Automated Enzyme Assays

Automated enzyme assays LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY Edited by T. S . WORK - N.I.M.R...

Author: D.B. Roodyn

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Automated enzyme assays

LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY

Edited by T. S . WORK - N.I.M.R., Mill Hill E. WORK - Imperial College London

Advisory board G. POPJAK - U. C. L. A . S. BERGSTROM - Stockholm K. BLOCH - Hurvard University P. SIEKEVITZ - Rockefeller University E. SMITH - U. C. L. A . E. C. SLATER - Amsterdam

NORTH-HOLLAND PUBLISHING COMPANY -AMSTERDAM * LONDON

AUTOMATED ENZYME ASSAYS

D. B. Roodyn Department of Biochemistry University College London

1970 NORTH-HOLLAND PUBLISHING COMPANY -AMSTERDAM

*

LONDON

0 1970 North-Holland Publishing Company All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanicaI,photocopying, recording or otherwise, without the prior permission of the copyright owner Library of Congress CataIog Card Number: 68-54514 ISBN North-Holland: 0 7204 4206 0

Published by: NORTH-HOLLAND PUBLISHING COMPANY - AMSTERDAM

Sole distributors for the U.S.A.and Canada: AMERICAN ELSEVIER PUBLISHING COMPANY, INC.

52 VANDERBILT AVENUE, NEW YORK, N.Y. 10017

This book is the pocket-edition of Volume 2, Part I, of the series ‘Laboratory Techniques in Biochemistry and Molecular Biology’. Volume 2 of the series contains the following parts: Part I Automated enzyme assays by D. B. Roodyn Part n Cellulosic ion exchangers by E. A. Peterson

Printed in The Netherlands

Contents

Preface .

............................. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter I .Principles and terminology of enzyme automation . . . . Chapter 2 . Semi-automatic methods . . . . . . . . . . . . . . .

26

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

26 29

2.1, Detection of enzyme-catalyzed reactions 2.2. Examples of semi-automaticmethods .

7 9 13

Chapter 3. Automatic methods illustrated by the Techniconsystem

.

35

3.1. The Technicon autoanalyzer . . . . . . . . . . . . . . . . . . 3.1.1. Proportioning pump 3.1.2. Sampler 3.1.3. Manifold . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.5. Recording devices . . . . . . . . . . . . . . . . . . . . . . 3.1.6. Dialyzer . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.7. Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.8. Heating bath . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Automation of a simple-enzymeassay . . . . . . . . . . . . . . 3.2.1. The manual method . . . . . . . . . . . . . . . . . . . . . 3.2.2. Dilution factors and fractional volumes 3.2.3. Adaptation to the autoanalyzer . . . . . . . . . . . . . . . 3.3. The sampling process 3.4. Interference by protein 3.4.1. Continuous dialysis 3.4.2. Continuous filtration . . . . . . . . . . . . . . . . . . . . .

.

35 36 37 37 37 38 38 38 39 39

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

.

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

40 41

42 50 54 54 55

.


4

.

Chapter 4 Interrupted-Jow and discrete-samplingsystems

....

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

4.1. Interrupted-flow systems 4.2. Discrete-sampling systems

.

................ 5.1. Molar conversion factors and enzyme units . . . . . . . . . . . . Chapter 5 Single-enzyme analysis

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

5.2. Determination of incubation time 5.3. Measurement of the progress curve 5.4. Relation between enzyme activity and protein concentration 5.5. Enzyme monitoring

.

59 59 63

68 68 71 75 80 81

..........

87

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

87 92 96 98

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

102

Chapter 6 Multiple-enzyme analysis (M.E.A.) 6.1. Principles of multiple-enzyme analysis (M.E.A.). 6.2. Enzyme patterns . . . . . . . . . . . . . . . 6.3. Enzyme gradients 6.4. Multi-channel analyzers

Chapter 7. Enzyme characterization

.........

. . . . . . . . . . . . 102 106 ..... 107 ........................ 108 ...................... . . . . . . . 108

7.1. Substrate specificity and Michaelis constants 7.2. p H optimum . . . . . . . . . . . . . . . . . . . . . 7.3. Enzyme stability 7.4. Temperature optimum 7.5. Plots of multi-parameter studies . . . . . . . . . .

Chapter 8. Calculation of enzyme activities from instrument 114 readings . . . . . . . . . . . . . . . . . . . . . . . 114 ........................ . . . . . . . . . . . . . . . 118 . . . . . . . . . . . . . . 119 122 ....................... . . . . . . . . . . . . . . . . . . . 124 . . . . . . . . . . 125 . . . . . . . . 126 131 .................... . . . . . . . . . . . . . 131

8.1. Data processing 8.2. Calibration and calculation of results 8.2.1. Molar conversion-factor calculation 8.2.2. Incubation time 8.2.3. Line-volume calculations 8.2.4. Calculation of concentrations of line reagents 8.2.5. Gradient calibration and gradient-making systems 8.2.6. Protein concentration 8.2.7. Sample pattern and theory of sampling 8.2.8. Calculation of enzyme activity

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

138

5

CONTENTS

......

140

9.1. Flow system . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Properties of the system . . . . . . . . . . . . . . . . . . . . . 9.2.1. Incubation time . . . . . . . . . . . . . . . . . . . . . . 9.2.2. Fractional-line volumes 9.2.3. Sample interaction and wash characteristics . . . . . . . . . . 9.2.4. Gradient-making system . . . . . . . . . . . . . . . . . . 9.2.5. Preparation of reagents . . . . . . . . . . . . . . . . . . . 9.3. Computer program . . . . . . . . . . . . . . . . . . . . . . . 9.3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . 9.3.2. Molar conversion-factor calculation 9.3.3. Incubation time and chart-speed determinations 9.3.4. Line-volume calculations . . . . . . . . . . . . . . . . . . 9.3.5. Line-reagent and reaction-mixture calculations . . . . . . . . . 9.3.6. Stock solutions . . . . . . . . . . . . . . . . . . . . . . 9.3.7. Gradient calibration 9.3.8. Assay conditions . . . . . . . . . . . . . . . . . . . . . . 9.3.9. Protein concentration 9.3.10. Sample pattern . . . . . . . . . . . . . . . . . . . . . . 9.3.11. Results 9.3.12. Computer sub-routines . . . . . . . . . . . . . . . . . . . 9.4. Example of the use of the above system . . . . . . . . . . . . . .

140 143 143 144 144 145 148 149 149 149 150 150 150 150 150 151 151 151 152 152 152

Chapter 9. Generalized systemfor enzyme automation

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

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

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

Conclusions and future prospects

. . . . . . . . . . . . . . . . . 165

. . . . . . . . . 168 Appendix II . Terminology used in enzyme automation . . . . . . . 173 Appendix III . Apparatus used in enzyme automation . . . . . . . 176

Appendix I . Published automated enzyme assay#

111.1. Analytical systems and instruments

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

111.1.1. Automated analytical systems . . . . . . . . . . . . . . . . 111.1.2. Spectrophotometers. colorimeters and their accessories 111.1.3. Other automatic sensing devices . . . . . . . . . . . . . . . 111.1.4. Dispensers. diluters, pumps and sampling systems . . . . . . . 111.1.5. Reagents 111.2. Addresses of firms

.....

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

176 177 182 187 189 190 191

6


.

Appendix ZV Computer program for generalized enzyme automation system . . . . . . . . . . . . . . . . . . . 193

. . . . . . . . . . . . . . . . . . . 193 . . . . . . . . . . . . . . . . . . . 195 . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

IV.1. Input: general instructions Iv.2. Input: detailed instructions IV.3. Program

............................ Subject index . . . . . . . . . . . . . . . . . . . . . . . . . . .

References

208 218

Preface

This book is primarily intended to be of some help to those who wish to free themselves of the burden of repetitive manual enzyme assays. Because of the current rapid developments in the field of biochemical automation, it is not intended as a comprehensive account of all the available systems, but rather as a guide to present trends. The literature in the subject is scattered over a wide range of journals, technical reports, brochures, reports of meetings and many other publications that are not usually found in department libraries. I must therefore apologize for any serious deficiencies in the text or for incompleteness in the material presented in the appendices on methods and instrumentation. I have illustrated the text heavily from experiments drawn from my own experience with the Technicon autoanalyzer, which has inevitably placed considerable weight on continuous flow methods. However, I have tried to overcome this bias by treating the subject in as general a manner as possible, emphasizing the principles of the various analytical approaches, rather than the detailed procedures. In such a young subject there is still no generally accepted terminology, and from necessity I have developed my own, which I include in an appendix. There is still much to be done in elaborating a theoretical basis for the subject of automated enzyme assay, and I have made a hesitant start in this direction in ch. 8. I have included a generalized system of analysis, with a full computer program, at the end of the

I

Sublecl index P. 218

8


book in the hope that the approaches I have evolved in my own work may be of some use to others. As I have intimated in the last chapter, it is possible that the rapid development of automated techniques in enzymology will have a revolutionary effect on the subject of Biochemistry as a whole and on related disciplines, and may well drastically change the working patterns of scientists in these fields. Combined with the inexorable developments in computer technology, these advances may well result in systems of biochemical analysis that are quite foreign to our present generation of biochemists. Perhaps this book may be of some help in the runaway world in which we may soon find ourselves.

November 1969

D. B. Roodyn

Introduction

Although the study of enzyme catalyzed reactions has been a central part of Biochemistry since its earliest days, it is only recently that methods have been developed which can largely eliminate the tedium of repetitive enzyme assays. At the present time there is considerable interest in automation of enzyme assay and many alternative systems have appeared on the market recently. Although the design of these systems differs considerably, the principles involved in their use are common to all. The aim of this book is to discuss these principles so that whatever the actual instrument used, the reader will be able to develop an automated assay with maximum ease and precision. A list of published automated assays is given in appendix I. At the moment, the majority of such assays have been performed with the Technicon autoanalyzer. For convenience, therefore, I have illustrated the text with various experiments from our laboratory using the Technicon system. However, the results and methods have been presented in a general manner applicable to any automated system. Once the principles of enzyme automation have been acquired, the reader may well find it more convenient to develop his own automated assay, rather than to search the literature for published methods to adapt to his own specific problems. A generalized method for automated enzyme assay is presented in detail in ch. 9 which applies to many current manual assays. The range of instruments now available for automated enzyme 9

Subfect Jndex P. 218

10


assays is shown by the material in appendix 111. There are several reviews on the subject (Bodansky and Schwartz 1961; Stein et al. 1965; Schwartz and Bodansky 1966,1968; Wacker and Coombs 1969). There is a wealth of information in the various reports of the Technicon symposia (‘Automation in Analytical Chemistry’, Mediad Press, New York, 1965-1967) and the Technicon Corporation has also published two useful bibliographies (‘Bibliography of Automated Enzyme Research’ 1965; ‘Technicon Autoanalyzer Bibliography 1957/ 1967’). The use of various biochemical analyzers has recently been reviewed by Buckley (1969). Reference to these sources and the detailed references in appendix I will help the reader in automating manual assays. It is probable that the introduction of fully-automated enzyme assay systems into the general field of enzyme research will have a dramatic effect. It is now possible to perform in a day experiments that would have previously taken weeks. This may be illustrated by an example of one of our experiments.* Suspensions of cells of four different mutants of yeast were disrupted with glass beads in a high speed shaker. The homogenates so obtained were then assayed in a dual-channel multipleenzyme analyzer. Four enzymes were estimated, each at 4 different protein concentrations and for 2 different times of incubation. Each assay had an appropriate blank assay. The results were taken from the recorders and fed into a digital computer which determined in each case the enzyme activity in pmoles/ml, pmoles/ml/min and pmoles/mg dry wt./min. In these calculations the computer used information on instrument calibrations and molar extinction coefficients that was obtained before the assay. The total number of assays, including blanks, was 256 and about 30 instrument calibration values were used. Since the computer corrected for instrument zeros, and blanks, and interpolated molar extinction coefficient values, the number of computational steps was several thousand. All the results were tabulated and printed in graphical form, with suitably calculated scale divisions.

* Experiments reported from our laboratory were supported by grants from the Science Research Council.

INTRODUCTION

11

All these operations were performed in about 5 hr, and during much of the time the operator was free to do other tasks. A reasonable estimate for the time required for a similar ‘manual’ experiment would have been about a week to 10 days. The full potentialities of such systems are perhaps not yet appreciated. Clearly, where routine and standard assays are performed on large numbers of enzyme samples (as in clinical laboratories) automation with computer processing of data must result in a great improvement in the operational efficiency of the laboratory. The use of enzyme assays on tissue fluids as a diagnostic aid is now well established (Quastel 1961) and it is in the clinical field that automated analytical methods have had their greatest impact so far (Whitby et al. 1967). There is little doubt that the introduction of these methods in clinical laboratories has been most beneficial. For example, Robinson et al. (1 966) have compared manual, ‘work-simplified’, and autoanalyzer techniques by work-study methods. They conclude that where there is a large number of assays to perform, the autoanalyzer is greatly superior in accuracy and reproducibility. This was found to be mainly due to the effect of tedium on the human operator during repetitive assays. (Of course it does not follow that automation is always better. For example, Nothstein and Ellerbrook (1962) used the autoanalyzer reagents for the assay of serum alkaline phosphatase to develop a rapid manual method that they regarded as more convenient than the automated procedure!) We can say therefore, that certainly in clinical laboratories, automated methods have in general proved themselves. The impact of fully-automated analytical systems on pure research laboratories has not yet been fully felt. Since such laboratories have their share of tedium no doubt the benefits will be as great. I must clarify a bias in this book. It is addressed mainly to those who wish to assay enzymes, rather than to enzymologists. I hope I will not cause any offence by saying that not all biochemists who estimate enzymes are enzymologists. Going further one might venture to suggest that not all those who estimate enzymes are even biochemists. Although this state of affairs may be disturbing to some purists, it is not at all suprising, or even unwelcome. Enzymes are major compoSubject index P. 218

12


nents of living matter. Their activity, distribution and interaction are subject to the most complex influences and are fundamental to the behaviour of all cells. Used as ‘markers’, they can monitor the sedimentation of sub-cellular particles with great precision. In clinical assays, changes in the enzyme activity of tissue fluids may be the first indication of pathological change. Their levels in micro-organisms may determine and act as indicators of the nature of the fermentation products. Changes in their electrophoretic pattern may reveal genetic abnormalities. Study of their activity during cell differentiation and morphogenesis may reveal fundamental processes in cell growth. Thus, the determination of enzyme activities in living systems is a fundamental and universal weapon of all biologists. Industrial enzymology is now expanding rapidly and attracting wide interest, including soappowder manufacturers. If we define enzymology as the study of the structure and function of enzymes with a view to explaining the latter in terms of the former, we must conclude that it is responsible for only a part of the total volume of work now being carried out on enzymes. For this reason, this book is designed for the non-specialist whose main aim is to determine the level of enzyme activity in suitable preparations of biological materials. For a general treatment of enzymes, the reader is recommended to read ‘Enzymes’ by Dixon and Webb (1964). Other useful books are ‘An Introduction to the Study of Enzymes’ by Gutfreund (1965) and ‘Structure and Function of Enzymes’ by Bernhard (1968). There are also several detailed textbooks on enzyme assay in general. These include ‘Methods of Enymatic Analysis’ edited by Bergmeyer (1968) and the series ‘Methods in Enzymology’ edited by Colowick and Kaplan (1955 onwards). A useful source of references on automated enzyme assays is ‘Analytical Abstracts’, published by the Society for Analytical Chemistry.

CHAPTER 1

Principles and terminology of enzyme automation

Apart from enzymes which have characteristic adsorption spectra (such as the cytochromes), the overwhelming majority of enzymes are estimated by means of their catalytic activity. Provided that the enzyme is saturated with substrate, the rate of reaction is directly proportional to enzyme concentration (see Bodanksy 1959) for a useful introduction to enzyme kinetics in relation to problems of enzyme assay). The rate of reaction is the rate of disappearance of substrate or appearance of product. A plot of concentration of substrate or product against time is called a progress curue and the rate is the slope of this curve. For a variety of reasons (including depletion of substrate, possible product inhibition, thermal inactivation or change in pH) the progress curve is seldom linear over its entire course. It is therefore usual practice to measure the initial slope of the progress curve. Thus, valid enzyme assays require that the enzyme be fully saturated with substrate and that the initial rate is measured. It is not always possible to work under zero order conditions,i.e., under conditions in which the substrate concentration does not affect the rate of reaction. For example, cytochrome oxidase and catalase assays are based on a first order reaction, with the rate of reaction falling continuously as the substrate is depleted.In these situations, the enzyme activity must be expressed in terms of the first order velocity constant. Whilst it is desirable to perform assays under theoretically acceptable conditions, it may not always be essential. If the aim of the experiment is to determine the absolute enzyme activity of a sample in terms of 13

Sublccr index P. 218

14


pmoles/min, then these criteria have to be fulfilled. However, there are many situations in which the point of interest is not so much the absolute activity, as the change in relative activity. For example, if one is following the inactivation of an enzyme with time, the results may be expressed adequately as percent of initial value. During purification of an enzyme, the values of interest are the relative specific activities of samples and starting material. If one is studying levels of enzymes in a bacterial culture under different growth conditions, again percentage changes are of greater interest than absolute values. In these situations, the prime requirement is that the reading obtained shouldbeproportiona1 to enzyme concentration. Whilst one can assure this by working with a fully-saturated enzyme and measuring initial rates, it may be possible to obtain proportionality with respect to enzyme concentration without fulfilling either of these criteria. An example is given in fig. 5.9. where one can obtain proportionality with non-linear progress curves. As regards substrate concentration, Bodansky (1953) developed a method whereby meaningful measurements could be made at sub-optimal substrate concentrations. The autoanalyzer was first calibrated with a standard reference enzyme, and then all unknown enzymes were estimated by determining the amount of reference enzyme which would produce the same change in substrate concentration in the same time. Pitot et al. (1965) discuss the effect of order of reaction on automated assays and assess the various criteria that should be met in such assays. One may conclude, therefore, that although it is preferable to use saturating amounts of substrate and to measure true initial rates, it may be possible in certain other circumstances to make useful measurements. This applies particularly to continuousflow systems, in which measurements of true initial rate may be difficult (see 0 5.2). In general all enzyme assays consist of the following operations : 1. The reagents are prepared from solids or stock solutions. These include buffer, substrate, and any co-factors required for the reaction to proceed. 2. Reagents are mixed in suitable proportions to give the reaction mixture.

PRINCIPLES AND TERMINOLOGY

15

3. The enzyme is mixed with the reaction mixture and incubated in some suitable incubation vessel. 4. The progress of the reaction is followed either by the disappearance of substrate or the appearance of product(s). The reaction is followed by some suitable sensing device or sensor*, which can detect and measure the physical or chemical changes that occur during the reaction. 5. The response of the sensor is recorded in some way so as to provide a permanent record of the reaction. If the sensor contains a device for making this record (e.g. an amplifier connected to a pen recorder) it may be called an autosensor*. 6 . The reaction is stopped. 7. Calculations are performed on the record so as to convert instrument readings into suitable units, such as pmoles/min. The sequence of events is shown in fig. 1.1, and the extent to which the events are automated in various systems is shown in fig. 1.2. The order of events may be changed for various reasons. For example it is usual practice to add substrate to reaction mixture and start the reaction by addition of enzyme. This is because many enzymes are unstable if incubated in the absence of substrate. However, if the enzyme preparation is a crude tissue extract it may well contain significant amounts of ‘endogenous’ substrates. In such cases, more meaningful assays are obtained by short pre-incubation in the absence of substrate. Each assay has its own particular difficulties. For example, if the enzyme preparation is a suspension of particles (e.g. mitochondria) it may be necessary to prevent aggregation or clumping of the particles during the assay. Thus the stirring must be sufficient to maintain a homogeneous reaction mixture, but not too violent to cause damage to the particles. The problem of blanks with crude enzyme preparations, such as serum, can often be serious and appropriate allowance has to be made for such factors as the presence of endogenous substrates, instability of substrates under the conditions of incubation (e.g. the See appendix I1 for terminology used in this book. Subject index p . 218

16


Stock solul ons and solids -~~ Calculate dilution of stock or required weight of solid. Prepare solutions ~

1

Reagents reagents and enzyme to give required assay conditions. Mix reagents in Reaction mixture e.g., time of incubation, temperature

I

Incubation vessel ~

I

Select suitable settings on sensor, e.g., wavelength, slit width

Sensor Adjust recording device, e.g., baseline and chart speed of recorder

Record Calculate enzyme activity in suitable units from values on record, e.g., from

I

Final result From result decide conditions required for next assay

Next experiment

Fig. 1.1. General sequence of events in enzyme assays. Main events areindicated by arrows, and detailed operations are given in rectangles. Fig. 1.2. Methods of automating sequence of events given in fig. 1.1. Main events are as in fig. 1.1. Methods of automating the various operations are given in rectangles.

+

PRINCIPLES A N D TERMINOLQGY

17

Stock solutions and solids Weighing by automatic balance. Dilutions by autodiluters or proportioning pumps. Calculations by computer c

Reagents Dilutions by autodiluter or proportioning pumps. Mixing by jets or mixing coils. Addition of samples by samplers. Calculations by computer

Selection of settings done by operator. In ‘closed loop’ system could be done by on-line computer. Transfer to sensor by probes or pumps

,

-

Variety of recording devices available to monitor output of sensor. Adjustments done by operator but could be done by on-line computer

Fig. 1.2.

I

Subject index R. 218

18


spontaneous hydrolysis of labile esters), the release of interfering substances from the enzyme preparation itself (e.g. the release of bound inorganic phosphate from the enzyme during a phosphatase assay) and, of course, drift in the recording or sensing device (e.g. drift in the base-line, or slow deposition of protein on optical surfaces). One of the great advantages of automated methods of enzyme assay is that it is very easy to make large numbers of measurements of these blanks at various stages of the assay with little addition to the workload, a situation that is not the case in manual assays. It should be noted that enzyme assays are essentially repetitive operations. Thus after the operations 1-7 above have been completed, they are repeated with modification in one of the operations. For example, if the aim is to determine the enzyme activity in a number of different samples, such as serum samples in a clinical laboratory, the cycle would be repeated with new enzyme samples added at stage 3. If several substrates are used for each enzyme preparation, as in multiple-enzyme analysis (see below), an ‘inner’ cycle would be used so that each enzyme preparation would be tested against a cycle of reaction mixtures. We may now consider different types of assay in more detail. Assays may be divided into two primary classes: ‘single-enzyme analysis’ (S.E.A.) and ‘multiple-enzyme analysis’ (M.E.A.), depending on whether one or several enzymes are being assayed. Multiple assay methods are relatively recent and are still in the process of active development (see ch. 6 ) and the bulk of the literature on enzyme assays is orientated towards the assay of individual enzymes. S.E.A. may be used for a variety of purposes, as follows. It often happens that a newly discovered enzyme, or a known enzyme in a new tissue, needs to be ‘characterized’. This usually entails measurement of Michaelis constant (I&,), p H optimum, temperature optimum, stability at 0 “C and during the assay, substrate specificity, response to inhibitors etc. Such studies proceed by using the same enzyme and varying the composition of the reaction mixture, the concentration and type of substrate, and the conditions of incubation. They are usually intended to provide what are called ‘optimum’ conditions of assay and are primarily performed for reasons of convenience, as a prelude to further studies


19

on the enzyme. More detailed and extensive studies of this nature amount to pure enzymological research, and have, of course, given us valuable information about the mechanism of enzyme action. However, as mentioned in the introduction, treatment of such detailed work is outside the scope of this book. Some systems for the automation of the simpler stages in the characterization of enzymes are given in ch. 7. Another obvious use for S.E.A. is in enzyme purification. This needs little discussion. Steps 1-7 are repeated using samples of enzyme at different stages of the purification process. This type of work introduces the question of determination of protein concentration during enzyme assays. An estimate of protein concentration is of the greatest importance in interpretation of enzyme assays. In enzyme purification, the value needed is the specific activity, which is the enzyme activity per unit of protein. There has been a great deal of confusion about enzyme units, and the International Union of Biochemistry suggested in 1961 that the units should be pmoles per minute per mg protein (’Enzyme Nomenclature’, 1965). To quote the 1965 report: ‘One unit (U) of any enzyme is that amount which will catalyse the transformation of 1 micromole of substrate per minute ... under standard conditions.’ Also: ‘The concentration of an enzyme in solution (as distinct from its purity) will normally be given as units per ml.’ ‘The specijic activity of an enzyme preparation is defined as units per mg protein. It is directly related to the purity of the preparation .’ Another measurement that may be made is pmoles of substrate transformed per ml of reaction mixture in a given time (i.e. other than 1 min). This value, which I will call ‘total reaction’, is useful in automated systems in which the precise time of incubation may be difficult to determine. Its use sometimes has advantages. For example, suppose we are following a reaction by measuring reduction of mM NAD’ at 340 nm. The total reaction will give a measure of percent reduction of NAD’; thus a value of 0.9 pmoles/ml will correspond to 90% reduction. If the total reaction value is divided by the incubation time, this relationship is obscured. If the incubation time were 2 min, the Subject index P. 218

20


enzyme concentration (in pmoles/ml/min) would be 0.45 units. The degree of purification of an enzyme is assessed from specific activity values, and the amount of enzyme, or its recovery during purification from concentration and total reaction values. In work on crude enzyme preparations, such as suspensions of subcellular particles, the measurement of both specific activity and total reaction or enzyme concentration is essential to assess the recovery of particles, the cross-contamination between particles, and the general validity of the fractionation scheme. (An analysis of the types of enzyme assay performed in sub-cellular fractionation is given in Roodyn (1965a).) It is generally essential to have some parameter to which we relate enzyme measurements. This is usually total protein, although in some situations, for example in rapidly dividing cells in which the overall composition of the cell changes during growth, enzyme activity per unit DNA may be more meaningful. In other situations, enzyme activity per unit dry weight, or per unit volume of culture, or per cell may be preferable. Even the measurement of specific activities on a protein basis may have its hazards, because of the difficulty of obtaining an accurate estimate of protein. Thurman and Boulter (1966) discuss various methods for estimating proteins and it is clear that no singlemethod is completely satisfactory.Methods based on total nitrogen give results that depend on the amino acid constitution and amide content of the protein. Colorimetric methods, such as the Folin-Ciocalteau or biuret methods, give different colour yields with different proteins and are subject to interference by a variety of agents. The measurement at 280 nm is notoriously unspecific. Dry weight measurements are affected by the degree of hydration which is not easy to control. In fact, it may not be too extreme to say that the only really meaningful measure is the total amino acid composition of the protein! At the moment, measurement of enzyme activity and protein are carried out as separate operations, often at different times. However, automated systems for the simultaneous measurement of both protein and activity have been described (e.g. Scheuerbrandt 1965) and their more general use would be of great benefit.


21

Perhaps the most common use for automated S.E.A. is what may be called enzyme sample determination. Most of such work is at present performed in clinical laboratories, where methods particularly suited to the tissue fluids under test have been evolved. Although pure and applied research laboratories often need to estimate numbers of enzyme samples, there is generally not the steady and routine flow of material to be tested that there is in clinical laboratories. Again, in the research laboratory, it is more usual to alter the conditions of assay from one experiment to another, and detailed inspection of the progress curve is more frequently required. For these reasons, the tendency in such situations has been to develop ‘semi-automatic’ systems (see ch. 2) in which there is a considerable degree of human intervention in the assays. However, it may well be that the development of flexible and generalized enzyme automation systems (see ch. 9) may facilitate the process of ‘enzyme sampling’ in general. Indeed it may not be unusual in the future for several laboratories to share a central ‘enzyme assay centre’, analogous to a computer centre, in which samples brought with a particular assay program are analysed automatically by a specialist staff not under the direct control of the user. The possible implications of central analytical laboratories is discussed later in ‘Conclusions and future prospects’. All the applications given above in S.E.A. can also be performed on groups of enzymes by multiple-enzyme analysis, M.E.A. This approach is relevant when the sample contains several enzymes, and it is suprising how frequently this occurs. Tissue fluids, homogenates, suspensions of sub-cellular particles, impure enzyme preparations, microbial cultures, may all be subjected to such analysis. Being a multiple analysis, particular problems arise in the assay procedure which must often strike a balance between practicality and precision. It is often difficult to arrange assay conditions which are optimum for every enzyme in the group, and sometimes it may be necessary to work under sub-optimum conditions in order to gain the advantages of a group assay. The subject is discussed in detail in ch. 6 ; I will merely stress at this point that the ability to estimate groups of enzymes automatically adds another dimension to the techniques of Subiecf index p . 218

22


enzyme assay and opens up the possibility of performing extremely complex analyses of biological materials. I would like now to try to define more precisely what I mean by an ‘automated’ enzyme assay. Strictly speaking, a fully automated assay is one in which dl1 the processes (1-7, pp. 14-15, and fig. 1. l ) are performed automatically. If the analyzer is connected to a digital output device and hence to a computer, the operator merely introduces reagents and enzyme into the machine and in due course receives a print-out of the enzyme activity in suitable units. As such systems are by no means universal, 1 would call them ‘fully-automated assays’, to distinguish them from the more common ‘automated assays’, in which data processing is not performed ‘on-line’. An automated assay system must prepare the reaction mixture from suitable stock solutions, add the substrate, equilibrate at the required temperature if necessary, add the enzyme, mix it with reaction mixture, carry out the incubation, with stirring if required, follow the course of the reaction, either by directly measuring some property of the reaction mixture, or by stopping the reaction, removing the protein and examining the filtrate or dialysate, and, finally, record the reaction in some permanent form (e.g. on a pen recorder or paper tape). The task of the operator is to prepare the reagents and enzyme, start and stop the analysis, and perform the required calculations on the final record (this may then be done ‘off-he’by an automated data processing system). A ‘semiautomated assay’ is one in which only some of the above operations are performed automatically. For example, if the reaction mixture, substrate and enzyme are pipetted manually into the cuvette of a recording spectrophotometer, the assay may be regarded as semiautomated, even though the progress curve is followed automatically (in one, or several cuvettes). There have recently been developed a large number of devices of varying complexity for dilution and sampling of reagents; methods based on such apparatus are sometimes called ‘work-simplified’. The combined use of such methods with semi-automated assays blurs the distinction between semi- and full automation. At the moment, however, the distinction is fairly clear and provides a useful basis for treatment of the subject. Probably in


23

the near future laboratories will have equipment which allows a continuous progression from manual to automated. The ‘manual assay’ is reasonably easy to define as one in which none of the operations 1-7 are performed automatically. For example the reaction mixture and enzyme are pipetted into a test-tube, shaken on a water bath, deproteinized, centrifuged, and the supernatant removed and read in a manual colorimeter. However, again the division between ‘manual’ and ‘automatic’ pipetting has become blurred recently. For example, Harrison (1966) describes a semi-automatic pipette to deliver 106) pl and 200 pl samples with & 1% accuracy. The BTL Dilutronic 250 (Baird and Tatlock Ltd., Chadwell Heath, Essex) contains two recycling syringes: one takes up sample into the sample pipette, and the other takes diluent into the diluent pipette; sample and diluent are then expelled into the receiving vessel, with a predetermined dilution (see appendix 111 for further details). Schwartz and Bodansky (1963) talk of three stages in automation. In stage I the reaction mixture is prepared manually and the measurement of enzyme activity is recorded in an instrument. In stage 11, the reaction mixture is prepared automatically. Stage 111 ‘... would incorporate feedback devices for controlling and correcting the action of the instrumental components and transformation of the enzyme activities into numerical values’. These correspond roughly to my three classes of ‘semi-automated’, ‘automated’ and ‘fully automated’. However, stage 111 differs in the important respect that it introduces the concept of feed-back control. This raises the possibility of a level of automation far above even the fully automated systems. There is much current discussion of ‘closing the loop’, i.e., developing a system in which an on-line computer adjusts the operation of the instrument on the basis of the output from the instrument and not by command from the operator. At a simple level, the analyzer could decide whether to repeat an assay or alter the assay conditions (perhaps by increasing the enzyme concentration if tl e rate is too low). However, at a higher level, the system could perform multiple assays, arrange its own work-load, data-processing and filing systems, and control its operation not only on the basis of the immediate result, but also by access to Subiecr index P. 218

24


a central store of analytical data. For example, the general request may be: ‘Of the enzyme assays available, find out which enzyme in this sample is most active and determine its characteristics.’ The system would rapidly scan the assays built into it, determine the most active enzyme, refer to its information store as to the properties of that enzyme, prepare appropriate reaction mixtures and carry out a flexible analysis of the enzyme based on its response to each condition of assay. Because of the very high ‘data-yield’ of fully-automated systems, it may become increasingly difficult for the human operator to assess and collate the output of the analytical system and he will inevitably have to rely to a greater extent on the computer in this task. Thus, what at the moment appears speculative, may well become a reality because of the problems of processing large amounts of analytical data. Another division of enzyme assays is into ‘continuous-flow’ and ‘interrupted-flow’methods. In the former method, all the operations proceed sequentially and without interruption. These systems, at first sight a little strange to the biochemist, are in fact interesting analogues of steady-state systems in the living cell. In the interruptedflow systems, some of the operations, i.e., preparation of the reaction mixture and introduction of the enzyme are carried out sequentially. The flow system is then stopped and the reaction is monitored with a stationary reaction mixture in the reaction vessel, which is usually the cuvette of a spectrophotometer. Such systems have great advantages for following progress curves and hence determining true initial rates. A third system has recently been widely introduced. This is the so-called ‘discrete-sampling’ analyzer. Each assay is performed in its own reaction vessel (usually a disposable plastic tube) with reagents introduced at various time intervals by suitably positioned automatic pipettes. These analyzers generally are able to operate with very little inter-sample contamination. In general it is difficult to monitor the progress curve continuously with continuous-flow analyzers or discrete-sampling systems, although there have been various arrangements to do this, at least in a somewhat primitive fashion (e.g. by the measurement of a small number of


25

different incubation times with different sensors). It is surprisingly difficult to obtain detailed information about the entire progress curve with available continuous-flow systems. This is because the enzyme and substrate are reacting in a continuously flowing stream. The only way to follow the course of the reaction is to set up ‘monitoring points’ along the stream. A complete sensing device is required at each monitoring point, since the same device cannot be used at the same time at more than one place. This would make the cost of obtaining, for example, six readings along the progress curve quite considerable. Thus at present, cofitinuous monitoring is best done in interruptedflow systems.

Subjecf index p . 218

CHAPTER 2

Semi-automatic methods

2. I . Detection of enzyme-catalyzed reactions In most semi-automatic methods the reaction mixture is prepared manually. A suitable volume is placed in the reaction vessel, the enzyme is added manually, mixed with the contents of the reaction vessel and then the sensor is allowed to follow the course of the reaction automatically (auto-sensor). A large number of methods have been used to follow enzyme-catalyzed reactions since almost any chemical or physical change that can be measured in a recording instrument may be used. For full details of the methods used the reader is referred to the textbooks mentioned at the end of the introduction. Gutfreund (1965) gives a useful account of the various approaches, and some of his examples will be considered. The following brief account is not intended as a comprehensive survey, but merely as a demonstration of the wealth of methods available. The most usual method is to measure changes in light absorption and a variety of instruments have been used for this purpose, from simple filter colorimeters t o sophisticated spectrophotometers. Changes in visible or ultra-violet light occur either as a direct result of the reaction or as a consequence of chemical or physical treatments carried out on the reaction mixture. Perhaps the commonest measurements are at 650-700 nm for the blue complex formed after the reduction of phosphomolybdate in the assay of inorganic phosphate, and at 340 nm to follow the reduction of NAD+ or NADP'. Many reactions have been linked either directly or indirectly to such measure26

SEMI-AUTOMATIC METHODS

27

ments. Esterases are often measured by using substrates with chromogenic substituents (e.g. nitrophenyl groups) which give characteristic colours after release from the substrate, as in the use of p-nitrophenyl phosphate in the assay for phosphatases. There are many systems available for monitoring changes in light absorption, and a commonly used instrument is the Gilford 2000 multiple sample absorbance recorder (appendix 111, p. 183). Wood and Gilford (1961a) give full details of such a system. If the enzyme has a characteristic absorption spectrum itself, as with haem proteins, it is possible to assay the enzyme, and even to follow the formation and disappearance of the enzyme-substrate complex by direct spectrophotometric measurement. A semi-automated system based on spectrophotometric measurement is described by Henry et a]. (1963) for use in clinical enzymology. Another important method of studying enzyme-catalyzed reactions is by measuring changes in fluorescence. In his excellent book on ‘Fluorescence Assay in Biology and Medicine’, Udenfriend (1962) discusses the use of such measurements in enzymology and demonstrates their widespread application and great sensitivity. Fluorimetry is also used to measure common substrates and co-factors, by the use of specific enzymes. As with light absorption methods, there is a great range in sophistication of the instruments used. Fluorescence spectroscopy is extremely sensitive and is useful, for example, in estimating reduced pyridine nucleotides. Duysens and Amesz (1957) used it to follow levels of NADH in living cells. For rapid reaction studies, stopped-flow fluorimetry is of use (see, for example, Harvey 1969, and Chen et al. 1969). A variety of electrode systems have been used, including electrodes sensitive to changes in concentration of dissolved oxygen, COz, Na+, or K + ions and, of course, H + ions. As an example, Severinghaus and Bradley (1958) described a double electrode for measuring pOz and p C 0 2 in biological fluids. The oxygen electrode is widely used in studies on mitochondria and oxidative enzymes. In p H measurements, the actual change in p H may be followed; in a more convenient method the p H is maintained constant by the continual addition of acid or alkali, and the volume of titrating fluid plotted against time gives a Subject index P. 218

28


measure of the rate of the reaction (see for example the rapid pH-stat assays described by Dyson and Noltmann (1965). Gutfreund and Hammond (1963) in a discussion on the use of pH assays to follow enzyme activity stress the sensitivity and simplicity of such methods. Other physical methods have been used. For example, changes in the heat content of the reaction mixture may be exploited, as in the calorimetric measurements of biological oxidations by Estabrook and Gutfreund (1960). Magnetic measurements may be used, for example in a study of myoglobin by Brill et al. (1960). There have been many studies on enzymes using NMR (nuclear magnetic resonance); Shulman (1962) summarized the use of electron and nuclear spin resonance in biology and gave some examples of its application to enzymology (see also Beinert and Palmer 1965). A very sensitive method for enzyme assay that is being used increasingly is to measure the conversion of a radioactive substrate to a radioactive product (for a detailed account see Reed 1968). The radioactive product is separated from the substrate by a variety of means, and if the separation is efficient (i.e. there is no contamination with residual substrate which would invalidate the assay), the sensitivity is quite remarkable. For example, with a substrate of specific radioactivity 62 mC/milliatom, one can detect 3 x 10- l 2 moles of product! The method has certain difficulties however. Apart from the problem of achieving a clear separation between substrate and product, the specific activity of the substrate must be known precisely, and the substrate must be extremely pure. (I am not aware of any fully-automated methods based on radioassay. However, if developed, their potential should be very great.) There is now a considerable literature on the separation of enzymes by various forms of electrophoresis, for example on gels or cellulose acetate strips. These studies are of particular importance in the study of isoenzymes. Usually the enzymes are detected qualitatively by treatment with a reaction mixture or dye that gives a coloured or UV-absorbing product, but quantitative methods may be used. As an example, Opher et al. (1966) studied the isoenzymes of serum lactate dehydrogenase by strip electrophoresis. They incubated the strips


29

with substrate and tetra-nitro-blue tetrazolium. This was reduced to a purple formazan by the action of the enzyme, and the amount of reduction was proportional to enzyme concentration. Quantitative determination of the coloured area was carried out with a densitometer; the assay may therefore be regarded as a semi-automated procedure.

2.2. Examples of semi-automatic methods These, then, are some of the methods available for the study of enzyme activity. How are they used as semi-automatic methods? In the simplest device, a temperature-controlled cuvette containing reaction mixture is placed in a recording spectrophotometer. A ‘base-line’ is obtained for any absorption by the reagents and then substrate and enzyme are added. For example a suitable volume of buffer is placed in the cuvette, NAD’ and malate are then added, followed by malate dehydrogenase. A trace of increase in absorbance at 340 nm with time is obtained (fig. 2.1) and the malate dehydrogenase activity may be

Chart divisions (inch)

Fig. 2.1. Example of automatic recording of progress of an enzyme catalyzed reaction. Semi-automatic assays for malate dehydrogenase with a recording spectrophotometer. The reaction mixture contained malate and NADf at pH 9.9. At arrows, successive samples obtained during chromatographic purification of the enzyme were added to a succession of cuvettes manually. The vertical lines were caused by closing the shutter between the addition of samples. (See Roodyn et al. (1 962) for details.) Subject index I. 218

30


calculated from the initial slope of the curve. This type of system is very useful if one wishes to make various additions while the reaction is in progress, with the amount and type of substance added depending on the initially observed rate. It is used a great deal, for example, in studies on mitochondria1 metabolism with the oxygen electrode. Quite complex traces may be obtained in this way. Provided that the reaction does not proceed too rapidly, the research worker can alter the composition of the reaction mixture at will by addition of small volumes of reagent while observing the progress of the reaction on the chart. For example, he can increase the amount of a possible inhibitor until the required inhibition is reached. On the basis of the instrument response, he can add any of his other reagents in any sequence at any time. This type of ‘man-machine’ interaction can be very fruitful. In more fully automated systems the sequence of operations has to be predetermined to a far greater extent, with the machine operation having to be clearly programmed before the assay begins. It would be useful to be able to alter the operation of the machine according to the results being obtained (see ‘closing the loop’, p. 23). However, the controls of almost all analytical systems at the moment are still under operator rather than computer control. Patient (1960) discussed the general problem of the relative advantages of partial and complete automation by taking examples from the processes involved in the manufacture of penicillin. It was found that total automation was expensive and required extensive instrumentation and control processes. However, if only those parts of the system that required a high degree of precision were automated and other activities (such as the loading of tube-racks) given to unskilled operators, a less expensive and more flexible system was possible. Thus in all automated systems a balance must be struck between the ideal and the practical. The introduction of systems for recording the reaction in a single incubation vessel was clearly an advance on manual methods. However, even in such simple systems, it does not follow that the manual approach is always inferior. Unless one has as many sensors as samples to be assayed, it could well be quicker to prepare a rack of test-tubes manually and incubate them all simultaneously, rather than to carry


31

out the assays sequentially with a recording device. For example, let us suppose that a given enzyme requires 3 min to give a convenient reading. If 100 samples are to be analysed, the time for sequential analysis is at least 300 min, not allowing for the time taken to load each sample into the recording device. If it takes one minute to pipette the sample and reaction mixture into a test-tube, about 100 min may be spent preparing racks of tubes manually. These could all be incubated simultaneously (perhaps for 5 min). If the tubes could be read manually at a rate of one every 30 Eec, the entire manual assay could take about half the timc of the semi-automatic assay. If the rate of reaction is slow, and it takes 10-20 min to obtain a satisfactory reading, the difference in time becomes even greater. To overcome this difficulty, manufacturers of recording spectrophotometers have introduced automatic sample changers of various degrees of complexity. Usually, about 4 cuvettes are loaded into the machine on a movable carriage, and each cuvette is moved in front of the light path at intervals determined by some attached control device. An early model for an automatic cuvette positioning attachment for a spectrophotometer is described by Wood and Gilford (1961b). It is usually difficult, or impossible, to add reagents to the cuvettes of the automatic sample changer, while the reaction is in progress. (Some instruments with this facility have become available recently : see appendix 111.) These systems are therefore useful when the assay procedure is reasonably standard and there is no need t o add reagents as the reaction progresses. Some automatic sample changers operate with flow cells, but usually the cuvettes are filled manually. Generally it is difficult to stir the reaction mixture in the cuvettes, although recently Beckmann-Spinco Instruments have described a system which does this (Kintrac VII: see appendix 111, p. 182). An automatic stirring device for four cuvettes has also been described by Lysionok and Flynn (1969). Since the cuvette itself is the reaction vessel, certain limitations are imposed on the type of reaction that can be followed spectrophotometrically. For example, continuous aeration of the liquid would be impossible because of light scattering from the bubbles. There may also be problems with temperature control and Subject index P. 218

32


with turbidity of the enzyme if it is bound to cell particles. The AmincoChance dual-wavelength spectrophotometer is designed to measure optical density changes in very turbid solutions (appendix 111,p. 184). It is theoretically preferable to carry out the enzyme reaction in a separate vessel and to pump a small fraction of the reaction mixture continuously through a continuous flow autosensor. The reaction vessel could then be designed specifically for the type of enzyme reaction followed and limitations imposed by the geometry of the cell carriage would be overcome. It should be noted that automation of the sensing device does not completely remove the tedium of repetitive assays, and as we have seen, in some cases may have certain disavantages. Nevertheless such instruments represent an important half-way stage between manual techniques and full automation by eliminating human error from the actual measurement of the enzyme-catalyzedprogress curve. It is thus becoming increasingly rare to see biochemists rapidly jotting readings as they simultaneously follow a stop-watch and the needle of the spectrophotometer, Several recording manometer systems have been described, usually based on the well-known Warburg manometer. For example Raikhman (1966) described a system in which the level of fluid in the manometer is detected by a suitable electrode system (see appendix 111, p. 188, for other examples). In many ways such instruments have advantages over recording spectrophotometers. A larger number of samples can be assayed at the same time. Temperature control is rigorous and it is possible to agitate and gas the vessels during the course of the reaction. It is interesting how fashion may sometimes dictate the equipment purchased. Many ingenious and fruitful techniques of manometry were developed before 1939 to follow enzyme reactions, for example using bicarbonate buffers in a COz gas phase to monitor acid-base changes (Dixon 1951). These methods are now generally neglected in favour of the recording spectrophotometer. Perhaps a more widespread use of recording manometers, or devices such as the Gilson respirometer (see appendix 111, p. 188) will correct this tendency. The oxygen electrode attached to a suitable circuit and recorder is a


33

semi-automatic device that has had a great impact on our knowledge of the biochemistry of respiratory systems. The reaction mixture is fully aerated and then the vessel is usually sealed from the air after addition of enzyme, so that as the reaction proceeds the oxygen concentration in the reaction mixture falls. This means that after a time, measurements are being made a t low oxygen tensions, and by the end of the experiment the vessel becomes virtually anaerobic. Thus experiments performed immediately after sealing the reaction vessel are not necessarily identical to apparently similar experiments performed later. This again illustrates the difficulty that may arise from having to use the sensor as the reaction vessel. Pressman (1967) employed a multiple incubation vessel into which are inserted an oxygen electrode, and electrodes for measuring H + , Na+ and K+ ions. At the same time the turbidity of the preparation can be measured. The plot includes a trace in which the rate of oxygen uptake is obtained by a simple electronic circuit that differentiates the signal from the oxygen electrode. With the availability of multichannel recorders and X-Y plotters, further development of such ‘multi-parameter’ sensor systems will be facilitated. By providing simultaneous measurement of different properties of the reaction mixture, more meaningful data may be obtained than from sequential measurements. For certain enzymes, particularly the flavoproteins and cytochromes, direct measurement of the spectrum is extremely useful. There have been many studies on the spectral changes that occur during the catalytic cycle of such enzymes, and these changes have been most useful in the study of the formation of enzyme-substrate complexes (for example see the high-resolution spectrophotometric studies of Chance (195 1) with catalase). Lundegardh (1959) described the spectrophotometric techniques available at that time for the study of respiratory enzymes in the living cell, and since then more complex systems have been developed. The study of enzyme-substrate complexes also calls for extremely rapid measurements. These are often performed in a stopped-flow apparatus which can measure reactions that occur in the millisecond range. For example, Gibson and Milnes (1964) give the Subject index p . 218

34


detailed design of a stopped-flow apparatus for following reactions with half-times of 5 msec upwards. The main features in the design of the Durrum stopped-flow apparatus are given in appendix 111, p. 184. One of the problems of studying enzyme-catalyzed reactions is that if the protein concentration is too high the rate may become too high to measure. Thus most studies are made at low enzyme concentration and high substrate concentration. However, in the cell, the reverse situation may be nearer reality, and it is important to understand the processes that occur at high enzyme concentrations. Eigen and Hammes (1963) in a general review on the study of fast reactions in enzymology describe the use of relaxation methods. In these the effect of a sudden perturbation to the system is examined (Eigen 1968), and it is possible to perform such studies at the desired high enzyme concentration. In a general sense, one could regard the monitoring of a reaction mixture with an autoanalyzer as a semi-automatic method, with the analyzer acting as a chemical sensor. For example Lenard et al. (1965) followed the progress of amidase- and peptidase-catalyzed reactions by pumping the reaction mixture continuously into an autoanalyzer that carried out the ninhydrin reaction. At the moment such systems are not widely used. However, if the reaction mixture could be monitored at the same time by other sensors (e.g. various electrodes) most useful analytical methods would result. For example, simultaneous monitoring of oxygen concentration and levels of inorganic phosphate in mitochondria1 suspensions would provide a continuous measure of P/O ratios. Thus the combined use of multi-xensor reaction vessels with chemical analyzers could greatly increase the productivity of semi-automatic methods.

CHAPTER 3

Automatic methods illustrated by the Technicon system

3.1. The Technicon autoanalyzer In compiling appendix I, the author has found that, a t the moment, the majority of published methods for enzyme automation are based on the Technicon system. It is therefore convenient to introduce the principles of automated assay by reference to this system. Several continuous flow systems have been described in the literature. For example, Jonnard (1960) described a ‘Random Selection System for Dynamic Biochemical Analysis’ which consists of a fluid processing system, mixer, sampler, colorimeter, various programmers, an automatic titrator, amplifier and recorders. However, the most commonly used continuous system is the Technicon autoanalyzer based on the method of Skeggs (1957) in which the earliest designs of sampler, proportioning pump, dialyzer, heating bath and colorimeter are given. The Technicon system has been used for enzyme assays for at least 10 years (e.g. Marsh et al. 1959) and appendix I demonstrates the range of enzymes that have been automated with this method. As with much current equipment, the Technicon system is based on the use of ‘modules’. Instead of purchasing a single complex instrument that is specifically designed for the required purpose, one can purchase sub-systems which may be assembled in various combinations to suit individual needs. Also, other equipment may be combined with one or more of the modules. The Technicon modules that are generally used for enzyme assays are as follows: 35

Subjecr index P. 218

36


3.1.1. Proportioning pump A series of plastic tubes are clamped against a flat surface by means of two end blocks and are compressed by rotating metal bars. The volume of fluid pumped by each tube, or line is determined by the internal diameter of the pump tubing. If the pump tube is L cm in length and has a diameter of d cm, the flow rate for a bar moving along the tube at b cycles/min is given by

LV =lrd2bL/4,

(3.1)

where LV is flow rate or line volume in ml/min. Since many different TABLE3. I Pumping rates with Technicon tubing. (Values refer to clear standard tubing used with a standard-speed proportioning pump. Pumping rates should not fall below the minimum value or rise above the maximum value. The expected rate is given by the nominal value, and above 0.32 ml/min there is usually little deviation from this value.) Colour code

Orange black Orange red Orange blue Orange green Orange yellow Orange white Black Orange White (clear) Red Grey Yellow Yellow blue Green Purple Purple black Purple orange Purple white

Pumping rate (ml/min) Minimum

Nominal

Maximum

0.005

0.015 0.030 0.050 0.10 0.16 0.23 0.32 0.42 0.60 0.80 1 .oo 1.20 1.40 3.00 2.50 2.90 3.40 3.90

0.029 0.048 0.072 0.128 0.19 0.27 0.36 0.47 0.66 0.87 1.08 1.28 1.49 2.10 2.63 3.03 3.54

0.016 0.032 0.075 0.13 0.19 0.28 0.37 0.54

0.73 0.92 1.12

1.31 1.90 2.37 2.77 3.26 3.15

4.05

THE TECHNICON SYSTEM

31

diameters of tubing are available, a wide range of volumes may be delivered at one pumping speed. The available range for the ‘standard’ proportioning pump is given in table 3.1. With a variable speed pump, however, any pumping speed may be obtained (see 0 9.2.2). 3.1.2. Sampler A circular table carries up to 40 cups and a probe dips into the cups at pre-determined time intervals. In the early sampler module (sampler I) the probe can sample at 20, 40 or 60 cups/hr, and between samples the probe aspirates air for half the sampling time. Thus at 60 cups/hr the probe is in the sample for 40 sec and in air for 20 sec. The improved sampler (sampler 11) has a wider range of sampling times and has a system for washing the probe between samples. It can also be refrigerated so that it is preferable for enzyme sampling. A larger sampler is available, but is not generally used for enzyme assays. 3.1.3. Manifold This is a system of tubes, glass joints, mixing coils and other devices that is attached to the pump, receives the various reagents and the samples, mixes them in various proportions determined by the line volumes in the proportioning pump and then delivers the mixtures to various peripheral devices. The manifold also usually contains waste tubes returning from these devices to the input (or ‘venous’ side) of the pump and hence to the waste receiver or drain. The manifold is usually mounted on a plastic tray fitting over the pump and can be detached more or less intact, so that the same analyzer may be used for different assays. The term manifold is sometimes also used in the more general sense to describe the entire flow diagram of the analytical system. 3.1.4. Sensors A variety of continuous-flow sensors are manufactured by Technicon specifically, but provided that a de-bubbling device is fitted, most other continuous-flow sensors may be used. The most commonly used device is the Technicon double-beam colorimeter which selects wavelengths by suitable filters. This is generally adequate for most Subierr index P. 218

38


assays in which some coloured product is formed and may also be used for measurement of NADH and NADPH, although this is rather at the optical limits of the lamp. For UV measurements, or more precise spectral measurements, a spectrophotometer is required. The other device of increasing use in enzymology is the continuous-flow fluorimeter, and Technicon have recently introduced an improved version with greater stability and a very small flow cell. Alternatively various electrodes or flame photometers may be used. The factors of importance in selecting a continuous-flow autosensor for automation are the sensitivity, the retention volume of the flow cell and the stability. 3.1.5. Recording devices These are usually supplied with the sensor, although flexible arrangements are possible. For example, White and Gauger (1968) have used the Gilford Model 300 spectrophotometer with a Technicon autoanalyzer and ratio recorder. The commonest recording device is the pen-recorder ; Technicon supply one-, two- or three-pen recorders. It is becoming increasingly common to have some form of digital output (e.g. paper tape), either in addition to, or instead of, the pen recorder. These systems are discussed in detail in § 8.1.

3.1.6. Didlyzer This dialyzes the stream from the manifold against an appropriate medium and is used to separate low-molecular weight substances (such as phosphate) from protein for subsequent assay. The efficiency of concurrent dialysis is generally not very high, so that it cannot be used to separate all the low-molecular weight materials from the protein. (Much more efficient dialysis can be achieved by ‘counter current’ dialysis in which the donor and acceptor streams flow in opposite directions. However, in this case it would not be possible to perform assays on material crossing the dialysis membrane.) 3. I . 7. Filter In some cases it is preferable to precipitate the protein and examine


39

the filtrate, rather than use the dialyzer. The continuous filter performs this operation, and is often used for phosphatase assays. 3. I .8. Heating bath This constitutes the reaction vessel. It consists of coils of many feet of glass tubing which act as time-delay coils. The bath usually contains two separate coils which may be used in series or parallel. Coils of different sizes are available, so that the retention time may be varied using a constant flow rate from the manifold. For long incubation times, more than one heating bath may be used. There are many other devices in the Technicon system (e.g. an automatic digestor for the dermination of total nitrogen or phosphorus) and the reader is referred to the many brochures of the company for full details. Because of its widespread use, I will try to keep to the terminology used by Technicon and will use the term ‘flow system’ to describe the overall arrangement of the modules.

3.2. Automation of a simple-enzyme assay In the remaining chapters, I will often illustrate the various systems by examples from work from my laboratory. Many of the results were obtained in preliminary experiments during the development of the various systems and their scientific interest is of little relevance to this book. For this reason, I have sometimes simply given instrument readings rather than absolute values, and have included few details as to composition of reaction mixtures and source and preparation of enzymes. Most of the work has been with various ferricyanide reductases of yeast, and the enzyme preparations were usually homogenates obtained fiom yeast that had been disrupted with glass beads. In some experiments yeast mitochondria, kindly prepared by Dr. L. A. Grivell, were used. A useful way to introduce the reader to the Technicon system in particular, and continuous-flowautoanalysisingeneral, is to describe in detail a typical manual assay (of e.g. L(+)-lactate dehydrogenase of yeast). The enzyme is assayed by following the reduction of potassium ferricyanide by lactate at neutral pH. The Sublecr index p . 218

40


ferricyanide absorbs strongly at 420 nm and none of the other reactants or products, including ferrocyanide, absorb strongly at this wavelength. In order to avoid complications due to the need for deproteinization, I will consider the simplest case in which a soluble extract is used. 3.2.1. The manual method The manual assay is as follows. A reaction mixture is prepared so as to give final concentrations, after addition of a suitable volume of enzyme, of 0.05 M potassium phosphate pH 7.4, 2.0 mM sodium L( +)-lactate and 0.4 mM K&(CN),. Enzyme is added to give a final concentration of 50 pg protein/ml, and after 10 min at 30 "C the fall in absorbance at 420 nm is measured. In detail, the actual assay proceeds in the following stages: 1. The stock solutions are prepared. (A stock solution is any solution that is used to prepare a reagent. The reagent may either be a suitably diluted stock solution, or may be a mixture of various stock solutions.) 2. The reagents are prepared by dilution of the stock solutions. The convenient stock solutions in this case are 1 M potassium phosphate¶ 50 mM lactate and 10 mM ferricyanide. Four reagents are prepared: A: 0.075 M potassium phosphate pH 7.4; B: 0.015 M sodium L(+)-lactate pH 7.4; C: 3 mM K&!(CN),; D: enzyme solution, 0.75 mg protein/ml. 3. The reagents, apart from the enzyme solution, are mixed to form a reaction mixture by adding 4 ml of (B) and 4 ml of (C) to 20 ml of (A). 4. Two test tubes each containing 2.8 ml of the reaction mixture are equilibrated in a water bath at 30 "Cfor 5 min. 5. Enzyme (0.2 ml of (D)) is added rapidly to each tube, the contents are mixed thoroughly, the E420 of the first tube is read immediately and the second is placed in a waterbath for 10 min. 6. The E420 of the second tube is read. 7. From the difference in values between the tubes and the known protein concentrations, the enzyme activity is calculated in terms of pmoles/min/mg protein. (The automation of calculations is discussed fully in ch. 8.)

41


3.2.2. Dilution factors and fractionul volumes In order to clarify the subsequent discussion it is useful to tabulate the various initial and final concentrations and the volumes used (table 3.2). This table contains two new terms, the dilution factor and the fractional volume. These are used in automated systems, particularly for calculation of reagents required for a given reaction mixture. TABLE3.2 Example of a simple manual assay: reagents used. Component

Volume of Initial con- reagent centration (ml)

K phosphate pH 7.4 0.075 M Na L(+)-lactate 0.015 M K3Fe(CN)s 3.0 mM Enzyme 0.75 mg/ml Total

2.0 0.4 0.4 0.2

3.0

Final concentration

Dilution Fracfactor tional volume

0.05 M 2.00 mM 0.40 mM 50.0pg/ml

1.5

7.5 7.5 15.0

0.67 0.13 0.13 0.067 1.ooo

The dilution factor measures the extent to which a component in a stock solution is diluted in the final reaction mixture. It is: initial concentration of component final concentration of component

9

and is equal to: final volume of reaction mixture volume of stock solution added The reciprocal of this value is the fractional volume, and measures the contribution a particular reagent makes to the total volume of the reaction mixture. When these terms are applied to an automated system each reagent is identified by the line in which it is pumped. In the Technicon system, the line is the particular tube in the proportioning pump that is connected to the reagent. However, in a general sense it is any tube, probe or pump that connects a given reagent to the analytical system. For automation, we therefore use the terms SubjecI index p . 218

42


line dilution factor and fractional line volume. For convenience these may be abbreviated to LDF and FLV respectively. 3.2.3. Adaptation to the autoanalyzer Let us now see how the above simple assay may be adapted to the autoanalyzer. A simple flow-system to perform the assay is shown in fig. 3.1. Phosphate and lactate are pumped into the system and mixed in a mixing coil (MC,). In order to produce a bubble pattern we introduce a stream of air at the same time. We thus have the arrangement: line 1 : substrate; line 2: buffer; line 3: air. Having mixed the substrate with the buffer, we now add the ferricyanide in another line (line 41, referred to as the co-factor line. Another mixing coil is used t o provide the complete mixed reaction mixture, ready for injection of the enzyme stream. Of course it would have been possible to prepare the mixture of buffer, substrate and co-factor in bulk, manually, before the assay, and in practice it is advisable to keep the number of lines

MC, -I//-

MC,

MC, ///P

1-

Substrate

2-

Buffer

3-

Air

4-

Co - f actor

5-

Enzyme

w*

Colorimeter Recorder

Fig. 3.1. A simple flow system for enzyme assay. D: de-bubbler; FC: flow cell; HB: heating bath; MC: mixing coils (MC3 is jacketed to operate at the temperature of the heating bath); W: waste.


43

to a minimum. However, the system shown in fig. 3.1 is a useful one, since it may be used as a generalized manifold for many enzyme assays as discussed in detail in ch. 9. Enzyme is introduced through line 5 and the reaction starts as the enzyme mixes with the reaction mixture. This occurs in a jacketed mixing coil (MC,) which is at the temperature of the heating bath, making calculation of the time of incubation somewhat easier. The reaction mixture containing enzyme now enters the heating bath where the bulk of the reaction takes place. The effluent from the heating bath then passes directly to the de-bubbler. This is an Fshaped glass joint. Gas plus liquid are pumped into the lower bar of the F and the vertical limb is connected to the flow cell. The upper bar is connected to waste. The flow rates in the various pump-tubes are so chosen that the volume of fluid being drawn through the flow cell is slightly less than the volume of fluid entering. The excess fluid, plus gas-bubbles, rises to the upper bar of the F and hence to waste. The effluent from the flow cell is returned to the venous (or input) side of the pump. (The terms ‘venous’ and ‘arterial’ are useful in describing autoanalyzer systems. They remind us that the autoanalyzer is essentially a closed circulatory system operating under pressure caused by the pump, with some similarities to vascular systems. For example once we have opened the system to atmospheric pressure we can only obtain accurate flow rates if the fluid is returned to the venous side, just as it is impossible to control the flow from an open blood vessel.) The problems of bubbling and de-bubbling arise from the fundamental design of the autoanalyzer. The stream of bubbles prevents smearing of samples during their passage through long lengths of tubing (Skeggs 1957). The efficacy of the bubble train is shown in fig. 3.2, in which samples were run with and without bubbling. Some authors have described flow systems that do not use gas-segmentation. For example Hicks and Updike (1965) describe an automated procedure for estimation of lactate dehydrogenase. However, the sample traces presented suggested that the system had poor wash characteristics. The theory of sample interaction and wash is discussed in 9 8.2.7. Mixing coils in the Technicon system operate by the action of the Subiecr index p. 218

44


bubbles rising and falling as the fluid passes through the coil. More rapid mixing may be achieved by so-called ‘jet-mixers’ in which fluid is forced through a tube with a series of constrictions that produce turbulent flow. The action of this device is shown in fig. 3.3. In most systems, the time spent in the mixing coil is about 1 min; mixing is usually complete within 30 sec. A1

A 2

0

A3

m

S -

0 0

B1

L

82

L

Fig. 3.2.Effect of gas-segmentation on the shape of thesample peak. The flow system in fig. 3.1. was used with a sampler attached to the substrate line (line 1). Water was placed in lines 2, 4,and 5, and three identical samples of 2 mM potassium ferricyanide were placed in the sampler, with water in alternate cups. Al-A3: peaks obtained with air in line 3,giving the usual stream of air-bubbles (air-segmentation) ; Bl-B3: peaks obtained with water in line 3.There was no stream of air bubbles. However, with the type of sampler used (sampler I) there was a pulse of air between samples caused by the sample crook being in air; C143: the sample probe was dipped manually into the samples, with rapid transfer so as to eliminate any air bubbles in the stream; S: steady state with sample probe in 2 mM potassium ferricyanide. (Note that poor sampling is shown by failure to approach the steady state value, and spike-shaped sample peaks.)


45

Inner segments (0.035)

Fig. 3.3. Design of jet mixer. Fluid flowing through the mixer is confronted with repeated sudden alterations in tube diameter. This produces marked turbulence and hence good mixing. (From Pitot et al. (1968), by courtesy of the authors.)

The requirement for a bubble train plus the rather stringent requirements of the de-bubbler cause some difficulties in the design of more complex manifolds. This is because few sensors, if any, can operate on a stream of fluid interrupted intermittently by bubbles. For example, a continuous-flow spectrophotometer would give a very irregular trace that would be impossible to interpret. In fact, if the waste return from the de-bubbler is not adjusted correctly occasional bubbles may pass through the colorimeter, spoiling the trace. As a result, in order to use a sensor one must de-bubble. This usually involves an additional waste line to the manifold, hence increasing its complexity. Also, once de-bubbled, smearing of the sample occurs rapidly and the lead from the de-bubbler to the sensor must be as short as possible. Any further operations on the stream must be done on a re-bubbled stream, which entails returning the unsegmented line to the venous side of the pump. During this process smearing can occur, with loss of resolution. The re-bubbling requires another gas line, so that insertion of an extra sensor into the system requires a new waste line and gas line, and also results in possible loss of sample resolution. A method for making measurements on an intermittent stream of liquid and gas would greatly simplify many Technicon systems. Recently, Technicon have introduced a device for producing a very regular bubble pattern. A small clamp rises and falls over the gas inlet line at regular intervals. With such a regular pattern it might be possible to have a time-controlled sensor which would only make measurements when fluid was passing through the system. Having explained the general design and principle of operation of Subject index P. 218

46


the flow system, I will now consider the choice of pump tubing. In the manual assay we mixed buffer, substrate, co-factor, and enzyme solutions in the proportions by volume of 2.0 :0.2:0.2 :0.2. We could try to find pump-tubing that had precisely these relative flow rates, i.e., one tube that delivered 2 ml/min and three tubes that deliver 0.2 ml/min. However, it is usually better to have flow rates in the various tubes nearer to each other than these ratios. This is because fluid injection from a slow stream into a fast stream is often imperfect and can lead to distortions in flow. Also we have to allow for the gas line and for loss of fluid at the de-bubbler. It is rather difficult to select an exact time of incubation. The reaction vessel has a fixed volume and with the standard pump one cannot vary the flow rate continuously. The retention time is determined by the ratio of vessel volume to flow rate (see Q 8.2.2) and hence cannot be varied continuously either. (This is possible, however, with a variable speed pump, as discussed in Q 9.2.1.) Fortunately, an important advantage of automated systems resulting from the high degree of reproducibility of the operations is that actual time of incubation is generally not critical, provided that it is reproducible. In our example, however, we will try to obtain an incubation time fairly near that used in the manual assay. To do this we may either use a total flow rate of about 2 ml/min with one coil in the heating bath, or about 4 ml/min with two coils. It is often difficult to choose between the alternatives of a slower flow with a smaller vessel and a faster flow with a larger vessel. In general, the smearing of samples is less if the length of tubing in the delay coils is kept as small as possible, but lower flow rates produce less eficient washing of joints and connections. The ideal system would have a fast flow rate and a small retention volume. However, this will reduce the amount of reaction occurring, and may lead to difficulties in measuring the reaction because of insensitivity of the sensor. In our case we will use the faster flow rate with two coils. Best bubble patterns are obtained with the gas line pumping 4 to 3 of the total fluid pumped, so that in our example a convenient ratio would be: air 1 ml/min; total liquid 3 ml/min. For convenience of design we will use lines with the following flow rates: buffer line 1.2


47

ml/min; substrate line 0.6 ml/min; co-factor line 0.6 ml/min; and enzyme line 0.6 ml/min. Having established these arbitrary line volumes we now work back to calculate what reagents may be connected to these lines to give a reaction mixture of the required final concentrations of each component. These reagents are conveniently called line reagents to distinguish them from the stock solutions from which they are prepared (see #9.2.5,9.3.5,9.3.6, and appendix IV). We now prepare a table similar to table 3.2 except that we now use line dilution factors (LDF) and fractional line volumes (FLV). (Note that for the purposes of calculation of reaction mixture concentrations, the gas lines are ignored. This is permissible since generally the gas does not enter into the composition of the reaction mixture. If it did, for example in C02/HC03- systems, due allowance would have to be made for this.) It is useful to specify line numbers by a subscript so that LDF, and FLV, are the values for the nth line. (A detailed treatment of the calculations is given in § 8.2.3.) Here, one may merely note that the final concentration of a component is equal to the product of its concentration in the line reagent and the fractional line volume of that line. Table 3.3. shows the arrangement of pump tubing for automation of our example. This system may be used for any assay in which buffer, substrate, co-factor, and enzyme are mixed and incubated for 5-10 min (i.e., with one or two coils in the heating bath). To prepare the appropriate line reagents all we have to do is multiply the required concentrations by the appropriate LDF values. For example, in an assay for malate dehydrogenase, the final reaction mixture consists of 0.05 M Tris/HCI, p H 7.6; 15 mM potassium oxaloacetate; 0.2 mM NADH; and 15 ,ug/ml of protein. The automated assay would have 0.125 M Tris/HCl in line 1,0.075 M potassium oxaloacetate in line 2, 1.0 mM NADH in line 4 and 75 pg/ml protein in line 5. More complex reaction mixtures are made by adding other reagents to the buffer or co-factor lines providing that the mixtures are compatible and stable. Thus it is more convenient to use a standard generalized assay system and alter the concentrations of the line reagents than to use a different pumping scheme for each assay. (A table of ratios of pumping rates Subject index p. 218

48

TABLE3.3

Reagents and pump tubing for automatioil of assay in table 3.2. (Flow system in fig. 3.1 used, with standard speed pump.)

Fractional line volume (FLV)

K phosphate Na L(+)-lactate Air KsFe(CN)e Lactate dehydrogenase

0.125 M 10.0 mM

1.2 0.6 1 .O 0.6 0.6

2.5 5.0

0.4 0.2

2.0 mM 0.25 mg/ml

Waste Total (excluding gas and waste lines)

2.0 3.0

-

-

5.0 5 .O

0.2 0.2

-

-

1 .O


Line dilution factor (LDF)

Buffer Substrate Gas Co-factor Enzyme

Final concentration in reaction mixture

Line flow rate (ml/min)

6

Initial concentration of line reagent

5

Component pumped by line

4

Description

Line no. 1 2 3


49

TABLE 3.4 Ratios of pumping rates with technicon tubing.* B/A B: 0.32 0.42 0.60 0.80 1.00 1.20 1.60 2.00 2.50 2.90 3.40 3.90 A ~~

0.32 0.42 0.60 0.80 1 .00 1.20

I .60 2.00 2.50 2.90 3.40 3.90

~

1.00 0.76 0.53 0.40 0.32 0.27 0.20 0.16 0.13 0.11 0.09 0.08

* See table

1.31

1.00 0.70 0.52 0.42 0.35 0.26 0.21 0.17 0.14 0.12 0.1 1

1.87 1.43 1.00 0.75 0.60 0.50 0.37 0.30 0.24 0.21 0.18 0.15

2.50 1.90 1.33 1.00 0.80 0.67 0.50 0.40 0.32 0.27 0.23 0.20

3.12 2.38 1.67 1.25 1.00 0.83 0.62 0.50 0.40

0.34 0.29 0.26

3.75 2.86 2.00 1.50 1.20 1.00 0.75 0.60 0.48 0.41 0.35 0.31

5.00 3.81 2.67 2.00 1.60 1.33 1.00 0.80 0.64

6.25 4.76 3.33 2.50 2.00 1.67 1.25 1.00 0.80 0.55 0.69 0.47 0.59 0.41 0.51

7.81 5.95 4.17 3.12 2.50 2.08 1.56 1.25 1.00 0.86 0.73 0.64

9.06 6.90 4.83 3.62 2.90 2.41 1.81 1.45 1.16 1.00 0.85 0.74

10.62 8.09 5.67 4.25 3.40 2.83 2.12 1.70 1.36 1.17 1.00 0.87

12.19 9.28 6.50 4.81 3.90 3.25 2.44 1.95 1.56 1.34 1.15 1.00

3.1 for colour code. Determine the required ratios of reagents e.g.

1.0:0.5:0.1:0.05. Select a convenient pumping rate for 1.0 and read-off the pump

tubing giving the nearest ratios. Thus if 3.90 ml/min, is chosen for 1.0, the nearest tubing to give the above ratios are 3.90 ml/min, 2.00 ml/min, 0.42 ml/min, and 0.32 ml/min. (Tubing of flow rate less than 0.3 ml/min is not included in table because of variation in observed flow rates.)

is presented in table 3.4.) There is usually nothing fundamental in the relative volumes of reagents that are used in manual assays, and they are often dictated by simple factors such as the availability of standard pipettes. It should be stressed that the above example, although dealing specifically with the Technicon system, is applicable to all automated systems, whether the reagents are mixed by proportioning pumps, individual pumps, automatic pipettes or other devices. It is still necessary to set the instrument in such a way as to deliver required volumes from different reagent bottles and unless the device exactly copies manual methods the above treatment is required.

Subiecr index P. 218

50


3.3. The sampling process The sampling process is fundamental to all automatic assay systems. I n the Technicon system the sampler module introduces components into the stream in a repetitive but discontinuous way. An examination of its characteristics is relevant to the problem of sampling in general. The sampler may be used simply to introduce different samples of the same enzyme into the analyzer (‘enzyme sampling’), to test a mixture of enzymes with different substrates (multiple-enzyme analysis) or to make various additions to the reaction mixture during an assay (e.g. addition of inhibitors or activators). Recently there has been a tendenC

t

J

0 N P

0

0

w

3 !J

0 N

4

d

Time-

Fig. 3.4. Analysis of enzyme samples. Four different preparations of yeast L(+)lactate dehydtogenase were placed in sample cups, using the flow system in fig. 3.1 with a sampler attached to line 5. The reaction was followed by fall in E420 on reduction of ferricyanide.


51

cy for manufacturers of automatic spectrophotometers to develop automatic sample changers to be used with their equipment (see appendix 111, p. 185). Usually they are designed simply to introduce the sample directly into the flow cell for measurement, and there is no provision for mixing reagents between sampling and reading in the sensor. However, such samplers could very easily be adapted for automated enzyme assay by using a suitable multi-channel pump and mixing chamber (see appendix 111, p. 189, for details of such pumps). The action of a simple enzyme sampler may be seen as follows. Line 5 in the manifold shown in fig. 3.1. is connected to a sampler operating at 40 cups/hr with a sample/wash ratio of 2: 1 (i.e., 1 min of sampling is followed by 30 sec of wash). Several enzymes placed in the cups of the sampler will then produce several peaks (fig. 3.4). Increasing the sampling speed increases the sharpness of the peaks, until eventually they do not reach the ‘steady-state’ level. There is an element of judgement as to what constitutes the best sampling rate. Although it is true that fast sampling can result in values less than the steady state and also in greater contamination between samples, these errors may be acceptable provided that they are reproducible. The absolute rate of sampling is an important factor in automation, and it may be better to accept a certain loss in precision in order to improve the rate. The theory of sampling is discussed in more detail in 0 8.2.7. During the assay, reagents pumped between samples, i.e., in the wash period, are wasted. A significant saving of expensive reagents, substrates or co-factors may be achieved if they are sampled in phase with the enzyme samples. Thus Hopkinson and Lewis (1967) developed an automated system for phosphoglucomutase in which a double sample probe was used, one for the enzyme sample, and one for the expensive substrate, glucose-1-phosphate. Discrete-sample analyzers (see 0 4.2) are really an extension of this approach, in that all the components of the reaction mixture are introduced into the reaction vessel in a discontinuous fashion, so that there is no flow of reagents between enzyme samples. The question of cost of reagents is not unimportant, particularly for large automated systems, and the use of multiple-sampling is certainly useful in reducing these costs. Subject index p . 218

52


The shape of the sample peak is an interesting index of the performance of the analytical system. In a perfect system with no smearing or cross-contamination, the peak would be a perfect square-topped curve. However, in practice the upswing and downswing parts of the curves are spread in a manner discussed in more detail in 0 8.2.7. If we simply introduce a marker substance (e.g. potassium ferricyanide) into the sampler we would obtain a typical curve of the shape shown in fig. 3.5a. If an enzyme is sampled, this would correspond to the rise and fall in enzyme concentration. However, unless the autoanalyzer is measuring the enzyme directly by some property (such as a characteristic absorption band) the actual record obtained will be that of enzyme actioity rather than absolute concentration. Since in general the activity should be proportional to concentration, the curve with the enzyme should have the same sample shape as that with the marker. However, this would not be so if there were deviations from linearity in the assay. For example, let us assume that the relationship between observed rate and concentration is as in fig. 3.5b. The actual rate observed on the analyzer with a sample of shape as in fig. 3.5a would be that shown in fig. 3 . 5 ~ .It can be seen that there is an apparent (a )

(b)

~

'0

10

.

n

1

Time (min)

2

(C

p 10

1

~

n 1 2 Enzyme concent r a t ion

OO

.I Time ( m i d

Fig. 3.5. Distortion of sampling peak due to lack of proportionality to enzyme concentration. (a) Typical sampling curve for rise and fall in enzyme concentration, assuming a half-wash time of 10 sec (see ch. 8). (b) Non-linear relation between observed activity andenzymeconcentration. (c) Sampling peakexpected with(b). (The units of enzyme activity and concentration are arbitrary, and all values are hypothetical.)

53


broadening of the curve, and this would result in less resolution between samples. Because the pen moves rapidly in the upswing and downswing of the sample peak, there have been few precise studies on the shape of the peak during enzyme sampling. However, much useful information may be derived from such a study. For example precise comparison of the traces with markers and enzyme catalyzed reactions, perhaps with faster recorders, or deliberately poor sample resolution, would give an automatic record of the relation between rate and enzyme concentration. The sampler I1 module has facilities for refrigerating the enzyme samples (Tappel 1965) and for stirring enzyme suspensions before sampling, so that such studies are quite feasible. In multiple-enzyme analysis, the substrate is placed on the sampler table, and aspirated into a continuous stream of enzyme. Again, close examination of the shape of the peak could well yield much information. Fig. 3.6 shows three curves as follows: (a) a typical curve with a marker, as in fig. 3.5a; (b) a hypothetical plot of substrate concentration against enzyme activity, using the usual MichaelisMenten relationship; and (c) the expected curve if the substrate is sampled over the range shown in (b). Again distortion of the curve occurs, and in this case precise comparison of (a) and (c) would

[k c

0

c

L c

n

oo

1

Time (min)

-2 0 0

8

16

Substrate concentration

Fig. 3.6. Distortion of sampling peak due to Michaelis effect. (a) Typical sampling peak for rise and fall in substrate Concentration, as in fig. 3.5a. (b) Hypothetical plot of enzyme activity against substrate concentration (Km = 4 arbitrary units, Vmsx = 10 arbitrary units). (c) Sampling peaks expected with (b). (Units for all scales arbitrary, and all values hypothetical.) Subject index P. 218

54


enable one to calculate curve (b) and hence the Michaelis constant and maximum velocity of the reaction. It is clear, therefore, that precise study of the shape of the sample peak could well yield fruitful results.

3.4. Interference by protein I will now discuss the problem of interference by protein in the enzyme assay. In general it is preferable not to remove protein, but to work under conditions in which the enzyme is sufficiently active for the concentration to be kept low during the assay. However, sometimes this is not possible. The Technicon system uses two modules, the dialyzer and the continuous$lter. Recently a discontinuous centrifuge for deproteinizing blood has been described by Quickfit & Quartz (see appendix 111, p. 181). In this the sample to be centrifuged is introduced into the drum centrifuge. After centrifuging a probe removes the supernatant fluid and the sediment is sucked off to waste so that the centrifuge is ready for the next sample. 3.4.1. Continuous dialysis The Technicon dialyzer (fig. 3.7) has a suitable membrane clamped between plates over which the protein flows. There are four streams, two input and two output. The donor stream passes material across the membrane to the acceptor stream so that there are input and output donor streams and input and output acceptor streams. (I have avoided the term ‘dialyzate’ as this is ambiguous.) For example, to dialyze a solution of serum albumin and glucose against phosphate output

Input

t-

Fig. 3.7. Principles and proposed nomenclature of Technicon dialyzer. The dialyzed material passes from the donor to the acceptor stream.


55

buffer in order to estimate glucose in the absence of protein, the input donor stream would contain the glucose-protein solution and the input acceptor stream phosphate buffer. Unfortunately, the dialyzer has a low efficiency, usually between 10 and 20%. Thus, although the output acceptor stream will be free of serum albumin, the output donor stream will still have a high glucose concentration, With 10% efficiency, the output acceptor stream will have about 1/10 of the glucose concentration of the output donor stream. This makes calculations of absolute enzyme rates with the dialyzer somewhat difficult. A disadvantage of the dialyzer is that it may alter its properties during the course of the assay. The efficiency of dialysis is affected, for example, by the relative ionic strengths of donor and acceptor streams. If these were to alter during the assay, for example by oxidation of an organic ion to C 0 2 and water, one may have different rates of dialysis as the reaction proceeds. For the dialyzer to work efficiently there must be good bubble patterns in the donor and acceptor streams. It is also preferable if the donor and acceptor streams have fluid segments that are roughly of the same length. Finally, the dialyzer has fine grooves in the dialyzer plate for the passage of fluid. If the enzyme solution is not perfectly free from debris, sediment accumulates particularly at the input nipples. This leads ultimately to complete ‘thrombosis’ with a dramatic bursting of the input leads from their nipples. 3.4.2. Continuousfiltration For the above reasons, the continuous filter is often preferable. Its design and method of operation are shown briefly in fig. 3.9. Fluid containing protein and low molecular weight material is passed into the mixing chamber where it is agitated with a stream of precipitant, such as 5% trichloroacetic acid. The mixture then drips onto a moving strip of filter paper and some of the filtrate (F) is returned to the analyzer. The filter differs from the dialyzer in that the concentration of low-molecular weight substances is the same on both sides of the filter. In calculation of enzyme rates, therefore (ch. 8), one has merely to allow for the dilution caused by the addition of the precipitating Subiecl index D. 218

56


1

1

Substrate Buffer Co-factor Enzyme Buffer G a5

Colorimeter Recorder

Fig. 3.8. Flow system for removal of protein by dialysis after enzyme sampling. After the reaction has taken place in the heating bath the reaction mixture is dialyzed against an air-segmented stream of buffer. The output acceptor stream (see fig. 3.7) is passed to the sensor. D: de-bubbler; FC: flow cell; HB: heating bath; JMC: jacketed mixing coil; S: sampler; W: waste. 7

JMC

MC

MC

Substrate Buffer Co- factor

Enzyme Mixer

Colorimeter

Trichloroacetic acid

Recorder

Fig. 3.9. Flow system for removal of protein by filtration after enzyme sampling. The reaction mixture is mixed with a de-proteinizhg agent and passed through the continuous filter to the sensor. (Symbols as in fig. 3.8.)


57

agent. The filter has an effect on the sharpness of the peak, and assays run with it generally have to operate at slower sampling rates. Heinicke et al. (1967) present a useful table of the properties of different filter papers and their efficacy in removing protein precipitates. Burns and Lazer (1965) used the filter to assay polynucleotide phosphorylase and Hopkinson and Lewis (1967) to assay phosphoglucomutase. A flow system for multiple phosphatase analysis using the continuous filter has recently been developed in our laboratory (Jowett and Roodyn 1969). To conclude this chapter, two analytical systems are given for the simple assay discussed at the beginning of the chapter, with enzyme sampling and removal of protein. In the first (fig. 3.8), the sampler is connected to the enzyme line, and enzyme samples are placed in the sampler cups. The effluent from the heating bath is passed into the input donor stream of the dialyzer. The input acceptor stream is 0.05 M phosphate buffer, and after dialysis the output donor stream is allowed to go to waste and the output acceptor stream is passed through the colorimeter, and thence to waste. The arrangement with the continuous filter is similar (fig. 3.9) except that the input acceptor stream contains 5% trichloroacetic acid. This chapter has not listed different detailed assays, but has tried to give a general picture of the processes involved in automating relatively standard enzyme assays. The adaptation of manual methods to automatic systems is not always as simple as the above account may indicate. For example, reagents may not behave the same way in the coils and plastic tubes of an autoanalyzer as they do in the test tube. Schwartz (1967) gives some examples of these problems. For example, in an assay for phosphohexose isomerase, veronal acetate buffer precipitated when acid was added and blocked the manifold. Use of Tris buffer instead solved the problem. In the manual assay the resorcinol formed was dissolved in ethanol. In the autoanalyzer this solvent boiled and the resulting vapour interrupted the flow and gave irregular traces. Substitution by ethylene glycol solved the problem, although it gave a lower colour yield. I have found that use of aminonaphthol sulphonic acid in phosphate determinations causes Subject index p . 218

58


blockage of the lines, which is avoided by using ascorbic acid as a reducing agent. One must also consider the possibility of surface denaturation of enzyme during the assay. It is also difficult to automate methods in which the enzyme preparation is very viscous, for example with bacterial lysates containing much DNA. It is therefore necessary to consider each case on its own merits, but in the light of the general principles indicated above. (A detailed example of an automated enzyme assay is given in 0 9.4.)

CHAPTER 4

Interrupted -flow and disCrete-sampling systems

4.1. Interrupted-flow systems We have seen in ch. 2 how the semi-automatic methods rely on manual loading of the autosensor. Interrupted-flow methods perform this automatically, i.e., the reaction mixture is prepared, mixed with enzyme and pumped into the sensor, usually the flow cell of a recording spectrophotometer. Once the reaction mixture is in the sensor, the flow of fluid is stopped and the course of the reaction is followed on a stationary reaction mixture. After a predetermined time, flow of fluid recommences, the sensor is washed free of old reaction mixture, new reaction mixture plus enzyme is introduced and the next assay is performed. The great advantage of such a system is that true initial rates may be measured. This is shown in fig. 4.1. In (a) four enzyme reactions are followed in sequence by filling the autosensor with the first reaction mixture plus enzyme, monitoring the reaction for 4 min, washing out the reaction mixture, filling with the next reaction mixture plus enzyme and so on until all four enzymes are measured. Thus a complete progress curve is obtained in each case for a period of 4 min each. In (b) the same reactions are followed in a continuous-flow system. Enzyme and reaction mixture are fed in continuously and the reaction is measured 1 min after enzyme was mixed with substrate. The four values obtained are given by the vertical bars and are seen to fall within the linear regions of all four progress curves in (a). However, if the continuous-flow system is arranged so that the reaction is measured 4 min after mixing, as in (c), only two enzymes (E, and E4) 59

Subject index P. 218

60

._ 73 c

(51

m

a,

LK


4

Fig. 4.1. Advantages of monitoring progress curve compared to single time-points assays. (a) Four enzymes assayed by interrupted-flow method. (b) The same enzymes assayed by a continuous-flow system with a single time of incubation and measurement of 1 min. (c) Continuous-flow system with single time of incubation and measurement of 4 min.

are measured in the linear region, assays for E, and E, giving low relative values, particularly for E,, Unfortunately, the advantage of being able to determine the initial rate is offset by the relative slowness of interrupted-flow methods if the reaction cannot be measured in a short incubation time. For example, if it takes 1 rnin to prepare the reaction mixture, and a reasonable reading is obtained after only 2 rnin of incubation, the total time for 50 samples is 2 hr 30 rnin using interrupted flow. With a continuousflow method that also requires 1 min to prepare the reaction mixture and has a 2 rnin retention time, the total time of assay for 50 samples is only 53 min (assuming a sampling rate of l/min). The time gained by the continuous-flow method is due to the fact that the analytical system is not halted during the assays. The gain in time with continuous-flow assay becomes much greater if the sample activity is not very high, or the method of detection not very sensitive. If 20 min are required to obtain a valid reading, the interrupted-flow method above

INTERRUPTED-FLOW AND DISCRETE-SAMPLING SYSTEMS

61

will take 17 hr 3 min for 50 samples. With continuous flow, 21 min will elapse before the first sample is measured, but thereafter samples will be obtained at the rate of one per min, so that the total time will be only 1 hr 11 min! The rate of analysis with the interrupted-flow method is improved somewhat if more than one sample can be read at a time (e.g. if a spectrophotometer with an automatic cell changer is used with 4 cuvettes) but in general the method can only operate a t a rate of analysis comparable to continuous-flow methods if the time of incubation is small. Where the actual rate of analysis is not a critical factor, clearly measurement of the entire progress curve on a stationary reaction mixture has great advantages. Continuous monitoring of enzyme activities is difficult to perform with interrupted-flow systems because of the intermittent flow of enzyme. As a simple example of the method, an interrupted-flow system using two pumps is shown in fig. 4.2. The reaction mixture is pumped into a temperature-controlled reaction vessel, the enzyme is added manually, and the mixture is then pumped out from the vessel through a continuous-flow colorimeter. A typical trace is shown in fig. 4.3. The

-

Pump 2

n

W -

II

Enzyme (add manually)

0.8ml/min

I

Pump1

n

vessel

Fig. 4.2. Semi-automatic interrupted-flowsystem. The assays are performed by the following sequence of operations: (1) Turn on pump 1 for 50 sec to fill jacketed reaction vessel with reaction mixture. (2) Turn on pump 2 to flush flow cell with reaction mixture for 20 sec. Turn on recorder. (3) Remove stopper of mixer vessel and rapidly blow in enzyme from a micropipette. (4) Follow reaction for 2 min. (5) Turn off recorder, wash out reaction vessel. Return to (1) for next assay. Subiecr index p . 218

62


I 0 [ L C U

4 4

h

4

0

OD

4

f N 0

LL'

1 rnin

-

Time b Fig. 4.3. Trace obtained with semi-automatic interrupted-flow system of fig. 4.2. RM: addition of reaction mixture for assay of L(+)-lactate dehydrogenase. (Reading corresponding to I pmole/ml calculated by calibrating recorder with G ,electron acceptor used in the assay.) Enzyme: standard solution of K ~ F ~ ( C N )the suspension of yeast mitochondria kindly prepared by Dr. L. A. Grivell. Numbers refer to pl of enzyme added.

system has the advantage of not using the colorimeter cuvette as a reaction vessel. It was, in fact, operated manually by following a sequence of times on a stop-watch. However, it is a relatively simple matter to connect the pumps to suitable timers which would control their operation. One of the difficulties of these systems is surging of the reaction mixture caused by suddenly stopping its flow. In particular, surge in the cuvettes can produce erratic readings. Pollard (1964) has described an interrupted-flow system in which a simple solenoid-operated clamp stops the flow into the cuvette. The most detailed work with interrupted-flow systems has come


63

from Pitot and his associates. Pitot and Preis (1964) described a system called a ‘combination unit’ that consisted of a Gilford 2000 multi-sample absorbance recorder (see appendix 111, p. 183), a Beckmann DU monochromator with thermostatted chamber, a Technicon large sampler and a Technicon proportioning pump. The system is described in detail by Pitot et al. (1965, 1966). The enzyme samples to be assayed are placed in the sampler table and various lines to the pump feed in buffer, co-factor, and substrate. According to a predetermined time schedule, the reaction mixtures are mixed with enzyme and pumped into the flow cells of the Gilford 2000 system. The pump and sampler are then stopped by relays controlled by a timing unit and the reaction is allowed to proceed in 4 cuvettes. After a suitable time, the pump and sampler are started again and new enzyme samples are introduced. The recorder trace obtained resembles that obtained by semi-automatic methods and the system allows inspection of the entire progress curve and selection of values from the linear region of the curve. It has been applied to the assay of many enzymes (see appendix I). Recently Pitot et al. (1968) have described a more complex system (fig. 4.4) with facilities for automatic data processing.

4.2. Discrete-sampling systems Recently, several automatic systems have appeared on the market which are intermediate between interrupted-flow and continuous-flow analyzers. One of the earliest of these was the ‘Robot Chemist’ and examples of its use in enzyme assay are given by Morgenstern et al. (1965c, 1966a, 1967), and Klein et al. (1966). Although the various discrete-sample analyzers differ greatly in the physical arrangement of their component parts and their overall complexity, they are all based on the following principle: A train of reaction vessels moves continuously through the incubation bath. The train may be linear or circular, with samples being transported either in linked chains of vessels, or more commonly on circular turn-tables. At various points in the train, automatic pipettes introduce reagents and enzyme into Subject index P. 218

64

AUTOMATED ENZYME ASSAYS 4

I

+

-

4

.

7

1

-

Upper level Lower level

1

2’

~

ervoir

Fig. 4.4. Manifold for multiple assays using four sample lines. Reagents plus enzyme are pumped from the reservoir and sampler on the right and mixed by ‘jet mixers’ which have a series of constrictions to produce turbulent flow. They are pumped into the flow cells of the spectrophotometer for monitoring of thereactions, and subsequently to waste. ‘Upper and lower levels’ refers to positions of lines in the proportioning pump. (From Pitot et al. (1968), by courtesy of the authors.)

the reaction vessels, with stirring usually effected by the swirling caused by injection of fluid. At a later point in the train, sample probes aspirate the reaction mixture into the sensor to measure the reaction. In some systems, the reaction vessel itself passes into the sensor and acts as cuvette. However, more often the sample probes are connected to a continuous-flow device in the sensor. After measuring the reaction, the reaction mixture is either passed to waste, or in some systems returned to the train for further processing. The volumes of reagent and enzyme are determined by settings on the automatic pipettes and it is usual to have pipettes in the 100 pl range for enzyme samples, and 1-5 ml range for reagents. The incubation time is determined by the

65


Reagents

- - - /

-

.

I

--I ,

-

- - --

Samples t r a i n

- - -

-

and /or printer

- -

I

- -

React ion t r a i PI Incubation vessel

Fig. 4.5. General principles of discrete-sampling analysis. A linear or circular train of enzyme sample cups moves past an automatic pipette PI which aspirates a suitable volume (A), moves its position (B), and delivers the enzyme sample (C) to a train of reaction vessels. After a suitable time of temperature equilibration, reagents are introduced by one or more automatic pipettes (Pz)and the reaction is started. A final probe (P3)transfers some of the reaction mixture into a continuousflow sensor. In some analyzers, the cups in the reaction train are optically standard and act as cuvettes of a colorimeter, so that no transfer a t P3 is necessary. Usually the sample and reaction trains are separate but sometimes they are on a single turn-table. The times between sampling, addition of reagents, and measurement of reaction are determined by the distance between probes and rate of movement of trains. (See appendix 111 for details of specific instruments.)

distance between the last pipette to produce the reaction mixture (usually the enzyme pipette) and the sensor, and of course by the rate of movement of the train. The principles of discrete sampling are shown in fig. 4.5. Usually, the discrete sampler gives a single time of incubation. However, the time course of the reaction could be followed in principle by placing sensor probes along the train, or by mixing enzyme and substrate rapidly just before the sensor probe and following the course of the reaction while the reaction mixture is in the sensor. It is still somewhat early to assess the merits of discrete-sample systems as compared to the older continuous-flow systems, particularly of Technicon. The various instruments vary greatly in design, and Subject index I . 218

66


some are not specifically intended for enzyme assays. In general the method has the theoretical advantage of greatly reducing, if not eliminating the problem of inter-sample contamination, provided that enough sample is used, so that higher sampling rates are possible (e.g. up to 300/hr). In addition, there is less danger of surface denaturation of enzymes since the surface over which the enzyme flows is reduced. It is rather surprising that very few authors have reported significant differences between results with the Technicon system and manual methods. This would indicate that the bubble pattern is not as great a source of surface denaturation as expected. However, there have been some reports of difficulties with the Technicon system. An extreme case was the report of Kirk (1967) that glutamotransferase appeared to be strongly and specifically adsorbed onto the glass surfaces of the autoanalyzer. However, discrete-sampling systems have some lengths of tubing through which the enzyme must flow, so that the problem may still exist there. The absence of the bubble pattern makes for easier monitoring of reaction mixtures, but the sensor probes must then be reasonably near the reaction vessel, thus making the instrument less flexible. This lack of flexibility probably is the major disadvantage of the systems so far produced. Apart from multi-channel systems, all parts of the train must be exposed to the same incubation conditions. There are some systems for transferring reagents from one turn-table to another, but the instrument is specifically engineered for this purpose. Some discrete samplers use disposable reaction vessels; others use a train of vessels that are washed in a ‘tube laundry’ of some sort. Here there is a danger that an occasional vessel may become contaminated. Since the blank assay would be carried out in another such vessel, it may be difficult to detect random contamination. Most of the discretesample analyzers do not give a continuous record of the progress of the reaction in the vessel, or indeed of the flow of reaction mixture into the sensor. Usually the sensor is connected to some digital output device and the value is printed out. This is a rather serious limitation in routine analytical work, since faulty loading of a tube, or even an empty tube, would give a spurious value that may not be obvious. A


67

half-empty sample cup in the autoanalyzer, however, would give a narrower peak than usual. The problem of drift and blanks is perhaps greater in the discrete-sample system. With continuous-flow systems it is possible to have continuous assessment of blanks and base line drift with the actual reagents and reaction vessel used, since all samples use the same stream; in discrete systems an error that occurred with one reaction vessel may not be repeated with another. From the point of view of instrument design and maintenance, discretesample systems have more moving parts than the continuous-flow ones and hence a greater risk of instrument failure. Such failures may be more difficult to detect than in the autoanalyzer. For example, in the assay described in 4 3.2 changes in the pumping rate of any of the lines is immediately detected by a change in the base-line at 420 nm. A discrete-sample analyzer giving digital values may not give such a clear and immediate indication. In spite of all these difficulties, there is little doubt that the discretesample analyzer will be of great value, particularly in the analysis of samples by routine and well established methods. Problems in calibration, detection of blank drift and occasional failure of components in the train are not insurmountable. Perhaps the greatest problem is the lack of flexibility, but again perhaps improved modular construction will be of help. Details of discrete-sampling systems are given in appendix 111.


CHAPTER 5

Single-enzyme analysis

In this chapter 1will discuss in more detail methods in which a single enzyme-catalyzed reaction is studied. Most of the examples will be with continuous-flow systems, but the arguments apply in many cases to all types of analyzer. The usual use of single-enzyme analysis (S.E.A.) is to measure the activity of a number of samples, for example in discrete serum samples, or in the effluent from a column. Another common use is to employ the reaction to estimate substrate, e.g., the determination of alcohol by alcohol dehydrogenase.

5.1. Molar conversionfactors and enzyme units The operation and calibration of enzyme analyzers is affected to some extent by the way activities are finally calculated. As mentioned in ch. 1, the most acceptable method is to express results as pmoles of substrate transformed per min and for specific activities as pmoles per min per mg protein. In order to do this, certain absolute measurements have to be made on the instrument, perhaps the most difficult of these being a precise measure of incubation time. However, it may be acceptable to use arbitrary units. For example, one may simply say that an instrument reading of 1.0 corresponds to one enzyme unit. For this unit to have any meaning, however, we would have to establish that theie was a linear relationship between instrument reading and enzyme concentration. This could be done by analyzing a range of enzyme concentrations introduced either manually or with a suitable 68

SINGLE-ENZYME ANALYSIS

69

gradient-making device (see $8 6.3 and 7.5). If the curve so obtained were non-linear, we would have to use it as a ‘calibration curve’ for each assay and calculate values by graphical or numerical interpolation. An improvement on the use of totally arbitrary units is to use so-called ‘standard’ enzymes which are commercially available and sometimes used in clinical enzymology. Since even the most stable enzymes may be inactivated under certain conditions, it is bad practice to assume that the value on the label is correct. If facilities are available, the standard should be checked either by the usual manual assay or by a semi-automatic method and a calibration curve constructed in terms of the true activity of the standard. In general the use of ‘standard’ enzymes is probably best avoided. Related to the question of standards is that of the units in which results should be expressed. In 1960 King and Campbell recommended the use of pmoles/ml as standard units, expressing these units in concentrations per ml or per litre. The subject of units in clinical enzyme assays is really in a sorry state! For example, King and Campbell converted various units to the suggested standard system of pmoles/min/litre. For alkaline phosphatase, 1 King-Armstrong unit/ 100 ml is equivalent to 7.1 standard units and 1 Bodansky unit/lOQ ml to 5.35 standard units; for amylase, 1 Somogyi unit/lOO ml is equivalent to 2.06 standard units. We thus have different units for different enzymes, and for the same enzyme estimated in different ways. Radin (1967) discusses in a general article the use of standards in enzyme assays and strongly supports the recommendations of the International Union of Biochemistry, i.e., to use pmoles of substrate transformed/ min. It would appear that in clinical chemistry the problem is still not fully resolved. Given that it is preferable to express results in absolute units, what is necessary to convert an instrument reading on an automated analyzer to such units? The first requirement is to know the relationship between instrument reading and pmoles of substrate or product. Although some enzyme reactions are based on the formation of rather illdefined coloured complexes, it is generally possible to define the reaction in rigorous chemical terms, by the correct use of standards. SubjecI index P. 218

70


Thus, although the precise chemical nature of the blue product formed after reduction of phosphomolybdate may not be known, the assay can be calibrated with known amounts of inorganic phosphate. The correct procedure is to calibrate the instrument with a known, stable and well characterized substance. In the case of spectrophotometric assays using standard flow cells of known optical path, published values of molar extinction coefficients may be used, although it is as well to ascertain that the instrument used does in fact give the published absorbance values. In the Technicon system it is difficult to calculate values from published molar extinction coefficients because of the difficulty in obtaining an accurate measure of the true path length of light in the tubular flow cell, which relies on internal reflectance to increase the effective light absorption. Even with more standard cells, however, the observed absorption will depend on the sharpness of the absorption band of the chromophore and the spectral resolution of the instrument. Thus, most instruments give similar values with phosphate standards using the reduction of phosphomolybdate as an assay because the coloured complex has a flat absorption spectrum in the blue region. However, measurements based on the CI band of cytochrome c at 550 nm require considerable accuracy in the wavelength calibration of the instrument and will vary with the width of the waveband of incident light. For these reasons, it is preferable to use the general factor relating readings to molarity which I call the molar conversion factor (MCF) ; this is defined as the concentration of solute in ,umoles/ml that corresponds to an instrument reading of 1.O. In spectrophotometric assays it is directly related to the molar extinction coefficient as discussed more fully in 0 8.2.1. However, it may be used for any assay in which response of the instrument can be related to concentration of a known substance. If the instrument gives a linear response, one simply calculates one MCF value and multiplies all readings by it to give the final concentration in pmoles/ml. This is done in the computer program in appendix 1V. If there is no linear or other predictable relationship between reading and concentration, a series of MCF values must be obtained over the range of assay, and the appropriate


71

value selected for a given reading by graphical methods or linear interpolation, preferably by computer. The calibration curve may be carried out by preparing a series of standard solutions manually and placing them in the sensor. However, if the instrument is fully calibrated (see Q 8.2.3) in terms of the flow rates in the various lines, the calibration curve may be performed automatically, using a gradientmaking system. In the actual enzyme assay, it is important to establish that the changes in instrument reading are due to changes in the concentration of the substance being measured and not to other effects. For example, if a mitochondria1 enzyme is being assayed, swelling of the mitochondria caused by addition of substrate may give a fall in absorbance that is unrelated to the transformation of that substrate. For this reason it is important to use correct blanks and to measure instrument zeros before and after the assay.

5.2. Determination of incubation time A more difficult problem than the calculation of MCF is the determination of the precise time of incubation. This does not arise in semiautomated methods or in interrupted-flow systems, since the progress curve is obtained automatically, and the only conversion necessary is from chart divisions to minutes or seconds. In the continuous-flow analyzer and probably in the discrete-sample analyzer certain problems occur. These are best illustrated by reference to the simple assay in Q 3.2. The enzyme is pumped through the enzyme line and meets the stream of reaction mixture on entering the jacketed mixing coil MC, in fig. 3.1. The reaction starts as soon as the enzyme and reaction mixture streams mix. Also, since in the flow system shown the reaction is not stopped at any particular time by a precipitant or filter, it will continue while the reaction mixture flows from the heating bath, where the bulk of the reaction occurs, to the colorimeter. We can measure the time taken for the reaction mixture to reach each point by using a short pulse of a suitable marker (e.g. strong ferricyanide solution for 2 sec). The time of passage through the jacketed mixing coil is 55 sec, through the two coils of the heating bath is 11 min 2 sec and from the Subjecl index P. 218

12


heating bath to the colorimeter is only 43 sec. Since this last value is relatively small, we can assume that there was negligible cooling of the reaction mixture in this time and we can add the time to the total time in the heating bath. The time of incubation in the mixing coil before the heating bath is very difficult to evaluate. This is because part of the measured time in this coil is spent in (a) raising the temperature of the enzyme to that of the jacketed coil, (b) mixing the enzyme and reaction mixture, and (c) increasing the enzyme concentration until it reaches steady state. By dipping the enzyme line into a standard solution of marker, we can measure the ‘enzyme input’ curve precisely, i.e., determine how the

Y

0

t

I

I

1

2

Dye enters r e a c t i o n vessel

I

I

3 4 Time (min)

I

5

I

6

t

Dye e n t e r s sensor

Fig. 5.1. Error in estimate of incubation time caused by approach of enzyme concentration to steady state. A flow system with 6 min retention time in the incubation vessel was calibrated by placing dye in the enzyme line. The plot of effective enzyme concentration against time is given. The system has a half-wash time of 15 sec (see ch. 8). The total activity is proportional to the product of enzyme concentration and time, i.e., the area under the curve. If one ignores the hatched area, i.e., if one assumes that the enzyme concentration reached steady state instantaneously, the error is 5.7%. If one deducts two half-wash times (30 sec) from the retention time, the error is only I .7 %. (A system virtually reaches steady state in four half-wash times - see 8 8.2.7.)


13

enzyme concentration rises in the coil. In an example of this (fig. 5.1) it can be seen that the enzyme has virtually reached its steady-state concentration in 90 sec. Assuming that the input curve is a straight line we could deduct half this time from the incubation time. It is difficult to measure the time for temperature equilibration in the coil. However, if the enzyme contributes only a small percent to the total volume of the reaction mixture, the effect is probably not great. With some effort, these factors could be measured precisely. The jacketed mixing coil as a whole only contributes a maximum of 7% to the rota1 incubation time, so if we arbitrarily assume that the effective incubation time in this coil is 50% of the measured delay time in it, our total error would not be very great. We may measure retention times by pumping markers through the systems and following their progress with a stop watch. A useful way of doing this is to introduce a marker into one of the lines for a very short time and to place a shutter between the light source and the flow cell momentarily as the marker passes the point being measured. This causes a vertical mark on the trace of the pen recorder, and when the marker ultimately reaches the colorirneter the sudden rise in absorbance clearly marks its entry into the flow cell. The distance between this mark and that caused by the shutter is measured, and the retention time calculated from the chart speed (see 5 9.3.3, and appendix IV, pp. 196,202). Another method is to determine the volume of the various parts of the system by filling them with fluid and pumping the contents into a measuring cyclinder. From the rate of pumping of fluid and gas we could then calculate retention times. Since the gas will expand in the heating bath the retention time would be affected by the temperature of the bath. The observed effect, however, is very small (fig. 5.2). The volume of the flow cell in the Technicon autoanalyzer (and indeed most well-designed autosensors) is small relative to the total volume of fluid-flowing, and retention time in it may be ignored for most purposes. However, this is not so with very slow flow rates (e.g. 1 ml/min or less). To conclude, with ‘average’ systems pumping 3-6 ml fluid per min and 1-2 coils in the heating bath, errors due to smearing of the enSubiecl index P. 218

74


0 N c

m

% m

60-

>

+ 0

s

40-

.-

.I-

I

40

,

50

I

60

Bath temperature

70

("C)

Fig. 5.2. Effect of bath temperature on retention time. The manifold consisted simply of a 2 ml/min fluid line pumping water and an 0.8 ml/min air line. The observed effect (solid circles) is greater than would be expected from simple expansion of the gas (open circles), possibly because of change in peripheral resistance caused by a fall in viscosity. Note that a change of 1 "C in bath temperature in the range 30-40 "C gives only a 0.13 % change in retention time. (See Barrera et al. (1969) for a similar curve.)

zyme front, mixing in pre-heating bath coils and flow through the sensor are probably less than 5%. They are not strictly errors, but rather reproducible and systematic delays in the flow system which result in an effective loss in incubation time. The position is that although the effective incubation time may not be known to an accuracy of greater than 5y0,its reproducibility is far greater than this. This situation is satisfactory for most circumstances. However, if a precise estimate of the incubation time is required the following procedure is necessary. The assay is first carried out manually or semi-automatically. If possible, the enzyme plus reaction mixture should be introduced into the sensor of the actual autoanalyzer so that the progress curve can be determined with the same instrument and reagents. The

SI NGLE-ENZY ME ANALYSIS

75

period of time over which the assay is linear is determined and the assay is then repeated on the autoanalyzer. From the plot of reading against time on the manual or semi-automatic method, the time corresponding to the reading obtained with the autoanalyzer may be calculated. Once this has been done with one enzyme with a given flow system, the value for incubation time so obtained may be used for all other assays. Another method is to perform the assay with no coils other than the pre-heating bath coil and to substract the value so obtained from the value obtained with the complete system. In this way the unknown effective time of incubation in the mixing coil is eliminated from the calculations. A progress curve obtained by running the same assay with 0, 1,2,3, and 4 coils (fig. 5.3) illustrates the method of correcting for any incubation that occurs before the heating bath.

Incubation time (min)

Fig. 5.3. Measurement of progress curve by varying number of heating bath coils. incubationwith no heatingbath(mixingcoi1only), 1,2, 3, and 4 coils in two heating baths in series. L(+)-lactate dehydrogenase of yeast measured by reduction of potassium ferricyanide.(From Roodyn 1969.)

5.3. Measurement of the progress curve This leads to the difficult problem of determining the full progress curve in a continuous-flow analyzer with a reasonably simple and Subject index P. 218

76


convenient flow system. One solution, which is rather expensive, is to use a multi-channel analyzer with different times of incubation in each channel. A simple dual-channel analyzer for this purpose is shown in fig. 5.4 and really consists of two manifolds of the basic design in fig. 3.1. Another design is to have one manifold with two colorimeters in series (fig. 5.5). By using a previously determined zero-time reaction we can obtain a three-point progress curve with such systems. (Note, however, that a sigmoid curve may also fit a three-point curve.) A 'family' of three-point progress curves is shown in fig. 5.6 where lactate dehydrogenase was studied at various temperatures. The method revealed that the assay was only linear below 40 "C,because of inactivation in the heating coils at the higher temperatures. Other workers have used dual-channel autoanalyzers for similar reasons. Schuel et al.

Fig. 5.4. Dual-channel multi-enzyme analyzer. The lower channel has one coil in the heating bath, the upper two. As a result the time of incubation in the lower channel is approximately half that in the upper. B: buffer; CI, CZ:colorimeters; D: de-bubbler; HB: heating baths; MI, Mz: mixers; MC: mixing coils; PI,Pz: proportioning pumps; S: sampler; R1, Rz: recorders; W: waste. (From Roodyn 1967a.)

77


COL 2

Fig. 5.5. Alternative-flow system to give two incubation times. After the reaction mixture has passed through the first heating bath (HB 1) the de-bubbled stream (D) passes through the first colorirneter (COL 1). The remainder of the gas-segmented stream passes through the next bath (HB 2) and colorirneter (COL 2). An enzyme gradient is generated by the two mixers (Mi, Mz) and a pump. G R : gradient tube; JMC: jacketed mixing coil; MC: mixing coil; S: sampler; W: waste; W1, W2: waste lines. (From Roodyn 1969.)

Temperature of assay ("C)

3 * Lu

03-

2

r 1.

._

Y 02-

!i

c

w

-

0

8 Incubation time (min)

12

Fig. 5.6. Examples of three-point progress curves obtained with a dual-channel analyzer. L(+)-lactate dehydrogenase assayed at different temperatures. Note that the reaction was not linear at 40 and 45 "C.(From Roodyn 1969.) Subject index P. 218

78


-

Pump 1

su B

1

G -co -E

Fig. 5.7. Four-channel enzyme analyzer. The enzyme, buffer, co-factor and substrate streams are all split into 4 channels with 0, &, 1 and 1& coils, respectively. The system requires 2 standard proportioning pumps, two heating baths, and a method of measuring four absorbance values (i.e., four colorirneters or a 4-cell continuous-flow sample changer in a spectrophotometer). The system is continuous flow and will give four-point progress curves (or five-point curves if one includes previously determined zero-time values). B : buffer; c1-4: colorimeters; CO: cofactor; E: enzyme; G: gas; HB: heating bath with size of &il; JMC: jacketed mixing coil; MC: mixing coil; SU:substrate; W: waste.

SINGLE-ENZYME AhALYSIS

79

(1964) used a dual-channel system for assay of cytochrome oxidase, with short incubation times, 20 sec in one channel and 120 sec in the other. Pitot et al. (1965) used a two-channel system in which the delay times in each channel were chosen from the linear region of the progress curve that had been determined previously by manual methods. The two streams passed through a dual colorimeter and the difference in reading between them was taken as a measure of the rate. More complex systems are possible using more sensors; fig. 5.7 shows a fourchannel system, combining the designs in figs. 5.4 and 5.5. Each channel has to be calibrated separately in terms of line dilution factors and molar conversion factors. Without computer processing the calculation of results in such systems can become burdensome. An alternative system that resembles the action of a discrete-sample analyzer is shown in fig. 5.8. The entire reaction mixture is placed in the sample cup and by using two 3.9 ml/min lines connected in parallel, the entire contents of the cup are pumped into the reaction vessel in

Fig. 5.8. Alternative method of operating the autoanalyzer for enzyme assays. Cups containing 1.6 ml of complete reaction mixture are loaded in the sampler, (Sl), and emptied by two wide pump tubes into a jacketed reaction vessel (R), stirred by a magnetic stirrer (M). At the same time another sampler (SZ) working in synchrony with SIis used to load 0.4 ml enzyme sample into the reaction mixture. The cups in S1 and SZ are emptied after 0.2 min pumping and no more fluid is pumped into R. The contents of R are emptied continuously through the flow cell (FC) of the colorimeter (COL) at a rate that gives a continuous reading for over 3 min. At the end of this time, R becomes empty and the next aliquots of enzyme and reaction mixture are pumped in from the samplers. (The number on each line indicates the flow rate in ml/rnin). Subjecl index p. 218

80


0.2 min. At the same time a small volume of enzyme is pumped into the vessel from a line controlled by the same sampler crook. The reaction vessel is emptied continuously through the flow cell at a rate which allows 3 min for incubation before the vessel runs dry. After another minute, the next sample and reaction mixture are pumped in. In this way, a continuous family of progress curves may be obtained without actually stopping the flow of the instrument, as in the interrupted-flow methods. However the method suffers from the same disadvantage of being a sequential assay which must have short incubation times for a reasonable overall rate of sampling. Several authors have described the use of the sampler I1 module of the Technicon system as the reaction vessel and the progress curve is followed as the cup is emptied. For example, Berry and Walli (1966) measured lactate dehydrogenase in serum by this method and determined the reaction rate from the sample peak, by selecting a region corresponding to steady-state enzyme concentration. A similar method has been employed by Brown and Ebner (1967).

5.4. Relation between enzyme activity and protein concentration A second important criterion for valid enzyme assay, perhaps more important than measurement of the progress curve, is that the rate should be measured under conditions in which it is proportional to enzyme concentration. In fact, although preferable, it is not essential to have linear progress curves in order to have proportionality to enzyme concentration. Thus, in fig. 5.9 some hypothetical reactions that are far from linear with respect to time are still proportional to enzyme concentration. Nevertheless, such proportionality is not always easy to achieve and, for technical reasons, is usually checked less often than the progress curve. In manual assays this check is usually carried out by repeating the assay at 2 or 3 different protein concentrations for each sample. Sometimes, authors establish the range of linearity at the beginning of a series of experiments and assume that all subsequent values with a single protein concentration that fall

81

SINGLE-EN,ZYME ANALYSIS

in this range are linear. With an automated assay system one can perform such calibration readily by placing the enzyme in a linear gradient-making system. In fact, it is easier to check proportionality to enzyme concentration on a routine basis than to follow the progress curve. (The use of gradient-making systems in this respect is discussed more fully in $9 6.3, 7.5, and 8.2.5; see also Roodyn and Maroudas (1968).)

5.5. Enzyme monitoring Having discussed some of the problems and principles involved in S.E.A. I would like to discuss its use in ‘enzyme monitoring’. At the

l

L

Fig. 8.8. Shape of sample peaks at different sampling speeds determined directly. Results obtained by sampling at rates of 70,60, 50,40,30and 20 per hour respectively, from left to right.

CALCULATION OF ENZYME ACTIVITIES

135

practice it is not necessary to reach complete steady state before taking the next sample, and most analytical systems accept some sort of compromise in order to have a reasonable sampling rate. For example, in fig. 8.8, sampling a t 70/hr would give acceptable results even if the peak is not quite at steady state. Errors due to fluctuations in sampling time become greater, however, the further one is from the steady state. Thus in the example in fig. 8.6, a 1 Eec error after 5 sec would change the sample peak from about 40% to 50% of steady state, i.e., a difference of about 20 %. However, a similar error after 40 sec would have no measurable effect on the size of the peak. Sampling error can result from faults in the mechanism of the sampler, but can also be due to variations in the depth of fluid in the sampler cup, particularly if the sampler probe enters the sample rather slowly. In general, therefore, it is better to work reasonably near steady state, although Reid and Wise (1967) have carried out several chemical assays on the autoanalyzer at sampling rates that gave peaks well removed from steady state. They claimed good reproducibility, but mentioned difficulties in wash-out between samples. The shape of the sample peak is directly related to the washing eficiency (WE) of the system. I will define this as the per cent return from steady state to zero concentration in the time interval between samples. The sample carry-over is (100- WE). Thus in our example above (fig. 8.6) the washing efficiency would be 65% for a sampling rate of 6/min, 90% for a rate of 3/min and 99.8% for a rate of l/min. The corresponding sample carry-over values would be 35, 10 and0.2%. Once the wash characteristics of the system are known, one can chose a convenient sampling rate, and also correct samples for crosscontamination. (The analysis of overlapping peaks is discussed generally in ‘Techniques in Amino Acid Analysis’ 1966.) In a train of samples, the reading obtained will be caused by the sample itself and the residual amount of previous sample(s). Thus for sample number SN giving a reading of RSN, the true reading RgN is given by (8.19) The possible error depends not only on the washing efficiency of the subject index P. 218

136


system but also on the difference in concentration between successive samples. For a given value of WE, one can determine the maximum ratio of readings of two successive samples that would give an acceptable error. For example, with a washing efficiency of 99% and a maximum acceptable error of 5%, the ratio R,,-,/RSN must not be greater than 4.75. Thus if this ratio were only 1.5 we would be well within our desired margin of error, and indeed could operate with a system giving only 97% rather than 99% washing efficiency. To summarize, the error in a reading due to contamination with a previous sample depends on the wash characteristicsof the system, the sampling rate and the ratio of concentration of subsequent samples. Thiers et al. (1966) have made an important study of the sampling process in continuous-flow systems. They made the interesting, and very useful, observation that the changes in concentration from one steady state to another obeyed first-order kinetics. Thus, when the sample probe is moved from sample to water the concentration of sample in solution falls exponentially, i.e., dC/dt = - kC,, where C, is the concentration at time t. If the sample probe is moved from water to a sample that would give a final steady state concentration of C,,, the ‘rise curve’ has the characteristics given by: dC/dt=k(C,,- CJ.

(8.20)

The rise and fall curves were followed experimentally and could be resolved into a lag phase and an exponential phase. The exponential phase could be defined by a term called ‘half-wash time’ or W, which is analogous to the half-life of a radio-isotope and is the time taken to change the concentration at a given time to a concentration half-way between it and the final steady-state concentration. Thiers et al. (1966) give examples of various analytical systems that have been analyzed in this way. For example, an alkaline phosphatase system had a lag of 8 sec and a W, of 21 sec. Their conclusions may be confirmed by fuIther analysis of the results given in fig. 8.6. The lag period was not measured, but if the value log,,(loO-~o steady state) is plotted against time, a straight line is obtained giving a W, of 5.8 sec. The ralue of W, would clearly depend on the flow rate, the faster the flow


137

Total f l u i d flow r a t e (rnl/rnin) Fig. 8.9. Effect of total fluid-flow rate on half-wash time (W*).System in ch. 9 used at different flow rates. Approach to steady state determined in each case as in fig. 8.6. and W* calculated as described in text.

the better the wash. This is confirmed by an experiment shown in fig. 8.9. Given a knowledge of W, we can calculate what time interval between samples is required to give a required washing efficiency. (One can also express the time in number of samples at various sampling speeds.) For example, if we only require a washing efficiency of 90% we can sample at 20 or 30/hr even with a system that has the very poor half wash time of 60 sec. However, if our analysis demands a washing efficiency of 99.9% (i.e., only0.1% carry over) we need a W , of 40 sec at the most to sample at 20/hr. If we sampled in the same system at 70/hr, we would need a W, of only 5 sec. In the above treatment the component in the sample peak may either be an enzyme (enzyme sampling), a substrate (multiple-enzyme analysis) or a co-factor (enzyme characterization). The shape of the peak will therefore reflect the relationship between enzyme activity (as measured by the response of the sensor) and enzyme, substrate or coSubiecr index p . 218

138


factor concentration. Examples of effects of these relationships on the shape of the peak have been given in 5 3.3 and it was pointed out that rigorous analysis of the shape of the sample peak may be a rapid way of obtaining useful information about the enzyme-catalyzed reaction. The shape of the peak is also determined by the recording characteristics of the sensor (e.g. maximum rate of pen movement in a pen recorder), and these matters are discussed by Jonnard (1960). Probably most autoanalyzer peaks are not amenable to precise analysis because the recorder used to follow the rise and fall curves had too slow a response. However, it is possible that future experiments with more rapid recording systems will exploit the possibilities of this approach. 8.2.8. Calculation of enzyme activity We now come to the final calculation of enzyme rates. If the reading is R and the blank B, the total reaction (TR) in pmoles/ml substrate transformed (see ch. 1, p. 19) is given by

TR =(R - B) AMCF.

(8.21)

The rate (RA) in pmoles/ml/min is given by RA=(R-B)*AMCF/IT,

(8.22)

where IT is the incubation time and AMCF the average molar conversion factor. The specific activity (SA) in pmoles/mg protein/min is given by SA =(R - B). AMCF/PCRM *IT,

(8.23)

where PCRM is the protein concentration in the reaction mixture. If the protein concentration in the enzyme line is PCEL and the line volume of the enzyme line is FLV,, SA =(R-B)*AMCF/PCEL*FLV;IT,

(8.24)

and if the enzyme line is pumped into a gradient-making system and the gradient dilution factor corresponding to the reading is GDF, SA =(R - B) * AMCF/PCEL. FLY, *IT* GDF.

(8.25)


139

These calculations assume that the reading R is on the linear portion of the progress curve. If this is not so, a series of values of R and IT must be determined and the true rate calculated either from the slope of a plot of T R against IT or as the intercept at IT =O on plots of RA or SA against IT. Space does not allow a full treatment of the further kinetic calculations that may be made with the results of such rate measurements. Cleland (1963, 1967) reviews statistical methods for analyzing kinetic data and discusses fitting such data not only to the conventional Michaelis-Menten formula, but also to more complex expressions. Thus, if the velocity is v, the maximum velocity V , the substrate concentration S and a, 6, c and d are constants, data may be fitted to any of the three following expressions

and

v = VS/(u+ s),

(8.26)

u = VSz/(d+2bS+ S’),

(8.27)

.

u = V(dS+ S2)/(c+bS+ P)

(8.28)

Cleland (1967) also gives a FORTRAN program for fitting such data by the method of least squares. The statistical treatment of data from enzyme rate measurements is also discussed in detail by Johanson and Lumry (1961). In the above treatment I have considered the various calibrations and calculations in a stepwise manner, and in practice it is useful to perform them in this way. However, it is possible in some cases to combine all relevant expressions given above to produce one rather complex general expression which may be used to calculate a single conversion factor. Multiplication of a given value of (R-B) by this factor would then give the final result immediately. However, this only applies to relatively simple systems with no gradients. Also, in much of the treatment above I have ignored corrections due to instrument zeros and various blanks. A completely comprehensive analysis would take all these into account. However, for most purposes the above treatment should be adequate, particularly if used in conjunction with the computer program in appendix IV. Subject index P. 218

CHAPTER 9

Generalized system for enzyme automation

The previous chapters have shown the variety of approaches that have been used to automate enzyme assays. In this chapter, I describe a generalized system based on the combined use of a Technicon autoanalyzer and the University of London ATLAS computer that is sufficiently simple and flexible to be of use in a large number of enzyme assays. In order to illustrate the principles of instrument calibration and calculation of data described in the previous chapter, I will describe the system in detail, giving a typical experiment as example.

9.1. Flow system The flow system is shown in fig. 9.1. Substrate, buffer, co-factor and enzyme are mixed in that order, and the mixing coil after addition of the enzyme is temperature-jacketed. Since most of the experiments performed with this system have been with M.E.A. the sampler module is connected to the substrate line. However, for analysis of enzyme samples it would be connected to the enzyme line. A sample gradientmaking system is incorporated in the manifold (lines GR and 4, and mixing vessels MI and M,) and is connected to the enzyme line (4). For substrate or co-factor gradients it should be connected to lines 1 and 3 respectively. A variable-speed proportioning pump is used and the speed is controlled by a dial with arbitrary divisions from 0-100. The volume of fluid pumped in the range 30-100 divisions is directly proportional to the number of divisions and the rate of compression 140

Water

Variable speed

P u m p

J MC

Buffer

P S -G

Gas Co-factor

3

Enzyme

Colorimeter

r--7 I I Recorder

7 7 I

I1

(Mixer 21

IMixer 1

I

Fig. 9.1. Flow system of generalized enzyme analyzer. The variable speed pump and gradient-making system allow for selection of different incubation times and protein concentrations (see text). The line volumes are given in table 9.2. D: de-bubbler; G: gas line; GR: gradient line; HB: heating bath; JMC: jacketed mixing coil; pH: pH meter with flow electrode; PS:pulse suppressor; S: sampler; and W: waste.

142


of the tubes by the rollers (fig. 9.2). The reaction mixture passes into a heating bath, usually operated with a single coil, and thence into a suitable autosensor, usually a colorimeter and pen recorder. It is useful to keep the physical arrangement of the system reasonably undisturbed for different assays and to change the assay conditions by altering the pumping rate, volume of fluid in the gradient mixing vessel (mixer l), composition of the line reagents, sample pattern, incubation temperature, and, with a colorimetric assay, the wavelength of the filters used. For example, although it is possible to change the pump tubing for different assays, it is more convenient to use a standard pumping system as far as possible and to adjust the concentration of reactants in different assays by varying the dilution of stock solutions. The computer program (appendix IV) is used to calculate these dilutions. One can develop ‘families’ of assay systems

-.-

6-

c

E

> E

5-

v

t

-30;

U

-102 .w 3 r

.w

e

030

4b

sb

7b

eb

0

50 90 108g Pump position ( a r b i t r a r y divisions)

Fig. 9.2. Volume of fluid pumped and rate of compressionof tubes at different pump positions. o : tube compressions/min. 0 : volume of fluid pumped. Flow system in fig. 9.1 used.

143

GENERALIZED SYSTEM

based on the invariant physical arrangement of the analyzer. Some of the instrument calibrations will apply to all assays (e.g., the absolute volume of the reaction vessel); others will vary for each assay (e.g., the molar conversion factor for the given reaction). However, even such calibrations may be stored for further assays. For example, calculations of retention times for a particular series of pump speeds may be stored in the computer and called upon where appropriate. In this way it becomes progressively easier to change from one assay to another or to develop new assays.

9.2. Properties of the system Some of the properties of this system will now be given. 9.2. I . Incubution time

With a fixed volume of reaction vessel, the retention time should be inversely proportional to the total flow rate of liquid plus gas (eq. (8.7)). Assuming that liquid and gas are always pumped in the same proportion, the retention time should also be inversely proportional to the total flow rate of liquid. This was found to be true over the range 20 r

-._ c

E

v

?._! c C

0 ._ c m

n

3 U

C

I

a

Pump position (arbitrary divisions)

m

Fig. 9.3. Effect of pumping speed on retention time. Flow system in fig. 9.1 used. Subjrcr index P. 218

144


tested (fig. 9.3), indicating that the pressure in the system does not build up excessively at high pumping rates. 9.2.2. Fractional line volumes The system was calibrated by marker solutions as described in 0 8.2.3, and the results for the different pump speeds are shown in fig. 9.4. The fractional line volumes were similar at all speeds, with only one line deviating slightly at the lowest speed. This means that the same line 51-

reagents may be used at all speeds to give the desired reaction mixture, so that the time of incubation may be varied in a continuous way simply by altering the speed of the pump. This is a very useful feature of the system, since it enables one to perform a preliminary assay at high speed in order to select a speed that will give readings in the desired range. 9.2.3. Sample interuction and wush characteristics In changing the pumping speed, however, one must not forget that the wash characteristics deteriorate at lower flow rates. This is shown in fig. 9.5 in which the rise curves of the sampling process are shown

145

GENERALIZED SYSTEM

f (4.1)

m

I

0.5 1 Time ( m i d

(5.5)

(4.7)

(6.1)

I

0.5

5-&-

Fig. 9.5. Rise curves at different pumping speeds. Flow system in fig. 9.1 used. The results are expressed as % of the steady-state value. The numbers in brackets refer to the total rate of fluid in ml/min.

for different pump speeds. It is, therefore, important to select a sampling rate that gives the desired resolution a t the pumping speed used. To find a convenient sampling rate for routine work, the carry-over from one sample to the next was measured at a variety of pumping and sampling rates. Between total fluid flow rates of 1.8 ml/min and 5.5 ml/min, it was found that sampling at 40/hr always gave less than 1% cross-contamination. 9.2.4. Gradient-making system In the manifold shown in fig. 9.1., the gradient-making system uses the variable speed pump. As a result, changing the pump speed will change the ratio : volume pumped into mixing vessel volume of fluid in mixing vessel Thus the steepness of the gradient will vary with the pump speed. Since the reaction mixture has the same composition at all pump speeds this provides a suitable way of selecting a range of gradient-dilution factors. Subiecr indexp. 218

146


The other way to vary the steepness of the gradient is to alter the initial volume of fluid in the mixing vessel. An experimentally determined family of curves of gradient-dilution factor against cup number with 25 ml in the mixing vessel, is given in fig. 9.6. (Similar families of curves have been obtained with a range of volumes in the mixer.)

Fig. 9.6. Plots of gradient dilution factor against cup number at different pumping speeds with 25 ml in mixer. Flow system in fig. 9.1. used, with a sampling rate of 40/hr. The numbers in brackets refer to the pump position. (See fig. 9.2 for the rate of fluid flow corresponding to these positions.)

147

GENERALIZED SYSTEM

We have said above that the incubation time may be varied by changing the pumping speed. This may be done if the enzyme concentration is constant throughout the assay, i.e., if the gradientmaking system is not used for the enzyme. However, if there is an enzyme gradient, changing the pump speed also changes the effective enzyme concentration in the assay, since this is directly related to the gradient-dilution factor. The total reaction observed is proportional to the product of the incubation time and the enzyme concentration. Since these vary in opposite directions, changing the pump speed does not have a dramatic effect on the instrument readings obtained. In fig. 9.7 are shown the effect of pumping rate on steepness of the gradient (curve A) and on the product of gradient dilution factor per cup and the incubation time (curve B). This curve is a measure of the total enzyme activity. If one wishes to vary the incubation time with0.06~

0.04U

\

0

c

U

m .+ c .-

-5 0.02._ -0 Q c .-

U

L Is1

a

0-

I

30

I

I

40 50 60 70 Pump position (arbitrary

80

I

I

90

100

divisions) Fig. 9.7. Effect of pumping speed on gradient dilution factor system in fig. 9.1 used with 50 ml in mixer 1. A gradient dilution factor/cup is the slope of a plot of gradient dilution factor against cup number (cf. fig. 9.6). (A) d gradient dilution factor/cup, at a sampling rate of 40/hr. (B) Product of A gradient dilution factor/cup and incubation time (proportional to the total enzyme activity). Flow rates at various pump positions are given in fig. 9.2. Subircf index P. 218

148


out changing the enzyme concentration one can either alter the volume of fluid in the gradient-mixing vessel (mixer 1) or, more conveniently, alter the dilution of enzyme in the ‘stock’ vessel (mixer 2). A curve to be used in order to select a suitable dilution of stock enzyme to achieve this is shown in fig. 9.8. Thus one can vary the incubation time, the protein concentration and the type of gradient used very easily with the above system.

‘30

40

50

60

70

80

90

100

Pump position (arbitrary divisions)

Fig.9.8. Method of obtaining constant protein concentration at all pump speeds. V is volume of enzyme to add per unit final volume of fluid in mixer 2 of fig 9.1. The value at pump position 100 is taken as 1.O. Flow rates at the various pump positions are given in fig. 9.2. Values of Vempirically calculated from calibration of gradients (figs. 9.6 and 9.7).

9.2.5. Preparation of reagents The calculations for this are performed by the computer. First of all it is decided which reagents are to be pumped in which lines. The concentrations of suitable stock solutions, the molecular weights of the solutes, where relevant, and the required final composition of the reaction mixture are entered into the computer with the line volume calibrations. Two tables are printed out, one in the form of a weighing sheet for the preparation of stock solutions and the other giving instructions for the preparation of each line reagent from the appropri-

GENERALIZED SYSTEM

149

ate stock solution. Since the pumping characteristics may vary from week to week, the latter table will give different dilution instructions after re-calibration of the instrument.

9.3. Computer program The full program and detailed input instructions are given in appendix IV. Here I will describe the program in a general way, showing how it is used in conjunction with the ‘hardware’ of the autoanalyzer system just described. The program is divided into sections (see appendix IV. 1 for instructions for moving from section to section). They are as follows : 9.3.1. Introduction

General comments of any length about the assay system may be entered here. It is convenient to prepare a detailed account of the system at this stage so that before each group of assays the operator may have precise instructions. At the moment, I have such information stored on cards. However, the most convenient arrangement would probably be to have it stored on magnetic tape in the computer, so that whenever a particular assay or type of assay is requested, full details may be provided. 9.3.2. Molar conversion-factor calculation

For spectrophotometric assays the name of the chromophore, the wavelength used, the optical path and a calibration curve with different concentrations of chromophore are entered here. The program calculates the molar conversion factor for each reading and determines the range of chromophore concentrations over which this factor does not vary by more than 5 % between readings. These values are averaged and used for all subsequent calculations. The maximum value within the linear range is stored and if any subsequent values exceed this a warning notice is printed. The section could be used for any instrument readings in which the concentration of solute is known. Subiecr indexp. 218

150


9.3.3. Incubation time and chart-speed determinations The instrument is calibrated for chart speed and incubation time as explained in the detailed example given below. These two values are printed out if so required. 9.3.4. Line-volume cahkdtions Details of the total line volume, the expected line volumes and the readings and blanks during calibration are entered here. The fractional line volume, line-dilution factor and observed flow rate are calculated for each line. If the observed value deviates from the expected value by f10% a warning notice is printed. The expected line volumes may either be the maker’s specifications, or more usefully, the values obtained in the previous line calibrations. In the latter case, the warning notice tells of any deviation in the functioning of the instrument from one assay to the next. 9.3.5. Line-reagent and reaction-mixture caIcuIations Details about the reagents pumped in each line, their final required concentrations and their concentrations in stock reagents are entered here. The computer prints out the dilution of stock solution required to give the correct final concentration in the reaction mixture, taking into account the observed line volumes calculated in the previous section. Thus, if one of the lines begins to pump poorly, the dilution of stock reagent would be adjusted so as to give the correct final concentration. 9.3.6. Stock soIut ions Molecular weights, required volumes and concentrations of each stock solution are entered here and a weighing sheet is printed showing the required weight for each solution with a space for entering the weight of the vessel. These weighing sheets may be accumulated and used repeatedly. 9.3.7. Gradient calibration The reading and blank with undiluted marker are enteredhere, followed by the gradient-calibration readings as the marker solution is pumped

GENERALIZED SYSTEM

151

into the gradient-making system. The gradient dilution factor is calculated for each reading and the results are printed as a table and a graph. 5.3.8. Assay conditions Comments about the assay, such as temperature and sampling rate are conveniently entered here. If one is preparing a detailed report or paper, it is convenient to enter textual material here, so that the final computer output will contain text, tables and figures. 5.3.9. Protein concentration Three possible situations are dealt with. In the first (‘CONSTANT’) no gradient-making system is used and the protein concentration is the same in all assays. This is used in simpler M.E.A. systems or during enzyme characterization studies. The protein concentration in the reaction mixture is then calculated from the protein concentration in the enzyme line and the fractional line volume of that line. In the second situation (‘VARIABLE’) the protein concentration in each sample is different, as in enzyme sample determination. In this case the protein concentration in the reaction is calculated for each sample from the initial protein concentration in the sample cup. (A system is under development in which the protein concentration will be determined by an on-line protein analyzer and the data froni this fed in at the same time as the enzyme results.) In the third situation (‘GRADIENT’) the protein concentration in the reaction mixture varies because of the action of the gradient-making system. This is the case in most recently developed M.E.A. systems. The protein concentration is then calculated from the initial protein concentration in the stock solution of enzyme dripping into the gradient-mixing vessel, and the gradient dilution factor for each reading calculated previously. The various protein concentrations are printed as tables, and where appropriate, as graphs of protein concentration against reading number. 5.3.10. Sample pattern Details of the samples are entered here including the concentration of Subjecr index n. 218

152


any components in the samples if known. Thus if the sample pattern consists of substrates for M.E.A., initial substrate concentrations are entered. The sample pattern is printed out with initial and final concentrations.

9.3.1 I . Results These are calculated for a variety of different situations. Allowance is made for the three types of protein concentration calculations mentioned in § 9.3.9. In addition, calculations are made in a slightly different way if the protein concentration is not entered. Not all assays use the sampler module and these also have a slightly different way of calculation and print-out. In all cases the results are calculated for each reading in terms of pmoles/ml of reaction mixture and pmoles/min. Where the protein concentration is known they are also calculated as pmoles/mg protein/min. All results are printed as a table and appropriate comments may be inserted in the legend. For each of the above situations, the instruction ‘GRAPH’ allows a plot of both pmoles/ml and pmoles/mg protein/min against reading number. Again, comments of any length may be inserted in the legend. The graphs are plotted as percent of maximum value in the given set of readings, and at the same time suitable scale divisions on the ordinate scale are inserted in absolute units. 9.3.12. Computer sub-routines The program given in appendix IV includes two sub-routines. The first (‘COMMENT’) allows one to print out any material of any length either to check the data before running on the computer, or simply for record purposes (‘LISTDATA’ entry). The second subroutine (‘PLOT’) will produce a graph of any series of values against reading number as described in the previous section.

9.4. Example of the use of the above system Details of a typical assay will now be given; it is a multiple-enzyme assay for three dehydrogenases, following the reduction of ferricyanide

GENERALIZED SYSTEM

153

at 420 nm. The reaction mixture required is 0.4 mM K,Fe(CN),, 0.05 M K phosphate p H 7.4, and any of the three substrates 2.0 mM Na L( +)-lactate, Na D(-)-lactate or Na succinate. The reaction takes place at 37 "C and the protein concentration should be in the range 0.1-2.0 mg/ml and the time of incubation should be about 5-10 min. The instrument is first thoroughly washed with 1 N NaOH, water, 1% Triton-X-100 and water, and the flow cell is disconnected from the heating bath. A series of solutions of increasing concentration of potassium ferricyanide are then pumped directly through the flow cell and the reading and blank recorded. The flow cell is then re-connected and marker (0.1 M potassium ferricyanide) is introduced into the enzyme line for about 5 sec, with all other lines in water. When the marker enters the jacketed mixing coil before the heating bath, a shutter is momentarily placed in front of the sample beam of light in

C h a r t divisions

Fig. 9.9. Method of calibrating gradients. 2 mM potassium ferricyanide is placed in the gradient-making system and water is placed in odd-numbered cups in the sampler. The even-numbered cups are left empty. The ferricyanide is diluted as the water is sampled and the gradient dilution curve is formed by joining the troughs (dashed line). Numbers on the trace refer to the cup number. (From Roodyn 1967b.) Subject indes P. 218

154


the colorimeter. This causes the pen recorder to mark a clear line on the chart. When the marker enters the flow cell a rapid upward trace on the recorder marks the end of the incubation. The distance between these lines is measured and the chart speed is determined by measuring the distance between two lines marked 5 min apart. (These measurements are usually repeated three times and the average values used.) The line volumes are then determined by placing each line in turn in 1 mM potassium ferricyanide for 5 min, followed by 5 min in water. The base-line and reading for each line is recorded and the total volume of fluid pumped by the analyzer is measured by collecting all the output from the waste lines for a known time in a measuring cylinder. (Mr. G. K. Jowett in our laboratory has recently suggested a simpler method of line calibration : Each line in turn is connected to a burette on the input side of the pump, and the time taken to empty a given volume into the pump is recorded.) The gradient-making system is calibrated by placing 25 ml distilled water in the gradient-making vessel (mixer 1) and 2 mM potassium ferricyanide in the stock vessel (mixer 2). Water is placed in all sample cups and the sample-wash line is disconnected. As soon as the ferricyanide starts to drip into the gradient-making vessel, the sampler is started from the wash position, at 40 samples/hr. An example of the resultant trace is shown in fig. 9.9. (For clarity, the calibration was performed with water in alternate cups.) The trough value for each cup is measured, as well as the initial and final blanks. When the gradient is completed, 2 mM ferricyanide is run directly into the enzyme line, and the reading and blank recorded. The above data about molar conversion factors, incubation times, chart speed, line volumes and gradient calibration are entered into the computer with details of the composition of the reaction mixture and the molecular weights of the solutes for the stock solutions. The output is shown as follows: molar conversion factors and incubation time in table 9.1, line volume calculations in table 9.2, stock solutions in table 9.3, line reagents in table 9.4, and gradient dilution factors in table 9.5. and fig. 9.10. From this information, the line reagents and sample table are prepared and a suitable dilution of enzyme is placed in the stock mixing vessel at 0 "C.The assay is then started by connect-

155

GENERALIZED SYSTEM

TABLE9.1 C

A

L

I

B

R

A

T

I

O

N

MOLAR C O N V E R S I O N F A C T O R C A L C U L A T I O N CHROHOPHORE CONCENTRATION IUMOLESIMLI

READING

BLANK

0.100 0.200 0.300

0.144

0.019

0.271 0.396 0.520 0.643 0.768 0.065 0.955

0.019 0 .019 . . 0.019 0.020

0.400 0,100 0.bOO

0.700 0.000

A R E A D I N G OF

MOLAR C O N V E R S I O N FACTORIUHOLESIML THAT G I V E A R E A D I N G O F 110

0.800 0.7Pl 0.796 0.798 0.803 0.802

0.020

0.020 0.021

1.0 IS E O U I V A L E N T I0

YITH b P I T Y L E N G T H O F 1 5 . 0 HM.

CHROHOPHORE C O N C E N T R A T I O N U P T O T H E CHROMOPHORE IS I( F E R R I C Y A N I D E

0.828 0.057

0 . 8 1 O U H O L E S / M L A T 420,O MU THE READINGS I R E PROPORTIONAL 0.800 UMOLES/ML.

TO

S T b U D A R O S O L U T I O N S PUMPEO O I R E C T L Y I N T O T H E F L O W C E L L OF T H E C O L O R I M E I E R . I N C U B A I I O Y T I M E AN0 CHART SPEEO C A L C U L A T I O N T H E C H A R T YAS R U N FOR 6 1 0 P I I N S . A N D M O V E 0 4.5 CMS. FOR I N C U B A T I O N T I M E MEASUREPINT Y E R E 4 . 0 CM. APART T H E C H A R T S P E E D Y A S 0.753 CHIWIN. AND THE INCUBATION

MARKS

TM IE

5 , s HIN.

V A R I A B L E S P E E D PUMP S E T A T P O S I T I O N 7 0

TABLE 9.2

L I N E VOLUME C A L C U L A T I O N LINE NUMBER

1

**.

**.

LINE COLOUR YYITE

YbRNlNG L I N E 2 GREEN 3 YHITE YARNING LINE 4 YELLOY

CALIBRbTIONREADING

BLANK

0.193

0.029

FRACTIONAL LINE VOLUME

0.113

LINE

D I L U T I ON FACTOR

...

d151

1 H A 5 MORE T H A N 1 0 P E R C E N T P U I l P l N G ERROR 0.4P3 0.029 0.153 2.51 0.189 0.029 0.149 6.70

3 HAS MORE T H A N 1 0 P E R C E N T L U M P I N G ERROR *a. 0.313 0.029 0.265 3.77

OBSERVED LINE VOLUfIE I*L/MINl

EXPECTED LINE VOLUPIE IMLIMINI

0.b2

0.55

1.76

1.84

0,)s

0.bL

i.08

O B S E R V E D TOTAL L I N E VOLUME I S 4.06 M L I H I N . E X P E C T E D T O T A L L I N E VOLUME O B S E R V E D V A L U E IS 1 0 0 . 2 P E R CENT OF E X P E C T E D

1.11

IS

LINE ACcUnAcv I P E R CENT OF T Y E O R V I

112.9

95.5

110.2

96.9

4.05MLIMIN.

T H E O R E T I C A L V A L U E S C A L C U L A T E D FROM H I K E R ' S S P E C I F I C A T I O N S , P U M P S P E E D O F S T A N D A R D T E C H N I C O N PUMP A N 0 O B S E I I V E O SPEEO O F V A R I A B L E S P E E O PUMP. I T Y E P U M P I N G ERROR I S P R O B A B L Y B E C A U S E T H E PUMP IS N O T R U N AT I H E S T A N D A R D I.CC" =-EL".

I H E M A N I F O L D U S E D Y A S AS F O L L O Y S L I N E NO.

COLOUR

COMPONENT I N L I N E

01 02

YHlTE S U E S T R A T E S t I N S I M P L E R I I MODULE GREEN BUFFER 0 GAS BLUE 03 YHlTE CO-FACTOR 04 'IELLOY ENZ'IME,FROM O R A D l e N T M A K I N P SYSTEM. YHlTE OR O R I D I E N T L I N E n T O G R A D I E N T M A K l N O SVSTEM. Y PURPLE BLACK YASTE L I N E T H E SAMPLER WAS O P E R A T E D A T I 0 S A L P L E S P E R H O U R , Y I T H WATER I N THE MASH V E S S E L . T H E H E A T I N G BATH Y A 5 A T 37 c.


156


TABLE9.3 PREPARATlON OF SOLUllONS COMPONENT OF STOCK SOLUllON

MOLECULAR Y E I O H T OF COMPONENT

W fERRlCVANlOE

CONCENTRATION OF COMPONENT I N STOCK SOLUTION

0.010

329.3

UNITS OF CONCN.

VOLUME Or STOCU S O L U l I O N REPUlREO IML.)

MOLAR

2001000

YEIOYT OF CONPONENT (OM)

YEIOHT Of VESSEL YEIOHT OF COMPOhCNT

_-__--_._ 0.65860

SUM

L l r ) L A C l A l E - C I SALT

296.2

0.050

2501000

MOLAR

Y E l O H l OF VESSEL YEIQHT OF COMPONENT SUM

Dl-)LACTATE-CA

SALT

290.2

0.050

MOLAR

210,000

_____ -__3.70210

YElOHT OF VESSEL Y E l Q H l Of COMPONENT

3.62750

SUM YElQHT Of

0.050

118.1

SUCCINIC ACID

250,100

MOLAR

VESSEL

YEIOHT OF COMPONINT

1.4?625 _____-___

SUM

1Sb.l

KH2POI

1.000

5000.000

MOLAR

WEIPHT O f VESSEL YEIOHT Of COMPONENI

-------.6110.50000

SUM

E l LACTATES CONVERTED TO NA S A L T S BY TREATMENT Y l T H NASPO4. SUCClNlC ACID NEUTRALlZED Y I T H NAOY. KH2PO4 ADJUSTED T O PH 7.4 Y l l H KOH.

TABLE 9.4 L I N E REAQENT AND REACTION MIXTURE C A L C U L A l t O N LINE NUMBER

COMPONKNT I N REACT.IDN MIXTURE

1 NA Ll.) LAClAlE I NA D ( - 1 LACTATE 1

2 3

NA SUCCINITE K PHOSPHATE PW 7,4 KIFECN6

CONCENTRATION OF COMPONENT I N 1l)LINE i2)REACTION 131STOCK MIXTURE SL)LUTION 13.07S 2.000 10.000 2.000 50.000 13.073

REAOENT 15.073 0.116 2.610

z.000 0,050

0.400

1o.ooo

1.000 10.000

UNITS

OF

CONCN.

MlLLIM MILLIM MlLLIM MOLAR

MILLIM

TABLE 9.5 ORADIENT CALIBRATION READIN0 NUMBER

REIDlNQ

TO PREPARE L I N E REAOENT MAKE I A I M L . OF STOCK UPTO IA) (0)

QRADlEHT DILUTION FACTOR

65.37 65.31 65.37

577.59 134.00

250.00 250.00 250.00

5000.00 ¶OO.OO

151

GENERALIZED SYSTEM

.

0

-It c

e a t

.

. .

D

.

mOt

.

D

.

C t

O

O

. .

t

.

O

.

In.

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

..

. . ... .. ... .. . ... .. -. ... . ... .. . .... . ... .. ... .. . .... ... . ...

C

.

v +

L D

C

n

" W

C O

a Y a

o

z . i

O

.

*)+

O

.

N t

O

.

d l

O t

.

z D

.

i

a

...

...

.. .. ... ... .. ... . ... . ... . .. .. ... ... ... ... .. .. .. ... ... ... .. .. .. ... ... .. .. .. .. ... ..- ... . . . ... ... ... .. .. .. ... ... ... . . . ... ... ... .. .. .. ... ... ... ... ... ... . . . ... ... ... ... ... .. ._. ... .. *. .. .. .. .. . . . .... .... ... . . .. .... .... ... . . .. ... ... ... . . . ... ... ... . . . ... ... ... .. .. .. .. .. .. ... ... ... .. .. ..

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

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

t

... ... ... . . . ... ... ... . . . ... ... ... . . . .. .. .. ... ... ... . . . .. .. .. ... ... ... .. .. .. .. .. .. .. .. .. ... ... ... .. .. .. . . . .. .. ... .. .. .. .. .. .. _ .. ... ... ... .. .. .. .. .. .. ... ... ... .. .. ... ... .. .. ... ... . . . ..... ..... ..... . . .

.

.. . ... .. ... ... ... .. ... ... .. .. . ... .. .._ . ... ... ... . ... .

.

-

. ... .. .

.. .

... .. ...

.. .. .. .. .. . .. .. .. ... .. .. .. .. .. .. .. ... ... ... .. .. .. . . . .. . . .. .. .. .. ... ... . . . . .~. . .. .. .. ... .. .. . . . .. .. ... ... ... .. .. .. .. .. .. .. .. .. .. ... ... ... . .. .. .. ... ... ... .... .. .. .. .. .. .. . . . . . . . . . . . .. .. .. .. .. ... ... ... ... ... ... ... *. .. .. .. . . . . . . ... ... ... ... ... ... ... .. . . . . . .. .. .. .. .. ... ... -... ... ... ... ... ... ... ... ... .. .. .. .. _ . .. . . . ... ... ... ... ... .._ .. . ... ... ... ... ... .. .. ._ . .. .. .. . ... ... ... ... ... ... ... ... ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . .. .. .. .. .. .. .. . ... ... ... ... ... ... ... .... . . . . . . . .. .. .. .. .. .. .. ...

.. . ... .. .

.. . ...

...

f

. ...

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

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

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

. ... ... .

.

. ... ... .

.

. ... ... .

. ... ... .

. . . .

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

u s o r .

E

C

D .

O i

r r

t (

-

N *

.. +

. ..... . - . ... . . . ...

...

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

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

...

n

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

D r

(

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

O r

O l

.. .. .

. .

. .

. ... * . .. .. .-. * ..

.. ... . ...

.

. ... .. ...

.

. . .._ .. .. .. ... ..

O C E - 4 . 4

d P

..

..

..

.

. *

. ... ... . . * ... .. .. . t .. ... . .. . t ..

.. .. ... .... + ... .-.. * ....

... ... ... . . . . . . .. ... ... ... ... . . . . _ .. .. . .. _.. .. .. .. .. .. .. .. .. .. ... ... ... . . . . . . . f

L

+

..

O

.

D

.

0

m

-

.

0

.

Z

.

O

z

0

I

. . .

>

m

.

.

m -

-

. .

0

U

.

w m

. . ..

I

. -

> m Y

.

Z

-

x

+

... .. N O

Fig . 9.10. Plot of gradient dilution factor against reading number (computer output).

Subjecr index p . 218

158


ing the appropriate reagents to the various lines, checking that the base-line is steady and starting the sampler as soon as the stock enzyme starts to drip into 25 ml of diluent (0.3 M mannitol) in the gradient-mixing vessel. The readings are taken from the steady-state values with each peak. The base-line rises during the assay because of absorption at 420 nm caused by the steadily rising enzyme concentration. If this rise appears to be linear, it is possible to use only initial and final blank values, using the computer to calculate the blanks for each sample by linear interpolation. However, it is more precise to measure the true blanks by drawing a line connecting all the blank samples in the assay and dropping a perpendicular line from the steady state portion of each peak to this base-line in order to calculate the true blank for each reading. The method of doing this is shown diagrammatically in fig. 9.1 l . The following information is then entered into the computer: the initial protein concentration in the stock mixing vessel, the nature and initial concentration of substrates in the sampler cups, the readings and blanks for each cup, and comments on the experiment where appropriate. The output is as follows: protein concentrations for each reading (table 9.6 and fig. 9.12), details of the sample pattern and substrate concentrations in the reaction mixture (table 9.7), the actual results of the enzyme assays (table 9.8), and graphs of enzyme activity in pmoles/ ml and pmoles/mg protein/min against reading number (figs. 9.13 and 9.14). This example has been given in detail in order to show the flexibility of the system, and also the wealth of data from instrument calibrations and readings that may be handled by the computer. The close interaction between computer program, autoanalyzer and human operator has its difficulties. In the above system, the data were punched manually on IBM cards and batch-processed on a large central computer. This lead to delays that were particularly annoying when an error had been made in input. Ideally, the analyzer should be on-line to a large computer capable of handling a program of the size shown in appendix IV. However, the recent expansion in small, relatively cheap, on-line computers may provide an alternative, more practical scheme. In

GENERALIZED SYSTEM

159

Fig. 9.11. Method of calculating blanks. (a) With blanks that increase or decrease linearly, linear interpolation is carried out, using the initial (I) and final (F) blank values. (b) With a non-linear change in the blank, blank values are obtained for each peak by dropping a vertical line (arrows) to the line connecting the various blanks interposed between the readings (dotted line). B : blank; R: reading. (The traces are hypothetical.) TABLE9.6 P R O T E I N CONCENTRATlPNS T H E E N Z Y M E H l S e N Z Y M E SAMPLE 1 6

UITH I N I T I A L P R O ~ E I NC O N C E N l R A T l O N OF

1.050MG1ML

P R O T E I N CONCENTRATION I N REACTION M I X T U R E DURING GRADIENT REIDING NUMBER

GRADIENT O I L U l I O N FACTOR 0.062 0.098

0.136

P R O T E I N CONCN. I N REACTION MIXTURE 0.017 0.027 0.030

6 7

0.173 0.209 0.243 0.278

0.040 0.050 0.068 0.077

0

0.316

o.ow

0.345 0.379 0.414 0.446 0.403 0.5011 0.542 0.573 0,604 0.635 0.660 0.702

0.096 0,106 0.115 0.124 0.134

1

2

3 4 5

9

10 11 12

13 14

15 16 17 10 19 20

O.l*l

0,151 0.159 0.160 0.177

0.104 0.195

Subjecr index P. 118

160


TABLE 9.7 SAMPLE PATTERN SAMPLE NUMBER

1 2 3 4 5 6 7

1 9 10

11

SAMPLE COMPONENT NUMBER

1 2

3 4

1 2

3 4

1 2

3

12 13 14

4

15 16 17

3 4

10 19 20

1 2

1 2

3 4

NA LI.) LACTATE NA 01-) L A C T A T E NA SUCCINATE YATER NA L l * I LACTATE NA 0 1 - ) L A C T A T E NA S U C C I N A T E YATER N A LI.1 LACTATE NA 0 1 - 1 L A C T A T E NA S U C C I N A T E HATER NA L l f ) L A C T A T E NA 0 1 - 1 L A C T A T E NA S U C C I N A T E YATER NA LI.) LACTATE NA 01.) L A C T A T E NA S U C C J N A T E HATER

MET1100 FOR L'OADINO S A M P L E T A B L E WBSTRAiE

C O N C E N T R A T J O N OF S A M P L E COMPONENT I N

S A M P L E COMPONENT

LINE

REACTION

13.100 13.100 13.100 0.000

2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000 2.004 2.004 2.00* 0.00.0 2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000

13.100

13.100 13.100 0.000 13.100 13.100 13.100 0.000 13.100 13.100

13.100

0.000 13.100

13.100

13.100

0.000

UNITS OF CONCN.

MIXTURE

MiLLin MILLIR MILLIR MlLLlR MILLIN MILLIU MlLLlI MILLIM MILLII MILLIM MILLIM MILLII MILLIM MILLII MlLLlM

-

CUPS

0 1 05 0 9 1 3 1 7 2 1 NA L l * I LACTATE N A DI-I L A C T A T E 0 2 06 1 0 1 4 1 0 2 2 NA SUCCJNATE 03 0 7 11 1 5 1 9 2 3 YATER 0 4 08 1 2 16 20 2 4 STOPS AT CUPS 2 0 AND 40 THIS A L L O Y S FOR THO ASSAYS P E R P L A T E

25 26 27 28

2 9 33 37 30 34 31 3 1 35 3 9 5 2 36 4 0

TABLE 9.8 PATE 1 5 / 5 / 6 9 IXPT. 01 RESULTS

OF E N Z Y M E ASSAYS

l E A D l N Q SAMPLE S A M P L E COMPONENT NUMBER COMPONENT NUMBIR

f

1

2. 3

2 3

4 3

4 1

6 7

2 3

a

6 ta

.

1

2

11

3

13

4 L 2

12

".,En

ii'Ci.1

J

LACTATE NA 01-) L A C T A T E NA SUCCINATE HATER NA L l t l L A C T A T E NA Dl-I L A C T A T E NA SUCCINATE

4

HATER

14 15 16 17

3 4

18

2

19 20

NA L l r ) LACTATE NA 01-1 L A C T A T E NA S U C C I N A T E YATER NA L l t I L A C T A T E NA DI-I L A C T A T E NA SUCCINATE YATEll NA L l r ) LACTATE NA D l - ) LACTATE HA S U C C I N A T E

1

CONCN, OF UNITS SAMPLE OF COMPONENT CONCN. I N REACTION MIXTURE 2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000 2.004 2.004 2.004 0.000

MlLLlM MILLIM MILLIM

READING BLANK

01461

0.479

ENZYME A C T I V I T Y UMOLESlML UMOLESIML UMOLEIIMP. IMIN PROTElN/MlN

-0.OlY

-0.003 -0.009

-

-0.001 -0.000

-

-0.010 -0.025 -0.002 -0.000

MILLIM MILLIM MlLLIM

-0.016

MILL111 MILLIH MILLIM

-0.034 -0.002 0.000 -0.021 -0.044 -0.002 -0.000 -0.027 -0.056 -0.002

-

MlLLlM MILLIM MILLIM

-

MILL111 MILLIM MlLLIM

-

T A E L E 95 ENZYME PATTERN I N D L U C O S l REPRESSED CELLS. ( V A L U E S I R E N E O A T I V E V E C A U S E THE D I S A P P E A R A N C E OF K F E R l C V A N J D E W E A S U R E U I N THE A S S A Y . I

0.000

IS

-0.07

-0.33b

-0.020 -0.009 -0.168 -0.340 -0.019 -0.00Y -0.168 -0.325

-0.011

0.002 -0.158 -0.314 -0.016

-o.ooa

-0.16L -0.316 -0.008 0.000

161

GENERALIZED SYSTEM

.. ... ..

... ... ... ... ... .. . .. ... ... ... ... ... ... ... ... ... ... ... ... . + . . . . . . . . . . . . . . . . . . . ... ... ... ... ... ... .. ... ... ... ... ... ... ... ... ... ... ... ... . .. .. .. .. .. . . . . . .. .. .. .. .. .. .. .. .. . . . . . . . .: .: .: .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. , .. .. .. ... ... .. . .. . .. ... ... ... ... ... ... ... ... ... ... ... ... . ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . .. .. .. .. .. .. .. . , .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . ... ... ... ... ... .. . .. ... ... ... ... ... ... ... ... ... ... .. ... ... . . .. .. .. .. .. .. . . . . . .. .. .. .. .. .. .. .. .. .. .. . ... ... ... ... ... ... ... . .. . ... ... ... ... ... ... ... ... ... ... ... .. . . . . . . . . . . . . . . . . . . . . . ... ... ... ... ... ..... ... ... .. . ... ... ... . ... ... ... ... ... ... ... . .. .. .. .. .. .. . . . . . .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . .. . .. .- .> .: . :. :. :. :. :. : : : : : : : : : .. .. .. .. .. .. . . . .. .. .. .. ... ... ... ... ... ... ...... . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . . . . . . . . . . . . . . . . . . . n . . . . . . . . . . . . . . . . . . . . . . ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . z . .: . : . : . : . : . :. :. :. :. :. :. .: .: .: .: .: .: .: . : : . .. ... ... ... ... ... ... ... . .. . ... ... ... ... ... ... ... ... ... ... ... .. 0 ,-$* D

-

0

P ,

0

I

0

I

O I

I..

p. u m

x

2 3

" z

.

I

0

w

p.

I-

-"

Y)

z

Automated Enzyme Assays

Enzyme Assays: A Practical Approach (The Practical Approach Series, 257)

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Enzyme Assays: A Practical Approach (The Practical Approach Series, 257)

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Stem Cell Assays

Cell Biology Assays: Proteins

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Miracle of Enzyme Indonesian

Enzyme catalysis in organic synthesis

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