela Polyoma-transformed B HK Rat liver

a Rabbit: Peripheral lymphocyte Thymocytes Human: Peripheral lyrliphocyte

Cell type

+

Ficoll 400, isoosm.

+

Sucrose, hyperosm. Ficoll 400, isoosm.

+

Sucrose, hyperosm.

+

Dextran 40, hypoosm.

Gradient solute osmolarity

4.6 4.8 4.66 4.78 4.05 8.2 4.1 8.58 4.6

6.4 -5.11 6.5 -5.05

6.35-4.79 5.6 -4.73 6.85-5.32 6.36-4.85

Several peaks

Several peaks Several peaks

Focusing pH (PI)

24

48

Time (hr)

Isoelectric Focusing of Mammalian Cells in Natural pl3 Gradients

TABLE IV

Sherbet (1978)

Sherbet et al. (1972)

Leise and LeSane (1974)

References

CD

oo F

Peripheral lymphocytes (rat) Chicken embryonic: kidney muscle neural retina pigmented retina liver heart brain fibroblast Rat embryonic: kidney liver fibroblast adult rat liver Chicken: bursa thymus Rat embryo Rat ernbryo+HSV Boar sperm(atozoa) Sperm.-seminal vesicles Sperm. t seminal plasma Bull sperm. Rabbit sperm id. off-season Ram sperm:

Meningioma Fetal brain

Human astrocytoma class I1 to IV

Ficoll 400, isoosm.

Ficoll 400, isoosm. t

+

Ficoll400, hypoosm (?)

t

Ficoll 400, isoosm.

Idem

Idem

Sucrose hyperosm., HEPES/MES

+

Ficoll400, isoosm.

+

Ficoll 400, isoosm.

3.18 4.52-4.69 4.47 4.62 6.5 4.5 5.8 5.0 6.8 5.05

4.7 4.8 4.58 4.48

4.35 4.28 4.61 3.89 4.60 4.20 4.38 4.1

4.73 4.38 4.6-3.8 T and B cells

from 4.4 to 5.05

3

2-4 B

3 B 2 E

4.5

25

0.5 E

(Continued)

Hammerstedt et al. (1979a)

Moore and Hibbit (1975)

Thompson et al. (1978)

Rani et al. (1982)

Rao et al. (1979)

Rao (1978)

Hirsch et al. (1977)

Sherbet and Lakshrni (1974)

CD

~

~

citratekitric acid ~~~~~~

Ficoll 400, isoosm.

1

Ficoll 400, isoosm.

t

I

3.5 and 4.7

6.6, 7.5 5.5

4.3 5.2 5.5

4.20

5.2 4.7 4.8

AUXILIARY

SCALE

,-STEM

d

f

J

/

d

IMMERSION LINE

\

I

I

?

J

MAIN SCALE

t

1

Figure 7. Features of liquid-in-glass thermometers.

294

R. L. BERGER, T. R. CLEM, v. A. HARDEN, AND B.

w. MANGUM

only scale. The auxiliary scale is a short scale, including within its range a reference temperature point such as the ice point, which provides a means for checking the calibration of the thermometer. This scale is added when a suitable reference temperature is not included in the range of the main scale. The expansion chamber is an enlargement at the top end of the capillary bore, having a volume equivalent to not less than the volume of a 20-mm length of the capillary. The expansion chamber is provided to prevent the build-up of excessive pressures in gas-filled thermometers as the thermometric liquid advances toward the top of the scale. The contraction chamber is an enlargement of the capillary bore which reduces a long length of capillary or prevents contraction of the entire liquid column into the bulb. This chamber is introduced below the main scale or between the main scale and an auxiliary scale. The accuracy attainable with a liquid-in-glass thermometer is limited by the characteristics of the thermometer itself. Instability of the thermometric liquid, nonuniformity of capillary bore, and inaccuracies in scale graduation are the important factors. Uncertainties in corrections for the emergent stem may greatly limit the accuracy of partial-immersion thermometers. Generally, partial-immersion thermometers are assigned an uncertainty of +0.3"Cin their calibration, whereas total immersion thermometers may have an uncertainty as small as +O.O3"C.Observer errors add to the uncertainty but with care these can usually be made relatively small. Since a considerable amount of material has been written about the proper calibration and use of liquid-in-glassthermometers (Wise, 19'76), we will not elaborate on all the corrections that must be applied to the indicated temperature values obtained in measurements.

2. Dial Thermometers There are many types of dial thermometers, including liquid, liquidvapor, or gas filled, and their use is very widespread. These thermometers usually consist of a bulb connected via a capillary tube to a Bourdon tube, which is attached through some mechanism to a movable pointer on a scale. The liquid most commonly used in dial thermometers is mercury. Their inaccuracy may range from 2 to 5°C; however, their reproducibility is usually considerably better than that.

3. Bimetallic-Strip Thermometers The principle of the expansion of a solid is employed in bimetallic-strip thermometers, which are comprised of strips of two different metals bonded together, side by side. Since the metals selected for this use have

TEMPERATURE MEASUREMENT I N BIOCHEMISTRY

295

different coefficients of expansion, the bimetallic strip will bend with a change in temperature and this can be used to move an indicator along a scale or to open and close electrical contacts. The latter operation forms a thermostat for use with a furnace or oven. The inaccuracy of bimetallicstrip thermometers may be as much as several degrees Celsius (2-5°C) but their reproducibility can be better by a factor of 10. 4. Gas Thermometers

The most frequently and conveniently used gas thermometer is the constant-volume thermometer, which utilizes the changes in pressure to indicate changes in temperature (Guildner and Thomas, 1982). Another type of gas thermometer is the dielectric-constantgas thermometer (Gugan and Michel, 1980). Measurements in this case depend on the change of the dielectric constant with gas density and, thus, are intensive in their nature, in contrast to the extensive quality of regular gas thermometry. A reference temperature is required for the operation of gas thermometers. Recent gas thermometry results (Guildner, 1980) have uncertainties of about 0.002"C associated with them, but such small uncertainties are very difficult to obtain.

5. Resistance Thermometers There are many different types of resistance thermometers, with each type being most suited for use in a particular temperature region. Included among the resistance thermometers are platinum, copper, nickel, rhodium-iron, and the semiconductor thermometers (arsenic-doped germanium, thermistors, carbon). The quality of each of these types of thermometers varies with the method of its construction. The reproducibility of the highest-quality thermometers of each of the above-named types can be as good as +O. 1 mK in their appropriate temperature range. The industrial-grade-qualityresistance thermometers may be reproducible to only a few tenths of a kelvin.

6. Thermoelectric Thermometry Thermocouples are used for measurements of temperature from a few millikelvins to above 2800°C. The basic thermocouple (Guildner and Burns, 1979)consists of two wires of different materials that, when joined together at one end (the hot junction, say) and connected to a voltage measuring instrument at the other end (the cold junction, say), will produce a voltage that will be a smooth function of the temperature difference between the two junctions. Various pure elements and alloy combinations can be used to form couples that are best suited for particu-

296

R. L. BERGER, T. R. CLEM, v. A. HARDEN, AND B. w. MANGUM

lar temperature regions. The most common types of thermocouples are listed in Table 111. Thermocouples are probably the most widely used industrial thermometers because they are inexpensive, rugged, longlasting, and suitable for continuous recording and/or control of temperature. Since thermocouples can be made very small, they can respond rapidly to fast changes in temperature. Because of the nature of thermocouples, an uncertainty of 2O.l"C is about the best that can be attained, even with calibrated thermocouples.

7. Radiation Thermometers Radiation thermometers were developed for measuring temperatures higher than 1064°C; they have the advantage that they are noncontact thermometers. Optical pyrometers measure apparent temperatures of objects by comparing the radiation from the objects over a narrow wavelength band with that of a standard, preferably using a photoelectric detector for the comparison. Corrections for the emissivity of the source must be made to determine the temperature; the preceived temperature may be, and usually is, lower because all of the heat is not radiated. Total-radiation pyrometers measure the whole spectrum of energy radiated by the source. They are less accurate than optical pyrometers but can measure much lower temperatures (of the order of 100°C). This type of pyrometer also requires emissivity corrections. Another technique utilizing radiation from an object as a means of measuring its temperature is that of thermography, that is, the mapping of surface temperature distributions over extended areas. This is fairly widely used in the medical field for the detection of tumors near the surface of the skin and in industrial applications for detection of hot spots, such as defective insulators on power lines, defects in furnace walls, and areas of heat leaks in buildings. Usually, comparison measurements, rather than the actual determinations of temperature values, are made in thermography. Resolutions of 0.05"C to 0.1"C are attainable under the best conditions. Another noncontact technique for measuring high temperatures involves Raman spectroscopy, in particular the nonlinear process known as coherent anti-Stokes Raman spectroscopy (CARS) (Radiation Thermometry, 1982). This technique is finding practical applications in measurements of temperatures of flames (in internal combustion engines, in jet engines) and of hot gases. The imprecision of such temperature measurements is generally a few percent. Recently, a fluoroptic thermometer (Wickersheim and Alves, 1979; Cheng, 1981) has become commercially available. The temperature sensor is comprised of a rare-earth phosphor or mixture of phosphors which,

TABLE 111 Thermocouple Characteristics Thermocouple combinations Type designation"

Temperature range ("C)

B

0 to 1820

E

-270 to 1000

3

-210 to 1200

K

-270 to 1372

R S

T

Materials Platinum-30% rhodium v. platinum-6% rhodium Nickel-chromium alloy v. a coppernickel alloy Iron v. another slightly different copper-nickel alloy Nickel-chromium alloy v. nickelaluminum alloy Platinum-13% rhodium v. platinum. Platinum- 10% rhodium v. platinum. Copper v. a copper-nickel alloy.

-50 to 1767 -50 to 1767 -270to 400

SINGLE-LEG MATERIALS

... N

The negative wire in a combination ...P The positive wire in a combination Platinum-nominal 6 wt% rhodium BN Platinum-nominal 30 wt% rhodium BP EN or TN A copper-nickel alloy, constantan: Cupron? Advance: Thermokanthal JN'; nominally 55% Cu, 45% Ni; often referred to as A d a m Constantan EP or KP A nickel-chromium alloy; Chromel," Tophe1,b T-1: ThermoKanthal KP"; nominally 90% Ni, 10%Cr A copper-nickel alloy similar to, but not always interchangeable with, EN and JN TN; SAMA specification Iron: ThermoKanthal JP," nominally 99.5%Fe JP A nickel-aluminum alloy: Alumel," NiAl,b T-2,d ThermoKanthal KN"; KN nominally 95% Ni, 2% Al, 2% Mn, 1% Si High-purity platinum RS,SN Platinum-13 wt% rhodium RP Platinum-10 wt% rhodium SP Copper, usually Electrolytic Tough Pitch TP The use of trade names does not constitute an endorsement of any manufacturer's products. All materials manufactured in compliance with the established thermoelectric voltage standards are equally acceptable. "The letter type, e.g., Type T, designates the thermoelectric properties, not the precise chemical composition. Thermocouples of a given type may have variations in composition as long as the resultant thermoelectric properties remain within the established limits of error. 'Registered trademark-Wilbur B. Driver Co. "Registered trademark-Kanthal Corp. dRegistered Trademark-Driver-Harris Co. "Registered Trademark-Hoskins Manufacturing Co.

297

298

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

when excited by the appropriate radiation, exhibit fluorescence and, in addition, have that fluorescent output concentrated into a few sharp emission lines that can be easily separated with narrow-band interference filters. The fluorescence of each of the phosphors has some lines that are a function of temperature and some that are not. The separation, detection, and calibration of the ratio of the temperature-sensitive to the ternperature-insensitive fluorescent lines yield a thermometer over a given temperature range. By taking the ratio, the thermometer becomes essentially independent of the exciting-source fluctuations in output. The exciting (input) and the fluorescent (output) radiation are conducted from and to an optoelectronic device by means of optical fibers. This provides the very important feature of electrical isolation. These thermometers must be calibrated. The manufacturer-quoted imprecision is 20.1"C for a 1-sec measurement time. The probe size is less than l-mm diameter. Although a wide temperature range of operation is possible, the commercial units have a range of from -50 to 200°C.

8. Noise Thermometers Johnson-noise thermometers (Ohte and Iwaoka, 1982) are based on the measurement of the Johnson-noise power or Johnson-noise voltage, and they are now commercially available.Johnson (i.e., thermal) noise voltage is the small fluctuating voltage generated in any electrical conductor by the random motion of the electrons (charged particles). The extent of the motion of the electrons is a function of temperature and, thus, the voltage generated is related to thermodynamic temperature. Noise thermometers may be used from temperatures of a few millikelvins to over lOOO"C, but the commercially available instruments are not suitable for use over this entire range. The inaccuracy of noise thermometers is typically 20.2 to +0.5%, although in specialized cases, they can be considerably better. 9. Resonance Thermometers

Thermometers that are based on the temperature-dependent, resonant frequency of a material are very attractive because the quantity measured is frequency. One such thermometer is the nuclear quadrupole resonance (NQR) thermometer (Ohte and Iwaoka, 1982; Utton, 1967; Ohte et al., 1979; Utton and Vanier, 1976). Nuclear quadrupole resonance is just nuclear magnetic resonance in the absence of magnetic fields. Nuclei with spin I 3 1 posses electric quadrupole moments that, through interactions with electric field gradients produced by valence electrons and by the surrounding ions in the crystalline lattice, cause a splitting of the nuclear energy levels in the absence of a magnetic field. It is the temperature

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

299

variation of these splittings, and hence of the resonance frequency, that gives rise to NQR thermometry. An excellent feature of the NQR thermometer is that the thermometric property involved is a fundamental property of a substance, a unique frequency-temperature relationship that must be established only once and is always thereafter applicable for that substance.Thus, once the frequency -temperature relationship has been determined for a suitable sensor material, such as KC103, that calibration will apply to all other samples of that material provided that the material has been prepared with consistent purity. This, then, eliminates the need to calibrate each thermometer individually as is required for most practical thermometers. Another advantage is that frequency can be easily and accurately measured and the thermometer can be easily made a part of an automated system for temperature monitoring and control. Through the use of standard frequency broadcasts by NBS, the accuracy of the frequency counter used in making measurements can be easily checked. The interaction of the nuclear electric quadrupole moment of 35Cl (1=3/2) of KC103 with the nonhomogeneous field, produced mainly by the valence electrons but with some from the surrounding ions, produces a splitting of the I = 3/2 energy level into two energy levels, each degenerate with respect to the sign of the magnetic quantum number, mz.These energy levels are separated by an energy hv where the frequency v is given by

where e is the electronic charge, (2 the nuclear electric quadrupole moment, q,, the component of the electric field gradient tensor along the principal axis, and h is Planck's constant. The effects of an asymmetric electric field have not been considered here. It has been found that fluctuations in the orientation of the electric field gradient tensor due to torsional vibrations of the C103 group of KC103 account for the temperature dependence of the NQR frequency at low temperatures through changes in the values of the qu. Above 80 K, however, this does not adequately account for the variation since an expansion of the lattice occurs and the molecular vibrations cannot be approximated by harmonic oscillators. As the lattice expands, the distance between the ions increases, causing an additional decrease of qu and an increase in the sensitivity of the thermometer. For KC103,the NQR of 35CIhas been studied from about 10 to 470 K. It was found that the resolution and accuracy of temperature measurements can be about 2 1 mK in the range from 50 to 470 K. Since the width

300

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

of the 35ClNQR line is approximately 500 Hz, at a resonant frequency of 28 MHz, a determination of the center of the line to only 1% (or 5Hz) gives 1 mK sensitivity. The reproducibility of pure strain-free samples of KC103 is d 2 1 mK near 300 K. Over the entire range of 10-470 K, the inaccuracy of a temperature measurement using this thermometer varies from about 1 to 10 mK, depending on the temperature. A commercial NQR thermometer is available and it is based on the NQR of 35Clin KC103, which has been the substance most studied. Another resonant-frequency thermometer is the quartz crystal resonator (Benjaminson and Rowland, 1972), which, if the crystal is properly cut, is quite linear from about 190 to 525 K. Although this thermometer has excellent resolution, it does exhibit hysteresis and drift. The principle of quartz crystal thermometry is based on the temperature dependence of the piezoelectric resonant frequency of a quartz crystal wafer of a given dimension. The angle of cut of the quartz crystal is selected to give as nearly a linear and yet sensitive correspondence between resonant frequency and temperature as possible. This angle of cut is referred to as an LC (linear coefficient) cut. The temperature sensitivity of the quartz crystal thermometer is about 1000 HzPC. Quartz crystal thermometry is naturally digital; hence, it is independent of lead resistance and does not require four-lead measurements. The resolution of the quartz thermometer depends upon the "counting period" or the integration time; for example, the least significant digit of a l-sec period corresponds to O.OOl"C, of a 10-sec period to O.OOOl"C, and of a 100-sec period to 0.00001"C. The inaccuracy of the quartz crystal thermometer is +O.04O0Cfor the temperature range from -50 to 150°C and +O.O75"C for the range from -80 to 250°C. The short-term stability is 5 X 10-6"C. The long-term stability of a quartz probe is +O.O03"C per month. The major source of error for absolute accuracy measurement is hysteresis of the quartz probe: st0.05"C from -80 to 250"C, *O.O2"C from -50 to 150"C,and ?O.OOl"C over any 10%span from -20 to 120°C. This frequency hysteresis of the probe is produced through mechanical coupling to leads and other effects.

10. Diode Thermometers Semiconductor p-n junction diode thermometers (Swartz and Gaines, 1972; Verster, 1972; Ohte et al., 1982)are becoming widely used throughout the range from liquid helium temperatures (1 K) to about 200°C. The diodes are currently made of germanium, silicon, or gallium arsenide. These thermometers are based on the principle that for forward-biased

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

30 1

p-n junction diodes, the current varies approximately exponentially with V / T ,where V is the voltage and T is the temperature. At constant current, the junction voltage decreases approximately linearly with increasing temperature. The emitter-base junction diodes of transistors have also been used as thermometers. The long-term reproducibility of diode thermometers is claimed to be kO.0 1°C or better. The instability problems that existed previously are claimed to have been solved. The short-term reproducibility is +O.OOl”C.

11. Liquid-Crystal Thermometers Liquid-crystal thermometers (Rozzell et al., 1974) have come into fairly widespread use in recent years. These thermometers rely on the change in wavelength of reflected light with a change in temperature. The liquid crystals or the liquid-crystal mixtures are selected such that the wavelengths of the reflected light are in the visible region of the spectrum, giving rise to a change in color with a change in temperature. T o serve as thermometers, the liquid crystals must be in their cholesteric phase, and their temperature range of operation is from about 0 to about 70°C. Liquid-crystal thermometers exhibit hysteresis, and care must be exercised in their use to prevent organic solvents from coming in contact with them.

12. Comments Each type of thermometer discussed above has its particular advantages and disadvantages and the selection of a thermometer for a given application must be based on the requirements of that application. Among the considerations that influence the choice of the thermometer are accuracy, sensitivity, reproducibility, size, temperature range, speed of response, durability, and cost. V. MODERN ELECTRONIC METHODS OF SENSOR MEASUREMENT

The great advances that have been made in analog and digital circuitry in the last 10 years have revolutionized our ability to make compact packages that will make measurements on virtually any sensor without “loading” or disturbing the sensor output. The type of instrument needed depends upon the sensor being used. In this section we discuss electrical resistance measurement devices for resistance temperature detectors (RTDs) such as thermistors and platinum resistance thermometers, and voltage mea-

302

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

surement devices for thermocouple sensors. There have been many publications that have addressed the classical methods of precisely measuring resistance (Wheatstone bridge) and voltages (potentiometers) (Sturtevant, 1973) and also (Laws, 1918). Consequently,we will not be concerned with those methods; instead, we are describing the types of instruments that a reader may find available commercially. We are mostly concerned with what could be termed “turn-key” systems that require minimal effort on the part of the user to obtain reliable temperature data. 1. Resistance Measurements

Assuming that the potential user has decided which type of RTD is to be used, the requirements for the readout instrument need to be assessed. The simplest and cheapest way to determine the temperature of an RTD is with an ordinary VOM (volt-ohm-miliammeter).This method, while cheap, is not very satisfactory for most applications. A VOM may apply a sufficiently large current to the sensor while its resistance is being measured to produce significant errors due to self-heating of the RTD. Also, this technique yields a value of resistance, and, therefore, a calculation must be made or a conversion table consulted to obtain a value for the RTD temperature. The classical methods of using bridges are cumbersome and, in addition, give only a value of resistance as a result. The accompanying table (Table IV) lists some representative RTD thermometers that are available commercially. This table includes examples of the simplest, cheapest units and also of the more expensive, more sophisticated units. Most instruments available today feature digital display, and since there is very little difference in price between analog and digital meters, there is littlejustification for selecting an analog readout, except for the case in which an instrument functions as a null detector. In addition to these ready-to-use thermometers, there also are available high-resolution, low-excitation power ac resistance bridges that can measure RTD resistances with moderately high accuracy (Berger et al., 1974). Such instruments cost about $2500. Since such instruments measure only a resistance value, the resistance-temperature (R vs. T ) relationships for the RTD must be known accurately to allow the temperature value to be accurately known. An example of this type of instrument is the Model LR-I 10, available from Linear Research, Inc., San Diego, California. A number of handbooks provide a further discussion of more modern bridges. (Handbooks).

2. Voltage Measurements If a decision has been made to use a thermocouple as a sensor, there is a wide range of voltage-measuring devices available from which to choose

Cole-Parmer

United Systems Corporation John Fluke Manufacturing Co.

-200 to +800°C

-190 to +750°C

2180A

K-8129-10

-100 to +600°C

5530

1°C

0.01"C

0.1"C

k 1.25%

+O.l"C

(- 100 to +400"C)

20.3"C

PLATINUM RESISTOR TYPE

2 10

995

860

618

20.15"C

0.05"C

0 to 51°C

46TUC

435

kO.25"C

20 to 43°C

49TA

Yellow Springs Instrument Co. Yellow Springs Instrument Co.

0.01"C

0.01"C 0.01"C

- 10 to + 110°C -10 to + 6 0 T

1006

Hart Scientific

1925

k0.04"C (- 100 to +250"C)

5000

0.01"C

- 183 to +650°C

4200

56-92

784

kO.45"C

~

Special characteristics or comments

~

85

Small, hand-holdable size Battery operation 4-Digit LCD display

142- 168 Analog recorder output 4-Digit LED display 60-600 5-Digit LED display IEEE-488 Bus capability Available, multiprobe accessory available

Recorder output 4-Digit LED display 125- 160 IEEE-488 Capability available 6-Digit LED display 800 IEEE-488 Capability available Recorder output 6-Digit LCD display 25- 100 Portable battery powered 4-Digit LCD display 25- 100 6 Input channels, recorder output, analog meter

Probe

Price U.S. Dollars Basic unit

Inaccuracy

~

k0.05"C k0.03"C

0.01"C

-30 to +lOO°C

5510

United Systems Corporation Instrulab, Inc.

Resolution

Temperature range

Model no.

Manufacturer or distributor

RTD Types, Thermistor Type

TABLE IV

304

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

to observe the thermocouple output. As with the RTD, a simple and inexpensive readout system could be obtained using a millivoltmeter or microvoltmeter. This method, however, would be unsatisfactory for most applicationsfor several reasons. Since most thermocouples produce about 20-50 pV/"C, a very sensitive meter would be needed. In addition, the user would have to consult a conversion table to determine the temperature difference, and the value of the temperature being measured could, in the best of circumstances, never be more accurate than that of the reference junction. It is possible to use an ice bath for the reference junction (see previous section). The classical method of using a potentiometer is very cumbersome and the result is a voltage, which presents the same problems as before. Table V lists some representative thermocouple thermometers that use voltage references. They are available commercially, are convenient to use, and will provide respectable resolution and accuracy. The accuracy specifications given in Table V pertain to temperature uncertainties on an absolute basis. For making relative or differential temperature measurements, such as in calorimetric applications, much higher-precision measurements are possible. Using a microvoltmeter or nanovoltmeter with appropriate low-noise and low-drift characteristics, it is possible to measure temperature differences in the microdegree range. Several electronic test instrument manufacturers produce units that are usable in this mode, an excellent example of which is the Keithley Model 181 Nanovoltmeter. Under the best conditions, this instrument can distinguish a change of 10 nV and has an input noise level of 30 nV peak-to-peak. These figures represent millidegree changes for typical thermocouples. Another class of voltage-output thermometer sensors is the silicon integrated-circuit temperature sensor, such as the National Semiconductor LM135. These devices function as Zenor diodes with a breakdown voltage directly proportional to absolute temperature at + 10 mV/K. Their inaccuracy is of the order of 2 1°C over a range of -55 to + 150°C when calibrated at +25"C. The voltage may be measured with an ordinary 3+ 1/2-digit digital voltmeter (DVM); the accuracy of the reading will be limited by the sensor accuracy. A similar thermometer is the Analog Devices AD590 Integrated Circuit Temperature Transducer. This device produces a constant current of 1 pA/K with an absolute accuracy of &0.05"Cover a temperature range of -55 to + 150°C.This current can either be measured directly by a DVM which has a low current-measuring capability, or the current can be used to develop a voltage across a precision resistor of high resistance and then that voltage measured with a 34-digit DVM.

u1

0

B,EJ,K, R,S,T

2 190A

2780-02

John Fluke Manufacturing Co.

United Systems Corporation

J,T,E,K

B,E,J,K, R,S,T

.

2165A

2T

BT

John Fluke Manufacturing Co.

K-8507-20

Cole-Parmer

2K

K-8505-20

870

Keithley

K-

Type

Cole-Parmer

K-8520-40

Model no.

Cole-Parmer

Manufacturer or distributor

0.1"C 1"C

0 to + 140°C Oto+400"C

-180 to +1372"C

-128 to +2471"C overall

0.1OC

0.1"C

1°C

1OC

1"C

-40 to 1999°C

+90 to + 175°C 0 to + l0O0C -100 to +20°C +25 to +45"C -200 to +2328"C overall

0.1"C

1°F

Resolution

-40.0 to 199.9"C

-60 to +2000"F

Temperature range

20.7"C

TYP +0.2 to 0.4%

r0.20"C TYP 2 1 to 22°C

2 1°C

r0.2% of full scale 2 1 digit

+- 1% of reading -+- 1 digit 0 to 1100°F ?0.25% of reading + 1°C

Inaccuracy

Thermocouple-Type Thermometer Systems

TABLE V

520 + 50 min for probe 995 + 50 min for probe 451 + probe

475 + 30- 110 for probe

495 + 30- 110 for probe

155 + 50-200 for probe 199 + 59 for probe

Price U S . dollars

5-Digit display IEEE-488 BUS CAP Multiprobe Acc: avail. 4-Digit LED display Panel mount

4-Digit display IEEE-488 BUS CAP

Battery operation Small size 34 Digit-LCD 2-probe cap Recorder output Small bench top unit LED display 3-Probe Cap Analog model Recorder output 2-probe cap

Battery operation Small size

Special characteristics or comments

306

R. L. BERGER, T . R. CLEM, V. A. HARDEN, AND B. W. MANGUM

3. Digital Recording Methods and Devices Quite often the measurement of temperature during the course of an experiment is a continuous process, and, in addition, it is not an isolated event but is to be related to other events. Consequently, it is desirable to have a method of recording the temperature readings over a period of time without operator attention. Many of the thermometers available accommodate this by providing some type of output that is directly related to temperature. A. ANALOG O U T P U T S

Many of the middle-price-range electronic thermometers available today have a recorder output. Typically, the voltage present there varies from 0 to 0.1 or 1 volt and is directly proportional to the temperature. Generally, this is intended for use with an analog strip-chart recorder to record temperature vs. time or with an x - j recorder to record temperature vs. some other related event. This signal could, however, be converted for use with a digital computer by using some type of analog-to-digital convertor that is connected to the computer. This convertor could be a digital voltmeter with a digital output. B. DIGITAL O U T P U T S

There are three methods in common use by which digital data are communicated between devices. In this section we will discuss these methods. They are; a) Binary Coded Decimal (BCD), b) RS232, and c) IEEE-488 GPIB. With each of these methods, the sending unit and the receiving unit must have the same type of communication port (Handbooks).

a . Binary-Coded Decimal (BCD). BCD output from a digital thermometer comprises four lines of data per digit, weighted in a 1-2-4-8 sequence. There are basically two methods for presenting the BCD data to external devices. One method is to have a set of four lines for each digit, with the total number of output lines equal to four times the number of digits. The other method is to multiplex all digits to the same four data lines and to provide control lines to allow command or determination of which digit is being presented at a particular time. These outputs usually have “TTL compatible” level voltages. TTL, which stands for Transistor-Transistor Logic, is a digital logic family that has been used extensively for the past 10 years. TTL levels are generally 0 +0.8 V for a binary 0 and +3.2 to +5.0 V for a binary 1 (although a 1 can be considered anything above +2.4 V). Each line of the four-digit data lines is called a bit (which is a contraction of binary digit) and the entire group of four is

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

307

referred to as a byte. When the BCD type of interconnection is referred to as a parallel interface, all four bits of each byte are put out at the same time on parallel lines; the multiplexed method is sometimes referred to as bit-parallel, byte-serial output if the digits (bytes) are output sequentially; or it is referred to as parallel output with hand shaking if the receiving unit tells the sender when to transmit the next byte.

b. RS232 SeriulZntetfuce. EIA RS-232-C is an Electronic Industries Association Standard (EIA, 2001 Eye St., NW, Washington, D.C. 20006) for the “Interface between Data Terminal Equipment and Communication Equipment Employing Serial Binary Data Interchange.” This interface technique is used in numerous applications in the electronics and computer fields for intersystem communications. For thermometers, the temperature data and possibly some identification information, is transmitted one character at a time, sequentially (or serially). Each character is transmitted as a series of, usually, seven ASCII-encoded (U.S.A. Standard Code for Information Interchange, ANSI x3.4-1977, American National Standards Institute, 1430 Broadway, New York, N.Y. 10018)pulses, plus synchronization pulses called start and stop bits. The rate of data transmission and the rate at which the receiver expects to receive it must be matched, and that rate is called the BAUD rate (bits per second). BAUD rates vary, at standardized values, from 110 to 19200 BAUD. Generally, thermometers using this form of data communications operate unidirectionally as far as data are concerned, but the RS232 connection may have some “hand-shaking” characteristics so that the source (the thermometer) will not be sending data when the receiver is not ready to accept it. c. ZEEE-488 General Purpose Interface Bus (GPZB). This is a relatively new technique for communication between electronic devices, and it is an IEEE standard (Institute for Electrical and Electronics Engineers, IEEE 488- 1978, “Digital Interface for Programmable Instrumentation,” IEEE, 345 E. 47th St., New York, N.Y. 10017).Devices with thiscapability generally are controllable via the IEEE-488 in the sense that ranges can be selected, functions can be changed, inputs selected, and so on. Up to 15 instruments, each with a unique address, may be connected together on one GPIB with a controller, thus allowing one computer or data acquisition device to accommodate many measurement devices. The use of GPIB communications requires very little input from the user to get the hardware operational. Most GPIB controllers use a high-level language, such as BASIC, which includes simple inpudoutput commands that simplify using instruments interconnected via the GPIB.

308

R. L. BERGER, T.R. CLEM, V. A. HARDEN, AND B. W. MANGUM

4. Temperature Controllers The control of the temperature of various measurement cuvettes, calorimeters, incubators, etc. has received a great deal of industrial attention in the last 10 years. Because of this, either complete controllers or various integrated circuits can be purchased which allow one to assemble controllers with almost any desired sensitivity or stability. In Table VI, we list a number of'controllers ranging in capability of control from ultrastable to +O.I"C. To use the table intelligently, however, an understanding of the problems associated with temperature control is extremely useful. A detailed discussion of this problem has been given elsewhere (Mudd et al., 1982). Basically, the problem involves operating the controller against at least a 5°C gradient. Thus, if the system being controlled is to be maintained at 25"C, some means must be provided to cool the surface of the device to 17°C since room temperature is often 22 to 25°C. This is most effectively done with a standard low-temperature bath. The controller must then add heat as needed. To do this, and maintain long-term stability, a proportioning system is used that provides a small amount of heating all of the time and adds proportionately larger or smaller amounts as needed. T o ascertain if the system is changing, a differentiating circuit is provided in the controller, and to determine the long-term trend, an integrating circuit is also included. Several such units are listed in Table VI. The controller discussed elsewhere (Mudd et al., 1982) will maintain a calorimeter within +O.OOl"C of a selected value for a number of hours, but it drifts by about t0.01"C over a period of a week. This can be corrected by using a comparison circuit and the digital, linearized thermistor thermometer (Berger et al., 1980).

VI. RECENT APPLICATIONS OF MODERN TEMPERATURE MEASUREMENT METHODS IN BIOCHEMISTRY 1.

Fast Stopped-Flow Thermal Measurements

Measuring the temperature of chemical reactions as a way of determining the extent of reaction has been used for almost 100 years (Callendar, 1902). An early application to the determination of the energy involved in the reaction of hemoglobin with oxygen was made by A. V. Hill (Brown and Hill, 1923). Roughton (Roughton, 1930, 1961) developed a continuous-flow system that was used to measure the reaction of hemoglobin with various ligands. Unfortunately, thermocouple insulation problems were very severe in Roughton's work since the slightest pinhole produced large streaming potentials in the thermocouples. In addition,

Proportional Thermistor or platinum 3002 Proportional Thermistor or platinum PTC 41 Proportional Thermistor or platinum Proportional Thermistor

Leeds and Northrup, Inc. Hart Scientific Company Tronac, Inc.

Commonwealth Technology, Inc.

pp

800 1000 1000 800

00.0 to 99.9"C - 10 to 100°C

-4.0 to 100°C

895

50 to 200°C

0 to 2000°F

0 to 600°C -200 to +20O0C

285

Proportional Platinum or RTD ordoff Proportional Thermocouple

420 1

235

223

185

Controller

620

Athena Controls, Inc.

Yellow Springs Instrument Omega

63-RC on/off

Yellow Springs Instruments Cole-Parmer

Temperature control range

Mercury -59 to + 150°C in glass *Thermistor -70 to + 260°C

Sensor

K2156 Proportional Thermistor -100 to +500"F or platinum RTD 72 Proportional Thermistor 0 to 120°C

K2149 on/off

Cole-Parmer

Type

Model no.

Manufacturer or distributor

50-300

+

.01"C

1°F

1°C 0.1"C

0.005"C

0.05"F

0.05"C

1"C

Stability

Adjustable dead band 0.1 to 30°C Digital readout of temperature, adjustable proportional bandwidth Microprocessor-based unit includes control algorithms and alarm functions

Easy to use, but only good in liquid medium

Special characteristics comments

+0.005"C

+O.O001"C

+O.OOOl"C with Model 408R Bath

+O.O05"C +O.O005"C with Model 5001 Bath

+0.001"

0.2% FS

O.l%FS 0.2% FS

0.5"C

0.05"F

0.05"C

1°C

Precision

50- 150 +0.25"C

50-150

40-100

40-100

40-90

40-90

60-90

55-96

Probe

Approx. price U.S. dollars

Temperature Controllers

TABLE VI

310

R. L. BERGER, T.R. CLEM, V. A. HARDEN, AND B. W. MANGUM

vast amounts of solution were needed. In later work in Roughton's laboratory, Berger started work on a stopped-flow calorimeter utilizing very small thermocouples and new coatings (Berger, 1963; Berger and Stoddart, 1965). At about the same time, Pinsent, Pearson, and Roughton (Pearson et al., 1954) and later Chipperfield (Chipperfield, 1966) with Roughton, greatly improved the continuous-flow calorimeter so that a kinetic experiment could be performed on 100 ml of each reagent. This was in contrast to a volume of only 0.5 ml of each reagent for Berger's system. For continuous-flow measurements, about 2 sec were required for each reading so that the speed of response of the detector was not so important. In the case of the stopped-flow method, however, the time resolution depended upon the sensor response. The need for a better detection system led to the examination of the problem of high noise and low stability of thermistors. As a result, a series of bead thermistors has been produced over the last 20 years that are very fast (circa 5-7 msec), glass encapsulated, have a noise figure that depends only on the Johnson noise, and are very stable. The Johnson-Nyquist Noise (Van Der Ziel, 1954) is given as: e2 =

4kTAfR

where e is the peak-to-peak voltage, k is the Boltzmann constant, T is the temperature in kelvin, Af is the frequency bandpass, and R is the resistance of the thermistor in ohms. Thus, for 1 ohm, 1 Hz, and room temperature e = dGZi-j%= 10-"volts

For a 10-kohm thermistor and a 100-Hz bandpass Thus with 0.3 V across the thermistor, and the usual 4% change in resistance per kelvin for the thermistor, a 1-mK change corresponds to a voltage change of 0.3 x 4 X V = 1.2 x V. Since the peak-topeak voltage is 1.4 times the root mean-square voltage, about 0.01 mK is the limit of detection at this bandpass. It is very difficult to produce the small, fast-response beads at a much lower resistance, but even if it were possible, the best signal-to-noiseratio for most solid-stateamplifiers is for input resistances greater than 1000 ohms. In addition, the inherent llf noise of the best solid-state amplifiers is of the order of 100 to 150 nV for this bandpass. A special ac amplifer was constructed for the work on stopped-flow calorimetry, and it is shown in Figure 8. It is discussed in detail elsewhere (Berger et al., 1974).A commercial version as mentioned earlier from Linear Research, operating at a somewhat lower bandpass,

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

311

but with excellent results, is available and is used for thermal titration (Watt et al., 1974) where a time resolution of 100 msec is adequate. Using this new detector and bridge, a stopped-flow calorimeter with a 2-msec dead time, a computer-corrected thermistor response of 1 msec, and a sensitivity of 0.1 mK has been constructed. Thus, for a chemical reaction with an enthaphy, Q, of 5 kcal/mole, we can calculate the temperature change, AT, from

Q = MC,AT (1 1) where M is the mass and C , the heat capacity, of the solution. Since both M and C, are approximately 1 for 1 ml of a water solution, the change, AT, will be about 0.125 m"C and thus we can detect about 25 nmol/ml.

2. Heat Conduction and Response Time Corrections for Thermal Detectors

Any heat-detecting device has a finite time response to a change in temperature, and it will, in general, lose some of the heat it has gained to its environment during the time of measurement. A detailed treatment of this problem has been given recently elsewhere (Davidsand Berger, 1964; Berger and Davids, 1965; Davids and Berger, 1969; 1982; Balko et al., 1981) and we will only outline the idea here. CALI BR AT10 W UNBALANCE

L

PMASL ADJUST

SlNL WAVE OSCILLATOR

SENSITIVE

OUTPUT

TIME CONSTANT

BALANCE METER

(A)

Figure 8. Fast, sensitive ac resistance bridge: (a) single-channel stopped-flow, I msec at 0.1 m"C sensitive; (b) differential bridge.

F3

I

w

Figure 8.

I

OSG MODULE

-

J

I

-

MoN'ToR'

+

1

AMPLIFIER MODULE

II

(B)

DlFFERENTIA L FAST THERMISTOR BRIDGE -SIMPLIFIED BLOCK DIAGRAM--

)IFF. A.C. BRIDGE MODULE

(Continued)

L

P

"8" THERMISTO

A" THERMISTOR

I

METER

FtJ

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

313

To test the response time of any detector, one normally challenges the detector with a step function. For example, consider a flow system (Balko et al., 1981) with a mixer and two driving syringes, an observation tube, and a thermistor as the detector as shown in Figure 9. If acid and base are mixed, a step increase in heat occurs because the reaction is so fast. Thus, 0.02M NaOH and 0.01M HCl mixed in the ratio of one to one will give a temperature rise of about 69 mK since AH for the heat of formation of water at 25°C and in 0.1M KCl is about 13.75 x lo3 cal/mol (Hale et al., 1963).T h e time it takes the temperature of the sensor to rise to lle of its total change is called the sensor's time constant, T. The time to rise half way is t1I2. Since most physical devices respond exponentially, at least to a first approximation, the output E may be expressed as

E = Eo[l - eWat]

(12)

when E = Eole, then a = 117. If we can adequately describe the system physically, we can correct this response time by at least a factor of 10. This is done using the D-B Finite Element Simulation Technique (Berger et al., 1982) or D-B FEST. A detailed analysis is given in (Davids and Berger, 1982; Balko et al., 1981). The basic idea is that if we consider a thermal sensor at temperature T, in contact with a solution whose temperature T , is changing with time due to solution heating during flow or a chemical reaction, then the change in temperature of the sensor in a short time At is

ATs = k(Tc - T,)At

(13)

where k is a constant that depends on geometry, thermal conductivities, densities, and specific heats of both the sensor and the solution. If changes in T , are due to a first-order chemical reaction then

Tc(t)= A (1 -

(14)

One can substitute Equation (14) into Equation (13), and take the limit as ATs and At approach zero. This gives ~

dTs dt

+ kT, = kA [ l - e-"'1

The solution of this is

(

a

Ts=A l + k-a Figure 10 shows the effect of this finite response time of a typical thermistor whose t 1 / 2 values are 10, 20, and 30 msec, respectively.

314

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM THERMISTOR

STOPPING BLOCK

100 PSI

STOPPING BLOCK

0

100 PSI

I

MIXER

~

THERMISTOR

(6)

Figure 9. Thermal stopped-flow system: (a) conventionalstopped-flow; (b) zero pressure drop stopped-flow.

A problem arises when the reaction is not a simple first-order reaction and heat is lost during the reaction. In this case, we invoke the D-B FEST Method. First, we must divide the system into small cells (qq).In this case, three cells will be adequate: solution, glass coating, and thermistor, all in axial symmetry. The generalization to more cells is straightforward. Gonsider the cells to be numbered 1, 2, and 3 with cell 1 representing the thermistor, cell 2 the glass coating, and cell 3, the solution in the observa-

TEMPERATURE MEASUREMENT I N BIOCHEMISTRY

315

' i i , 40

I

20

0.2

0.4

0.6

0.8

1.0

1.2

1.4 t(sec)

Figure 10. Calculated thermistor response to the temperature change obtained in a firstorder reaction for four different reaction half lives. From top to bottom, the reaction half lives are 10,30,100, and 300 msec, respectively. In each graph, the solid line represents the actual temperature change and the dashed, dotted and dot-dashed curves represent the response of thermistors with t1/2 = 10, 20, 30 msec, respectively.

tion tube. The temperatures of the cells will be denoted by Tq,where i refers to heat transferred in the radial direction a n d j heat transferred in the axial direction; TI^ corresponds to the temperature measured by the thermistor (this is the same as T, of Equation 16). The laws of heat conduction may then be used to calculate the temperature in the reaction chamber, TSj,from the series of measured temperatures, T y assuming that all changes in the temperatures of cells 1, 2, and 3 are due to conduction.

316

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

We now assume that each cell q, has a known specific heat Cp,, density

p,, and volume VY. We define B, as the effective conductivity between

cells q, and q2+ S , as the surface area between these cells, and X, as the separation of their centers. The time interval At is chosen to satisfy the stability criteria for the heat conduction calculation. For a tabulation of the temperatures of the cells at different times see Table VII. T11, T12, and TI3 are known from the measurement. By the laws of heat conduction, the amount of heat transferred between cells 1 and 2 is

This transfer of energy is responsible for the observed temperature change in cell 1; therefore,

Solving Equations (17) and (18) for T21,we get

T22can be obtained in a similar manner from T I 2and TI3.To determine TJ1from the changes in temperature of cell 2 during the time interval At, we have to include both the heat exchange between cells 1 and 2 and cells 2 and 3. This gives

This iterative scheme is used to obtain the reaction temperature from the temperature recorded by the thermistor. The reaction temperature T,(t) - T, at time t, is obtained from Equation (20). TABLE VII Temperatures of Cells at Different Times Temperature of cells Time

Cell 1

Cell 2

Cell 3

317

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

The effectiveness of the response time correction was illustrated by correcting the experimental results obtained with some known reactions by making computer reconstructions. It has been shown also that the technique is internally consistent by generating a simulated calorimeter output and using the correction program to retrieve the input kinetics. The calorimeter simulation program was used to calculate a thermistor output as a result of a fast reaction (step function temperature change in the observation tube. The output of this program was entered into the data reconstruction program, and the results obtained are presented in Figure 1 1. The inset in Figure 11 represents the reconstructed step function from the simulated exponential response of the thermistor. The open circles show the response curve obtained experimentally by the fast-plunge technique. The solid circles represent the reconstructed temperature change as experienced by the thermistor during the plunge. Figure 12 shows data reconstruction of an actual reaction trace obtained by mixing glycylglycine with CO:!in solution. In Figure 12a, the actual oscilloscope trace of the thermistor output (top curve) is shown along with the velocity transducer output (bottom curve). The vertical scale is 20 m"C per division. The thermistor used in this experiment had a response time tIl2 = 7 msed. In Fig. 12b, the computer reconstruction,

0.0 0.0

1

5.0

00

0 5 I

TIME

10.0

(MSEC)

10 I

15.0

I5

2.a

20.0

Figure 1 1. Data reconstruction of an actual thermistor response to a step-function temperature change obtained by plunging the thermistor into a beaker of water: (0)the measured response;(0)thedatacorrected for finite responsetime.(Insert) The reconstructionofa computer-generated response to a step-function temperature change. The reconstructed step function has only a slight distortion close to time zero.

FLOW

tTOPPED

z

(A)

59.8

46.2

35.8 rndegOC

23.8

11 - 9

STOP FLOW

000

000

.052

.lo4

SECONDS (B)

318

.156

* 209

.261

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

3 19

which has corrected for the thermistor response time, and the experimental data are shown; the curve on the right-hand side is of the observed data. A comparison of the two traces of Fig. 12, a and b indicates that the stopping of the flow and the beginning of the reaction, as revealed by the thermistor trace, is about 8 msec later than the stopping as shown by the velocity transducer. This discrepancy disappears when the data reconstruction procedure is used to obtain the true temperature change in the observation tube. This is shown in Figure 13,where the experimental data and the reconstructed data traces are presented. Here the start of the reaction and the time that flow stopped, as indicated by the flow velocity meter, correspond exactly. Thus, in this example, correcting for the thermistor response time not only shows a faster reaction, as expected, but also resolves an apparent inconsistency between the two traces by shifting the apparent start of the reaction to correspond with the true stopping time of flow. This analysis now makes it possible to make precise fast stopped-flow thermal reaction measurements. An interesting application of these methods has been the study of the isomerization of Cryptrand 21 1 in its reaction with Li and Ca ions by Liesegang (Liesegang, 1981).At the present time, we are using the system in the investigation of the reaction of COPwith hemoglobin (Berger et al., 1978).

3. Thermal Titration and Immobilized Enzyme Reaction Detection The use of modern temperature detection methods in thermal titration has increased considerably in recent years, with several commercial instruments now available. Marini and Martin have recently reviewed this field (Marini and Martin, 1979)extensively so that only a brief discussion will be given here. We have developed a combined pH- thermal differential titration apparatus that is modelled after our earlier single-cellsystem (Berger et al., 1974; Marini et al., 1980).Figure 14 shows the essentials of the instrument. The unique part of this device is that it is under microprocessor control. The computer starts the titration, records the data, and speeds up or slows down the titration automatically if the curve is changing too rapidly. Data-correction programs adjust for response time and Figure 12. Data reconstruction of an actual reaction trace obtained by mixing glyclyglycine with C o p in solution. (a) The actual oscilloscopetrace of the thermistor output (top curve) is shown along with the velocity transducer output (bottom curve). Vertical scale is 20 m"C per division. The thermistor used had a response t l l Z = 7 msec. (b) The computer reconstruction, which corrects for the thermistor response time, along with the raw data are shown. The curve to the right is the observed data.

320

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

Figure 13. Reaction of HCI and NaHCOs. (---) The experimental data; (-) puter-corrected curve.

the com-

thermal losses in the system. The sample size is 2-4 ml. Titration times are 120 sec (from pH 2 to pH 10 for a protein such as cytochrome C (Marini et al., 1980, 1980, 1981). In another somewhat similar area, immobilized enzymes have been used in columns with a thermistor as the reaction detector. Several reviews of this work have been published recently (Carr and Bowers, 1980; Klibanov, 1983). Berger and Everse have had success in immobilizing enzymes directly on a glass-coated thermistor bead, thus enabling clinical measurements in vivo of blood glucose from 25 to 1000 mg% glucose. The reaction uses an oxidase that produces hydrogen peroxide, which is then broken down by catalase, yielding an overall heat of 29 kcal/mole. By using a differential thermistor bead system, excellent results were obtained in blood. Unfortunately, the enzymes hydrolyze very quickly, thus greatly limiting the utility of this system. Vurek has used thermistor flakes to build a micro CO2 detection system (Vurek et al., 1975). In that system, differential measurements are used with LiOH on one flake. The reaction with C 0 2 produces about 110 kcallmol, thus making it very sensitive. A commercial version of this instrument is available (W. P. Instruments). One of the uses of new small, sensitive temperature sensors is in

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

32 1

Figure 14. Differential thermal-pH titration apparatus.

microcalorimetry. One such new sensor is made from n and p type bismuth-telluride grown as single crystals, cut, and fabricated as thermopiles where n type is connected to p type through a copper plate. These thermopiles exhibit a very high Seebeck effect, that is, circa 400 FVPC per junction. Figure 15 shows how they are mounted in a typical calorimeter application. Recent extensive reviews in this series (Spinks and Wadso, 1977; Jolicoeur, 1981) have described the large variety of uses of such thermopiles. We have recently described a new calorimeter specifically designed for biochemical use, which uses this type of thermopile (Mudd et al., 1982).A newly developed unit (Marlow Industries), with hundreds of thermocouples per square inch, has been tested and incorporated into a modified rotor of our calorimeter. The sensitivity is eight times higher than that of our first instruments and three times higher than that of the one described in reference (Mudd et al., 1982). Commercial versions of the NIH-developed fast, thermal stopped-flow, thermal titration, batch,

322

R. L. BERGER, T. R. CLEM, V. A. HARDEN, A N D B. W. MANGUM

r2*

Bismuth-Telluride Thermopile, 2 1, mountel in a heat conduction calorimeter. Figure 1 3 the leads; 18, the heater; 20, the copper cell holder; 19, the cell; and 22, the entrance channel.

and flow microcalorimeters are currently available (Commonwealth Technology). Finally, we mention several current applications somewhat outside of biochemistry in the usual sense. Thermography has slowly been coming to the fore. Many of the problems associated with the analysis of thermograms were treated at the Fifth International Symposium on Temperature (Plumb, 1972) in 1972 and new applications were discussed at the Sixth Symposium (Schooley, 1982) in 1982. Of perhaps more current interest is the greatly expanded interest in temperature measurement in hyperthermia and hypothermia. A recent New York Academy of Sciences conference has done an excellentjob of reviewing this (Ann. N.Y. Acad., 1980). Cetas also wrote a general review of thermometry in this field (Cetas, 1968). Perhaps the most exciting new method in thermometry is that of optical fluorescence, which we described earlier. Catheters, wholebody scanners, etc., have been made for use with this method. At this point, 0.01"C is probably the least imprecision that can be obtained with the commercial instrument (Luxtron), with data obtained every 0.1 sec. Improvements are likely, however, as needs are made known to the company. 4.

Standard Temperature Reference System for Biochemistry and Clinical Chemistry

In an earlier section, we discussed the IPTS-68 and mentioned several ways in which that scale can be transferred to the laboratory. While

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

323

platinum resistance thermometers, mercury-in-glass thermometers, and thermocouples may be used for that purpose, we believe that bead thermistors, such as the highly stable S-10 and S-15 (Thermornetrics, Inc., Edison, N.J. 08817) thermistors with any of several types of bridges and with several fixed-point reference cells, offer the most practical and convenient method of accomplishing this for the biochemical and clinical laboratories. Let us now examine the accuracy required with regard to absolute temperature for both equilibrium and kinetic measurement to be known to & 1%. [For a general discussion of the variation of reaction rate with temperature, see the article by Bunnett in Techniques in Chemistry,Vol. V I (Lewis, 1974).] Let us discuss the general reaction

kf A+BF=C kb

where kf is the forward rate constant, and Rb the back reaction constant.

where K is the equilibrium constant. I n general, the variation of the equilibrium constant, K, with temperature follows the Van't Hoff Law, i.e., d(ln K) - AH dT RTz

(23)

Upon integration from Ti to T f , we obtain

where Ti and T f are the initial and final temperatures. Assuming AH for the reaction enthalpy to be 12 kcallmol, we can calculate the error for a 0.1"Ctemperature change from, say, 30 to 30.1"Cand from 37 to 37.1"C. Remembering that for thermodynamic calculations we must use the Kelvin scale, the first temperature change is from 273.15 K + 30 = 303.15 K to 303.25 K and the second from 310.15 to 310.25 K. Taking the anti-log of equation (24)

324

R. L. BERGER, T. R. CLEM, V. A. HARDEN, A N D B. W. MANGUM

Thus, an error of 0.1"Cat 30°C leads to an error of 0.65%in estimate of concentration for a 12 kcaVmol reaction. At 37"C, the error would be a little less. Consequently, for equilibrium measurements one must know the absolute temperature to 0.03 K if a 1% uncertainty in the data is desired for comparisons between laboratories as recommended by IUB (Expert Panel, 1975). For reaction-rate measurements, the variations of reaction rate with temperature is usually expressed as the Arrhenius equation, which in its integrated form is

*

where k, i s the forward rate constant in Equation (21) (assuming the back reaction can be neglected). E is the activation energy and Zo is the collision factor, assumed to be temperature independent. Taking logarithms In kf

=

In ZO - EIRT

(27)

or log k, = log Zo - E12.303 RT

(28)

If one plots log k f against 1/T for a great many reactions, one obtains a straight line. Since the slope is -E/2.303 R , E is readily evaluated and is called the Arrhenius activation energy. R has the value of 1.987 cal/deg when E is in cal/mol. A great many reactions double their rate for a 10°C rise in temperature. If we take only two temperatures, it is convenient to use the expression

In doubling the rate from 25 to 35°C one has log 2k,, 2 and E = 0.30108 x 4.576 x 308.15 x 298.15110. Thus, E = 12656 cal/mol

- log k f T k = log (30)

For a reaction rate that goes up by a factor of 5, E would be 30,000 callmol, providing other changes, such as the ones Bunnett discusses in some detail, are not occurring. In most clinical chemistry and biochemical reaction studies, the rate is determined by observing the change in concentration. If in Equation (21) a thousandfold excess of A over that of B is used, then the rate of formation of C is independent of A or B and depends only on k f ; we call this a zero-order reaction and

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

325

dc

- = kg dt

In this case, the determination of c for a fixed time depends on knowing k f , whose value is temperature dependent. Thus, for E being 12,656 cal/mol, a change of 0.1"C at 30°C will produce a change in the ratio of kfTdkfT1 of

For a first-order reaction: dc

- = kfC dt

(33)

and

c = CO

(1 - eVkft)

(34)

where Co is the value of C at t equals infinity. Many reactions however, must be run in the so-called pseudo first-order mode, that is, A. >> Bo, by say 10 to 100, but neither can now be neglected. Under these circumstances

where A0 and Bo are the initial concentrations of A and B. This neglects the back-reaction. This is usually a reasonable assumption if A. is 100 times larger than Bo and only the first 25-40% of the reaction is used. The integrated form of this equation yields

In both of these cases, to a good approximation, the temperature effect is still mainly due to the variation of the rate constant and, thus, Equation (33) best describes the dependence of C on temperature. Essentially, this gives:

c

CO

[l

- e-kt]

and thus

= 1.007

(37)

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

326

Thus, for a change of 0.1"C at 30°C, the absolute temperature should be known to Ifr 0.03 K if 1% data are to be presented for comparisons between laboratories (Expert Panel, 1975). At the present time, to our knowledge, it is doubtful if anyone knows the reaction temperature in a cuvette to better than 0.1"C. Using the IPTS-68 and an S-10 thermistor as the transfer standard, together with the fixed points provided by the gallium melting point and the triple point of water [29.772 and O.Ol"C, respectively], an absolute temperature accurate to & 0.01"Cor better, can be realized in any laboratory in the world for under $5000. A very convenient IPTS-68 transfer standard is provided by the digitally linearized thermistor thermometer, fully described elsewhere (Berger et al., 1980).The latter instrument is a small computer (one card) that holds a constant power (8 pW) across an S- 10 Thermistor. The voltage divided by the current supplied to do this is a direct measure of the resistance of the thermistor on an absolute basis, since the measurement device is directly traceable to NBS standard voltage and current sources. A second input is provided so that a small P20 type probe (Therrnometrics, Inc., Edison, N.J.) as shown in Figure 16,can be used to measure directly the temperature in the reaction vessel. While this probe does not have the high stability of the S-10, it can be readily

1 L * PROG GAIN INST. AMP

CONTROLLED CURRENT SOURCE

HIGH QUALITY OP AMP

I

I

I SYSTEM BUS

PROCESSOR

MEMORY

110

I10

R S 232 OR TTY

I10

CRT DISPLAY

Figure 16. Digital Linearized Thermistor Thermometer traceable to IF'TS-68 to 2 0.002"C absoiute. Absolute "A to D", analog to digital; ''UO', input-output port.

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

327

compared with the S-10 and the fixed points, and the PROM reprogrammed, if necessary. A look-up table for every 0.1"C from 0 to 60°C (or 100°C) is placed in a small PROM [Programmable Read Only Memory]. An interpolation algorithm is then used to determine the temperature to the nearest 0.001"C. The equation used to do this (Kilibanov, 1983) is:

RT = R(25"C)exp (Bo +

+

$ + $1

(39)

This equation is fitted to the data on a large computer and the constants Bo, B 1 ,BP,and B3 determined. A look-up table is then generated and put on the PROM. Alternatively, one could have only the constants on the PROM and simply generate the look-up table on the RAM [powered by battery (CMOS RAM) so that it is done only once]. VII. CONCLUSIONS AND FORECASTS

In conclusion, the last 10 years has seen a greatly increased interest in temperature measurement, particularly in the use of thermistors in everything from clinical thermometers, thermal dilution catheters, waterbath regulators, hand-held digital thermometers, and, finally, an "absolute" temperature standard. We can look forward in the next 10 years to bringing temperature standardization to all areas of chemistry, biochemistry and clinical chemistry, as well as to the pasteurization and virus kill point for vaccines. For the present recommendations in biochemistry, see Expert Panel on Enzymes (1975). For the biochemist, the advance of new, sensitivetemperature-measuring devices comesjust at a time when large amounts of very pure proteins, nucleotides, DNA, RNA, lipids, etc., are becoming available. Thus, the ubiquitous nature of heat can be utilized, providing both the thermodynamic and kinetic data needed for any theory of biomolecular interaction. Research now in progress (Schutz et al., 1983) promises several orders of magnitude increase in sensitivity and of measurement speed, so that fast stopped-flow reaction studies on enzyme reactions can be conducted at micromolar enzyme concentrations with a resolving time of 100 psec. Acknowledgments

The authors wish to express their gratitude to Dr. Gearld W. Liesegang, formerly oft e National Heart, Lung, and Blood Institute and presently of Perkin- E mer Corporation, Norwalk, Connecticut, for helpful discussions in the course of preparing this paper; and to Mrs. Pauline Ballew for diligence and patience in typing the manuscript.

5t

328

R. L. BERGER, T . R. CLEM, V. A. HARDEN, AND B. W. MANGUM

References Ann N.Y. Acad. Sci. (1980), vol. 335. Balko, B., Berger, R. L., and Anderson, K. (1981), Rev. Sci. Instrum., 52, 888-894. Barnes, H. T. (1902), Phil. Trans. R. SOC.(London) A , 199, 149-263. Benjarninson, A. and Rowland, F. (1972), Measurement and Control in Science and Industry, vol. 4, Instrument Society of America, Pittsburgh, pp. 701-708. Berger, R. L. (1963), Temperature, Its Measurement and Control in Science and Industry, vol. 3, part 3, Reinhold Publishing Co., New York, pp. 67-77. Berger, R. L., Balko, B., Borcherdt, W., and Friauf, W. (1968), Rev. Sci. Instrum., 39, 486-493. Berger, R. L., Balko, B., Bowen, P., Paul, R., and Hopkins, H. P., Jr. (1978), Frontiers of Biologtcal Energetics, vol I (L. P. Dutton, J. S. Leigh, and A. Scarpa, Eds.), Academic Press, New York, pp. 698-706. Berger, R. L., Balko, B., Clem, T. R., and Friauf, W. S. (1982), Temperature: Its Measurement and Control in Science and Industry, vol. 5, American Institute of Physics, New York, pp. 897-910. Berger, R. L., Clem, T., Gibson, C., Siwek, W., and Sapoff, M. (1980), Clin. Chem., 26, 1813-1815. Berger, R. L., and Davids, N. (1965), Rev. Sca. Instrum. 36, 88-93. Berger, R. L., Fraiuf, W. S., and Casico, H. E. (1974), Clin. Chem., 20, 1009-1012. Berger, R. L., and Stoddart, L. C. (1965), Rev. Sci. Instrum., 36, 78-84. Berry, K. H. (1979), Metrologk, 15, 89. Bolton, H. C. (1900), Evolution o f t l u Thermometer, 1592-1743, Chemical Publishing Co., Easton, Pennsylvania, p. 18. Boyle, R. (1683), New Experiments and Observations Touching Cold, Richard Davis, London, p. 112. Brown, W. E. L. and Hill, A. V. (1923), Proc. Roy. SOC.(London) B , 94, 297-334. Brown, H. D. (1969), Biochemical Calm'metr), Academic Press, New York. Cajori, F. (1929), A Histoty of Physics, Macmillan, New York, p. 117, as quoted in Taylor, p. 251. Callendar, H. L. (1887), R . SOC.Phzlos. Trans. London, 178, 161. Callendar, H. L. (1902), Phil. Trans. R. SOC.(London) A , 199, 55-148. Carnot, S . (1824), Refictions sur la Puissance Motrice due Feu el sur les Machines Propres a developper cette Puissance, Chez Bachelier, Libraire, Paris. Carr, P. W. and Bowers, L. D. ( 1 9 8 0 Immobilized ~ Enzymes in Analytical and Clinical Chemistry, John Wiley & Sons, New York. Cetas, T . C. (1968), Thmapeutic Heat and Cold, 3rd ed. 0. F. Lehmann, Ed.), Williams and Wilkins, Baltimore, Maryland. Chappuis, P. (1888), Trav. et Mem. Bur. Int. Po& el Mesures, 6, 1. Cheng, A. F. (1981). Measurements and Control 15, 115. Chipperfield, J. R. (1966), Proc. R. SOC.LondonB, 164, 401-410. Cohen, M.E. and Drabkin, I. E. (1948). A Sovrce Book in Greek Science, McGraw-Hill, New York, pp. 326-334.

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Comit6 International des Poids et Mesures (1955),Procb-verbaux des Skances & i‘annke 1954, 24, 81-82, T20-T23, T44-T47; see also Comptes Rendus des Skances de la DixiZme Confkrence Gknkrale des Po& et Mesures, 1954, Gauthier-Villars et Fils, Paris. Commonwealth Technology, Inc., Alexandria, Virginia 22304 Comptes Rendus des Skances de la Septi2me Confkrence Gknh-ale des Po& et Mesures, 1927, Annexe 4, p. 94. Comptes Rendus des Skances de la Neuui2me Confkrence Gknkrale des Poi& et Mesures, 1948, Resolution 3, pp. 55, 63; Resolution 6, p. 64; Annexe 6, p. 89. ComptesRendus de la Treizihe ConfkrenceGtnkrale des Po& et Mesures, 1967 - 1968, Annexe 2, p. Al; Comitk Consultatif de Thermomktrie, 8e session, 1967, Annexe 18; and Metrologm (1969) 5 , 35. Comptes Rendus des Skances de la Quinzihe Confkrence G k n a l e des Po& et Mesures, 1975, Resolution 7, p. 105; Annexe 2, p. Al. Connolly, J. J. (1982), Temperature: Its Measurement and Control in Science and Industry, vol. 5, American Institute of Physics, New York, pp. 815-817. Cork, J. M. (1947), Heat, 2nd ed., John Wiley & Sons, New York, ch. 1. Davids, N. and Berger, R. L. (1964), Comm. ACM, 7, 547-551. Davids, N. and Berger, R. L. (1969), Cum. Mod. Biol., 3 , 169-179. Davids, N. and Berger, R. L. (1982),J. Biochem. Biophys. Meth., 6, 205-217. Expert Panel on Enzymes, Committee on Standards (IFCC) (1975), Clin. Chim. Acta, 61, F11-F24. Fahrenheit, D. G. (1724), Philos. Trans. R . Society (London), 33, 78. Fery, C. (1902), Compt. Rend., 134, 997. Gugan, D., and Michel, G . W. (1980), Metrologia, 16, 149. Guildner, L. A. and Burns, G. W. (1979), “Accurate Thermocouple Thermometry”, in High Temperatures-High Pressures 11, 183 and Manual on the Use of Thermocouples in Temperature Measurement (1981), American Society for Testing and Materials, Baltimore, Maryland. Guildner, L. A., and Edsinger, R. E., (1976), NBS J. of Research 80A, 703. Guildner, L. A. and Thomas, W. (1982), Temperature: Its Measurement and Control in Science and Zdustry, vol. 5, American Institute of Physics, New York, pp. 9- 19. Guildner, L. A. (1980), Accuracy of Realizing Thermodynamic Temperatures by Gas Thermometry, PTB-Mitteilungen 90, 41. Hale, J. D., Izaat, R. M., and Christensen, J. J. (1963),J. Phys. Chem., 67, 2605-2609. Handbooks: (a) Hewlett-Packard application Note No. 290, “Practical Temperature Measurements.” (b) Sheingold, D. H., Transducer Interfacing Handbook, Analog Devices, Inc., Norwood, Mass. 02062. (c) Omega Temperature Measurement Handbook (1982), Omega Engineering, Inc., One Omega Drive, Box 4047, Stamford, Conn. 06907. (d) Transcatalog (1983), Transcat, Box D1, Rochester, N.Y. 14606. (e) Cole-Parmer Instrument Catalog, Cole-Parmer Instrument Company, 7425 North Oak Park Avenue, Chicago, IL 60648. (0 NANMACTemperature Handbook, NANMAC Corporation, 9-1 1 Mayhew Street, Framingham Center, Mass. 01701. Hudson, R. P. (1980), Rev. Sci. Instrum., 51, 871. Jolicoeur, C. (1981), Meth. Biochem. Anal. 27, 171-287. Kelvin, L., (Sir William Thomson) (1891- 1894), Popular Lectures and Addresses, 3 vols., Macmillan, New York, 1, 13.

330

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

Klibanov, A. M. (1983), Sczence, 219, 722-727. Langley, S. P. (1881), A m . J . Sci., 21, 187. Laws, F. B. (1918), Electrical Measuremenh, McGraw-Hill, New York. Lewis, E. S. (Ed.) (1974), Techniques in Chemistry, vol. V I , 3rd ed., Wiley Interscience, New York, pp. 369-488. Lewis, G. N. and Randall, M. (1961), Thermodynamics, 2nd ed., revised by K. S. Pitzer and L. Brewer, McCraw-Hill, New York, p. 75. Liesegang, G. W. (1981),J. Am. Chem. Soc., 103, 953-955. Luxtron, Inc. 1060 Terra Bellas Ave., Mountain View, Calif. 94043. Mangum, B. W. (1974), Clin. Chem., 20, 670. Mangum, B. W. and Evans, G. A., Jr. (i982), Temperature: Its Measurement and Control in Science and Industry, vol. 5, American Institute of Physics, New York, pp. 795-801. Mangum, B. W. and Thornton, D. D., Ed. (1977), Th.e Gallium Melting-Point Standard, NBS Special Publication 48 1, Government Printing Office, Washington, D.C. Mangum, B. W. and Thornton, D. D. (1979), M e t r o l o p . 15, 201. Mangum, B. W. and Wise, J. A. (1974), Description and Use of Precision Thermometers for the ClinicalLaboratory, SRM 933 and SRM 934. NBS Special Publication 260-248, Government Printing Office, Washington, D.C. Marini, M. .A., Marti, G. W., Berger, R. L., and Martin, C. J. (1980), Biopolymers, 19, 885-898. Marini, M. A. and Martin, C. J. (1979), Crit. RPU.Anal. Chem., 8 , 221-285. Marini, M. A,, Martin, C. J.. and Berger, R. L. (1980). Biopolymers, 19, 899-91 1. Marini, M. A., Martin, C. J., Berger, R. L., and Forlani, L. (1981), Eiopolymers, 20, 22532261. Marlow Industries, Inc., 1021 South Juniper Road, Garland, Tex. 75042. M e t r o l o p , 12, 7, (1976). “The 1976 Provisional 0.5 K to 30 K Temperature Scale,” Metrolopa, 15, 65 (1979); also Procb-verbaux des Comitd International des P o d et Me.sures, 4 4 , 1 1 (1976). Middleton, W. E. K. (1966), The History of the Thennometer, Johns Hopkins University Press, Baltimore, Md. Mudd. C., Berger, R. L., Hopkins, H. P., Jr., Friauf, W. S., and Gibson, C. (1982),J. Biochem. Biophys. Methods, 6, 179-203. Ohte, A., Iwaoka, H., Mitsui, K., Sakurai, H., and Inaba, A. (1979), Metrolog-ia, 15, 195. Ohte, A. and Iwaoka, H. (1982), Temperature: Its Measurement and Control in Science and Indust?, vol. 5, American Institute of Physics, New York, pp. 1173- 1180. Ohte, A., Yamagata, M., and Akiyama, K. (1982), Temperature: Its MeasurementandControlin Science and lndustry, vol. 5, American Institute of Physics, New York. pp. 1197-1203. Pearson, L., Pinsent, B. R. W., and Roughton, F. J. W. (1954), Disc. Faruday Soc., 1 7 , 141 - 145. Plumb, H. H. (Ed.) (1972), Temperature, vol. 4, Instrument Society of America, Pittsburgh, Penna. in 3 parts, see Part 111. Preston-Thomas, H. (1972), Temperature, vol. 4, part I Instrument Society of America, Pittsburgh, Penna. pp. 4-7. Radiation Thermometry Session (1982), in Temperature: Its Measurement and Control in Science and Industry, vol. 5, American Institute of Physics, New York, pp. 575-629.

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

33 1

Reiser, S. J. (1978), Medicine and the Reign of Technology, Cambridge University Press, Cambridge, England, p. 112. Riddle, J. L., Furukawa, G. T., and Plumb, H. H. (1972), Platinum Resistance Thermometry, NBS Monograph 126, U.S. Government Printing Office, Washington, DC. Roller, D. (1960), The Early Development of the Concepts of Temperature and Heat: The Rue and Decline of the Caloric Theory, Harvard University Press, Cambridge, Mass., p. 13. Roughton, F. J . W. (1930), Proc. R. SOC.(London), A 126, 439-469, 470-486. Roughton, F. J. W. (1961), in Technique of Organic Chemistry,vol. V I I ,(A. Weisberger, Ed.) Interscience, New York, pp. 758-778. Rozzell, T. C., Johnson, C. C., Durney, C. H., Lords, J. L., and Olsen, R. B. (1974), J . Microwave Power, 9, 241. Sappof, M. (1980), Measurement and Control, 14, 144-149. Sarton, G. (1959), A History of Sczence, vol. 2, Harvard University Press, Cambridge, Mass., pp. 343-349. Schooley,J. F., Ed. (1982), Temperature: Its Measurement and Control in Sctence and Industry, vol. 5, American Institute of Physics, New York. Schuetz, L. S., Cole,J. H., Jarzynski, J., Lagakos, N., and Seebeck, T.J. (1826),Poqq. Annals, 6, 133, 263. Siemens, W. (1871), Proc. R . Sac. (London), 19, 443. Spinks, C. H. and Wadso, I. (1977), Methods of Biochemical Analysis, Vol.23 (G. Click, Ed.), John Wiley & Sons, New York, pp, 1- 159. Sturtevant, J. M. (1973), in Techniques in Chemistry, vol. I , Part 5 (A. Weissberger, Ed.), Wiley-Interscience, New York. Swartz,J. M. and Gains, J. R. (1972), Temperature: Its Measurement and Control in Science and Industry, vol. 4, Instrument Society of America, Pittsburgh, Penna., pp. 1 1 17- 1124. Taylor, L. W. (1941), Physics: The Pioneer Science, Houghton Mifflin Co., Boston, ch. 19. Thornson, W. (1848), Phil. Mag., 33, 313-317. Thomson, W. (1849), Tram. R. SOC.Edinb., XVI, 541-574; X X , 261-288. Thomson, W. (1854), Phil. Tram. R. SOC.(Lond.), 144, 321. Utton, D. B. (1967), Metrolopa, 3, 98. Utton, D. B. and Vanier, J. (1976),Imtrum. Technol., 2 3 , 47. Van Der Ziel, A. (1954), Noise, Prentice Hall, Englewood Cliffs, N. J., pp. 8-28. Verster, T. C. (1972),Temperature: Its Measurement and Control in Science and Industry, vol. 4, Instrument Society of America, Pittsburgh, pp. 1125- 1134. Vurek, G. G., Warnock, D. G., and Corsey, R. (1975), Anal. Chem., 47, 765-767. Watt, D., Berger, R. L., Green, D., and Marini, M. A. (1974),J.Clin. Chem.,20, 1013-1016. Wickersheim, K . A. and Alves, R. B. ( 1 979), Recent Advances in Optical Temperature Measurement, Industrial ResearchlDevelopment,21, 82. Wise, J. A. (1976), Liquid-in-Glass Thermometry,NBS Monograph 150, Government Printing Office, Washington, D.C. and references therein. W. P. Instruments, Inc., P.O. Bsx 3110, New Haven, Conn. 06515.

Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

Author Index Page numbers in italics indicate pages on which the full reference appears. Austin, R.H., 105, 116, 120, 121,125,

Abrudan, I., 253,266 Adam, G.,66,90,101 Adams, P.A., 39,101 Adamson, A.W., 66,72,101 Agmon, N., 123,137 Akerlund, H.E., 152,192 Akeroyd, R., 201,218,225 Akiyama, K.,330 Alben, J.O., 121, 124,137 Alberding, N.,120, 121,I37 Albertsson, P.A., 152,192 Alfano, R.R., 109,137 Alkaitis, S.A., 72,101 Allaz, M.J.,258,266 Allington, R.W., 150, 154,193 Aloni, Y.,185,195 Alpert, B., 126,137 Altamura, N., 261,265,266 Alten, J.A., 152, 164, 183,197,198 Alves, R.B., 296,331 Amann, R.P., 190,194 Amar-Costesec, D.,152,192 Ambrose, E.J., 150,192 Amidon, G.L., 25,40,I03 Ana, A., 237, 242, 243, 246,258,262,

127, 128, 135,137-138

Ayad, S.R., 185,194

Balko, B., 311,313,319,328 Bangham, A.D., 211,223 Bara, A., 237,242,243,246,258,262,

264,266

Baran, G.J.,154, 175, 178,195 Barenholz, Y.,216,223 Barnes, H.T., 328 Barnhmrn, M.G.,145, 150, 152, 154,

175, 178, 183,198

Barsukov, L.I., 216,223 Barter, P., 201,225 Barter, P.J., 213,223 Bartholdi, M., 127,138 BArzu, O.,231-233, 235,237,238,242,

243,246,248-250,253-255,258, 262, 264-266 Batenburg, J.J., 200,225 Bearden, 134,138 Beaufay, H.A., 152,192 Beck, G.,72,101 Beck, J.D., 182, 183,196 Beece, D.,120, 121, 124,137,138 Beeson, K.W., 116, 120, 121,137 Belch, A.C., 11,101 Bellanti, J.A., 189, 191, 194 Benesch, R., 235,236,264 Benesch, R.E., 235,236,264 Benga, G.,231-233,248,251,254,255, 264 Benjaminson, A., 300,328 Benson, D.M., 229,265 Benz, R., 57,101 Beohar, P.C., 189, 191,196 Bergelson, L.D., 201,216,223,224

264,266

Anderson, K., 311,313,328 Anderson, R.E., 213,224 Andersson, B., 152,192 Ann. N.Y. Acad. Sci., 322,328 Antonetti, A., 125,138 Antonini, E., 120,137, 230,235,261,

264

Applebury, M., 136,137 Archakov, A.H., 256,264 Astier, R., 125,138 Attardi, G.,185,195 Auk, K.A., 181,192

333

334

AUTHOR INDEX

Berger, R.L., 302, 308, 310, 31 1, 313, 319-321,328-331 Bergmeyer, H.U., 262,265 Berne, J.B., 7, 15, 20, 22, 103 Berry, K.H., 291,328 Berthet, J., 152, 192 Bienvenue, A., 200,226 BillesWUe, S., 254, 266 Blanchaer, M.C., 254,266 Bloemendal, H., 152, 192 Bloj, B., 201, 210,223 Bloomfield, V.A., 154, 175, 178,195 Boehmer, H., 180, 192 Boffoli, D., 261, 266 Bohme, H.J., 234,265 Bohn, B., 191, 195 Boltz, R.C.,143, 150, 152, 154, 156, 157, 167, 175, 176, 178, 183, 185, 189-192, 192-1 95, 197 Bondi, A., 25, 62, 101 Borcherdt, B., 328 Bdrjesson, B.W., 154, 161, 183, 193 Borza, V., 231, 237, 251,264 Bowen, P., 319,328 Bowers, L.D., 320,328 Bowne, S.F., 121, 124, 137 Boyle, R.,275, 328 Bozzato, R.P., 220,223 Bradford, M., 251,265 Brakke, M.K., 143, 150, 154, 193 Brierley, G.P., 229, 247,266 Brockerhoff, H.,214,225 Bronson, P.M., 150, 152-154, 161, 178, 181, 193, 198 Brooks, D.E., 148, 150, 177,193, I98 Brossmer, R., 191, 195 Brouillette, C.G., 37, 101 Brown, H.D., 328 Brown, R.P., 185, 197 Brown, W.E.L., 308, 328 Brubaker, L.H., 147, 193 Brumley, G.W., 200,225 Brunori, M., 120, 137, 230, 261,264 Buerkli, A,, 127, 138 Burns, G.W., 295,329 Burns, R.H., 228,267 Busslinger, M., 127, 138 Butler, M.M., 207, 223 Bysterbosch, B.H., 147, 148, 195 Bystrov, V.F., 216,223

Cajori, F., 274,328 Callendar, H.L., 279, 308,328 Campbell, H.D., 252,265 Capitano, N., 261,266 Capuano, F., 26 1,265-266 Caravaggio, T., 185,193 Cardinal, C.R., 75, 82, 102 Carmeli, C., 73, 101 Carnot, S., 278,328 Carr, P.W., 320,328 Cany, D.C., 161, 175, 177, 193 Cascio, H.E., 302, 310, 319,328 Cass, K.H., 234,267 Catsimpoolas, N., 143, 147, 150, 153-156, 158, 175, 176, 178-183,192-194, 196 Cetas, T.C., 322,328 Chan, S.S., 105, 116, 120, 121, 127, 137 Chance, B., 116, 138, 229, 247, 253, 258, 265,266 Chang, A.M., 135, I38 Chappuis, P., 279,328 Chataing, B., 213,224 Cheng, A.F., 296,328 Cherry, R.,127, I38 Chiancone, E., 235,264 Chipperlield, J.R., 310,328 Christensen, J.J., 313,329 Cioara, P.,242, 254,264 Cittadini, A., 258,265 Clark, L.C., 229,265 Clem,T.R., 308, 313,328 Cline, M.J., 182, 198 Cobon, G.S., 200,223 Cohen, L.K.,200, 213, 214, 218,223 Cohen, M.E., 272,328 Cole, J.H., 327,331 Collard, J.G., 152, 164, 183, 197, I98 Collie, C.H., 102 ComitC International des Poids et Mesures, 329 Commonwealth Technology, Inc., 329 Cmptes R d w de la Treidme Conftrence Gkdralt des Paids et Mesures, 1967-1 968, 28 1,329 Cmptes R d w des Stances de la N e u d m e Confhence G h k r a b &s Po& et Mesures,

1948,329

AUTHOR INDEX

C m p e s Rendus des Sparues dc In Quinrikne ConfketlEe G W a l e des Po& et Mesures, 1975, 282,329 Cmnptes Rendus des Sparues a% In Septit?me Confkrme G W a l e des Po& et Mesures, 1927, 281,329 Connolly, J.J., 286, 329 Conway, B.E., 20,101 Cork, J.M., 275,282, 329 Correa-Freire, M.C., 216,223 Corsey, R., 320,331 Crain, R.C., 200, 201, 207, 210, 217, 218, 223,223 Crambach, A., 153, 194 Cramer, R., 154, 156, 193, 196 Crawford, E.A., 161, 175, 177, 193 Cross, A., 132, 139 Crowfoot, P.D., 200,223 Czerlinsky, G.M., 44,101 Darnen, J., 213,223 Dancea, S., 251, 254, 255,264, 265 Dsnsoreanu, M., 237,238,242,243,246, 249,250, 258,262,264-266 Davids, N., 3 11, 3 13,328, 329 Davies, P., 229, 265 Davis, R.E., 154, 197 Debrunner, P.G., 116, 137 Defize, L.H.K., 211, 223 Degn, H., 229,265 De Jonge, H.R.,154,198 DeKouchkowsky, Y., 41,101 Delbruck, M.,66, 90, 101 De Leng, P., 145, 198 Deluca, N., 190, 194 DeMayer, L., 7, 61, 66, 69, 70, 72, 73, 101 Demel, R.A., 201, 211, 220, 221,223,226 httartog, M., 258,265 De Vaux, P.F., 200,226 DeVault, D., 117, 138 Dewanjee, M.K., 178, 183,194 Dhar, S., 189, 191, 196 DiCorleto, P.E., 201, 210, 221-223, 224 Diercksen, G.H.F., 21, 102 Dilger, J.D., 57, 102 Dill, K.A., 75, 101 Dixon, M., 228, 269 Docherty, J.J., 150, 152, 154, 178, 183, 185, 189, 190, 192,193, 195 Dodiuk, H.,23, 26, 101

335

Dombi, G.W., 145,197 Douady, P., 200, 201,224 Douzou, P., 117, 138 Downing, R., 116, 137 Drabkin, I.E., 272,328 Duckwitz-Peterlein. G., 2 16,224 Dudley, P.A., 213, 224 Durney, C.H., 301, 331 Duynstee, E.F.J., 46, 101 Dyatloviukaya, E.V., 201,224 Eaton, W., 109, 138 Ebrey, T., 138 Edridge, J.D.. 262,266 Edsinger, R.E., 291,329 Egbers-Bogaards, M.,182, 198 Ehnholm, C., 208,224 Ehrenson, S., 103 Eigen, M., 7, 44, 46, 61, 66, 69, 70, 72, 73,92, 101, 103 Eilenberg, G., 216, 224 Eisenstein, L., 116, 120, 121, 125, 137 Ek, K., 185, 197 Ellsworth, J.L., 212, 213,224 Erdy-Grutz, T., 14,101 Estabrook, R.W., 229, 253, 255,265 Evans, G.A., Jr., 286,330 Evans, W.H., 147, 152,193, 196 Expert Panel on Enzymes, Committee on Standards (IFCC) (1975), 324, 326,327, 329 Fahrenheit, D.G., 276,329 Fakharzadeh, F.F., 22 1, 224 Fatt, I., 229, 265 Feder, J., 256,265 Feinstein, M.E., 71, 101 Fendler, J.H., 102 Fenn, J.B., 20, 21, 103 Fery, C., 280,329 Fiess, H.A., 74, 78, 102 Fleming, G., 132, 139 Flory, P.S., 75, 101 Forlani, L., 320, 330 Fornerod, M., 254,266 Forster, Th., 8, 32, 36, 45, 46, 101 Frauenfelder, H., 1 17, 120, 121, 124, 137, 138 Fredriksson, S., 152, 193

336

AUTHOR INDEX

Frei, J., 238, 243, 254, 258, 265 Friauf, W.,302,308, 310,313,319, 321, 328,330 Friedman, J.M., 125, 138 Frornherz, P., 70, 82, 101 Fuller, N., 37-39, 67, 102 Fureinitti, P.S., 183, 293 Furukawa, G.T., 290,331 Gaal, O., 152,293 Gaines, R.A., 150, 152, 154, 178, 183, 189, 191-192, 193*195, 197. Gains, J.R., 300, 331 Galante, E., 185, 193 Calla, H.J., 216, 226 Geacintov, N., 127, 138 Gear, A.R.L., 161, 175, 177, 178, 193 Geiss, E., 150, 154, 295 Gershon, E., 50-51, 63, 67, 68, 74. 75, 78, 90,101, 102 Gerstman, B., 125, 238 Gerstman, B.S., 121, 138 Ghysen, W.E.J.M., 154,198 Gianazza, E., 185, 194, 197 Gibson, C., 308, 321,328,330 Gibson, Q.H., 120, I38 Gingel, D., 37, 67, 202 Giniget, R.,50-51, 63, 67, 68, 74, 75, 78, 90,102 Gitanda, V., 183,197 Glaeser, R.M., 158, 178, 195 Glau, J.F.C., 201,225 Glesmann, M.C., 67, 203 Glietenberg, D., 16, 101 Goldman, Y.E., 120, 138 Goldschmidt, R.,65, 102 Goldstein, 134, 138 Goldstein, L.,66, 71, 101 Good, D., 121, 124,137-138 Good, R.A., 154, 178, 180-183,193, 196 Gordon, E.E., 258,265 Gojatschko, L., 213,223 Goselle, U . , 36, 101 Gotto, A.M., Jr., 215, 225 Gould, A., 214,225 Gouterrnan, M., 120,138 Gratzel, M., 72, I02 Gray, I., 189, 191, 194 Green, D., 31 1,332 Green, D.E.,207,224 Gregory-Dewey, T., 43, I01

Greig, R.G., 185, 194 Griffith, A.L., 143, 150, 153-155, 175, 176, 178-181, 183, 192-194, 196 Grignon, C., 103 CroUman, A., 15, 101 Grosbois, M.. 200. 201,224 Gross, E.L., 158, 196 Grover, R., 189, 191, 197 Grunwald, F., 46, 101 Guerbette, F., 200, 201, 224 Guerrieri, F., 261,266 Gugan, D., 295, ?29 Guildner, L A . , 291, 295,329 Guillouzo, A., 143, 297 Gunsalus, I.C., 121, 125, 137, 139 G ~ p t aS., , 154, 178, 180-183,193,196 Curd, F.R.N., 66,67,102 Gutman, M., 7, I 1, 25, 33, 45, 46, 50-51, 57, 63, 65, 67, 68, 73-75, 78, 90, 101-103

Haar, M.P.,7, 15, 102 Hagdahl, L., 145, 154, 197 Hagen, A., 262,265 Hagenberger, B., 177,198 Haglund, H., 159, 184, 185, 194 Hale, J.D., 313,329 Halsall, B., 145,195 HaIsaU, H.B., 145,197 Hamberger. L.,259,265 Hammerstedt, R.H., 150, 152, 154, 138, 183, 185, 186, 189-191,192-294 Hamrnes, G.G., 43,101, 103 Hanania, G.I.H., 66, 67, 102 Hannig. K., 143-145, 148, 167, 174, 176, 180, 184,194, I98 Hansen, E., 147, 184, 194, 198 Haraux, F., 41, I01 Hards, J.M., 177, 198 Harmony, J.A.K., 213,224 Harned, H.S., 150, 194 Hart, A.A.M., 152, 164,197 Hartmann, W., 216,226 Harvey, M.S., 201, 207,224 Hasted, J.B., I02 Hauser, M., 7, 15, 36, 87, 101, I02 Hay, S., 190, 194 Hayson, D.A., 148, 150, I94 Heidrich, H.G., 148, 176, I94 Heirwegh, K.P.M., 145, 195

AUTHOR INDEX Hellings, J.A., 209, 218,221,224 Helmkamp, G.M., Jr., 201,207,209,218, 219,222,224 Henry, E., 109,138 Henry, U.P., 37,67,103 Herlitz, H., 259,265 Hershkowitz, S., 128,138 Hibberd, M.G., 120, I38 Hibbit, K.G., 160, 189, 191,195 Hickson, D., 215,225 Hill, A.V., 308,328 Hill, W., 113, 138 Hiram, S., 247,265 Hirorni, K.,138 Hirsch, R.L., 189, 191,194 Hjerten, S., 144, 147, 180,193,194 Ho, C., 66,67,73,78,103 Ho, N.T., 66,67,73, 78,103 Hochstrasser, H., 154,194 HodArnau, A.,251,254,255, 264,265 Hoffman, E., 234,265 Hofrichter, J., 109,138 Hogan, M.,128,138 Hollande, E., 143,197 Honig, B., I38 Honjo, I., 255,265-266 Hopfield, J.J., 121, 123, 125, 134, 137-138 Hopkins, G.J., 213,223 Hopkins, H.P., Jr., 308,319,321,328, 330 Horowitz, P., 113,138 Horwath, C.,25,40,102 Hostetler, K.Y., 218,225 Hudson, R.P., 291,329 Hughes, L.B.,201,212,225 Hughes, M.E., 204,207,222,223,226 Hultborn, R.,259,265 Hunter, S.J., 183,193 Huppert. D., 7, 11, 25,33,45,46,57,65, 74,78,101-103 Hurley, J., 138 Hyden, H., 259,265 Hyman, J., 137,138 Hymer, W.C., 150, 152, 154, 178, 183, 191, 192,193,197 Ihm, J., 213,224 Iizuka, T.,125,I39 Imai, K., 247,265

337

Inaba, A., 330 Ireland, J.F., 8,102 Ivanof, A., 237,243,265 Iwaoka, H., 298,330 Izaat, R.M., 313,329 IZZO,G., 261,265-266 Jackson, R.L.,212,224 Jahn, T.L.,145,198 Jain, M., 249,265 James, K., 229,266 Jarzynski, J., 327,331 Jaworowski, A., 252,265 Jebeleanu, G., 231,232,235, 264,266 Jobsis, F.F., 229,266 Johnson, C.C., 301,331 Johnson, L.W., 207,224 Jolicoeur, C.,321,329 Jones, J.L., 37,101 Jones, M.N., 185,194 Jonsson, M., 152,194 Jovin, T.,153,194 Jovin, T.M.,127,I38 Jung, S.,235,236,264 Jungalwala, F.B., 213,225 Just, W.W., 184-186,194 Kader, J.C., 200,201,218,224 Kagedal, L.,146,196 Kalsbeek, R., 201,211,223 Kamp, H.H., 201,207,209,217,218, 221,224,225 Kanety, H., 101, I02 Kano, K., I02 Kaplan, J. H., 149, 150,194,198 Kaplan, S., 200,213,214,218,223 Kapoor, N.,182, 183,196 Karohl, J., 127,I38 Karuzina, I.I., 256,264 Kasper, A.M., 209,222,224 Kaufman, K.J., 25,103 Kaufrnan, S., 252,265 Kebarle, P., 20,21,102 Keevil, T.,230,265 Keith, A.D., 185, 186, 189, 190, 194 Kelvin, L., 272,329 Kevan, L., 72,75,102 Kezdi, M.,254,265 Kipp, J.B.A., 198 Kirkwood, J.G., 67,103

338

AUTHOR INDEX

Kitamura, 0.. 255,266 Klaning, W.K., 65, 102 Klein, U.K.A., 7, 15, 36, 87, 101, 102 Klibanov, A.M., 320,330 Kloosterman, A.D., 201,207,224 Klou, I.M., 74, 78, 102 Knopp, J.A., 229,265 Kobdinsky, L., 229,266 Kolin, A., 143, 144, 147, 150, 180, 193-1 95 Kolodney, E., 7, 11, 74, 78, 102 Kooi, M.W., 198 Kopperschlanger, G., 234,265 Korohoda, W., 178,195 Kosower, E.M., 23,26, 101, 102 Kotani, M., 247, 265 Kraaipoel, R.J., 191, 195 Kraan, W., 152, 198 Kracek, F.C., 15,102 Kraemer, W.P., 21, 102 Krase, W., 7, 61, 66, 69, 70, 72, 73, I01 Kntmrine, P.H., 144, 177,198 Kufera, P., 259, 260,266 Kukulinsky, N.E., 190-192, 193 Kunze, K., 229,266 Kunze, M.E., 183, 197 Kuroda, M.V., 247,265 Kutschker, A., 16,101 Kuzmin, V., 127, 139 Laas, T., 146, 196 Labonowski, J.. 178, 195 Lachich, V., 65, 102 Lagakos, N.. 327,331 Lakowicz, J.R., 126, 138 Lakshmi, M.S., 143, 160, 188, 189, 191, 197 Landel, A.M., 144, 150,195 Landriscina, C., 214,225 Langille, F.A., 150, 154, 193 Langley, S.P., 279, 330 Larsson, C., 152, 192 L~SCU, I., 235,238,243,265-266 Laurent, T.C., 146, 196 hvalette, D., 121, Z38 Laws, F.B., 302,330 Leaback, D.H., 161, 195 hcarpentier, Y.,125, 138 Leise, E.M., 185, 188, 191, 195 h Neveu, D.M., 37, 67, I02

Lengyel, S., 14, 101 Lerner, H., 154, 194 Lerner, K., 145, 154, 197 Le Sane, F.,185, 188, 191, 195 Lessler, M.A., 229, 266 Lester, R.L., 207,224 Leuking, D.R., 200, 213, 214, 218,223 Lewis, E.S., 323,330 Lewis, G.N., 278,330 Li, A.S.W., 72, 75, 102 Liebich, H.G., 174, 180, 198 Liesegang, G.W., 319,330 Lijklema,J., 148, 196 Lillie, R.S., I95 Lim, T.K., 154, 175, 178,195 Linderstram-Lang, K., 187, 195 Lindqvist, L., 127, 137 Linnane, A.W., 200,223 Lipari, G., 61, 70,77, 103 Lis, L.J., 37, 67, 102, 103 Lloyd, D., 229,266 Loeb, A.L., 148, 198 Longmuir, I.S., 229, 265-266 h s , J.A., 175, 195 Lords, J.L., 301,331 Lorusso, M., 261,266 Louie, M.K., 143, 150, 154, 167, 175, 176, 183,193 Low, M.G., 223,224 Lu, A.Y.H., 255,266 Lubbers, D.W., 229,247,266 Lurnb, R.H., 200,201,207,224,225 Lumry, R., 139 Lundsgaard, J.S., 229,265 Lutter, R., 152, 183, 198 Lutton, C., 200, 201,224 Luxtrom, Inc., 330 Maasse, G., 7, 61, 66, 69, 70, 72, 73, 101 McAlister, M., 37, 67, 102 McCoy, G.D., 256,266 McCray, J.A., 120, 138 McDonald, D., 132, I39 MacDonald, R.C., 216,224 McGuire, J.K., 185, 195 Machida, K.,214, 222, 224 McKay, H.A.C., 202,224 McLaughlin, D., 137, 138 McLaughlin, S.A., 57, 101, 102 McLean, L.R., 209,225

AUTHOR INDEX McQuame, D.A., 87,192 McVittie, L., 212,224 Maeda, T., 224 Maillard, M., 254,266 Makin, H.L.J.. 262,266 Mangum, B.W., 286,287,289,290,330 Manske, W., 191,195 Manson, W., 154,I95 Mantsch, H.H., 254,265 Marcolin, H.-E., 125,138 Marden, M.C., 121, 124,137 Margel, S., 184,197 Margoliash, E.,25 1,266 Marini, M.A., 311,319,320,330-331 Markert, M., 237,238,243,258,261, 265-266 Marks, G.M., 183,197 Marlow Industries, Inc., 330 Marti, G.W., 320,330 Martin, C.J., 319,320,330 Martin, F.J., 216,224 Martin, J.L., 125,138 Mason, D., 154,195 Mason, H.S., 230,265 Massey, J.B., 215,225 Matheka, H.D., 150, 154,195 Matinca, D., 253,266 Matthew, J.B., 66,67,102 Mazliak, P., 224 Medgyesi, G.A., 152,193 Megli, F.M., 214,225 Mehrishi, J.N., 183, 185,195, 197 Mehta, A., 189, 191,197 Mel, H.C., 158, 178,195-197 Melander, W.,25,40,102 Merola, A.J., 229,247,266 Merzbacher, E., 132-133, 138 Metrologia 12,7 (1976),282,330 Metrologia 15,65 (1979),330 Metz, R.J., 200,225 Meuwissen, J.A.T.P., 145,I95 Meyer, B., 148,194 Micale, F.J., 144, 177,198 Michalik, M., 178,195 Michel, G.W., 295,329 Middleton, W.E.K., 273,330 Migus, A., 125,138 Milito, R.P., 150, 152, 154, 178,183,191, 192,193,197 Miller, J.D., 87, 102

339

Miller, R.G., 147, 195 Miller, T.Y., 185, 190-192, 193,195 Mitsui, K.,330 Moh, P.P., 121, 124,137 Molnar, I., 25,40,102 Monitto, C., 128,138 Montes, A., 201,212,225 Moonen, P., 201,225 Moore. H.D.M., 160, 189, 191,195 Morimoto, H., 247,265 Mortell, R., 150, 152, 154, 178, 183, 191, 192,193 Morton, R.E., 201,213,218,225 Mottola, H.A., 262,267 Mudd, C., 308,321,330 Mukerjee, P., 75,82,102 Munteanu, R., 237,242,243,246,258, 264 Muresan, L., 231-233, 237,243,248,251, 253-255,262,264,266 Murphy, M., 200,223 Nachliel, E., 7,25,33,50-51, 63,67,68, 70,72,74,75,78,90,101, 102 Narayana, P.A., 72,75,102 Nason, P., 145,195 Naughton, M.A., 153,194 Neihof, R., 195 Nelemans, S.A., 217,218,224 Nerren, B.H., 184,197 Nessi, P., 254,266 Newton, M.D., 103 Ng, T.C., 37,I01 Nichols, A.V., 158,197 Nielsen, S.O., 187,195 Nobel, P.S., 158,195, 196 Nordlund, T.M., 116, 120, 121,I37 Nossal, G.J.V., 180,192 Notter, M.F.D., 150, 152, 154, 178, 183, 191, 192,193 Oarga, M., 235,237,243,253,265-266 Ohnishi, J., 213,214,225 Ohnishi, S., 214,222,224 Ohte, A., 298,330 Oldenborg, V., 200,225 Olsen, R.B., 301,331 Ondrias, M.R., 125,138 Op den Kamp, J.A.F., 210,222,225 Opelz, G., 182,198

340

AUTHOR INDEX

Orbach, N., 23,26,102 Oshawa, T., 255,266 Oshino, N.,229,265-266 Oshino, R., 229,265,266 Ott, H.W., 110, 135,138 Ott, M.G., 143,197 Ottolenghi, M., 23, 26,65,102, 138 Overath, P., 216,224 Overbeek, J.T.G., 147, 148,195-197 Owen, B.B., 150,194 Ozawa, K., 255,265-266 Packer, L., 158,I96 Pande, S.V., 254,266 Papa, S.,261,265-266 Parish, G., 127,138 Parsegian, V.A.,3749.67,102, 103 Pascher, G.,171, 172, 174,180,I98 Pattnaik, N.M., 201,212,225 Patumraj, K.,214,225 Paul, R., 319,328 Pearson. L., 3 10,330 Perry, S., 147,197 Pertoft, E.A., 146,196 Perutz, M.F.,120, 121,137 Peters, K.,136,138 Petersen, L.C., 229,265 Peterson, E.A., 147, 152,196 Pettersson, S., 152, 170,194, 197 Phillips, M.C., 209,225 Phillips, R.A., 147,195 Philpot, J.S.L., 143, 158,196 me&, N., 175,196 Pines, E., 7,25,45,46,50-51, 57,63,65, 67, 70,72, 75,90,101, I02 Pinsent, B.R.W., 310,330 Plank, L.D., 183,197 Platsouas, C.D.,153, 154, 178, 180-183, 192,193,196 Plesset, M.S., 145,196 Plumb, H.H., 290, 322,330,331 Polak, F., 152,198 Polson,A., 156,196 Ponder, E., 150,I96 Poorthuis, B.J.H.M., 201,210,218,225 Popescu, O.,235,266 Porath, J., 147, 180,193 Porumb, H., 235,253,266 Possmayer, F., 207,225 Post, M.,200,225 Poulis, M.I., 252,265

Poulos, A.T., 127,138 Pownd, H.J., 215,225 Poyart, C., 125,138 Pradac, J., 175,196 Pradacova, J., 175,196 Resecan, E.,253,266 Pressman, B.C., 229,266 Preston-Thomas, H., 281,300 Pretlow, T.G., 181,196 Pretlow, T.P., 181,I96 Prouzova, O.,175,196 Puijk, W.C., 201,225

Quagliariello, E., 214,225 Racker, E., 256,266 Raddacz, E.,259,260,266 Radiation Thermometry Session (1982), 296,330 Radin, S., 200,225 Raftery, M.A., 185,195 Rajaram, O.V.,201,225 Rand, R.P., 37-39, 67,102,103 Randall, M., 278,330 Rani, S., 189, 191,196 Rao, K.V.,143, 188,189,191,196,197 Rao, M.,7, 15,20,22, 103 Redi, M.H., 121,I38 Regis, J., 213,223 Reinisch, L., 120, 121, 124,137, 138 Reiser, S.J., 274,331 Rembaum, A., 184, 197 Remenyik, C.J., 145,I97 Rentzepis, P., 109, 117,137, 138 Resch, R.C., 256,266 Reschke, R., 125,I38 Reuss, F.F., 197 Reynolds, A.H., 109, 117, 121, 124, 137-138 Rice, S.A., 11, 101 Richardson, L.S., 150, 152, 154, 178, 183, 191, 192,I93 Richter, P.H., 61,103 Riddle, J.L.,290,331 Riggs, A.,234,266 Righetti, P.G., 185, 187, 192,193-195, 197 Rilbe, H.,152, 170,177,194,197 Ritson, M.D., 102 Robbi, M., 152,192 Robbins, S.G., 178, 182,I96

AUTHOR INDEX Robinson, M.E., 200,225 Roller, D., 273-275,331 Roos, D., 175,195 Roozendaal, K.J., 182,198 Rosano, L., 71,I01 Roseman, M.A., 215,225 Rosen, P.J., 147,197 Rosenfeld, T., 138 Rossi-Bernardi, L., 235,264 Roughton, F.J.W., 308,310,330,331 Rousseau, D.L., 125,138 Rowland, F., 300,328 Rozzell, T.C., 301,331 Ruenwongsa, P., 213,225 Ruppel, D., 216,226 Russell, B., 156,I96 Russu, I.M., 66,67,73, 78,I03 Sahgami, T., 208,225 Sakura, H.,330 Sands, F., 249,265 Sapoff, M., 308,328 Sappof, M., 287,331 Sarfaty, G.A., 154,183,193 Sarton, G., 272,331 Sartory, W.K., 145.I97 Sasaki, T., 208,225 Sato, K.,258,266 Satre, M., 232,264 Scarpa, A., 255,258,265-266 Sceats, M.C., 11,101 Schabel, F.M., 147,197 Scherphof, G., 213,223 Schindler, F.,229,266 Schneider, G., 127,138 Schooley,J.F., 322,331 Schuldiner, S., 195 Schullman, S.G., 7,103 Schulz, J., 234,265 Schumaker, V., 145,195 Schumaker, V.N., 145,197 Schurr, M., I38 Schutz, L.S., 327,331 Schwedes, J., 145,195 Scott, A., 137,138 Scott, K.M., 229,247,266 Seaman, G.V.F., 177,193,194,198 Searcy, J.Q., 20,21,103 Searles, L.L., 221,224 Seebeck, T.J., 327,331 segrest, J.P., 37,I01

34 1

Serwer, P., 150,197 Shapiro, Y.E., 216,223 Sherbet, G.V., 143, 148,159,160, 184-188, 191,197 Shore, W.S., 67,103 Shortman, K., 180,192 Shoup, D., 61,70,77,103 Siemens, W.,279,331 Simonetti, A.L.M., 41,103 Singh, H.,213,225 Siwek, W.,308,328 Skeggs, L.T., 154,194 Skrabut, E.M., 180,193 Slaby, F., 214,225 Slater, E.C., 228,266 Sligar, S.G., 139 Sluyser, M., 152,198 Smets, L.A., 182,198 Smith, K.K., 25,I03 Smits, P., 201,225 Smolka, A.J.K., 184,197 Snow, T.R., 229,266 Snyder, R.S., 185,195 Sole, R., 61,I03 Somerharju, P.,214,225 Sommer, J., 109,138 Sorenson, L.B., 120,137,138 Sorof, S., 143,197 Spinks, C.H., 321,331 Spiro, T., 125,139 Stakelberg, M., 16,101 Standish, M.M., 211,223 Starlinger, H., 229,247,266 Stauffer, J.F., 228,267 Steere, R.L., 154,197 Stein, G., 65,102 Stellwagen, E., 234,267 Stockmayer, W.H., 61,103 Stoddart, L.C., 310,328 Stone, A.L., 185,I97 Streibel, M.J., 143, 150, 154, 156, 167, 175,176,183,193 Sturtevant, J.M., 302,331 Subramaniam, S.,214,225 Suzuki, R., 258,266 Svendsen, P.J., 156,I97 Svensson, H., 145,154,156,167,193, 194 Swanson, S.A., 67,103 Swam,J.M., 300,331 Szabo, A., 61,70,77,I03

342

AUTHOR INDEX

Takabi, T., 43,103 Takasan, H., 255,265 Tanaka, T., 213,214,225 Tanford, C., 66-68, 70,71,103 Tanizaw, H., 23,26,102 Tao, T., 127,139 Tarmure, C., 229,237, 238, 243,248, 254,264-266 Taylor, L.W., 272,276,331 Teerlink, T., 218,225 Telia, M., 235,237,243,265,266 Tenforde, T., 158, 178,195 Thines-Sempoux, M., 152,192 Thomas, J.A., 143,197 Thomas, J.K., 72,101 Thomas, P., 103 Thomas, W., 295,329 Thompson, C.J., 150, 152,154,178, 183, 185, 189-192,193,195 Thompson, S.T., 234,267 Thompson, T.E., 215,216,223,225 Thompson, W., 207,223 Thomson, A.E.R., 185,195 Thomson, W., 278,281,331 Thornton, D.D., 287,290,330 Tdinca, R.,254,264 Timmerman, A., 150,178,198 Timofeeva, N.G.,201,224 Tinker, D.O., 220,223,223 Tippets, R.D., 158,I97 Tipps, R.W., 185,195 Tipton, K.F., 249,267 Tiselius, A., 143,197 Todd, P., 143,150, 152,154, 156, 157, 167, 175, 176,178, 183, 185, 186, 189-192,192-195, 197 Tolben, W.R., 256,265 Tong, L.K.J., 67,103 Trautwein, A., 125,138 Trentham, D.R., 120,138 Tsao, F.H.C., 213,225 Tsuiki, S., 258,266 Tulp, A., 145, 150, 152, 154, 164, 175, 178, 182, 183,197,198 Urnbreit, W.W., 228,267 Utton, D.B., 298,331 Uzgiris, E.E., 149,150,194,198 Valeri, C.D., 180, 193 Valmet, E., 156,197

Valvani, S.C., 25, 40,I03 Van Assendelft, O.W., 230,236,267 Van Beck, W.P., 152, 182, 183,198 Vancea, D., 253,266 van Dam, K., 41,103 Van Deenen, L.L.M., 201,207,209-21 1 , 217,218,221,223-225 Van den Besselaar, A.M.H.P., 219,225 Van den Eeden, A.L.G., 21 1,223 Van der Hoff, J.W., 144, 177,198 van der Krift, T.P., 218,225 Van Der Ziel, A., 310,331 Van Duin, A.M., 191,195 vanColde, L.M.G., 200,225 Vanier, J., 291,331 Van Os, C.H., 154,198 Van Oss, C.J., 144, 150, 152-154, 161, 177, 178, 181,193,198 Van Regenmortel, M.H.V., 154, 156,198 Vass, Sz., 87,103 Vassar, P.S., 177,198 Vaughan, W.M., 229,267 Vaz, W., 127,139 Vereczkey, L., 152,193 Verster, T.C., 331 Vilah, C., 143,197 Vishniac, W., 229,267 Vitktorov, A.V., 216,223 Vogel, H., 127,139 Volkers, A., 32,36,45,46,101 Volkova, V.I., 216,223 Von Korff, R.W., 249,265 Vriend, G., 218,219,221,223,226 Vurek, G.G., 320,331 Wadso, I., 321,331 Wagner, G., 174,180,198 Walasek, O.F., 251,266 Waldeck, D., 132,139 Walters, J.A.L.I., 154,198 Wang, J., 128,138 Warach, J.B., 201,224 Warnock, D.G., 320,331 Warren, P.J., 262,266 Warshel, A., 7, 15,103 Watan. H.,247,265 Waterman, M.R., 230,267 Watkins, J.C., 211,223 Watson, R.H., 150,197 Watt, D., 311,331 Weber, G., 126,138,229,267

AUTHOR INDEX

Weir, E.A., 196 Weirsema, P.H., 148, 196, 198 Weller, A., 7, 8, 37, 61, 103 Wells, J.R., 170, 182,198 Werner, G., 184-186,194 West, S.B., 255,266 Westerhoff, H.V., 41, 103 Westerman, J., 201, 218, 219, 221, 223, 225-226 Westra, J.G., 152, 183, I98 Whipple, C.G., 145, 196 White, G.H., 201,225 Wibo, M., 152,192 Wickersheim, K.A., 296,331 Williams, G.R., 229, 247, 253,265 Williams, J., 229, 266 Williams, J.M., 150, 154, 175, I93 Williams, N., 229, 266 Winet, H.,145, 196, 198 Wirth, H., 148,194 Winz, K.W.A., 200, 201, 206, 207, 209-211.214, 217-219, 221, 223, 223-226 Wise, J.A., 286, 289, 294,330-331 Wolff, M. Ch., 262, 267 Wortis, H.H.,143, 175, 176, 178, 183, 194

343

W.P. Instruments, Inc., 331 Wrigley, C.W., 161, I95 Wu, L.N.Y., 200,225 Wyatt, P.A.H., 8, I02 XU, Y.H., 216,226

Yalkowsky, H.S., 25, 40, I03 Yamada, M., 213,214,225 Yamagata, M., 330 Yamamoto, H., 125, 139 Yapel, A.F., 139 Yonetani, T., 125,139 Young, I.G., 252,265 Yue, K.T., 121, 124,137,138 Zborowski, J., 21 1, 220, 221,223,226 Zeiller, K., 147, 148, 167, 171, 172, 174, 176, 180,194, I98 Zettergren, J.G., 196 Zhirnov, G.F., 256, 264 Ziegler, D.M., 207, 224 Ziegler, H.,216, 226 Ziljstra, W.G., 230, 236,267 Zilversmit, D.B., 200, 201, 204, 206-208, 210, 212,213,217,218,221-223, 223-226

Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

Subject Index Absolute temperature, concept of, 277 Absorption, monitoring triplet state population by, 128-130 Activation energy spectrum, 121-124 possible origin, 123-1 24 Adsorbed Bromo Creson Green, simulation of protonation, 75-78 Alkalinization pulse, proton emitter, 62-63 Amontons, Guillaume, 276 Amplification, signal, 112-1 13 Analog Devices AD590 Integrated Circuit Temperature Transducer, 304 Analog outputs, electronic thermometers, 306 Analytical electrophoresis, cells in density gradient, 174-175 column with laser beam, 175 discontinuous density interface, 175 transanalyzer, 174-1 75 Anisotropy decay, 127-128 Antigen-sensitized vesicles, 208 Assays, to quantitate lipid transfer activity: effects of composition of assay mixture, 218-223 separation, 203-206 specific, 206-2 14 spectroscopic, 2 14-2 17 Barothermoscope, 273 Bimetallic-strip thermometers, 294-295 Binary-coded decimal (BCD), 306-307 Black, Joseph, 278 Bolometers, 279 Boltz-Todd device, 156-157 Bone marrow cells, 182-183 Boyle, Robert, 274-275,276, 281 Biichler Polyprep, 153-156

Calibration, thermometer, 287 Callendar, H.L., 279 Carnot, Sadi, 278-279 Castelli, Father Benedetto, 273 Celsius, Anders, 276 Celsius scale, 276, 281 Chappuis, P.,279 Charge separation, intrinsic photoactivation, 132-135 conformation distribution, 133-1 34 instrumentation, 134-136 transfer band, 134 Charles, J.A.C., 277 Chemiosmotic hypothesis (Mitchell theory), 2 Christian of Lyons, 276 Clausius, Rudolf, 278 Coherent anti-Stokes Raman spectroscopy (CARS), 296 Comitt International des Poids et Mesures (International Committee on Weights and Measures), 278 Concentrated salt solution, proton dissociation, 15-21 Conformation distribution, electronseparation, 133-134 Controllers, temperature, 308, 309 Cryogenic techniques, laser photolysis, 116120 helium cryostat, 116-1 17 optical configuration, 118 sample preparation, 117-1 18 temperature measurement and control, 118-1 19 Cryostat optics, optical path, 118 Curie Law, 280 Dalence, Joachim, 275 Dalton, John, 277

345

346

SUBJECT INDEX

Density gradient electrophoresis, separation of mammalian cells, 141-198 apparatus, 152-175 analytical electrophoresis, 174-1 75 introduction, 152-153 preparative electrophoresis columns, 153-174 applications, 177-184 lymphoid, blood, and hemopoietic cells, 177-183 miscellaneous, 183 experimental conditions, 175-1 77 density solute, 175-1 76 electrodes, 176 electroendoosmosis, 177 ionic composition, 176 power supply, 176 future prospects, 184 history, 143 introduction, 142-143 isoelectric focusing, 184-192 applications, 187- 192 introduction, 184-186 lack of theory, 186-187 theory, 143-152 electrophoretic effects, 147-1 50 gravity effects, 144-147 miniaturization principle, 150-152 Dial thermometers, 294 Digital outputs, electronic thermometer, 306-307 Diode thermometers, 300-301 Discontinuous density interface, electrophoresis and, 175 Dissipation of energy, law of, 278 Electron spin resonance (ESR), 214 Electrophoresis columns, 153-1 74 Boltz-Todd device, 156-1 57 Biichler Polyprep, 153-156 ISCO column, 161 LKB model, 159-160 quickfit column, 161 separation chamber according to TULP, 162-174 compact device, 166-1 74 with movable electrodes, 162-166 small-size focusing chamber, 160-161 STAFLO, 158 Van Oss and Bronson device, 161-162

Electrophoretic effects, mammalian cells, 147-150 Electrophoretic separation of erythrocytes, 178 murine spleen B and T cells, 179 Eivius, 276 Erythrocyte membranes, transfer of radiolabeled phospholipids between vesicles and, 210 Erythrocytes, 177-178 Excited molecules, proton dissociation, 4 Fabri, Honore, 275 Fast stopped-flow thermal measurements, 308-3 11 Ferdinand 11, Grand Duke of Tuscany, 274 Fery, C., 280 Flashlamp-pumped organic dye laser, 109-1 10 Fluorescence, phospholipid exchange activity, 214-216 Focusing chamber, small-size, 160-161 Galileo Galilei, 271-272 Gallium, 287,289-290, 292 Gas thermometers, 295 Gay-Lussac, L.J., 277 Gravity effects, density gradient electrophoresis of mammalian cells, 144-147 Ground-state anion of proton emitter,

4546

Ground-state compounds, dynamics of protonation: excitation pulse, 5 geometry of excitation and probing beams, 6 measuring equipment, 5-6 monitoring light, 5 Helium cryostat, 116-1 17 Helmholtz, Hermann von, 278 Heme proteins, 120-125 activation energy spectrum, 121-124 possible origin, 123-1 24 kinetics, 120-121 molecular and spin tunneling, 124-125 Heron of Alexandria, 272 Hess, Victor, 278

SUBJECT INDEX

High-molecular-weight structure, kinetics of protonation, 66-84 effect of charge on rate, 69-73 postprotonation reaction, 73-84 uncharged target adsorbed on uncharged carrier, 68-69 High-speed transient recorder, 113-1 15 Hooke, Robert, 274-275 Human hemoglobin derivatives, spectral characteristics, 230 Human liver homogenates, cytochrome oxidase activity, 250-251 Huygens, Christian, 274-275 Hydrogen peroxide-generating oxidases, determination of, 249-250 Hydroxypyrene trisulfonate: fluorescence decay time, 27 lifetime of excited anion, 28 macroscopic rate constants, in water and protein complexes, 31 steady-state fluorescence emission, 25 time-resolved fluorescence, 28 IEEE-488 General Purpose Interface Bus (GPIB), 307 Immobilized enzyme reaction detection, thermal titration, 319-322 Incubations, unilamellar vesicles, 21 1 International Practical Temperature Scale (IPTS), 279, 281 International Practical Temperature Scale of 1968 (IPTS-68), 282-285,286 fixed points, 283 future improvements and extensions, 284 secondary reference points, 284 thermodynamic laws, 285 International Temperature Scale of 1927 (ITS-27), 280-28 1 Intrinsic photoactivation, 132-136 charge separation, 132-135 conformation distribution. 133 instrumentation, 134-136 transfer band, 134 proton pumping, 136 ISCO column, 161 Isoelectric focusing (IEF), 184-192 applications, 187-192 introduction, 184-186 lack of theory, 186-187 in natural pH gradients, 188-190

347

Isolated cells, oxygen consumption, 255-258 Johnson-noise thermometers, 298 Joule, James, 278 Kelvin, Lord, 278, 281 Kelvin thermodynamic scale, 281 Langley, S.P., 279 Large perturbation techniques, laser photolysis, 119-125 general considerations, 119-120 heme proteins, 120-1 25 activation energy spectrum, 121-124 kinetics, 121 molecular and spin tunneling, 124-125 Laser-induced proton pulse: effect of buffer on proton cycle, 90-98 three-compound system, 94-98 two-compound systems, 91-93 introduction, 2-3 methodology and instrumentation, 3-6 protonation of ground-state compounds, 4-6 proton dissociation from excited molecules, 4 protonation of high-molecular-weight structure, 66-84 effect of charge on rate, 69-73 postprotonation reaction, 73-84 uncharged target adsorbed on uncharged carrier, 68-69 proton dissociation, 6-22 in classical chemistry, 6-7 in concentrated salt solution, 15-21 effect of pK on rate of, 10 effect of solvent on rate of, 10-15 rate determination, 7-10 proton transfer, surface of macromolecular structure, 84-90 reactions, 22-66 with its emitter, 2 2 4 3 with molecular proton detector, 43-66 Laser photolysis, 105-139 intrinsic photoactivation, 132-136 charge separation, 132-135 proton pumping, 136 introduction, 106- 107

348

SUBJECT INDEX

Laser photolysis (Continued) pulsed lasers. 107-1 11 techniques: cryogenic, 116-1 19 hrge perturbation, 119-125 signal acquisition, 111-1 16 triplet-state probes, 125-132 anisotropy decay, 127-128 importance of long lifetimes, 125-127 instrumentation, 130-132 population, monitoring, 128-1 30 Le Chatelier, H.,280 Leains, vesicles sensitized to, 208-209 Linnaeus, Carolus, 276 Lipid transfer activity, 199-226 assaying crude extracts, 2 17-2 18 effects of composition of assay mixture, 218-223

acceptor and donor particles, 220-222

exchangeable lipid pool, 222-223 physical state of substrate lipid, 222 ratio of acceptor to donor particles, 219

general considerations, 202-203 introduction, 200-202 separation assays, 203-206 cross-contamination of isolated particles, 204-206 nonexchangeable markers, 203-204 specific assays, 206-214 microsomes-mitochondria, 206-208 vesicles, 208-2 13 spectroscopic assays, 2 14-2 17 electron spin resonance, 214 fluorescence, 2 1 4 2 16 nuclear magnetic resonance, 2 16 Liquid-crystal thermometers, 30 1 Liquid-in-glass thermometers, 293-294 LKB column, 159-160 Localized chemiosmosis or “local pH,” 41 Lymphoid, blood, and hemopoietic cells, 177-1 83

bone marrow cells, 182-183 erythrocytes, 177-1 78 peripheral blood cells, 180-182 platelets, 177 spleen cells, 178-1 80 thymocytes, 183 tonsillar cells, 182

Mammalian cells, density gradient electrophoresis, 141-148 apparatus, 152-1 75 analytical electrophoresis, 174-1 75 introduction, 152-1 53 preparative electrophoresis columns, 153-1 74

applications, 177-184 lymphoid, blood, and hemopoietic cells, 177-183 miscellaneous, 183 experimental conditions, 175-177 density solute, 175-176 electrodes, 176 electroendoosmosis, 177 ionic composition, 176 power supply, 176 future prospects, 184 history, 143 introduction, 142-143 isoelectric focusing, 184-192 applications, 187-192 introduction, 184-186 lack of theory, 186-187 theory, 143-152 electrophoretic effects, 147-150 gravity effects, 144-147 miniaturization principle, 150- 152 Maria of Alexandria, 272 Mayer, Alfred, 278 Mercury thermometers, 279 Microsomal oxidases, measurement of, 255-256

Microsomes, 206-208 Miniaturization principle, 150-152 Mitchell theory (chemiosmotic hypothesis), 2 Mitochondrial respiration, 253-255 Mode-locked CW dye laser, 110 Molecular proton detector, reaction of proton, 43-66 alkalinization pulse, proton emitter, 62-63

direct proton exchange, 57-62 ground-state anion of proton emitter, 4546

limitations and inaccuracies, 63-66 macroscopic parameters, 65-66 reactrants concentration, 63-65 pH indicators, 46-57

SUBJECT INDEX

Molecular tunneling process, 124-125 Monolayer vesicle, 211-2 12 Multilamebr vesicle, 210-2 1 1 preparation of, 21 1 Myoglobin. 120, 122 NAD-linked dehydrogenases, 252-253 2-Naphthol-3,5-disulfonate, fluorescence emission spectra, 33 National Bureau of Standards, 286,287 Negatively charged vesicles, 209-2 10 Nitrogen laser, 110 Noise thermometers, 298 Nonexchangeable markers, lipid transfer activity, 203-204 Nuclear magnetic resonance (NMR), 216 Optical pyrometers, 280 Oxidative phosphorylation, 253-255 Oxygen consumption, measurement of,

227-267

applications, HbOn method, 250-263 calculation of experimental data,

244-247

comparative measurements, 244-246 at different pigment concentrations,

246

polarographic estimation, 246-247 hydrogen peroxide-generating oxidases,

249-250

instrumentation, 236-242 introduction, 228-230 purification, characterization, and storage of donors, 233-236 chromatographic purification of HbOp, 234-235 determination of HbOn concentration, 235-236 hemolysate preparation, 234 simultaneous measurement of several parameters, 247-249 technical procedure: repetitive measurements, 243-244 single measurements, 242-243 Oxyhemoglobin (HbOp) method: applications, 250-263 continuous measurement, in vitrocultured embryos, 259-260 cytochrome oxidase activity in human liver homogenates, 250-251

349

determination of substrate concentrations, 262-263 isolated cells, 256-258 microsomal oxidases, 255-256 microspectrophotometric assay of oxygen consumption, 258-259 mitochondrial respiration and oxidative phosphorylation,

253-255

NAD-linked dehydrogenases,

252-253

rapid functional transitions of respiratory systems, 261 rat liver phenylalanine hydroxylase activity, 251-252 consumption measurements, 242-244 groups, 230-233 major characteristic, 230 Peripheral blood cells, 180-182 Philon of Alexandria, 272 pH indicators, reaction of proton with,

46-57

dynamics in absence of direct proton exchange, 47-5 1 effect of initial conditions on macroscopic parameters, 5 1-57 pH jump: effect of buffer on proton cycle, 90-98 introduction, 2-3 methodology and instrumentation, 3-6 protonation of high-molecular-weight structure, 66-84 proton dissociation, 6-22 proton transfer, surface of macromolecular structure, 84-90 reactions, 22-66 with its emitter, 22-43 with molecular proton detector,

43-66

Phosphatidylcholinevesicles, 210 Phosphorescence, monitoring triplet state population by, 130 Planck's radiation law, 280, 282 Plasma lipoproteins, 212-2 I3 Platelets of man and rat, 177 Platinum resistance thermometers, (PRTs), 286, 287 Polarographic estimation, oxygen consumption, 246-247

350

SUBJECT INDEX

Postprotonation reaction, 73-84 adsorbed Bromo Cresol Green, 75-78 classification, 78-84 Proteins. lipid transfer activity, 199-226 assaying crude extracts, 2 17-2 18 effects of composition of assay mixture, 218-223 general considerations, 202-203 introduction, 200-202 separation assays, 203-206 specific assays, 206-214 spectroscopic assays, 214-217 Protonation: ground-state compounds, 4-6 excitation pulse, 5 geometry of excitation and probing beams, 6 measuring equipment, 5 - 6 monitoring light, 5 high-molecular-weight structure, 66-84 effect of charge on rate, 69-73 postprotonation reaction, 73-84 uncharged target adsorbed on uncharged carrier, 6 8 4 9 Proton cycle, effect of buffer on dynamics, 90-98 three-component system (proton emitter, detector, and buffer), 94-98 buffer protonation, 96-98 initial conditions, 96 simulative solution, 94-96 two-component systems (buffer and proton emitter), 91-93 Proton dissociation, 6-22 in classical chemistry, 6-7 in concentrated salt solution, 15-21 effect of pK on rate, 10 effect of solvent on rate, 10-15 excited molecules, 4 poorly hydrating site, 33-38 rate determination, 7-10 scheme I, 8-10 Proton emitter, 22-43 alkalinization pulse, 62-63 dissociation in poorly hydrating site, 33-38 ground-state anion, 45-46 reactions in small, open, hydrating microcavity, 24-33

steady-state fluorescence, 24-26 time-resolved fluorescence, 27-33 Proton pumping, intrinsic photoactivation, 136 Proton reactions, 2 2 4 6 detection with its emitter, 22-43 discussion and conclusions, 3 8 4 3 dissociation in poorly hydrating site, 33-38 small, open, hydrating microcavity, 24-33 molecular proton detector, 43-66 alkalinization pulse, proton emitter, 62-63 direct proton exchange, 57-62 ground-state anion of proton emitter, 45-46 limitations and inaccuracies, 63-66 pH indicators, 46-57 Proton transfer, surface of macromolecular structure, 84-90 Pt-lO%Rh/Ptthermocouple, 286 Pulsed lasers, 107-1 1 1 flashlamp-pumped organic dye, 109-1 10 mode-locked CW dye, 110 nitrogen, 110 Q-switched Nd : YAG, 107-109 rare-gas excimer, 109 shielding, 110-1 11 Purified lipid transfer proteins, 201 Q-switched Nd : YAG laser, 107-109 Quickfit column, 161 Radiation thermometers, 296-298 Rankine, W.J.M., 276 Rankine (or absolute Fahrenheit) scale, 276 Rare-gas excimer laser, 109 Rate constant, proton dissociation, 6-22 in classical chemistry, 6-7 in concentrated salt solution, 15-21 determination, 7-15 effect of pK, 10 effect of solvent, 10-15 Rat liver microsomes, oxygen consumption and NADPH oxidation, 257

SUBJECT INDEX

Rat liver phenylalanine hydroxylase activity, 251-252 Rkaumur, R e d , 276 Rkaumur scale, 276 Reference points, temperature, 287 Regnault, H.V., 277 Renaldini, Carlo, 275 Resistance measurements, thermometer,

302

Resistance temperature detectors (RTDs),

301-304

Resistance thermometers, 295 Resonance thermometers, 298-30 1 Respiratory systems, rapid functional transitions, 26 1 Rey, Jean, 273-274 Riiemer, Ole, 275-276 RS232 serial interface, 307 Rumford, Count, 278 Seebeck, T.J., 279 Sensor measurement, electronic methods,

301-305

Separation assays, 203-2 14 cross-contamination of isolated particles,

204-206

nonexchangeable markers, 203-204 Separation chamber device, 162-1 74 compact, 166-174 diagram, 167 membranes, 167-169 preparing gradient, 169-174 with movable electrodes, 162-166 example of separation, 165-166 Shielding, pulsed lasers, 110-1 1 1 Side-on photomultiplier tube, 112 Siemens, Sir William, 279 Signal acquisition techniques, laser photolysis, 1 1 1-1 16 amplification, 113 side-on photomultiplier tube, 112 transient recorders: high-speed, 113-1 15 with signal averaging and logarithmic time base, 116 Silicon integrated-circuit temperature sensor, 304 Small-size electrofocusing chamber,

160-1 61

351

Small unilammellar vesicle, 210-2 1 1 preparation of, 21 1 Solid-stem liquid-in-glass thermometers,

293

Spectrophotometrically calibrated substrates, oxygen consumption measurements, 244-246 Spectrophotometric oxyhemoglobin method, 227-267 calculation of experimental data,

244-247

comparative measurements, 244-246 at different pigment concentrations,

246

polarographic estimation, 246-247 hydrogen peroxide-generating oxidases,

249-250

instrumentation, 236-242 introduction, 228-230 purification, characterization, and storage of donors, 233-236 chromatographic purification of H b 9 , 234-235 determination of HbOp concentration, 235-236 hemolysate preparation, 234 simultaneous measurement of several parameters, 247-249 technical procedure, 242-244 repetitive measurements, 243-244 single measurements, 242-243 see d o HbOn method Spectroscopic assays, 214-2 17 electron spin resonance, 214 fluorescence, 214-2 1 6 nuclear magnetic resonance, 216 Spleen cells, 178-1 80 Stable-flow-free boundary electrophoresis (STAFLO), 158 Standard platinum resistance thermometers (SPRTs), 290-291 Standard temperature reference system,

322-327

Steady-state fluorescence, 24-26 Temperature measurement, 269-33 1 conclusions and forecasts, 327 digital recording methods and devices,

306-307

352

SUBJECT INDEX

Temperature measurement (Continued) electronic methods, 301-305 resistance measurements, 302 voltage measurements, 302-305 introduction, 270-271 practical standards, 286-292 recent applications, 308-327 fast stopped-flow thermal measurements, 30 1-3 1 1 heat conduction and response time corrections, 311-319 thermal titration and immobilized enzyme reaction detection, 319-322 scales, 281-285 thermometry, 271-281 types of thermometers, 292-30 1 bimetallic-strip, 294-295 dial, 294 diode, 300-301 gas, 295 liquidcrystal, 301 liquid-in-glass, 293-294 noise, 298 radiation, 296-298 resistance, 295 resonance, 298-300 thermoelectric, 295-296 Thermal detectors, heat conduction and response time corrections, 3 11-3 19 Thermal titration, immobilized enzyme reaction detection and, 319-322 Thermoelectric thermometry, 295-296 Thermometry, 271-281 Thermopiles, 279 Thymocytes, 183 Time-resolved fluorescence, 27-33 Tonsillar cells, 182

Transanalyzer, 174-1 75 Transient recorder: high speed, 113-1 16 signal averaging and logarithmic time base, 116 Transition measurements, lipid phase, 216-217 Triplet-state probes. 125-1 32 anisotropy decay, 127-128 importance of long lifetimes, 126-127 instrumentation, 130-132 population, monitoring, 128-130 absorption, 128-130 phosphorescence, 130 Tulp chamber, 162-174 compact device, 166174 with movable electrodes, 162-166 Tunneling processes, ligand-hemeprotein recombination work, 124-125 Unilamellar vesicle, 2 10 incubations, 2 11 Van Oss and Bronson device, 161-162 Vesicles, 208-2 13 antigen-sensitized, 208 monolayer, 211-2 12 negatively charged, 209-210 sensitized to leains, 208-209 unilamellar, 210-211 Voltage measurements, thermometer, 302-305 VOM (volt-ohm-miliammeter), 302 von Helmholtz, Hermann, 278 Yttrium-aluminum-garnet (YAG) laser, 4

Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

Cumulative Author Index, Volumes 1-30 and Supplemental Volume Acherman, C . J., see Engb, R. W . Alber&on, Per-&, Interaction Between Biomolecules Studied by Phase Partition .............................................. Alb-on, Per-&, Partition Methods for Fractionation of Cell Particles and Macromolecules ................................. Albertsson, P., Anderrcon, B., Larsson, C., and Akerlund, H., Phase Partition-A Method for Purification and Analysis of Cell Organelles and Membrane Vesicles ............................ Alcoch, Nancy W., and MacIntyre, lain, Methods for Estimating Magnesium in Biological Materials ............................ A&, E l k , and W m k , Warren E. C., Enzymatic Methods Used for Diagnosis ................................................ Ames, S t a n k R.,see Embree, Nonis D. Andcrsen, C . A., An Introduction to the Electron Probe Microanalyzer and Its Application to Biochemistry .............. Anderson, N. G.,Preparative Zonal Centrifugation ................. Andrews, P., Estimation of Molecular Size and Molecular Weights of Biological Compounds by Gel Filtration ........................ A d , Mzra, see Grossowicz, Nathan Asboe-Hanren, Gustau, see Blumenkrantz, Nelly Aspen, Anita J., and M&&, Alton, Determination of Transaminase . . Augwtimon, Kh-Bertil, Assay Methods for Cholinesterases ........ Determination of Cholinesterases ............................. Austin, Rob& H., see Chan, Shirk S. Awdch, 2. L., see McLaren, D.S. Baker, S . A., Bourne, E. J., and Whffen, D. H., Use of Infrared Analysis in the Determination of Carbohydrate Structure ........ Balk, M . Earl, Determination of Glutamic and Aspartic Acids and Their Amides ........................................... Barchns, Jack D., see Faull, Kym F. Barks&&, A. D., and Rosenberg, A , , Acquisition and Interpretation of Hydrogen Exchange Data from Peptides, Polymers, and Proteins Bhrzu, Octuvian, Measurement of Oxygen Consumption by the Spectrophotometric Oxyhemoglobin Method ................... B a d , W. S., and Greenway, R. M., Chemical Determination of Estrogens in Human Urine ................................... Bayer, Edward A., and Wilcheh, Meir, The Use of the Avidin-Biotin Complex as a Tool in Molecular Biology .......................

353

VOL.

PAGE

29

1

10

229

28

115

14

1

13

265

15 15

147 27 1

18

1

6 5

SUPP.

131 1 217

3

213

20

103

28

1

30

227

5

337

26

1

354 CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Bell, H e h H., see J q s , Lou& B . Benesch, Reinhold, and Benesch, Ruth E., Determination o f C H Groups in Proteins ........................................... Benesch, Ruth E., see Benesch, Reinhold Benson, E. M., see Storuich, C. A. B e n t b , J . A., Analysis of Plant Hormones ........................ Benzinger, T. H., see Kitzinger, Charlottc Berg, Marie H., see SchwaHz, Samvel Berger, Robert L., Ckm,Thomac R , Sr., Hardm, V u t h A. and Mangum, B.W., Historical Development and Newer Means of Temperature Measurement in Biochemistry.. ................... Bergmonn, Felix, and Dikrtkn, Shabtu?, New Methods for Purification and Separation of Purines .................................... Benon, S o h A,, see Yalow, Rosalyn S. BhaMi, Tarig, see Clump,J. R . B ~ c ~E .I M., , Determination of Carotene ........................ Binnmi3, W . T., Determination of Iodine in Biological Material ..... Bishop. C. T., Separation of Carbohydrate Derivatives by Gas-Liquid Partition Chromatography .................................... Btclckburn, S.,T h e Determination of Amino Acids by High-Voltage Paper Electrophoresis ........................................ Blmu, D. M.,see Holmes, K. C. Blumenkrantz, Nelly, and Asboe-Hansen, Gustav, Methods for Analysis of Connective-Tissue Macromolecules by Determination of Certain Constituents ......................................... Bodanshy, Oscar, see Schwartz, Morton K. Bosscnmain; Irene, see Schwartz, Samuel Bosshard, H a m RudoK Mapping of Contact Areas in Protein-Nucleic Acid and Protein-Protein Complexes by Different Chemical Modification ....................................... B o u h , Alan A., T h e Automated Analysis of Absorbent and Fluorescent Substances Separated on Paper Strips .............. Boulton, A. A., see Majn; J. R. Bourne, E. J., see Baker, S.A. Brantmarh, B. L., see L i d , N . 0. Brauser, BoUO, see Sics, Helmut Bray, H. G.,and Thmpe, W. V., Analysis of Phenolic Compounds of Interest in Metabolism ....................................... Brierby, G. P.,see Lessler, M. A. Broahen, R., and Jacobsen, J., Separation and Determination of Bile Pigments ............................................... Brodie, B m r d B., see Udenfrhd, Sidney B r o o k , Gory, Newer Development in the Determination of Cyclic AMP and Other Cyclic Nucleotides, Adenylate Cyclase, and Phosphodiesterase ....................................... B u d s , Carl A., Tiffany, Thomcrr O., and Scott, Charles D., The Use of a Centrifugal Fast Analyzer for Biochemical and Immunological Analyses ...................................... Bush, I. E., Advances in Direct Scanning of Paper Chromatograms for Quantitative Estimations .................................. Bush, 1. E., Applications of the R MTreatment in Chromatographic Analysis .................................... Erratum ....................................................

10

43

9

75

30

269

6

79

4

22

1 25 1

10

1

13

1

24

39

25

273

16

327

1

27

17

31

22

95

23

189

11

149

IS

357 497

14

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT 355 CaldweU, Karin D., see Giddings, J . Calvin Campbell, I. D., and Dobson, C. M., The Application of High Resolution Nuclear Magnetic Resonance to Biological Systems ... Carstensen, H., Analysis of Adrenal Steroid in Blood by Countercurrent Distribution .................................. Caster, W. O., A Critical Evaluation of the Gas Chromatographic Technique for Identification and Determination of Fatty Acid Esters, with Particular Reference to the Use of Analog and Digital Computer Methods .......................................... Chambers, Robin E., see Clamp J . R. Chan, Shirlq, S., and Amtin, Robert H., Laser Photolysis in Biochemistry .............................................. Chance, Britton, see Maehly, A . C. C h e , Aurin M., The Measurement of Luciferin and Luciferase .... Chinard, Francis P., and H e h , Leslie, Determination of Sulfhydryl Groups in Certain Biological Substrates .............. Christen P., and Gehring, H., Detection of Ligand-Induced and Syncatalytic Conformational Changes of Enzymes by Differential Chemical Modification ....................................... Clamp, John R., and Bhatti, T., and Chambers, R. E., The Determination of Carbohydrate in Biological Materials by GasLiquid Chromatography ...................................... Clark, S t a n k J., see Wotiz,Herbert H . Cleaary, E. G., see Jackson, D. S. Clem, Thomm R., Sr., see Berger, Robert L. Code, Charla F., and McIntyre, Floyd C., Quantitative Determination of Histamine ................................................ Cohn, Waldo E., see Volhin,Elliot Cotlove, Ernest, Determination of Chloride in Biological Materials ... Craig, Lyman C., and King, Te Piao, Dialysis ....................... see also King, Te Pi00 Crane, F. L., and DiuLy, R. A., Determination of Coenzyme Q (Ubiquinone) ................................................ Creech, B. G., see Homing, E . C. Creueling, C. R. and Dab, J . W., Assay of Enzymes of Catechol Amines ............................................ CUT, A. S., The Analysis of Basic Nitrogenous Compounds of Toxicological Importance .................................... Daly, J . W., see Creveling, C. R . Daviakan, Harold M., see Fkhman, William H , Davis, Neil C., and Smith, Emil L., Assay of Proteolytic Enzymes ..... Davic, R.J., see Stohtad, E. L. R. Davis, Robert P.,The Measurement of Carbonic Anhydrase Activity Dean, H. G., see Whitehead,J . K. Degn, H.,L u d g a a r d , J . S., Peterson, L. C., and Omuki, A., Polarographic Measurement of Steady State Kinetics of Oxygen Uptake by Biochemical Samples ............................... Dihstein, Shabtay, see Bergmann, Felix DiuCy, R. A., see Crane, F. L. Dinnnore, Howard, see Schwartz, Samuel Dkche, Za.chak, New Color Reactions for the Determination of Sugars in Polysaccharides ..................................... Dodgmn, K. S., and Spencer, B., Assay of Sulfatases ................

25

1

9

127

17

135

30

105

8

61

1

1

28

151

19

229

3

49

12

277 175

11

279

SUPP.

153

7

39

2

215

11

307

26

47

2

313 211

LO

4

356 CUMULATIVE AUTHOR IXDEX, VOLUMES 1-30 AND SUPPLEMENT Use o f Subzero Temperatures in Biochemistry: Slow Reactions .............................................. Dyer,John R., Use o f Periodate Oxidations in Biochemical Analysis Edwarh, M . A., see Sloruick, C. A. Elving, P.J., O'Rklly, J . E., and Schmakel, C. O., Polarography and Voltammetry of Nudeosides and Nucleotides and Their Parent Bases as an Analytical and Investigative Tool ................... Embree, Nmrir D., Ames, Stanley R., Lehman. Robert W., and. Harris, Philip L., Determination of Vitamin A ......................... Engel, L& L., The Assay o f Urinary Neutral 17-Ketosteroids ..... Engel, R. W., Salmon, W . D., and Ackennan, C.J., Chemical Estimation of Choline ........................................ Engeiman, Karl, see Lovenberg, S. W a k E m & , Lars, see Lindberg, OIa, Everse, Johannes, Ginsburgh, Char& L., and Kaplan, Nathan 0.. Immobilized Enzymes in Biochemical Analysis .................. Faull, Kym F., and B a r c h , Jack D., Negative-Ion Mass Spectrometry, Fused-Silica Capillary Gas Chromatography of Neurotransmitters and Related Compounds ..................................... Felber, J . P., Radioimmunoassay of Polypeptide Hormones and Enzymes ................................................ Fink, Freahick S.,see Kersey, Roger C. Fisher, Susan R., see Giddings, J . Calvin Ftihman, William H., Determination of f3-Glucuronidases ........... Fishman, Willram H., and Davidson, Harold M., Determination o f Serum Acid Phosphatases .................................... Fleck, A.. see Munro, H. N. FwsCn, Sture, and Lindman, B j h , Ion Bonding in Biological Systems Measured by Nuclear Magnetic Resonance Spectroscopy ........ Fraenhel-Conrat, H., Harris,J . Inran, and 4 , A. L., Recent Developments in Techniques for Terminal and Sequence Studies in Peptides and Proteins ...................................... Friedman, Sydnty M., Measurement of Sodium and Potassium by Glass Electrodes ............................................. Frisell, Wilhelm R., and Mackenzi.e, Co.ww G., Determination o f Formaldehyde and Serine in Biological Systems ................ Gale, Ernest F., Determination of Amino Acids by Use of Bacterial Amino Acid Decarboxylases .................................. Gar&& Sven, Determination of Hexosamines ...................... GasheU, Simon J., Analysis o f Steroids by Mass Spectroscopy ........ Giddings,J . Calvin, Myers, Marcus N.,Caldwell, Karin D., and Fisher, Susan R., Analysis of Biological Macromolecules and Particles by Field-Flow Fractionation ...................................... Gofman,John W., see Lalla, Oliver F. de Goklbmg, Nelson D., and OToole, Ann G., Analysis of Cyclic 3',5'-Adenosine Monophosphate and Cyclic 3',5'-Guanosine Monophosphate .............................. Grabar, P k e , Immunoelectrophoretic Analysis ................... Greenway, R. M., see Bauld, W. S. Gross, D.,see Whalky, H.C. S. & Grossman, Shlomo, Oestreicher, G u i h o , and Singer, T h P., Determination of the Activity of Phospholipases A, C, and D ....

Douzou, Pierre, The

22 3

40 1

21

287

4 1

479

1

265

25

135

29

325

22

1

15

77

4

257

27

289

2

359

10

71

6

63

4

6 29

285 289 385

26

79

20 7

1

22

177

111

43

1

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Grossman, S h k , and Zakut, R i m , Determination of the Activity of Lipoxygenase (Lipoxidase) .................................... Grossowicz, Nathan, and Ariel, Mira, Methods for Determination of Lysozyme Activity ........................................... Guhnun, Menachem, The pH Jump: Probing of Macromolecules and Solutions by a Laser-Induced, Ultrashort Proton Pulse-Theory and Application in Biochemistry .............................. Haegeie, Klaw D., see Thkrot,Jean-Paul G. Haglund, Herman, Isoelectric Focusing in pH Gradien-A Technique for Fractionation and Characterization of Ampholytes Haines, William J., and Karnemaat, John N., Chromatographic Separation of the Steroids of the Adrenal Gland ................ Hanessianr, Stephen, Mass Spectrometry in the Determination of Structure of Certain Natural Products Containing Sugars ....... Harden, Victoria A., see Berger, Robert L. Harris, J . Ieuan, see Fraenhel-Cunrat, H . Harris, Philip L., see Embree, NonG D. Heimegh, K . P . M., Recent Advances in the Separation and Analysis of Diazo-Positive Bile Pigments ............................... Helhman, Leslie, see Chinard, Francis P. Hermans, Jan, Jr., Methods for the Study of Reversible Denaturation of Proteins and Interpretation of Data ......................... Hater, Charles S., see Wikheh, Meir Hiromi, Keitaro, Recent Developments in the Stopped-Flow Method for the Study of Fast Reactions ............................... Hirschbein, L., and G u i l h , N., Characterization, Assay, and Use of Isolated Bacterial Nucleoids .................................. Hjertdn, S., see Porah J. Hjertdn, Stellan, Free Zone Electrophoresis. Theory, Equipment and Applications ............................................ Hjertdn, Stellan, Hydrophobic Interaction Chromatography of Proteins, Nucleic Acids, Viruses, and Cells on Noncharged Amphiphilic Gels ................................ Hoff-Jmgmen, E., Microbiological Assay of Vitamin BIZ ........... Holnon, Ralph T., Measurement of Lipoxidase Activity ............ Measurement of Polyunsaturated Acids ........................ Holmes, K. C., and Blow, D. M . , The Use of X-ray Diffraction in the Study of Protein and Nucleic Acid Structure ................... H m h , Jiri, Polarography of Proteins, Analytical Principles and Applications in Biological and Clinical Chemistry ............... H m i n g , E. C., Vandm Heuvel, W .J . A., and Creech, B. G., Separation and Determination of Steroids by Gas Chromatography ......... Hurvath, C., High-Performance Ion-Exchange Chromatography with Narrow-Bore Columns: Rapid Analysis of Nucleic Acid Constituents at the Subnanomole Level ........................ Huugh, Leslie, Analysis of Mixtures of Sugars by Paper and Cellulose Column Chromatography .................................... Hughes, Graham J . and Wilson, Kenneth, J . , High-Performance Liquid Chromatography: Analytic and Preparative Applications in Protein Structure Determination .............................. Hughes, Thomas R., and Klotz, Irving M., Analysis of Metal-Protein Complexes .....................................

357

25

303

29

435

30

1

19

1

1

171

19

105

22

205

13

81

26

137

28

297

18

55

27 1 2 4

89 81 113 99

13

113

19

435

11

69

21

79

1

205

29

59

3

265

358

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT

Humphrey,J . H., Lung, D. A., and P m y , W. L. M., Biological Standards in Biochemical Analysis ............................. Hutner, S.H., see Stokslad, E. L. R. Jackson, D. S.,and Cleaty, E. G., The Determination o f Collagen and Elastin .................................................. Jocobs, S., The Determination of Nitrogen in Biological Materials ... Jacobs, S., Determination o f Amino Acids by Ion Exchange Chromatography ............................................ Jmobs, Stanley, Ultrafilter Membranes in Biochemistry ............. Jacobsen, C. F.,Ltonk,J., Linderstrm-Lang, K., and Ottesen, M., The pH-Stat and Its Use in Biochemistry ........................ Jacobsen, J.. see B r h s e n , R. James, A. T.,Qualitative and Quantitative Determination of the Fatty Acids by Gas-Liquid Chromatography ......................... Jams, D o u g h R., and Lumty, Rufiu W., Recent Developments in Control of pH and Similar Variables .......................... James, Gordon T., Peptide Mapping of Proteins .................... Jques, Louis B., Determination of Heparin and Related Sulfated Mucopolysaccharides ................................ Japes. Louis B., and Bell, HeZen J., Determination o f Heparin ...... Jar&@, C., and Jar&t&y, O., Biochemical Applications of Magnetic Resonance ....................................... Jardetzhy, O.,see Jarhtzhy, C. Jenden, Donald J., Measurement o f Choline Esters ................. Johnson, George, Gel Sieving Electrophoresis: A Description of Procedures and Analysis of Errors ............................ Joliconcr, Cannel, Thermodynamic Flow Methods in Biochemistry: Calorimetry, Densimetry, and Dilatometry ..................... Jones, Richard T., Automatic Peptide Chromatography ............. JoscfSFon, L. I., and Logcrstedt, S.,Characteristics o f Ribonuclease and Determination of Its Activity .................................. Jukes, ThmMs H., Assay of Compounds with Folk Acid Activity .... Kabara, J . J., Determination and Localization of Cholesterol ....... Kalckar, Hennun M., see Plesnn; P a d Kapcllcr-A&, R., Determination o f Amine Oxidases .............. Kaplan, A., The Determination of Urea, Ammonia, and Urease .... Kamemaat, John N.,see Haines, William J . Kearney, Edna B., see Singer, T h P. Keenan, Robert G., see Saltunan, Bernard E. Kmey, Roger C., and Fink, Freo!mih C., Microbiological Assay of Antibiotics ................................................ King, Te Piao, and Craig, Lyman C., Countercurrent Distribution .... see also Craig, Lyman C. Kitzinger, Charlotte, and Benzinger, T. H., Principle and Method of Heatburst Microcalorimetry and the Determination of Free Energy, Enthalpy, and Entropy Changes ....................... Klotz, INing M., see Hughes, Thumas R. Kobayashi, Yutoka, and Maudrley, David V., Practical Aspects of Liquid-Scintillation Counting ................................. Kolin, Alexandn; Rapid Electrophoresis in Density Gradients Combined with pH and/or Conductivity Gradients ..............

5

65

15 13

25 241

14 22

177 307

4

171

8

1

29 26

137 165

24 7

203 253

9

235

SUPP.

183

29

25

27

18

171 205

9 2 10

39 121 263

SUPP, 17

35 311

1 10

53 201

8

309

17

55

6

259

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Kopin, Irwin J., Estimation of Magnitudes of Alternative Metabolic Pathways .......................................... K m , Edward D., The Assay of Lipoprotein Lipase in vivo and in vitro .................................................. KuAris, A., New Developments in Determination of Bile Acids and Steroids by Gas Chromatography ............................. Kunkel, Henry G., Zone Electrophoresis ........................... Kurnich, N. B., Assay of Deoxyribonuclease Activity ............... Lugerstedt, S., see Josefsson, L. I. Lulh, Oliver F. de, and Gofman,John W., Ultracentrifugal Analysis of Serum Lipoproteins .......................................... Laursen, Ruhurd A., and M a c U t , Werner, Solid-Phase Methods in Protein Sequence Analysis .................................... Lazarow, Amold, see Patterson, J . W. Leddicotte, George W., Activation Analysis of the Biological Trace Elements .............................................. Lehman, Robert W., Determination of Vitamin E ................... see also Embree, N I ~ YD.I ~ Lekr, Luis F.,see Ponh, Horacio G. Leon&,J., see Jacobsen, C. F. Le Pecq, Jean-Bernard, Use of Ethidium Bromide for Separation and Determination of Nucleic Acids of Various Conformational Forms and Measurement of Their Associated Enzymes ................ Lerner, Aaron B., and Wright, M . Ruth, in vitro Frog Skin Assay for Agents That Darken and Lighten Melanocytes ................. Lessler, M. A., Adaptation of Polarographic Oxygen Sensors for Biochemical Assays .......................................... Lessler, M. A,, and B w l e y , G. P.,Oxygen Electrode Measurements in Biochemical Analysis ...................................... L q ,A. L., see FraetUtel-Conrat, H. L q , H i h B., see Webb,Junius, M . Lindberg, O h , and E m t n ; Lars, Determination of Organic Phosphorus Compounds by Phosphate Analysis ................ Lindcrstrom-Lung, K., see Jacobsen, C. F. L i d , N.O., and Brantmrh, B. L., Preparation and Analysis of Basic Proteins ............................................. Lindman, B j h , see Font%, Sture Lissikhy, Serge, see Roche, Jean Long, D. A.. see Humphrey,J . H. Lovenberg, S. Walter, and Engelman, Karl, Serotonin: The Assay of Hydroxyindole Compounds and Their Biosynthetic Enzymes .................................................... Loveridge, B. A,, and Smales, A. A., Activation Analysis and Its Application in Biochemistry .................................. Lumry, Rujius, see Yapel,AnthonyF.,Jr. Lumry, Rujiw W., see Jams, D o u g h R. Lundquict, Frank, The Determination of Ethyl Alcohol in Blood and Tissues ................................................. Lunhgaard. J. S.,see Degn, H. McCarthy, W.J.. see Winefordner,J . D. Mach&& Werner, see Lawsen, Richard A.

359

11

247

7

145

14 1 9

325 141 1

1

459

26

201

19 2

345 153

20

41

8

295

28

175

17

1

3

1

14

79

SUPP.

1

5

225

7

217

360 CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Mclntire, Ftoyd C., see Code, Charks F. M a d w e , lain, see Alcock, Nancy W . Machmie, Cmmo G., see FriseU, WilhelmR. MacKenzk, S.L., Recent Development in Amino Acid Analysis by Gas-Liquid Chromatography .................................. McKibbin,John M.,The Determinarion of Inositol, Ethanolamine, and Serine in Lipides ........................................ Mchren, D. S.,Read, W . W . C.,A d h , 2.L.,and Tchalian, M., Microdetermination of Vitamin A and Carotenoids in Blood and Tissue .................................................. M c P h o n , Alexander, The Growth and Preliminary Investigation of Protein and Nuclei Acid Crystals for X-Ray Diffraction Analysis Maehly, A. C., and Chance, Britkm, T h e Assay of Catalases and Peroxidases ............................................. Majer, J . R., and BouUa, A. A., Integrated Ion-Current (IIC) Technique of Quantitative Mass Spectrometric Analysis: Chemical and Biological Application .................................... Malmtrimr, Bo C., Determination of Zinc in Biological Materials .... Mangold, Helmut K.. Schmid, H a r d H. 0..and Stahl, Egon, ThinLayer Chromatography (TLC) ................................ Mangum, B.W., see Berger, Rob& L. Margmhcs, Marvin, and V&e, Brrt L., Flame Photometry and Spectrometry: Principles and Applications ..................... Maudsb, David V., see KobaFhi, Y & Mcirter, A h , see As@, Anita J . Muhel, Raymond, see Roche, Jean Micklsen, O w , and Yamamoto,Richard S.. Methods for the Determination of Thiamine ................................... Miller, H& K., Microbiological Assay of Nucleic Acids and Their Derivatives ............................................ Mdner, Kelsty. see Ribi, Edgar Miwa, I., see Oh&, J . Montgomety,Rcx, see Smith, Fred Mullcr, Otto H., Polarographic Analysis of Proteins, Amino Acids, and Other Compounds by Means of the Brditka Reaction ....... Munro, H. N., and Fleck, A., The Determination of Nucleic Acids ... M9en, Marcus N., see G a n g s , J . Calvin NatCLron, Samuel, and W w m d ,William R., Determination of Elements by X-Ray Emission Spectrometry ..................... Neaq, Michael P., see Sktz, W . RudoY Neish, WilliamJ. P., a-Keto Acid Determinations .................. NoveU:, G. David, Methods for Determination of Coenzyme A ...... Oberlem, D d , The Determination of Phytate and Inositol Phosphates .......................................... Oestreicher, GuiUmno, see Grossman, Shlumo O h f a , J., and Miwa, I., Newer Developments in Enzymic Determination of D-Glucose and Its Anomers .................. O&m, K. G., Radiometric Methods for Enzyme Assay ............ Olson,0. E., Palmer, I. S.,and Whitehead,E. I., Determination of Selenium in Biological Materials .............................. O’Rcillg,J . E., see Elving, P. J . onnicrki, A., see Degn, H .

27

1

7

111

15

1

23

249

1

357

21 3

467 327

12

393

3

353

6

191

6

31

11 14

329 113

12

1

5

2

107 189

20

87

21 21

155

21

39

191

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT 361 OToole, Ann G., see Goldberg, Nelson D.

Ottesen, M., see Jacobsm, C. F. Ottesen, Martin, Methods for Measurement of Hydrogen Isotope

Exchange in Globular Proteins ................................ Pdmer, I. S.,see Olson,0. E . Parker, Reno, see Ribi, Edgar Patterson, J. W., and Lazarow, Arnold, Determination of Glutathione Peny, W. L . M., see Humphrey, J . H . Persky, Harold, Chemical Determination of Adrenaline and Noradrenaline in Body Fluids and Tissues ..................... Peterson, L. C., see D e e , H . P l e s w , Paul, and Kalckar, Hermun M., Enzymic Micro Determinations of Uric Acid, Hypoxanthine, Xanthine, Adenine, and Xanthopterine by Ultraviolet Spectrophotometry ........... Po&, Horacio G., and Lebir, Luis F., Measurement of UDPEnzyme Systems ............................................. Porath, J., and Hjerth, S.,Some Recent Developments in Column Electrophoresis in Granular Media ............................ Porter, Curt C., see Silber, Robert H . Pod&, M . D., Gel Electrophoresis in Buffers Containing Urea ..... Pourfarznneh, M., Kamel, R. S.,Landon, J., and Dawes, C. C., Use of Magnetizable Particles in Solid Phase Immunoassay ............. Ranflaub, Jurg, Applications of Metal Buffers and Metal Indicators in Biochemistry .............................................. Radin, Nonnan S., Glycolipide Determination ..................... Ramwell, P. W., see Shaw, Jane E. Read, W ,W. C., see McLaren, D. S. Ribi, Edgar, Parker, Reno, and Milner, Kekey, Microparticulate Gel Chromatography Accelerated by Centrifugal Force and Pressure Robins, Eli, The Measurement of Phenylalanine and Tyrosine in Blood .................................................... Robins, S.P., Analysis of the Crosslinking Components in Collagen and Elastin .................................................. R o c k ,Jean, Lissitzhy, Serge, and Michel, Raymond, Chromatographic Analysis of Radioactive Iodine Compounds from the Thyroid Gland and Body Fluids ....................................... Rock, Jean, Mickl, Raymond, and Licsitrky, Serge, Analysis of Natural Radioactive Iodine Compounds by Chromatographic and Electrophoretic Methods ..................................... Roe, Joseph H., Chemical Determinations of Ascorbic, Dehydroascorbic, and Diketogulonic Acids ..................... Rosmhrantz, Harris, Analysis of Steroids by Infrared Spectrometry .. Infrared Analysis of Vitamins, Hormones, and Coenzymes ...... Roth, Marc, Fluorimetric Assay of Enzymes ....................... Salmon, W. D., see Engel, R. W. Saltrman, Bemard E., and Keenan, Robert G., Microdetermination of Cobalt in Biological Materials ................................. Schayer, Richard W., Determination of Histidine Decarboxylase Activity ....................................... Determination of Histidine Decarboxylase ..................... Schmakel, C. O., see Eluing, P. J. Schmid, Harald H . O.,see Mangold, Helmut K.

20

135

2

259

2

57

3

97

10

107

9

193

14

455

28

267

3 6

30 1 163

22

355

17

287

28

329

1

243

12

143

1 2 5 17

115 1 407 189

5

181

16

273 99

SUPP.

362 CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Schuberi, Jack, Measurement of Complex Ion Stability by the Use of Ion Exchange Resins ......................................... Schuberth,Jan, see Sosbo, S. Bo Schultm, Ham-Rolf, Field Desorption Mass Spectrometry and Its Application in Biochemical Analysis ........................... Schwartz, Morton K., and Boda7uhy, Oscar, Automated Methods for Determination of Enzyme Activity ............................. Schwartz, Morton K., and B&mk>, Oscar, Utilization of Automation for Studies of Enzyme Kinetics ................................ Schwartz, Samuel, Berg, Marie H., Batsenmuier, Irene, and Dimmore, Howard, Determination of Porphyrins in Biological Materials .... Scott, Churlcs D., see Burt& Carl A. Scott, J . E., Aliphatic Ammonium Salts in the Assay of Acidic Polysaccharides from Tissues .............. ............. Seaman, G. R., see Stohtad, E. L. R . Sebaki, Walter, see Werner, Sigurd S e ih , N., Use of the Dansyl Reaction in Biochemical Analysis ...... Seitz, W. Rudolf, and Nearg, Michal P., Recent Advances in .Bioluminescence and Chemiluminescence Assay ................ Shuw, Jane E . , and Ramwell, P. W., Separation, Identification, and Estimation of Prostaglandins .................................. Shibata, Kaz w, Spectrophotometry of Opaque Biological Materials: Reflection Methods .......................................... Spectrophotometry of Translucent Biological Materials: Opal Glass Transmission Method . . .............................. Shore, P . A., Determination of Histamine ......................... Sies, Helmut, and Brawn; B o b , Analysis o f Cellular Electron Transport Systems in Liver and Other Organs by Absorbance and Fluorescence Techniques ..................................... Silber, Robert H.,Fluorimetric Analysis of Corticoids ............... Silber, Rob& H., and Porter,Curt C., Determination of 17,21Dihydroxy-20-Ketosteroidsin Urine and Plasma ................ Singer, T h u u P., see Grossman, Shlomo Singer, Thonrcrc P., Determination of the Activity of Succinate, NADH, Choline, and a-Glycerophosphate Dehydrogenases ...... Singer, Thomas P., and Kearney, Edna B., Determination of Succinic Dehydrogenase Activity ...................................... Sjouall, Jan, Separation and Determination of Bile Acids ........... Sheggs, Helm R., Microbiological Assay of Vitamin BIZ ............. Smales,A. A., see Loveridge, B. A. Smith, Emil L., see DaVrr, Neil C . Smith, Fred, and Muntg-, Rex, End Group Analysis of Polysaccharides ........................................... Smith, Lucilc, Spectrophotometric Assay of Cytochrome c Oxidase . . Smh, S. Bo, and Schuberth, Jan, Measurements of Choline Acetylase ............................................ Spencer, B., see Dodgson, K . S . Spcrty, Warren M ., Lipid Analysis ................................ Spink, Charles H.,and Wadso, Ingemar, Calorimetry as an Analytical Tool in Biochemistry and Biology .............................

3

247

24

313

11

211

16

183

8

22 1

8

145

18

259

23

161

17

325

9

217

7 SUPP.

77 89

26 14

285 63

4

139

22

123

4 12 14

307 97

3 2

153 427

SUPP.

275

2

83

23

1

53

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Stehl, Egun, see Mangold, Helmut K. St. John, P. A., see Winefordner,J. D. StokFtad, E. L. R., Seaman, G. R., Davis, R. J., and Hutner, S.H.,

Assay o f Thioctic Acid

.......................................

Stmvick, C. A., Benson, E. M., Edwards, M. A., and Woodring,M . J,, Chemical and Microbiological Determination of Vitamin Be ..... Strehler, B m r d L., Bioluminescence Assay: Principles and Practice Strehh, B. L., and Totter,J. R.,Determination of ATP and Related

Compounds: Firefly Luminescence and Other Methods ......... Swartz, Harold M., and Swartz, Sharon M., Biochemical and Biophysical Applications of Spin Resonance .................... Swartz, Sharm M., see Swartz, Harold M. Talalay, Paul, Enzymic Analysis o f Steroid Hormones ............. TcWiun,M., see McLaren, D. S. T h t ,Jean-Paul G., and Haegek, Khw D., Analysis of Morphine and Related Analgesics by Gas Phase Methods .................. Thicrs, Ralph E., Contamination o f Trace Element Analysis and Its Control .............................................. Thorpe, W . V., see Bray, H . G. Tqfany, Thonras O.,see Burtk, Carl A. Tinoco, Zgmuio, Jr., Application o f Optical Rotatory Dispersion and Circular Dichroism to the Study of Biopolymers ................ Tohdm$ SibyuC, The in d r o Determination o f Hyaluronidase ...... T O MJ,. R.,see Strehh, B. L. Treudwell,C. R., see Vahmtny,Gewge V. Tulp, Ahaham, Density Gradient Electrophoresis of Mammalian Cells ............................................ U h f k z d , Sidney, Wekbach, Herbert, and Brodie, Bernard B., Assay of Serotonin and Related Metabolites, Enzymes, and Drugs ........ Ushukov, A. N.,see Vaver, V. A. Vahouny,George V., and Treadwell, C. R., Enzymatic Synthesis and Hydrolysis of Cholesterol Esters ............................... Vallee,Bert L., see Margoshes, Marvin V a h Heuuel, W .J.A., see Homing, E. C. Van PiLrum, John F., Determination o f Creatinine and Related Guanidinium Compounds .................................... Vaver, V. A., and Ushakov,A. N.,High Temperature Gas-Liquid Chromatography in Lipid Analysis ............................ Venkateswarlu,P., Determination of Fluorine in Biological Materials .................................................... Vessey,D. A., see Zakim, D. Vestling,Carl S., Determination o f Dissociation Constants for Two-Substrate Enzyme Systems ............................... Volkin,Elliot, and Cohn, Waldo E., Estimation of Nucleic Acids ...... Volhweider, H. J., Visual Biochemistry: New Insight into Structure and Function of the Genome ........................ Wacher, Warren E. C., see A&, El& Wadro, ingemur, see SpinR, Charks H. Waldemann-Meyer,H., Mobility Determination by Zone Electrophoresis at Constant Current ...........................

363

3

23

12 16

183 99

1

34 1

29

207

8

119

24

1

5

273

18 1

81 425

30

141

6

95

16

219

7

193

26

327

24

93

10 1

137 287

28

20 1

13

47

364 CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Wang, C. H.,Radiorespirometry ................................. Webb,Juniuc M., and Lay,HiuOn B., New Developments in the Chemical Determination of Nudeic Acids ...................... Wed-Malherbe,H., The Estimation of Total (Free + Conjugated) Catecholamines and Some Catecholamine Metabolites in Human Urine ............................................ Determination of Catechol Amines ............................ Weikuin,BmG, Separation and Determination of Amino Acids and Peptides by Gas-Liquid Chromatography .................. W k b a c h , Herbert,see U d e n j d , S k h q Wetterau,John R., and Zilversmit,Donald B., Quanutation of Lipid Transfer Activity ............................................ Werner, Sigurd, and Sebald, Waltcs, Immunological Techniques for Studies on the Biogenesis of Mitochondrial Membrane Proteins . . WhauCjr, H . C. S. a'e, and G r m , D., Determination of Raffinose and Keatose in Plant Products .................................... Whqfen,D. H.,see Barker, S. A. Whitehead,E. I., see Olson,0.E. Whitehead,J. K., and Dean, H . G., The Isotope Derivative Method in Biochemical Analysis ......................................... W h i t e h e , M . W., and ZiMen, F., Isolation and Determination of Neuraminic (Sialic) Acids ..................................... Whaiford, William R., see Ndclson, Samuel Wilchek,Meir, see Bayer, Edward A. Wdchek,Meir, and Hater, Charles S., The Purification of Biologically Active Compounds by Affinity Chromatography ................ Willis,J . B., Analysis of Biological Materials by Atomic Absorption Spectroscopy ..................................... Wilson,Kenneth J., see Hughes, Graham J . Winefordw,J . D.,McCarthy, W .J., and St. John, P.A., Phosphorimetry as an Analytical Approach in Biochemistry ..... Winrlcr, Richard J., Determination o f Serum Glycoproteins ......... W d r i n g , M . J., see Sf&h, C. A. Wotiz,Herbert H., and Clark, Stanlcy J.. Newer Developments in the Analysis of Steroids by Gas-Chromatography ................... Wright, M . Ruth, see Lmrer,Aarm B. Yagi, Kunio. Chemical Determination of Flavins ................... Yapcl,Anthony F.,Jr. and Lumv, Rufw, A Practical Guide to the Temperature-Jump Method for Measuring the Rate of Fast Reactions ............................................... Yalow,Rosalyn S., and Berson, S o h A., Immunoassay of Plasma Insulin .............................................. Yamamdo, Richard S., see Muhehen, Olaf Zakim, D., and Vesscy, D. A., Techniques for the Characterization of UDP-Glucuronyltransfera~,Glucose-6-Phosphatase,and Other Tightly-Bound Microsomal Enzymes .......................... Z U h , F.,see Whitehouse,M. W . Zilvenmit, D d B.,see Wetterau, John R.

15

311

6

1

16 SUPP.

293 119

14

203

30

199

27

109

1

307

16

1

8

199

23

345

11

1

15

369

2

279

18

339

10

319

20

169

12

69

21

1

Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

Cumulative Subject Index, Volumes 1-30 and Supplemental Volume

Absorbent and Flumescent Substances, The Automated Anal* of, Separated on Paper Strips (Boulton) ............................. Activation Analysis and Its Application in Bhhemisgr (Loveridge and Smales) ...................................... Activahn Analysis of Biological Trace Elemen& (Leddicotte) .......... Adenine, Enzymu Micro Determi&, by Ultraviolet Spectrophotonuhy (Plesner and Kalckar) ......................................... Adrenal Gland, Steroidc of, Chromatographic Separation (Haines and Karnemaat) ..................................... Adrenal Steroids in Blood, Analysis of, by Counttmurrmt Distribdm (Carstensen) ................................................. Adrenaline, Chemical Determinution, in Body Fluids and Tissues (Persky) A f f i @ yChromatography, The Punjication of Biologicallr Active Compounds by Aliphatic AmmoniumSalts in the Assay of Acidic Polysacchdes from Tissues (Scott) .............................. Alterncrtve Metabolic Pathways, Estimutiun of Magnitudes of (Kopin) .... Amine Oxidascs, Delffnrinationof (Kapeller-Adler) .................. AminoAcid Analysis by Gas-Liquid Chrmahgraphy, Recent Developents in, (Mackenrie) ............................ Amino A&, Analysis by Means of Brditka Reaction (Miiller) .......... Amino Acids, Determination by High-Vohge Paper E&ctr@hessis (Blackburn) ................................................. Amino Acids, Determinution by Ion Exchange Chromatography (Jacobs)... Amino A&, Determination by Use of Bactnial Amino Acid Decarboxykues (Gale) ...................................................... AminoA d , Separation and Determination by Gas-Liquid Chromatography (Weinstein) .................................................. AmmoniumSalts, Aliphatic, in the Assay of Acidic Polysacchades from Tissues (Scott) ........................................... Ampholytes,A Techntipfor Fractionation and Charactetization through Grndieptts (Haglund) .................. Isoelechic Focusing in+H Analgesia, Analysis by Gas Phase Metftodr (ThCnot and Haegele) ...... Antibiotics, Microbiological Assay (Kersey and Fink) ................. Application of High R e s o l d m Nuclear Magnetic R e s m n c e to Biological Systems (Campbell and Dobson) ................................ Ascorbic Acid, Chemical Determinution (Roe) ........................ A h u Absorption Spectroscopy, Analysis of Biological Materials by (Willis)

365

VOL.

PAGE

16

327

5 19

225 345

3

97

1

171

9 2

127 57

8

145 247 35

11 SUPP. 27

1

11

329

13 14

177

4

285

14

203

8

145

19 24 1

1 1

153

25 1 11

115 1

1

1

366 CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT ATP. Determination of Firejly Luminescence (Strehler and Totter) ..... Avidin.Bwtin. Use of, As Tool in Molecular Biology (Bayer and Wdchek) .................................................... Bacterkd Amino And Decarboxylares in Determination of Amino A d (Gale) ...................................................... Basic Pro&’ns. Prepamtiun and Analysis o f (Lindh and Brantmark) .... Bile A d . Newer Dcvelqpmpnts in the Gm Chromatographic Determination of (Kuksis) ...................................... B 5 A d . Separatiun and Determination of (Sjovall) .................. Bile Pigments. Separation and Determination o f (Brodenen and Jacobsen) .................................... Biochnnical Applicatiollr of Magnetic Resonance (Jardetzky and Jardetzky) .................................... Biochenishy. Historical Developnuw and Nnvtr Means of Temperature Measurement in (Berger. Clem. Harden and Mangum) ........... BiochcniChy. Laser Photolysk in (Chan and Austin) ............... Biological Matmials. Analysis by A t a r u Absmprirm Spectroscopy (Wdlis) . Biological M a h i & . Determindion of Nitrogen in (Jacobs) ............ Biological M a h l s . Determination of Porphyrins in (Schwartz. Berg. Bossenmaier. and Dinsmore) ................................. Bidlogical M a h l s . Determination of Zinc in (Malmstrom) ........... Biological Materials. Methodsfw Estimoring Magnesium in (Alcock and MacIntyre) ...................................... Biological Mnloials. Micr&terminufiun of Cobalf in (Saluman and Keenan) ....................................... Biological Materials. Spectrophotonutty ofi Rt$ection Methods (Shibata) ....... ........................................ Spechophotamet?y of; Opal Gkw M e W (Shibata) ................................... Bwlogical Standards in Biochemical Analysis (Humphrey. Long. and Perry) ................................ Biological Systems. Determination of Sm’ne in (Frisell and Mackenzie) . . Biological Systems Ion Binding in. Memured by Nuclear Magnetic Resonance Spectroscopy (For&n and Lindman) ................... Biological Trace Elementc. ActiuatMi Analysis of (Leddicotte) .......... Bioluminescence Assay: Prin+&s and Practice (Strehler) .............. Bioluminescence and Chiluminescence Assay. Recent Advances in ...... Blood. Analysis of Adrenal Sttroids in. by CountcrGurrenl DishibutMn (Cartensen) ................................................. Blood. Detmnindion of Ethyl Alcohol in (Lindquist) .................. Determination of Adrenaline and Noradrenaline an

.

.

.

............................................

Body Fluidr. Chromatographic Analysis of Radioactive Iodine Cmpuundcf r m (Roche. Lissitzky. and Michel) .................. B q f f c . Containing Urea. Gel Electrophoresis in (Poulik) .............. Calorimehy as a n Analytical Tool in Biochnnistly and Biology ........... Carbohydrate. The Defermination of, in Biological MatniOls by Gas-Liquid Chromatography (Clamp. Bhatti. and Chambers) ................. Carbohydrate Derivatives. Separation of. by Gas-Liquid Partition Chronurtography (Bishop) ..............................

1

341

26

1

4 14

285 79

14 12

325 97

17

31

9

235

30 30 11 13

269 105 1 241

8 3

221 327

14

1

5

181

9

217

7

77

5 6

65 63

27 19 16 23

289 345 99 161

9

7

127 217

2

57

1 14 23

243 455 1

19

229

10

1

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT 367 Carbohydrate Structure. Use o f Infrared Analysis in Determination of (Baker. Bourne. and Whiffen) ................................ Carbonic Anhydrose Activity.Measurements of (Davis) ................. Carotene. Determination of (Bickoff) ............................... (Creveling and Daly) ......................................... Catalases. h a y of (Maehly and Chance) .......................... Catechol Amine Biosynthesis and Metabolism. Assay of Enzymes of Catechohmines and Catecholamine Metabolites. Estimation of Total (Free + Conjugated). in Human Urine (Weil-Malherbe) ............ Catcchol Amines. Determination of (Weil-Malherbe) .................. Cell Parttkles and Macromolecules. Partition Methodr f w Fractimution of (Albertsson) ................................................. Cellular Electron Transport Systems in Liver and Other Organs. Analysis 06bJ Abswbame and Fluorescence Techniques (Sies and Brauser) ........................................... Celluhe Column Chromatography. Analysis of Mixtures of Sugars bJ (Hough) .................................................... C&f'ugal Fast Analyzerfor Biochemical and ImmunologicalAnalyes. The Use of a ................................................. C&@gatiun. Preparative Zonal (Anderson) ....................... Chloride in BWl.gical Materials. Determination of (Cotlove) ............ Cholesterol. Determination and Microsc~@cLocalization of (Kabara) ..... Cholesterol Esters. Enzymatic Synthesis and Hydrolysis of (Vahouny and Treadwell) .................................... Choline. Chemical Estimation of (Engel. Salmon. and Ackerman) ..... Choline Acetyke. MeasurMnents o f (Sorb0 and Schuberth) ........... Choline Estm. Measurement of Uenden) ........................... Cholinesterases. Assay Methods f w (Augustinsson) ................... Cholinesterases. Determination of (Augustinsson) ..................... Chromatographic Analysis. A#licatM?ls of the R M Treahnent in (Bush) ... Chromatographic Analysis. A#lica&ns of &he RMTreatment in. Erratum (Bush) ...................................................... Chromatographic Analysis of Radioactive Iodine Compoundsfrom the Thyroid Ghnd and Body Fluids (Roche. Lissitzky. and Michel) ...... Chromatographic and Electrophoretic Methods. Analysis of Natural Radioactive Iodine Compovnds by (Roche. Michel. and Lissitzky) .... Chromatographic Separation of Steroids of the Adrenal Ghnd (Haines and Karnemaat) ..................................... Chromatography. Gas. in Detennination of Bile Acids and Steroids (Kuksis) ..................................................... Chr&graphy. Gas. Separation and Determinution of Steroids by (Homing. Vanden Heuvel. and Creech) ....................... Chromatography. Gas.Liquid. Determination of &he F q A d by (James) Chromatography. Gas.Liquid. Separati'm and Determination of AminoAcids and Peptides by (Weinstein) .................................... Chromatography. Gas-Liquid Partition. Separation of Carbohydrate Derivatives by (Bishop) ........................................ Chromatography. High-Pevjinmance Llprud: Analytic and Preparative APplUaiiOn in Protein Structure Determination (Hughes and Wilson) ........................................

3 11 4 SUPP. 1

213 307 1 153 357

16 SUPP.

293 119

10

229

26

285

1

205

23 15 12 10

189 271 277 263

16 1 SUPP. SUPP. 5 SUPP. 13

219 265 275 183 1 217 357

14

497

1

243

12

143

1

171

14

325

11 8

69

14

203

10

1

29

59

I

368 CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Chromatography. High Temperature Gas.L+. in L i e Analysis (Vaver and Ushakov) ........................................ Chromatography. Ion Exchange. Determinatkm o f Amino A& by (Jacobs) ..................................................... Chromatografihy. Paper and Cellulace Column. Analysis of Mixtures of Sugars by (Hough) ........................................... Chromatography. of Proteins. N w L k A&. V i w e s . and CeUs on Noncharged Amphiphilu Gels. Hydrophobic Interaction. (Hjerth) ..... Chromatography. Thin-layer (TLC) (Mangold. Schmid. and Stahl) .... Cobalf. Mirrodctrmrination of, in Bwrogical Matowls (Saltzman and Keenan) .................................................... CoenzymeA. M e w fm DetmninotiOn of (Novelli) .................. CoenzymeQ, DeknniMhbn of (Crane and Dilley) ................... Coenzymes.Infrared Analysis of (Rosenkrantz) ...................... Collagen and Elastin. Analysis of the Crosslinking Cmponenfs in (Robins) CoUugen and Elastin. The Detmnination of (Jackson and Cleary) ...... Color Reacfions. New. for Detenninatiun of Sugars in Polysaccharides (Dische) ........................................ ..I.. ........ Column Electrophoresis in Granular Media. Some R e c d Devekpneni3 (Porath and Hjerten) ......................................... Complexes, Metal Protein. Analysts of (Hughes and Klotz) ............ CmplexIon Solubcluy. Measurement Use of Ion Exchange Resins (Schubert) .................................................. Connective-TissueMacromolecules. Analysis by Detnnrifiation of C m i n Cm(Blumenkrantz and Ash-Hansen) ................. ConiaminaciOn in Trace Ekment Analjsis and Its Control (Thiers) ...... C&&. Fluorimehic Analysis of (Silber) .......................... Countercurrent Dirhibution (King and Craig) ....................... Countercurrent Dishibutia. Analysis of Adrenal Stctoidc in Blood by (Carstensen) ................................................. Creatinine and Rehated Gunnidinium C m p n u k . DetenniMEion of (Van Pilsum) ................................................ Current. Canstant. Mobiky Det?nni& by Zone Electrophoresis at (Waldmann-Meyer) .......................................... Cyclic 3'.5 '.Admasine M o m p h p b and Cyclic 3'J '-Gunmine M o n o p h a p h . Analysis of (Goldberg and OToole) .............. C ~ l i AMP c and Other Cyclic Nucleotdes. Adenykate Cyckase. and Phosphodiesterase. Newer Developmnrts in the DetmninOtimt o f (Brooker) ................................................... Cyckxhromec Oxidare. Spectrophoktric Assay of (Smith) ............ Dansyl Reaction. Use of the. in Biochemual Analysis (Seider) ........... Dehydroascorbic Acid. Chemical DctmninotMn of (Roe) ................ Dehydrogenases. Detmninatiun of the Actiwly of Succinate. NADH. Choline a-Clycerophphaie (Singer) .................................... Denaiuration. Reversible. of Proleins. Methodr of Study and Intngrccalion ofData fw (Hermans.Jr.) ..................................... Dmcilp Gradients. R a e Electrophoresis in (Kolin) ................... Deoxyribonuclease Actiwly. Assay of (Kurnick) ....................... Diagn~~ciC. Enzycrtrc Metha& o f (Amador and Wacker) .............. Ddysis (Craig and King) .......................................

26

327

14

177

1

205

27 12

393

5 2 11 5 28 15

181 189 279 407 329 25

2

313

9 3

193 265

3

247

24 5 14 10

39 273 63 201

9

127

7

193

13

47

20

1

22 2 18 1

95 427 259 115

22

123

13 6 9 13 10

81 259 1 265 175

89

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT 369

Diaw-Positive Bile Pigncents. Recent Advances in the Separahn and Analysis of (Heinvegh) ........................................ Dffraction. X.ray. in the Study of Protein and Nuchic Acid Structure (Holmcs and Blow) .......................................... Z.O K .rD .oZ se.t7.i Detmninativn in Urine and Plusma (Silber and Porter) ........................................... DikeiogulrmicAcid. Chemical Determination of (Roe) .................. Dissociation Conrtants. Determination 05for Two-SubstrateEnzyme Systnnr (Vestling) ................................................... Electron Probe Microanalyzer. A n Introduction to. and Its Applicahbn to Biochemistq (Andersen) ....................................... Electron Spin Resonance. Biochemical and Biophysical Applicdon o f (Swartz and Swartz) .......................................... Electrophoresis. Free Zone. T h e q . Equipment. and ApPlUatiuns (Hjerten) ................................................... Electrophoresis. Gel. in Buflers Containing Urea (Poulik) .............. Electrophoresis. Gel Sieving; A DesrriptMn of Procedures and Anulysk o f E r r a uohnson) ............................................. Electrophoresis. Paper. Detmninatbn of Amino A& at High-Voltage by (Blackburn) ................................................. Electrophoresis. Rapul. in Dencity Gradimts Combined with pH o d o r Conducrivaty Gradients (Kolin) .................................. Electrophoresis. Zone (Kunkel) .................................... Electrophoresis. Zone. Constant Cuwent Mobility Detmnination by (Waldmann-Meyer) .......................................... Electrophoresis in Granular Media. Column. Some Recent Deve@nmts (Porath and Hjerten) ......................................... Electrophoretic Methodr. Analysis of Natural Radioactive Iodine Comfiounds by (Roche. Michel. and Lissitzky) .............................. Elements. Determinution 05by X-Ray Evnission Spectrometry (Natelson and Whitford) ..................................... EnthaIpy and Enlropr Changes. Detnminahbn by Heatburst Microcuiurimetry (Kitzinger and Benzinger) ...................... EnzymaticM e w . in Diagnosis (Amador and Wacker) ............. Enzyme Actim$y.Rutonrated M e t h d for Detffninntion o f (Schwartz and Bodansky) .................................................. EnzymeAssay. R a d k t r i c Methods o f (Oldham) .................... EnzymeKinetics. Utilization of Autonuationfor Studies of (Schwartz and Bodansky) ..................................... Enzymes. Assay of in Catcclrol Amine Bimynthesisand Me&lism (Creveling and Daly) ......................................... Enzymes. Detection o f Lend-Induced and Syncatrrlytic C o n f m h d Changes of by D i f l e r d l Chemical Mod$cahbn (Christen and Gehring) ...................................... Enzymes. FZuorhetric Assay of (Roth) .............................. Enzymes. Immobilized. in Biochemical Analysis (Everse. Ginsburgh. and Kaplan) .............................. Enzymes. Proteolytic Assay of (Davis and Smith) ..................... Enzymes. Relaud to Serotonin. Assay of (Udenfriend. Weissbach. and Brodie) ..........................

22

205

13

113

9 1

139 115

10

137

15

147

29

207

18 14

55 455

29

25

13

1

6 1

259 141

13

47

9

193

12

143

12

1

8 13

309 265

11 21

211 191

16

183

SUPP.

153

28 17

151 189

25 2

135 215

6

95

370

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT

Enzyme Systems. Two Substrate. DetmniMtion of Dissm-ktion Conrtantsfor (Vestling) ................................................... EnzymicAnalysis of Steroid Honnoncs (Talalay) ...................... EnzymicDetenniMtMn of D-Glucose and Its Anomen. New Dtoelopmntts in (Okuda and Miwa) ........................................ Estrogens. Chemical Determination of, in Human Urine (Bauld and Greenway) ....................................... Ethanolamine. Determinolion oJ in Lipids (McKibbin) ................ F & y Acid Esters. A Critical Evaluation of the Gas Chromatagrophic Techniquefor Identij5cation and Detmnination 4 with PariinJar Refmeme to the Use of Analog and Digttal Computer Methods (Caster) FuUy A d . DelmniMtion by Gas-Liquid Chromatography (James) ...... Field Desorption M a s Spectrometly: Application in Biochemical Analysis (Schulten) ................................................... Field-Flow Fractionation. Analysis of Bwbgical Matromoleds and Particles By (Giddings. Myers. Caldwell. and Fisher) ............. Firejty Luminescence. DetnrniMtion of A T P by (Strehler and Totter) ... Flame Photometry. Principles and Applicalions (Margoshes and Vallee) Flavim. Chemical Determination of (Yagi) ........................... F1uid.s. Body. Chemical Determination of Adrenuline and Noradrenuline in (Persky) ..................................................... Fluids. Body. Chraurtographu Analysis of Radioactive Iodine Compounds from (Roche. Lissitzky. and Michel) ............................ Flumimetric Analysis of Cwticoidr (Silber) ...... Fluorine. Determinnfiun in Biological Uaierials (Venkateswarlu) ....... Folu Acid Actimty.Assay of Compounclr Wth uukes) .................. FmIdehyde. Determination ofi in Biological Systems (Frisell and Mackenzie) ....................................... F r a c W i u n of Cell Particles and Motromolecules. Partition Melhodr for (Albertsson) ................................................. Free EWB Changes. DetenniMtion by Heatburst Microcahimetly (Kitzinger and Benzinger) .................................... Frog Skin Assayfor Agents that Darken and Lightm Melanocyfa (Lerner and Wright) ......................................... Gas-Llguul Chromatography. The Determination in Carbohydrates and Biological MatPrialr (Clamp. Bhatti. and Chambers) .............. Gel Electrophesis in Buffers Containing Urea (Poulik) ............... f3-Glucuronidoses. Determination of (Fishman) ....................... UDP.GlucuranyLtramfmase. Glucose.6.Phosphatae. and Other Tightly-Bound Microsoma1 Enzymes. Techniquesfor the C h a r a c ~ i z a t i aof (Zakin and Vessey) . . . . . . Gluiumic and Aspartic A d and Their A d s . DetmniMcion of (Balk) .. Glulathionc. Determination of (Patterson and Lazarow) .............. Glycolipid DetnrniMtirm (Radin) .................................. Glycoprotcinr. Serum. Determinatiun of (Winder) ..................... Gradfents. Densily. Rapid Electrophesk in (Kolin) ................... Heatburst Microcalmimetly. Primiple ond Methads of. and DetermiMtion of Free Energy. Enthalpy. and Entropy Changes (Kitzinger and Benzinger) .................................... Heparin. Determination of Uaques and Bell) ........................

.

10 8

137 119

21

155

5 7

337 111

17 8

135 1

24

313

26 1 3 10

79 34 1 353 319

2

57

1 14 24 2

243 63 93 121

6

63

10

229

8

309

8

295

19 14 15

229 455 71

21 20 2 6 2 6

1 103 259 163 279 259

8

309 253

7

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT 371 Heparin. Deternination of (Jaques) ................................ HexosamincS. Determimation of (Gardell) ............................ High-Performance Ion-Exchange Chromatografihy with Narrow-Bore Columns: R a g Analysis of Nucleic Acid Constituents at the Subnanomole Level (Horvath) .................................. Histamine. Determinuha of (Shore) ............................... Histamine. Quantitative Determination of (Code and McIntire) ........ Histidine Decarbqlase. Determination of (Schayer) .................. Histidine Decarbqyhe Activiv. DetermiMh'on of (Schayer) ............ Hormones. Infrared Analysis of (Rosenkrantz) ....................... Hormones. Plant. Analysis of (Bentley) ............................. Hormones, Steroid. Enzymic Analysis of (Talalay) ..................... Hyalurmiduse. in vitro Determination (Tolksdorf) ................... Hydrogen Exchange Data. Acquisition and Interpretation of. from Peptidcs. Polymtrs. and Proteins (Barksdale and Rosenberg) ................ Hydrogen Isotope Exchange in Globular Proteins. M e wfor Mearurement (Ottesen) .................................................... Hydrophobic Inh-action Chromatography o f Prothns. Nucleic Acidr. and Celk on Noncharged Amphiphilic Gels. (Hjertkn) ................... Hypoxanthine. Enzymic M k o Determination. by UltraGolet Spectrophotmneby (Plesner and Kalckar) ......................... Immunoarsay of Plasma Insulin (Yalow and Berson) ................. 1rnmunoclcch.ophmetit Analysis (Garbar) ............................ Immunological Techniquesf m Studies 071 the Biogenesis of Mitochondd Membrane Proh'ns (Werner and Sebald) ........................ Infrared Analysis. Use of. in the Determination of Carbohydrate Stmcture (Baker. Bourne. and Whiffen) ................................ Infrared Analysis o f Vitamins. Hormones, and Cmzymes (Rosenkrantz) Infrared Spectromeq. Analysis of Steroids by (Rosenkrantz) ........... Inositol. Determination of, in L i w s (McKibbin) ..................... Iodine. in Biological Matnial. Determination of (Binnerts) ............. Iodine Compoud. Natural Radioactive. Analysis by Chromatographic and Electrophoretic Me& (Roche. Michel. and Lissitzky) ............ Iodine Compound. Radioactive. from Thyroid Gland and Body Fluids. Chromatographic Analysis (Roche. Lissitzky. and Michel) ........... Ion Binding in Biological Systems Measured by Nuclear Magnetic Resonance Spectrascopr (Forsen and Lindman) ................... Ion Exchange Resins. Measurement of Complex Ion Stability by Use of (Schubert) .................................................. Isolated Bacterial Nuclewids. Charactekatim. Assay. and Use of (Hirschbein and Guillen) ..................................... Isotope Derivative Method in Biochemical Analysis. The (Whitehead and Dean) ....................................... Kestose. Determination. in Plant Products (de Whalley and Gross) ..... a-Keto Acid Determinations (Neish) ................................ 17.Ketosteroids. Urinary Neutral. Assay of (Engel) .................... Lipme. Lipoprotein. Assay of, in vivo and in vitro (Korn) ............. Lip& Analysis (Sperry) ......................................... L i M s . Determination of Inosdol. Ethanolamine. and Serine in (McKibbin) ..................................................

24 6

203 289

21

16 5 9 8 1

79 89 49 99 273 407 75 119 425

28

1

20

135

27

89

SUPP*

3

SUPP*

3 12

7

97

69 1

27

109

3 5 2 7 22

213 407 1 111 251

12

143

1

243

27

289

3

247

28

297

16 1 5 1 7 2

1 307 107 459 145 83

7

111

372

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT

L i e Transfer Activity, Quaniitaiim of (Wetterau and Zilversmit) ..... Lip.p.0t.i. L i p e . Assay 05in vivo and in vitro (Korn) .............. Lipoprotkns. Serum. Ultraentrifugal Analysis (de Lalla and Gofman) . . Lipaxidare Actively, M e a s u r m of (Holman) ....................... Lipoqgenase (Lipoxidate). Detffminationof the Actidy of (Grossman and Zakut) ....................................... ~ -Counting. Practical ~ Aspecis o f ~ ' (Kobayashi and Maudsley) .................................... Lucrferin and Luafmase. M e a s u r e of (Chase) .................... LysozymeActivzty. Me& for Determination of (Grossowicz and Ariel) Magnesium Estimation. in Biological MatetiaD (Alcwk and Macintyre) MagnctiC Resonance. Biochemical App1icaiion.s of (Jardeuky and Jardetzky) .................................... Mammalian Cells. Dmnly Cradieni Electrophoresis of (Tulp) ........... Moss Spectrometty. Anulysir of Sieroids by (Gasdell) ................... Moss Specirometty. Fie12 Desorpiim: Appkation in Biochemical Anolysir (Schutten) ................................................... Mass Spectrometv in the Deierminatiun Df Structure of Certain Natural Products Containing Sugars (Hanessian) ......................... Mehmqtes. Darkening and Lighning. Frog Skin Assayfor (Lerner and Wright) ......................................... Metabolic Pathmys. Altmurtive. Estimation o f Magmtudes of (Kopin) ... Metabolism. A d + of Phenolic Compounds of Interest in (Bray and Thorp) .......................................... Metal ByFfers. Application. in B i o c h i s i t y (Raafiaub) ................ Metal Indicaiurs. AppliMfiMis. in Biocfrmtiriry (Raaflaub) ............. Metal-Protein Complexes. Analysis of (Hughes and Klotz) ............. Microbiabgad Assay of Antibiotics (Kersey and Fink) ................ Microbiobgical Assay of VitnwunBln (Hoff-Jorgensen) ............... Microbiological Assay of VitaminB 12 (Skeggs) ....................... Microbwbgical DeiermiMtMn of Vitamin B6 (Storvick. Benson. Edwards. and Woodring) .................... Muropariiculatc Gel Chromatography Accehated by Ceninfigal Force and Pressure (Ribi. Parker. and Milner) ............................. Mobthy. DeiermiMtMn by Zone Electr@hor& at C m t n n t Current (Wddmann-Meyer) .......................................... Moleculur S i x . EstinrcrCirm of. and Moleculur Weights of Biobgical Compounds by Gel F&& (Andrews) .......................... M w h i n e . and Relotcd Analgesics. A d p i s by Gas Phase M e W (Thtnot and Haegele) ........................................ Mucopolysacclwkk-s.Sqated. DetewniMtMn of (Jaques) .............. Negative-Ion Moss Spectromeiry. Fused-Silica CapilLaty Gas Chronurtogaphy of Neurotransmitters and Related Compounds ( F a d and Barchas) .......................................... Neuraminu (Sialic) A d . Isoldirm and Deiermtnaiiun of (Whitehouse and Zilliken) .................................... Nitrogen. DeunniMtimr in Biobgical MaktiaLc (Jacobs) ............... Nitrogemus Compounds. Basic. of TmiCobgacal Importance. Analysis 4 (Curry) ..................... ............................. N o r a d r d n e . Chemical Determination. in Body Flu& and Tissues (Persky) .....................................................

.

30

1 2

199 145 459 113

25

303

17 8 29 14

55 61 435 1

9 30 29

235 141 385

24

313

19

105

8 11

295 247

1 3 3 3 1 1 14

27 301 301 265 53 81 53

12

183

22

355

13

47

18

1

24 24

1 203

29

325

8 13

199 241

7

39

2

57

7

~

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Nucleic Acid. Strutture. X-ray oij'@ction in the Study of (Holmes and Blow) .......................................... Nuckic A d . Chemical DetenninaciOn of (Webb and Levy) ........... Nucleic A d . The Determination of (Munro and Fleck) .............. Nucleic A d . Estimation (Volkin and Cohn) ....................... Nuckic A d and Their Dnivdivcs. Mierobiobgical Assay of (Miller) .... Nucleic A d of Various ConformationalF a n s and Measurement of Their Associated Enzymes. w e of Ethidium Bromidefor Separation and Detenninution o f (Le Pecq) ..................................... Nucle& and Nucbotidcs and Their P a r d Bases (IJ an Amlyrical and Investigative Tool. Pokarografihy and Voltummehy of (Elving. OReilly. and Schmakel) .............................. Optical R o w Dispersh. Application of. and Circular Dichroism to the Study of B i o p o l (Tinoco. ~ Jr.) ............................... Organic P h p h Compounds. Determinution of. by P h p h a t e Analysis (Lindberg and Emster) ....................................... . . Pnialete. Use of. in Biochemical Analysis (Dyer) ........... oxrdatrons. Oxygen Electrode Measuremen& in Biochemical Analysis (Lessler and Brierley) ........................................ Paper Chromatogram. Direct Scanning of. for Quuntiidve Estimaticms (Bush) ...................................................... Paper Chromatography. for Analysis ofMiitures o f Sugan (Hough) ..... Partition Mclhodc for Fractionation of Cell Partkles and Macromole& (Albertsson} ................................................. Peptide Chroma&graphy. Autmnaeic (Jones) ......................... Peptide Mapping of Proteins (James) ............................... Peptides. Separation and Determinution. b Gas-Llquul Chromatography (Weinstein) .................................................. Pep&. T m t n a l and Sequence Studies in. Recent Developmtntsin Techniquesfor (Fraenkel.Conrat. Harris. and Levy) .............. Peptides and Amino A d in N m l Human Urine. Separation and @anttarion of (Lou and Hamilton) ............................ Pniodatc 0dahn.s. Use of. in Biochemical Analysis (Dyer) ........... Peroxiduses. Assay of (Maehly and Chance) ........................ Phase PartifimtiA Method for Purijication and Analysis of CeU O r g a n e k and Membrane Vesic&s(Albertsson. Andersson. Larsson. and Akerlund) ...................................... Phase Pmtition. I&action Between Biumoleculrr Studied by (Albertsson) Phenolic Cmpoundr of Interest in Metabolism (Bray and Thorp) ...... Phmylalaninc and Tyosine in Blood. The Measurement o f (Robins) ..... pH Gradients. IsoelectriC F&g in-A Techniquefor Fractiona&m and C h a r a c e of Amphlytcs (Haglund) ........................ pH Jump. The: Macromolecules and Solutions by a her-Induced. Ultrashort. Proton Puke. Probing of-Theq and Application in Biochemistry (Gutman) ........................................ pH and Similar Variables. Recent Developnuntr in Control o f uames and Lumry) .......................................... pH-Stat and I& Use in Biochemistry (Jacobson.LRoNs. Linderstrbm.Lang. and Ottesen) .............................. P h p h d c Analysis. D e t e d n & m o f Organic P h o s p h Compound by (Lindberg and Emster) .......................................

373

13 6 14 1 6

113 1 113 287 31

20

41

21

287

18

81

3 3

1 111

17

1

11 1

149 205

10 18 26

229 205 165

14

203

2

359

25

3

1

203 111 357

28 29 1 17

115 1 27 287

19

1

30

1

29

137

4

171

3

1

374 CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Phpholrpaccs. A. C. and D. DetmniMtion of Ihe Actiw of. (Grossman. Oestreicher. and Singer) .......................... Phphoritnehy. a an Analytical Approach in Biochmrtstry (Winefordner. McCarthy. and St.John) ........................ P h p h Compounds. Organic. Determi& of, by Phosphate Analyses (Lindberg and Ernster) ....................................... Ph&mwtty. Flame. Principles and A p p l i c h of (Margoshes and Vallee) ...................................... Phytclu and Inositol PhosphatcJ. the Delmnination of (Oberleas) ........ Plant Honnmrcs. Analysis of (Bentley) ............................. Plasma. Determination of 17.21.Dihydrq.2 O.Ketas&& in (Silber and Porter) ........................................... P&ma Insulin. lmmunwsray of (Yalow and Berson) ................ Polarographic Analysis of Proteins. Amino A d . and Other Compounds by Means of the Brdifka R e a c h (Miiller) .......................... Polarographic Oxygen Senson. Adaptation of. f m Biochemical Assays (Lessler) .................................................... Polysaccharidcs. Acidic. from Tissues. Aliphatic AmmoniumS a h in the Assay of (Scott) ............................................... Polysaccharides. End Croup Analysis of (Smith and Montgomery) ..... Polysaccharides. Sugars in. New C o h Reactionsfor Determination of (Dische) ................... .............................. Polpnsaturated Fatty A d . Measurement of (Holman) ............... P-hyrins in Biological Makrials. Determination of (Schwartz.Berg. Bossenmaier. and Dinsmore) ................................. Prostaglandins. Separation. Iahyication. and Estimatia of (Shaw and Ramwell) ......................................... Protein. S h t u r e . X-ray D j f f r a c h in the Study of (Holmes and Blow) Protein. Terminal and Sequence Studies in. Recent Developmen& in Techniques for (FraenkeLConrat.Hams. and Levy) .............. Protein-Nucleic Acid and Proiein-Protein Complcxcs by Djffer&l A r e a (Bosshard) ........ Chemical ModjFcation. Mapping of C&t Proh’ns. Analysis by Means of Brdi€ha Reactiun (Miiller) .............. Proteins. Bark. Preparation and Analysts of (Lindh and Brantmark) ... Proteins. Mitochondnal Membrane. Immunological TechnipucSfor St& on the Bwgmsts of (Werner and Sebald) ........................ ProtcinC Polarography OJ Analytical Principles and Applications in B i o l o g d and Clinical Chtmtstly (Homolka) ...................... Proteins. Reversible Dmaturation of. Methodr of Study and IntngrctatMn of Dato for (Hermans. Jr.) ..................................... P r o h n Sequence Analysis Solid Phase Methodr i n (Laursen and Machleidt) ..................................... Protcolytic Enzymes. Assay of (Davis and Smith) ..................... PunjicatiOn of BwbgicaUy Active Compounds AffinttyChromatography. The Purines. New Methodr for Purifcation and Separation of (Bergmann and Dikstein) ..................................... Quantitative Marc Spectrometric Analy~ts:Chemical and Biological Applicotiotrc. Inteqated Ion-Current (IIC) Techniqu of (Majer and Boulton) ......................................... Radioactive Iodine Compounds. frmn Thyroid Gland and Body Fluids. Chromatographic Analysis of (Roche. Lissitzky. and Michel) ........ Radioimmunwssay of Polypeptide Hormones and Enzymes (Felber) ......

.

.

22

177

15

369

3

1

3 20 9

353 87 75

4 12

139 69

11

329

28

175

a 3

145 153

2 4

313 99

a

221

17 13

325 113

2

359

25 11 14

273 329 79

27

109

19

435

13

81

26 2

201 215

6

79

21

467

1

243 1

22

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Radimespirmtry (Wang) ........................................ Raffinose. Determination in Plant Pr0dut.s (de Whalley and Gross) .... Rw.ns, Ion Exchange. M e a s u r d of Complex Ion Stability. by Use of (Schubert) .................................................. Resonance. Magnetic. Biochemical Applications of Uardetzky and Jardetzky) .................................... Ribmulease. Chracterizatim of. and Determination of Its Activity (Josefsson and Lagerstedt) .................................... RM Treatment. Applications in Chromatographic Analysis (Bush) ........ R ~ Treatment. A Applications in Chromatographic Analysis. Erratum (Bush) ...................................................... Selenium in Biological Materials. Determination of (Olson Palmer. and Whitehead) .............................. Serine. Determination of, in Biological Systems.(Frisell and Mackenzie) .... Serine. Determination of, in L i e s (McKibbin) ...................... Serotonin: T h Assay of Hydroxyindole Compoundsand Their B i o p t h t i c Enzymes (Lovenberg and Engelman) ........................... Serotonin and Related Metabolites. Enzymes. and Drugs. Assay of (Udenfriend. Weissbach. and Brodie) .......................... Serum Acid Phphatases. Determinations (Fishman and Davidson) ..... Serum Glycoproteins. Determination of (Winzler) ...... .............. Serum Lipoprotkns. Uhracentnfugal Analysis of (de Lalla and Gofman) -SHGroups in Protknr. Determination of (Benesch and Benesch) ..... Siulic Acids. see Neuraminu Acids Sodiumand Potarrium. Measurementsof, by G h s Electrodes (Friedman) ... Solid P h e Immunoclssay. Use of Magnetizable Particles in (Pourfarzaneh. Kamel. Landon. and Dawes) .................... Spectrometv. Infrared. Analysis of Steroids by (Rosenkrantz) ........... Spectromeq. Principles and Applications (Margoshes and Vallee) ...... Spectrometry. X-ray Emission. Determination of Elements by (Natelson and Whitford) ..................................... Spectrophotometric Assay of Cytochrume c Oxidase (Smith) .............. Spectrophotometric OxyhmwglobinMethod. Meamrement of Oxygen Consumption by (Barzu) ....................................... Spectrophotumetty. Ultraviolet. Enzymic Micro Determination of Uric A d . Hypoxanthine. Xanthine. Adenine. and Xanthoptm’ne by (Plesner and Kalckar) ........................................ Spectrophotometry of Opaque Biological Materials;RefEection Methodr (Shibata) .................................................... Spectrophotometty of Translucent Biological Materials; Opal Glars Method (Shibata) .................................................... Stamhrh. Biological. in Biochemical Analysis (Humphrey. Long. and Perry) .................................................. Steady State Kinetics of Oxygen Uptake by Biochemical Samples. Polarographic Measurement of (Degn. Lundsgaard. Peterson and Ormicki) ....................................... Steroid Hormones, Enzymic Analysis of (Talalay) ..................... Steroid. Adrenal. in Blood. Analysis by Counlercurrent Distribution (Carstensen) ................................................. Steroids. Analysis by Infrared Spectrometry (Rosenkrantz) .............. Stem&. Nmer Developments in the Analysis of. by Gas-Chromatography (Wotiz and Clark) ............................................

.

375

15 1

311 307

3

247

9

235

9 13

39 357

14

497

21 6 7

39 63 111

SUPP.

1

6 4 2 1 10

95 257 279 459 43

10

71

28 2 3

267 1 353

12 2

1 427

30

227

6

97

9

217

7

77

5

65

26 8

47 119

9 2

227 1

18

339

376 CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Steroids. Newer Developnrents in the Gas Chromabgraphic Determination of (Kuksis) ..................................................... Steroids. Separation and DetmniMtMn. by Gas Chromatography (Homing. Vanden Heuvel. and Creech) ................................. Steroids o f the Adrenal Gland. Chrvmahgraphic Separation (Haines and Karnemaat) ..................................... Stoppcd-Flow Method. Recent Deyelopmcnlc in. For the Study of Fast R e a c h (Hiromi) ........................................... Subzero Temprraiures in Biochemistry:S h R e a c h . The Use of (Douzou) ................................................... Succinic Dehydrogetwe Activrty. Determination of (Singer and Kearney) Sugars. A d y s i s of Mixtures. by Paper and CeUulare Column Chromatography (Hough) ...................................... Sugars. the Determination of Structure of Certain Natural Products Confuining Sugam (Hanessian) ................................. Sugars. in Polysaccharides. Detmnination. New C o b React- for (Dische) ..................................................... Su@ases. Assay (Dodgson and Spencer) .......................... Suljhydryl Groups. Detffminorionin Biological Substances (Chinard and Hellerman) .................................... Temperature-Jump Method for Measuring the R& of Fast R e a c h . a Practical Guide to (Yapel and Lumry) ........................... Thmnodynanric Flow Me& in Biochemistry: Caloritnctty. Dntrimetry and Dilatonrclty (Jolicoeur) ........................................ Thiamine. M e w for the Detmmhation of (Mickelsen and Yamamoto) Thiocfic Acid. Assay o f (Stokstad. Seaman. Davis. and Hutner) ....... Thyroid Gland. Chrmabgraphic Analysis of RadiwcfiveIodine Compounds from (Roche. Lissitzky. and Michel) ............................ TirSus. Aliphatic AmmoniumS& in the Assay of Acidic Polysaccharides from (Scott) .................................................. Tissues. Body. Chemical DetenninntMn of Adrenalim and Noradrenahne in (Persky) ..................................................... Tissues. Determidion of Ethyl Alcohol in (Lundquist) ................ Trace Elnnent Analysis. C o n t a m i e in. and Its Control (Thiers) ..... Transaminuse. Deknnination of (Aspen and Meister) ................ Uhqumme. Detmnination of (Crane and Dilley) .................... UDP-Enzyme Systam. Measurements of (Pontis and Leloir) ........... Ultraccntr@gal Analysk of Serum Lipoproteins (de Lalla and Gofman) Uhajiltrr Membranes in Biochemistry (Jacobs) ....................... Ultraviolet Spccbophotometry. E n z p k Micro Deknninations of Urit Acid. Hypoxanthine. X a h n e . Adenine. and Xadwpt.e&e by (Plesner and Kalckar) ........................................ Urea. Ammonk. and Ureate. The Determination of (Kaplan) ........... Urea. Gel Electrophoresis in B$fm Containing (Poulik) .............. Uric Acid. Enzymic Micro DelmniMtirmc. by Ultraviolet Spectrophotometry (Plesner and Kalckar) ........................................ Urinary Ne&al 17.Ke&steroids. Assay of (Engel) .................... Urine. Deknninaaon of 17.21 -Dihydrvqy-20-Ke&ster& in (Siber and Porter) ........................................... Urine. Human. Chemicut Determanation o j Estrogens in (Bauld and Greenway) .......................................

14

325

11

69

1

171

26

137

22 4

401 307

1

205

19

105

2 4

313 211

1

1

20

169

27 6 3

171 191 23

1

243

8

145

2 7 5 6 10 1 22

57 217 273 131 279 107 459 307

3 17 14

97 311 455

3 1

97 479

4

139

5

337

11

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Visual Biochemistly: New Insight into Structure and Function of the Genome (Vollenweider) ....................................... VitaminA. Determination of (Embree. Ames. Lehman. and Harris) ... VitaminA and Carotenoids. in Blood and Tissue.Micro&tennination of (McLaren. Read. Awdeh. and Tchalian) ....................... VitaminBe. Chemical and Microbiological Detennination of (Storvick. Benson. Edwards. and Woodring) ............................. VitaminB I Z .Microbiological Assay of (Hoff-Jorgensen) .............. VitaminBIZ.Microbiological Assay of (Skeggs) ...................... VitaminE Determination (Lehman) ................................ Vitamins. Infrared Analysis of (Rosenkrantz) ........................ Xanthine. EnzymicMicro Determination. by Ultraviolet Spectr-Ophotomehy (Plesner and Kalckar) ........................................ Xanthopterine. Enzymic Micro Determinations. UltravioLt Spectmpiwtometq (Plesner and Kalckar) ......................... X-Ray Diffraction. in the Study of Protein and Nwlkc Acid Structure (Holmes and Blow) .......................................... X-Ray D$fractk?n Analysis. The Growth and Preliminary Investigation of Protkn Nwlkc Acid Crystals for ................................. X-Ray Emission Spectrometry. Detennination of Elements by (Natelson and Whitford) ..................................... Zinc. DetenniMcion OJ in Biological Materials (Malmstrom) ........... Zone Electr@hesis (Kunkel) ..................................... Zone ELctrophuresis. at Constant Cuwent. Mobility Determination by (Waldmann-Meyer) ..........................................

377

28 4

201 43

15

1

12 1 14 2 5

183 81 53 153 407

3

97

3

97

13

113

23

243

12 3 1

1 327 141

13

47