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ADVANCES IN

Applied Microbiology VOLUME 36

This Page Intentionally Left Blank

ADVANCES IN

Applied Microbiology Edited by

SAUL L. NEIDLEMAN Vacaville, California

ALLEN I. LASKIN Somerset, New Jersey

VOLUME 36

Academic Press, Inc. Horcourt Brace Jovanovich, Publishers

San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @ Copyright 0 1991 BY ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. San Diego, California 92101 United Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road, London NWI 7DX

Library of Congress Catalog Card Number:

ISBN 0-12-002636-8 (alk. paper)

PRINTED IN THE UNITED STATES OF AMERICA 91929394

9 8 7 6 5 4 3 2 1

59- 13823

CONTENTS Microbial Transformations of Herbicides and Pesticides

DOUGLAS J . CORKAND JAMES P . KRUEGER I. Introduction ....................................................... I1. History of Microbial Conversions..................................... I11 Entry and Movement of Herbicides in the Environment ................ IV Taxonomy of Degradative Organisms ................................. V. Kinetics of Biodegradation by Microorganisms ........................ VI . Factors Affecting Biodegradation Kinetics ............................. VII . Biochemical Mechanisms of Aerobic Chloroaromatic Metabolism ....... VIII . Cometabolism ...................................................... IX Biochemical Mechanisms of Anaerobic Aromatic Metabolism ........... X. Molecular Biology of Degradative Microorganisms ..................... XI . Molecular Biology of Chloroaromatic Degradation ..................... XI1 Dicamba Biodegradation: A Case Study ............................... XI11 Growth Kinetics in Liquid Culture ................................... References .........................................................

. .

.

. .

1 2 4 5 7 10 21 29 32 36 37 44 49 63

An Environmental Assessment of Biotechnological Processes

M . S . THAKUR. M . J . KENNEDY.AND N . G . KARANTH I . Introduction ....................................................... I1. Ecological Consequences of the Release of Microorganisms ............. 111 Risk Assessment .................................................... IV Case Studies ....................................................... V. The Regulation of Biotechnological Processes ......................... VI . Conclusions ........................................................ References .........................................................

. .

67 69 73 75 80 82 83

Fate of Recombinant Escherichia coli K-12 Strains in the Environment

GREGGBOGOSIANAND JAMES F. KANE

. Introduction .......................................................

I I1. I11. IV. V. VI .

Construction and Properties of pBR322 ............................... Fate of E. coli and Related Organisms in Water ........................ Fate of E. coli and Related Organisms in Soil .......................... Fate of E . coli and Related Organisms in Sewage ....................... Fate of E . coli K-12in the Mammalian Intestinal Tract .................. V

87 89 100 104 108 113

vi

CONTENTS

.

VII Alternative Detection Methods for Recombinant Organisms in the Environment ....................................................... VIII . Conclusions ........................................................ References .........................................................

120 121 123

Microbial Cytochromes P-450 and Xenobiotic Metabolism

F . SIMASARIASLANI

. . . .

I Introduction ....................................................... I1 General Properties of Cytochromes P-450 ............................. 111 Microbial Cytochromes P-450........................................ IV Conclusion ......................................................... References .........................................................

133

134 139 173 174

Foodborne Yeasts

T . DEAK I. I1. I11. IV . V. VI .

Introduction ....................................................... Characteristics and Classification of Yeasts ............................ Ecology of Yeasts ................................................... Specific Habitats .................................................... Methods of Isolation and Enumeration ................................ Methods of Identification ............................................ References .........................................................

179 180 183

194 228 234 258

High-Resolution Electrophoretic Purification and Structural Microanalysis of Peptides and Proteins

ERIKP . LILLEHOJ AND VEDPAL s. MALIK I . Introduction ....................................................... I1. Polyacrylamide Gel Electrophoresis .................................. 111. Structural Analysis of Proteins Directly Eluted from One- and Two-Dimensional Polyacrylamide Gels ............................... IV . Structural Analysis of Proteins Electroblotted from One- and Two-Dimensional Polyacrylamide Gels ............................... V. Electrophoretic Micropreparative Procedures as Part of a Comprehensive Purification Strategy ................................................

280 281 290 303 315

CONTENTS

vii

VI . Applications of Microsequence Analysis of ElectrophoreticallyPurified Proteins ........................................................... VII . Quality Control of Recombinant Proteins .............................. VIII Prospective Directions .............................................. References .........................................................

318 323 327 329

INDEX ................................................................... CONTENTSOF PREVIOUSVOLUMES ...........................................

339 361

.

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Microbial Transformations of Herbicides and Pesticides DOUGLASJ. CORK*AND JAMES P. KRUEGER~ *Department of Biology Illinois Institute of Technology Chicago, Illinois 60616 'Fitch, Even, Tabin and Flannery Chicago, Illinois 60603 I. Introduction 11. History of Microbial Conversions 111. Entry and Movement of Herbicides in the Environment

IV. Taxonomy of Degradative Organisms V. Kinetics of Biodegradation by Microorganisms VI. Factors Affecting Biodegradation Kinetics A. Structure B. Solubility C. Adsorption/Desorption D. Adaptation Rate E. Moisture, Temperature, and Nutrients F. Rates of Chloroaromatic Degradation VII. Biochemical Mechanisms of Aerobic Chloroaromatic Metabolism A. Demethylation B. Dehalogenation C. Ring Cleavage D. Chlorocatechol Metabolism VIII. Cometabolism IX. Biochemical Mechanisms of Anaerobic Aromatic Metabolism X. Molecular Biology of Degradative Microorganisms XI. Molecular Biology of Chloroaromatic Degradation Cloning of Pseudomonas Genes in the Escherichia coli Vector, pUC XII. Dicamba Biodegradation: A Case Study XIII. Growth Kinetics in Liquid Culture A. Effect of Dicamba Concentration B. Dependence of Activity on pH C. Effect of Temperature D. Growth Kinetics in Soil E. Growth Chamber Study F. Field Study References

I. Introduction

The use of xenobiotics has increased during recent decades. Herbicide and pesticide usage has benefited modern society by improving the quality and quantity of the world's food supply, while keeping the cost 1 ADVANCES IN APPLED MICROBIOLOGY. VOLUME 36 Copyright 0 1991 by Academic Press, Inc. All rights of reproductionin any form reserved.

2

DOUGLAS J. CORK AND JAMES P. KRUEGER

of that food supply reasonable. However, increased usage of chemicals has resulted in environmental concerns. Microorganisms in soil and water can transform many synthetic organic chemicals. The development and integration of microbes or their activities with the use of herbicides and pesticides can enhance the beneficial effects of chemical usage while eliminating some of the environmental concerns. Microbes can provide a means to eliminate unwanted residues from the environment, protect previously susceptible crops from herbicide or pesticide damage, and provide a source of genetic material for the development of herbicide-resistant crops or pesticide-producing plants. A fundamental understanding of a microbe’s degradative kinetics under various conditions, its biochemical systems, and its molecular biology are vital in maximizing the potential benefits of its use. II. History of Microbial Conversions

Microbial transformations have long been beneficial to mankind. More recently, these transformations have had environmental and chemical applications. Over billions of years, microorganisms have evolved an extensive range of enzymes, pathways, and control mechanisms in order to degrade a wide array of aromatic compounds. Microorganisms have been isolated that can degrade benzene (Dagley et a]., 1964), phenol (Feist and Hegeman, 1969), naphthalene (Davis and Evans, 1964), salicylate (Chakrabarty, 1972), toluene (Chakrabarty, 1976), and p- and m- hydroxybenzoate (Johnson and Stanier, 1971). There are a few instances in which catabolic pathways have evolved that are specific for chlorinated substrates (Dorn and Knackmuss, 1978a; Gibson, 1978). Haloaromatic-assimilating strains have been obtained by (1)enrichments from nature, (2) in vivo genetic manipulations, and (3) in vitro genetic manipulations. Enrichments have been used to show the involvement of microbes in the degradation of synthetic compounds such as 2 &dichlorophenoxyacetic acid (2,4-D) (Bollag et al., 1968; Loos, 1975) 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (Rosenberg and Alexander, 1980), atrazine (Kaufman and Kearney, 1970),and some isomers of polychlorinated biphenyls (Ahmed and Focht, 1972).Pure cultures of bacteria that have been enriched for, isolated, and characterized can utilize 2chlorotoluene, 3-chlorotoluene, 3,4-dichlorotoluene, 2,4-dichlorobenzoate, 3 &dichlorobenzoate, and 5-chlorosalicylate as sole carbon and energy sources for growth (Vandenbergh etal., 1981; Pierce etal., 1983; Crawford e t a / . , 1979). The first report of in vivo construction of a catabolic pathway for the mineralization of chloroaromatic compounds was conducted with

TRANSFORMATIONSOF HERBICIDES AND PESTICIDES

3

Pseudomonas sp. strain B13 and Pseudomonas putida mt-2, using 4chlorobenzoate (Reineke and Knackmuss, 1978). Strains were isolated that could degrade 3- and 4-chlorobenzoate. Similar methods have been used by others to develop strains that grow on 3-chlorobenzoic acid (Chatterjee et al., 1981) and 3,5-dichlorobenzoic acid (Chatterjee and Chakrabarty, 1982). Techniques such as plasmid-assisted molecular breeding have been used to develop a microbe capable of degrading 2,4,5-T (Kellogg et al., 1981). Advances in technologies associated with molecular biology have resulted in the construction of haloaromatic-degrading organisms. Organisms have been constructed that have the ability to degrade chlorosalicylate and chlorobenzoate (Lehrbach et al., 1984). For example, a strain of Pseudomonas sp. has been manipulated to completely mineralize dichloronaphtalene or convert it to the corresponding dichlorosalicylate (Durham and Stewart, 1987). The bioremediation industry is an excellent example of the application of degradative organisms to environmental cleanup. Table I lists TABLE I COMPANIES DEVELOPING XENOBIOTIC-DEGRADING MICROBES Company/location

Application

Advanced Mineral Technology/Golden, Colorado Air Products and Chemicals/Trexlertown, Pennsylvania Amgen/Thousand Oaks, California Battelle/Columbus, Ohio Bioclean/Bloomington, Minnesota BiotechnicalCambridge, Massachusetts Chemical Waste ManagemenKhicago, Illinois Celgene/Summit, New Jersey Ciba-Geigy/Greensboro, North Carolina Detox/Dayton, Ohio Dow/Midland, Michigan Ecova/Redmond, Washington Flow/Orange, California G.E./Schenectady, New York Genex/Gaithersburg, Maryland Groundwater Technology/Norwood, Massachusetts Homestake Mining/Reno, Nevada IGTKhicago, Illinois

Heavy metals Organics Trichloroethylene Chlorinated aromatics Pentachlorophenol Phenol Toxic waste Chlorinated aromatics Herbicides Organics Chlorophenols Solvents Sewage Polychlorobiphenyl Toxic waste Solvents Cyanide Coal Tars, sulfides, chlorinated aromatics Dicamba Herbicides Chlorinated aromatics Herbicides

IITRUChicago, Illinois Monsanto/St. Louis, Missouri Occidental ChemicaUGrand Island, New York Sandoz Crop Protection/Des Plaines, Illinois

4


some of the companies involved in bioremediation and their applications. Others have used degradative organisms as a source of genetic material for the development of herbicide-resistant plants. Glyphosate and bromoxynil degradative genes have been transferred via Agrobacterium tumefaciens to produce resistant tobacco and other plant species (Stalker et a]., 1988).

Ill. Entry and Movement of Herbicides in the Environment

Herbicides enter the soil and water as a result of direct. application and runoff from plant surfaces, as an integral part of the weeds killed by them, via aerial transport and deposition because of earlier volatilization, via wind drift or wind-blown soil particles with adsorbed residues, from spillage, from water used to clean equipment, and from disposal of packing. Figure 1 illustrates the routes of entry of herbicides into the Aerial transport spillage, washing, and disposal of containers

Wind-drift Photodecomposition

-, mineralorganic

Transport in soil

s

.*-

Desorption and diffusion

Chemical transformation

Microbial transformation

Leaching

FIG.1. Entry and transformationof herbicides in the environment (Torstensson,1988).


5

environment and some of the transformations that take place. Once in the environment, herbicides are subject to photochemical, chemical, and biological effects capable of causing transformations in the compound’s chemical structure. Biological and nonbiological processes work together to degrade herbicides. In nature it is difficult to distinguish between the two modes of degradation in most cases (Ashton, 1982).Though some reactions are clearly nonbiological, such as photolysis, others, such as hydrolysis, can be either nonbiological or biologically mediated. Examples of reactions that can transform herbicides in the environment are shown in Table 11. Mineralization or complete biodegradation of an organic molecule in water and soil is almost always a consequence of microbial activity. Few abiotic mechanisms in nature totally convert organic compounds to inorganic products (Alexander, 1981).Whether or not a herbicide is adsorbed, absorbed, activated, inactivated, persistent, short-lived, mobile, stationary, or will eventually constitute a residue problem may depend upon its transformation by soil microorganisms. The microbial metabolism of herbicides can be classified as indicated in Table 111. IV. Taxonomy of Degradative Organisms

Microbes that are natural components of soil and water environments are potential agents for the biological transformations of aromatic compounds that enter the ecosystem. Microorganisms usually occupy a TABLE I1

REACTIONSTHATCANTRANSFORM CHEMICALS IN THE ENVIRONMENT Category Photo1ysis Hydrolysis Oxidation Dehalogenation Deamination Decarboxylation Methyl oxidation Hydroxylation Sulfur oxidation Reduction Oxime metabolism Ester cleavage C-N cleavage C-S cleavage C-Hg cleavage S-N cleavage

Example Aldrin Diazinon 2,4-D

Chlorophenols Aniline Bifenox Isopropylnaphtha Dicamba Aldicarb DDT Aldicarb Malathion Alachlor Benthiocarb Ethylmercury Oryzalin

Reference Matsumura (1982) Matsumura (1982) Sandrnann and Loos (1988) Steiert et al. (1987) Zeyer et al. (1985) Leather and Foy (1977) Yoshida and Kojima (1978) Smith (1974) Andrews et al. (1971) Pfaender and Alexander (1972) Jones (1976) Paris eta). (1975) Tiedje and Hagedorn (1975) Ishikawa et al. (1976) Kimura and Miller (1964) Golab et al. (1975)

6

DOUGLAS J. CORK AND JAMES P. KRUEGER TABLE 111 GENERAL CLASSIFICATION OF THE MICROBIAL METABOLISM OF HERBICIDES"

Reaction type Enzymatic

Nonenzymatic

a

Description Incidental metabolism: herbicide does not serve as an energy source Metabolism by generally available enzymes Metabolism due to generally present broad-spectrum enzymes (hydrolases, oxidases, etc.) Metabolism due to specific enzymes present in many microbe species Analog-induced metabolism (cometabolism) Metabolism by enzymes utilizing substrates structurally similar to pesticides Catabolism: herbicide serves as an energy source Herbicide or part of the molecule is readily available source of energy for microbes Herbicide is not readily utilized; some specific enzyme must be induced Detoxification metabolism Metabolism by resistant microbes Participation in photochemical reactions Contribution through pH changes Contribution through production of inorganic and organic reactants Contribution through production of cofactors

From Matsumura (1982).

volume of less than 0.1% of the soil, but are responsible for numerous transformations that cycle elements and energy in nature. Microbial densities may be as high as lo9 per gram of soil, with a biomass up to several tonnes per hectare (Torstensson, 1988). The microbial population exists in a dynamic equilibrium formed by interactions of abiotic and biotic factors that can be altered by modifying environmental conditions. Microbes are able to degrade a wide variety of chemicals, from simple polysaccharides, amino acids, proteins, lipids, etc. to more complex material such as plant residues, waxes, and rubbers. Some important degradative bacteria that occur in water and soil environments are described in Table IV. Pseudomonas strains are extremely common and are often the predominant members of the populations selected from natural sources, such as soil, polluted waters, and sediments, for their ability to grow on single compounds as sole carbon sources (Ribbons and Williams, 1982). Pseudomonas strains are facultative aerobes, as some can use nitrate as a terminal acceptor for a limited number of substrates. Aerobically grown pseudomonads can be described as Gram-negative unicellular rods,


7

TABLE IV CLASSIFICATION OF DEGRADATIVE BACTERIA THATOCCUR IN WATER AND SOIL' ~~~

Description

Family

Gram-negativeaerobic rods and cocci

Facultatively anaerobic Gram-negativerods Endospore-forming Grampositive rods and cocci ~

~

~

~~~~~

~

~

Genus

Pseudomonadaceae

Pseudomonas, Xanthomonas

Azotobacteraceae Rhizobiaceae Methylococcaceae Neisseriaceae -b Enterobacteriaceae

Azotobacter Rhizobium, Agrobacterium Methylomonas, Methylococcus Momxella, Acinetobacter Alcaligenes, Flavobacteriurn Escherichia, Enterobacter, Serratia, Proteus Aerornonas Bacillus

Vibrionaceae Bacillaceae ~

From Bergey's Manual of Systematic Bacteriology (1984). Affiliation uncertain.

with the long axis straight or curved, but not helical. They do not form spores, stalks, or sheaths. The energy-yielding metabolism is respiratory, never fermentative or photosynthetic. All use molecular oxygen as a terminal oxidant, except for several that can use denitrification as an alternative anaerobic respiratory mechanism. All are chemolithotrophs, though some are facultative chemolithotrophs that use Hzas an energy source (Stanier et al.,1966).The genus falls into five main groups by the criterion of ribosomal RNA homology [Table V). The predominant biological feature of the pseudomonads is their biochemical diversity [Stanier et a]., 1966). Strains of Pseudomonas spp. are capable of utilizing over 100 different compounds. Pseudomonas spp. have been reported as being capable of growing on alkanes, mono- and polycyclic hydrocarbons, salicylate, heterocyclics, phenolics, and aliphatic and aromatic halogenated compounds (Ribbons and Williams, 1982).Table VI lists some of the catabolic activities of Pseudomonas spp. and other degradative organisms. V. Kinetics of Biodegradation by Microorganisms

Microorganisms grow in a wide spectrum of physical and chemical environments. Growth of microorganisms and other physiological activities are a response to the physiochemical environment. Growth rate, like a chemical reaction rate, is a function of chemical concentration.


8

TABLE V CLASSIFICATION OF PSEUDOMONAS SPECIES INTO RNA HOMOLOGY GROUPS" rRNA homology group ~

% GC in DNA ~

67 59-63 61-62 61-66 63-64 66 67-68 65 69 69 67 62 62-64 66-67 66 67 66-68

1

2

3 4 5

a

Species

~~

P. aeruginosa P. fluorescens P. putida P. stutzeri P. rnendocina P. alcaligenes P. cepacia P. caryophylli P. pseudomallei P. mallei P. acidovorans P. testosteroni P. facilis P. diminuta P. vesicularis P. rnaltophilia Xanthomonas sp.

From Ribbons and Williams (1982).

The relationship between growth rate and substrate concentration can be described by the Monod model: where p is the specific growth rate, pmaxis the maximum specific growth rate, S is the substrate concentration, and Ks is a constant equal TABLE VI BACTERIA THATCANDEGRADE CHLOROAROMATIC COMPOUNDS Organism

Substrate

Reference

Alcaligenes denitrificans Pseudomonas cepacia Bacillus circulans Alcaligenes sp. Pseudornonas sp. Pseudomonas putida Bacillus brevis Alcaligenes eutrophus Flavobacterium sp. Arthrobacter sp. Pseudomonas sp. Pseudomonas maltophilia

2&Dichlorobenzoate 2.4.5-T Metolachlor 1,3-Dichlorobenzene Atrazine 3-Chlorobenzoate 5-Chlorosalicylate 2,4-D Pentachlorophenol 4-Chlorobenzoic acid 3,5-Dichlorobenzoate Dicamba

Van Den Tweel et 01. (1987) Chatterjee et al. (1982) Saxena et a]. (1987) DeBont et al. (1986) Behki and Khan (1986) Chatterjee et al. (1981) Crawford et al. (1979) Perkins and Lurquin (1988) Steiert et al. (1988) Marks et 01. (1984) Reineke and Knackmuss (1980) Fujimoto and Cork (1991)


9

to the substrate concentration when p = 0 . 5 ~ The ~ ~specific . growth rate can be calculated by the following equation: fd

= In 2Ip

where td is the time required to double cell mass or cell number. A graphic representation of the Monod model is shown in Fig. 2. Some physical and biological meaning can be attributed to the two constants in the Monod model. The value of P m m is the maximum specific growth rate in a given chemical medium at specified temperature and pH. The value of K, is inversely proportional to the affinity the microorganism has for the substrate (Wang et al., 1979). When the concentration of a utilizable organic substrate is considerably in excess of the bacterium’s K, value, logarithmic or exponential kinetics of growth occurs. If the cell density is so great that the quantity of substrate is insufficient to support a significant increase in cell mass, then the kinetics of disappearance of organic chemicals present at high levels (in excess of K,) is zero order, or linear with time. Two patterns of kinetics can be envisioned when a single bacterial species is provided with a mineralizable substrate at concentrations below the K, value. In the first pattern, there is no increase in cell number either because the concentration of substrate is too low to

Substrate Concentration FIG.2. The Monod model for microbial growth.

10

DOUGLAS J. CORK AND JAMESP. KRUEGER

support growth, or because the initial cell number is too large, relative to the quantity of organic compound, to permit an appreciable increase in cell mass. At constant biomass and limiting substrate levels, the rate is proportional to the concentration of substrate; this is typical of firstorder kinetics. In the second pattern, few cells of the active species are present initially. Under these conditions the bacteria will grow, but at a rate that falls constantly with diminishing substrate concentrations. A growth pattern in which there is an increasing cell number encountering a decreasing nutrient resource resembles the classical logistic growth curve (Alexander, 1985). The kinetics expected at different chemical concentrations and cell numbers are shown in Fig. 3. The shapes of the curves for chemical disappearance that coincide with these kinetics are illustrated in Figs. 4 and 5. VI. Factors Affecting Biodegradation Kinetics

The rate of microbial decomposition of a chemical in soil and in liquid medium is mediated by three factors: (1)the availability of the chemical to the microorganism or enzyme system that can degrade it, (2) the quantity of these microorganisms or enzyme systems, and (3) the

Monod nithout growtt

First order

/

order

Monod

Logarithmic

(with growth)

Initial Substrate Concentration

FIG. 3. Kinetic models as a function of initial substrate concentrationand bacterial cell density (Alexander, 1985).


11

Time FIG. 4. Disappearance curves for chemicals that are mineralized as related to firstorder, zero-order, and Monod growth kinetic models (Alexander, 1985). 1

Time

FIG.5. Disappearance curves for chemicals that are mineralized as related to logistic, logarithmic, and Monod kinetic models (Alexander, 1985).

12


activity level of these organisms or enzyme systems. The availability of a chemical to a microbial population in soil or liquid medium is determined by the physical properties of that chemical. The chemical’s structure and its resulting solubility in water, dissolution rate, and adsorption/desorption characteristics in soil are properties that determine availability (Bollag, 1974,Goulding et a]., 1988;Alexander, 1981;Stucki and Alexander, 1987;Ogram et a]., 1985).Biodegradation rates of available organic substrates have been shown to be directly related to microbial biomass and the activity of that biomass (Anderson, 1984).Environmental factors such as pH, temperature, soil moisture level, and soil composition are important regulators of microbial activity, and therefore of the degradation rate of a chemical (Vaishnav and Babeu, 1987). A. STRUCTURE The introduction of substituents on a benzene ring influences its degradation considerably. Minor alterations in structure frequently cause a drastic change in the susceptibility of such compounds to biotransformations. Introduction of polar groups such as OH, COOH, and NH2 may provide the microbial system a site of attack. Halogen or alkyl substitutions tend to make the molecule more resistant to biodegradation (Bollag, 1974).Degradation rates of chlorine-substituted aromatic compounds are dependent on the position of the substituents and the degree of substitution (Goulding et a]., 1988). Table VII shows that monochlorosubstituted isomers were generally degraded more rapidly than di- or trichloro derivatives. Monochlorophenols were more rapidly assimilated than monochlorobenzoic acids. Both 3,4- and 3,sdichlorophenols were more difficult to degrade than 2,3-,2,5-, and 2,6-dichlorophenols. Dichlorobenzene isomers were completely degraded whereas dichlorobenzoic acids were less efficiently removed due to the position of the chlorines.

.

B SOLUBILITY In general, compounds with low water solubility tend to be more resistant to microbial degradation than are compounds of higher water solubility. Chemicals having low solubilities in water may not provide sufficient carbon to support microbial growth. Low ambient concentrations of substrate may result in a decreased penetration rate into the cell and too few molecules per unit time to allow enough energy for the organism to maintain itself (Alexander, 1981).To mineralize or grow on substrates having low solubilities in water, microorganisms may require

TRANSFORMATIONS OF HERBICIDES AND PESTICIDES

13

TABLE VII DEGRADATION RATESAND PERCENTAGE REMOVALOF CHLOROSUBSTITUTED AROMATIC COMPOUNDSO Substrate

Removal (Yo)

2-Chlorobenzoic acid 3-Chlorobenzoic acid 2-Chlorophenol 4-Chlorophenol 2,6-Dichlorobenzoic acid 3,5-Dichlorobenzoic acid 2,6-Dichlorophenol 2,3-Dichlorophenol 2,5-Dichlorophenol 3,4-Dichlorophenol 3,5-Dichlorophenol l&Dichlorobenzene 1,3-Dichlorobenzene 1,4-Dichlorobenzene

100 100 100 100 52 81 100

100 100 44 52 100 100 100

Removal rate (mg/liter/hour)

Incubation time (hours)

4.10 1.57 5.10 4.80 2.57 1.65 4.80 4.60 2.10 0.53 1.04 NDb ND ND

96 96 96 96 168 168 72 72 144 168 168 96 96 96

From Goulding et al. (1988).

Rate not calculated.

some physiological adaptations. Bacteria may facilitate the uptake of poorly soluble compounds by producing emulsifiers [Guerra-Santos et ~ l .1984). , Modification of the cell surface may increase its affinity for hydrophobic substances and thus facilitate their absorption (Neufeld et QI., 1980) Organisms may grow only at the expense of the compound dissolved in solution. Therefore, the rate of dissolution of such chemicals would govern the rate of their biodegradation [Stucki and Alexander, 1987). C. ADSORPTION/DESORPTION The binding of xenobiotics to soil may involve various interactions such as ionic or covalent bonds, van der Waals forces, hydrogen bonds, charge transfer, and hydrophobic bonds. Each contributes not only to the binding but also to the extent of the subsequent release (Dec and Bollag, 1985). In most cases, herbicides and pesticides reversibly partition between the soil solution and soil organic matter (Karickhoff, 1981). The adsorption/desorption characteristics of a chemical in soil may determine its availability to degradative organisms. Comparison of the half-lives of the chemical with data describing its adsorption showed that there was a direct correlation between the amount of

14


chemical in solution and the rate of dissipation (Torstensson, 1988). When pesticides such as paraquat and diquat are intercalated into clay, they are isolated from the degrading organisms and protected form intracellular degradation (Burns and Audus, 1970;Weber and Coble, 1968).Other authors have indicated that pesticide sorption might either enhance or decrease microbial degradation rates in soil (Ogram et al., 1985).Because bacteria themselves may be sorbed, it is conceivable that bacteria and herbicides may be sorbed on adjacent locations on the soil surface, thereby facilitating the scavenging of the chemical by the sorbed bacteria. Three models have been proposed by Ogram et al. (1985)to describe the effects of sorption of bacteria and 2,4-D on the biological degradation rates of 2,4-D. The sorption of 2,4-D and bacteria were characterized by the following equations: For 2,4-D, S = KDC For bacteria,

Ns = KBNW where S and Ns are the amounts of 2,443 and bacteria, respectively, sorbed on soil; KD and KB are the respective sorption coefficients; and C and Nw are the solution-phase concentrations of 2,4-D and bacteria, respectively. The first model states that only 2,4-D in solution is degraded and that it is degraded only by bacteria in solution. The model is expressed as follows: dTldt = - K,CN,W where dTldt is the change in mass of pesticide over time, K, is the degradation rate coefficient, and W is the volume of water in the system. Model 2 states that bacteria in a given phase only degrade 2,4-D in that phase. This model is expressed as follows: dTldt =

-

(K,CN,W

+ K,,SN,M)

where K,, is the rate coefficient for degradation in sorbed phase and M is the mass of soil. The third model states that only 2,4-D in solution is available for degradation but bacteria in both sorbed and solution phases would be capable of degrading 2,4-D. This model is as follows: dTldt = - (K,CN,W

+ KswCNsM)

where K,, represents the combined effects of the rates at which sorbed bacteria encounter 2,4-D, take it up, and then mineralize it to COz.


15

If model 1 is correct, then K, (degradation rate coefficient) should be constant with varying soil/solution ratios and for different soils. For 2,4-D, K , increased with increasing soil/solution ratios. As the soil solution ratio increases, sorption of both bacteria and 2,4-D should increase. The increase in K, with increasing soil/solution ratios suggests that sorbed 2,4-D was being degraded or that sorbed bacteria may have been degrading 2,4-D. If model 2 accurately describes the situation, then K,, values (rate coefficient for degradation in sorbed phase) should be the same for all soils. As sorption of both 2,4-D and bacteria increased in various soils, the calculated values of K,, decreased, suggesting there may have been at least partial protection of 2,4-D from degradation when it was sorbed. , and K,, (combined degradation rate If model 3 is correct, then K coefficient) will be constant with varying soil types and soil/solution ratios. This model describes the data for 2,4-D. Sorbed 2,443 was completely protected from biological degradation and sorbed and solutionphase bacteria degraded solution-phase 2,4-D with almost equal efficiencies (Ogram et a]., 1985).

D. ADAPTATION RATE An understanding of adaptations of microbial communities to organic chemical exposure is critical for predicting chemical degradative ratios. The mineralization of many organic compounds by microorganisms is often preceded by an acclimation period. The acclimation period is the time interval during which biodegradation is not detected. The acclimation time required for a microbial population to degrade a chemical can be influenced by the rate and frequency of exposure to that chemical. Soil microorganisms have been shown to degrade 2,4-D more rapidly after repeated exposure to 2,4-D (Torstensson, 1988).A graphic representation of the effects of repeated applications of a herbicide is shown in Fig. 6. High or low concentrations of a chemical may increase the acclimation period. The acclimation period for degradation in soil of the herbicide picloram increased as its concentration increased (Grover, 1967).High concentrations of a chemical may be toxic or inhibitory to the microbial populations present. At low concentrations of the compound, the long acclimation may be the result of slow growth of the mineralizing organisms or low concentrations of substrate (Wiggins and Alexander, 1988). Additionally, evidence exists that one compound may shorten the acclimation period needed before another is degraded. The acclimation of sewage microflora to 3-chlorobenzoate or 4-

16


Time FIG.6. Effect of repeated herbicide application on acclimation (Torstensson, 1988).

chlorobenzoate reduced the acclimation time for mineralization of other monosubstituted aromatic hydrocarbons (Haller, 1978). The adaptation process may involve one or a combination of (1)induction or derepression of enzymes specific for degradation pathways of a particular compound; (2) a random mutation in which new metabolic capabilities are produced, allowing degradation that was previously not possible; or (3) an increase in the number of organisms in the degrading population (Aelion et al., 1987). An induction signal may come from the chemical substrate itself or from other chemicals present. It has been reported that the phenylurea herbicide monuron could induce an acylamidase in Bacillus sphaericus that is capable of hydrolyzing the herbicide linuron, although monuron itself is not a substrate (Engelhardt et al., 1973). An acclimation period may result from the time required for the appearance of a new genotype after a mutation or genetic exchange occurring during exposure to the compound. For example, adaptation of Acinetobacter calcoaceticus to the degradation of aniline has been attributed to a mutation in the natural population with the involvement of a plasmid-carried gene (Wyndham, 1986).Studies in the mineralization of 4-nitrophenol indicated that the acclimation time resulted from the time needed for a small population to become suffi-

17


ciently large to give detectable loss of the chemical (Wiggins and Alexander, 1988).In this study, the growth of mineralizing organisms was affected by predation by protozoa and competition for inorganic nutrients.

E. MOISTURE, TEMPERATURE, AND NUTRIENTS It is the environment that actually controls the biodegradation process and has a greater influence on the process than microorganisms per se (Vaishnav and Babeu, 1987).Inhibition of microbial activity by a low or high temperature and extremes of pH may result in the persistence of potentially mineralizable compounds. Soil composition, percentage of organic matter, nutrient levels, and moisture levels are important regulators of microbial degradative activity. The kinetics of 2,4-D degradation for soil samples incubated at four moisture tensions and four temperatures is shown in Table VIII. Degradation occurred by a slow first-order reaction (slow phase), which, under some conditions, was followed by a rapid first-order reaction (fast TABLE VIII EFFECTS OF SOILMOISTURE TENSION AND TEMPERATURE ON THE RATE OF 2,4-D DECOMPOSITION IN SOIL INCUBATED WITH 25 pg 2,4-D PER GRAMOF SOILa

Temperature (“Cl 20

27

30

35

a

Moisture tension

Decomposition rate (Ccg/g/day)

(bas1

Duration of slow phase (days)

Slow phase

Fast phase

0.10 0.33 0.50 1.00 0.10 0.33 0.50 1 .oo 0.10 0.33 0.50 0.00 0.10 0.33 0.50 1.00

36 >90 >90 >90 28 90 >90 >90 >63 >63 >63 >42 >42 >42 >42 >42

0.123 0.075 0.038 0.026 0.230 0.140 0.128 0.075 0.238 0.125 0.126 0.253 0.151 0.117 0.127 0.058

0.887

From Parker and Doxtader (1983).

* No fast phase observed.

-b

18


phase). The rate of decomposition of 2,4-D decreased with increasing soil moisture tensions for temperatures between 20 and 35°C. The decrease was a result of the reduced activity of the 2A-D-degradingmicroorganisms arising form decreased water availability and increased 2,4-D solution concentration (Parker and Doxtader, 1983). The effects of soil water content and soil temperature on the degradation of 2,4,5-T are shown in Table IX. The optimal temperature for 2,4,5-T degradation was 30°C and the optimal soil water content was 25% (Chatterjee et a]., 1982). Generally, chemicals were found to have biodegraded to a greater extent in waters enriched with both nutrients and microbes than in those receiving either amendment alone (Vaishnav and Babeu, 1987). Table X illustrates that effects of nutrient and microbial additions on first-order biodegradation rate constants and half-lifes of selected chemicals in natural waters. A sufficient microbial population capable of utilizing the chemical is also important for biodegradation. For example, the soil degradation of the herbicide 2,4,5-T was increased by increasing the concentration of a 2,4,5-T-degradingorganism (Table XI).

F. RATESOF CHLOROAROMATIC DEGRADATION Degradative organisms have been isolated that can transform a number of chloroaromatic compounds. The rates at which pure cultures of TABLE IX EFFECTS OF SOILMOISTURE AND TEMPERATURE ON THE DEGRADATION OF 2,4,5-T IN SOIL TREATED WITH 1000 pg/g 2,4,5-T AND INOCULATED WITHP. Cepacia AC1100"

Incubation temperature

Soil water

2,4,5-T

content

degradation

("C)

(%I

(%Ib

20 30 37 42 30 30 30 30 30

20 20 20 20 15 25 50 75 90

52 73 56 32 78 95 89 58 57

, From Chatterjee et al. (1982). As determined by GC analysis.

19

TRANSFORMATIONSOF HERBICIDES AND PESTICIDES TABLE X

EFFECTSOF NUTRIENT AND MICROBE ADDITION ON FIRST-ORDER RATECONSTANTSAND HALF-LIVES OF CHEMICALS IN LIQUIDMEDIUM" Acclimated microbes added

Nutrients and acclimated microbes added

NDb

ND

0.029 (24) 0.009 (77)

0.008 (87) 0.044 (16) 0.016 (43)

0.082 ( 8) 0.062 (11) 0.031 (22) 0.018 (39)

Nutrients added

Chemical Benzene t-Butylbenzoate Hexadecane Naphthalene

ND

From Vaishnav and Babeu (1987). Half-lives are given in parentheses. No difference from controls.

these organisms degrade various chlorinated compounds in liquid medium and in soil have been determined in a number of cases. Figure 7 shows the release of I4CO2from I4C-labeled 2,4-D incubated with a culture of Alcaligenes sp. Up to 40% of the substrate radiocarbon was mineralized to COz in 20 hours (Amy et al., 1985). A pure culture of Pseudomonas cepacia ACllOO has been isolated that is capable of growing on 2,4,5-T as its sole source of carbon and energy (Karns et al., 1984).After a 24-hour incubation of 0.1 mM 2,4,5-T with P. cepacia in liquid medium, 83% of the chloride was released. Soil contaminated with as much as 20,000 pg of 2,4,5-T per gram of soil showed greater than 90% degradation after six weekly treatments with P. cepacia. Furthermore, a strain of P. putida has been isolated that can degrade 3-chlorobenzoate (Chatterjee et al., 1981).After a 48-hour incubation of 7.5 mM 3-chlorobenzoate with P. putida, about 80% of the TABLE XI EFFECT OF MICROBIAL CONCENTRATION ON 2,4,5-T DEGRADATION IN SOILo Organisms per gram of soil ~-

2,4,5-T

degradation

(%Ib

~~

5 5 5 5

0

2.2 x 105 2.2 x 10" 2.2 x 107 ~~

Incubation period (days)

~

From Chatterjee et 01. (1982). As determined by GC analysis. Not detectable.

NDC 50 66 81

20

DOUGLAS J, CORK AND JAMESP. KRUEGER

Time (Hours)

FIG.7. Release of 14C02from a culture of Alcaligenes incubated with [14C]2,4-D (Amy et al., 1985).

chloride was released as inorganic chloride into the medium. Additionally, three species of Pseudomonas sp. were isolated that were capable of utilizing the herbicide atrazine as a sole carbon source (Behki and Khan, 1986). Strains were able to increase in cell number when incubated with atrazine as a sole carbon source (Table XII). Strains of Bacillus sp., Fusarium sp., and Mucor sp. have been identified that can transform the herbicide metolachlor, but cannot completely mineralize it (Saxena et al., 1987).Organisms were able to transform up to 70% of the metolachlor present at a concentration of 50 pg/ml. TABLE XI1

GROWTHOF PSEUDOMONAS STRAINS ON ATRAZINE AS A SOLE CARBONSOURCEO Viable celldm1 ( x 10’) Strain

Initial

After 14 days

192 195 555

1.48 3.10 2.42

19.40 16.72 14.90

a

From Behki and Khan (1986).

21


VII. Biochemical Mechanisms of Aerobic Chloroaromatic Metabolism

The majority of aromatic compounds are converted by bacteria to catechol or protocatechuate (Figs. 8 and 9). Catechol and protocatechuate become starting substrates for subsequent oxidative cleav-

06 CHOH-COOH

CO-COOH COOH

L- tryp tophan

anthracene

CO-CHZ-CHNH2-COOH

benzaldehyde

6 benzoate

benzene

formylkynurenine

1

0"'

CO-CH2-CHNHZ-COOH

L-kynurenine

phenol FIG.8. Aromatic compounds that can be converted to catechol (Gottschalk,1979).

DOUGLAS 1. CORK AND JAMES P. KRUEGER

22

co-coou

CI10H-COOH

OH

013

p-hydroxybenzoyl formate

p-hydroxy-lmandelate

HOCOOH

HO COOH

p- toluat e

OH

P-hY droxybenzaldehyde I

0

shikimat e

I

COOH

benzoate

quinate

OH

J

{OOH

o - O O H

OH

oti

ite

G OOHC H 3

benzoate

OH

protocatechuate FIG. 9. Aromatic and hydroaromatic compounds that can be converted to protocatechuate (Gottschalk, 1979).

age reactions. Chlorinated aromatic compounds can be converted to catechol, protocatechuate, or their corresponding chlorocatechol or protocatechuate by reactions described in Table 11. For example, various soil bacteria have been reported to cleave the ether linkage of 2,4-D to produce 2,4-dichlorophenol (Beadle and Smith, 1982). The catabolism of 2-chloroaniline involves 3,6-dioxygenation to yield 3-chlorocatechol (Latorre et a]., 1984). Also, nonselective dioxygenation was responsible


23

for the conversion of chlorinated benezoates into the respective catechols (Reineke and Knackmuss, 1978). Two processes that are especially important in the initial degradation of chloroaromatic compounds are demethylation and dehalogenation. A. DEMETHYLATION A demethylase enzyme that converts 4-methoxybenzoate to 4hydroxybenzoate has been characterized (Bernhardt et a]., 1975). The 4-methoxybenzoate-O-demethylase is a multienzyme system described as an iron-containing and labile-sulfur-containing monooxygenase. The demethylase enzyme consists of an NADH-dependent reductase and a monooxygenase. The NADH reductase contains FMN and an ironsulfur complex. The iron-sulfur complex appears to be essential for the catalytic function of the reductase and may mediate the transfer of electrons from NADH to the monooxygenase. The monooxygenase appears to be a dimer containing an iron-sulfur chromophore. A proposed mechanism of action for the demethylase enzyme system is shown in Fig. 10.

B. DEHALOGENATION A crucial point in the biodegradation of chloroaromatic compounds is the removal of halogen substituents from the organic compound. Dechlorination mechanisms can be classified as follows: (1) displacement of halogen through hydrogen, (2) displacement of halogen by hydroxyl, (3) oxygenolytic halogen-carbon bond cleavage, and (4) chloride elimination from nonaromatic intermediates. Halogen removal may occur at an early state of the degradative pathway with reductive, hydrolytic, or oxygenolytic elimination of the chlorosubstituent. Alternatively, nonaromatic structures may be generated that spontaneously lose the halide. Displacement of halogen through hydrogen is mainly an anaerobic process and is also referred to as reductive dechlorination. Anaerobic microbial consortiums have been shown to remove chloride without alteration of the aromatic ring (Suflita et al., 1982). Dechlorination occurred under methanogenic conditions, appeared to be enzymatic, and it occurred after induction and because of a low substrate K, of 67 pM. Loss of activity at temperatures above 39°C was observed, and the enzyme exhibited a high degree of substrate specificity. The reducing power required for reductive dechlorination was obtained from the hydrogen produced in the acetogenic oxidation of benzoate. Aerobic

24


I R-H

1

R H

s

-' 1 \\

' 'FeIII\'/'FeIII(II)< -S-

Reductase eFel I I

nooxygenase

(oxld ized)

T

1

1- '

H

H20

\

2e- 0 H H FIG.10. Proposed reaction mechanism of the 4-methoxybenzoate monooxygenase (0-demethylase)enzyme system (Bernhardt et al., 1975).


25

reductive dechlorination has also been demonstrated with a strain of Alcaligenes denitrificans incubated with 2,4-dichlorobenzoate (Van Den Tweel et al., 1987). It has been demonstrated that a halogen can be directly replaced on a benzene ring by a hydroxyl group (Johnston et al., 1972). Other studies have shown that 4-chlorobenzoate is converted to 4-hydroxybenzoate before being degraded via the protocatechuate pathway (Chapman, 1975). The mechanism of the dehalogenation process has been determined by experiments using "Oz and Hz180 (Marks et a]., 1984; Muller et a]., 1984). Data indicated that the dechlorination reaction utilizes water, not molecular oxygen, as the hydroxyl donor. The results showed that the conversion of 4-chlorobenzoate to 4-hydroxybenzoate proceeded via a hydrolytic cleavage of the carbon-chlorine bond. Oxygenolytic halogen-carbon bond cleavage is another mechanism to remove halogen substituents from haloaromatic compounds. The mechanism involves the fortuitous dehalogenation of the substrate by a dioxygenase enzyme. This method of dehalogenation has been demonstrated by a Pseudomonas sp. that degrades 2-fluorobenzoate (Milne et al., 1968).Dehalogenation was the result of nonselective dioxygenation by a benzoate 1,2-dioxygenase. The mechanism is illustrated in Fig. 11.

/"

NADHz

FIG.11, Dehalogenation by nonselective dioxygenationby a benzoate 1,2-dioxygenase (Milne et al., 1968).

26


Chloride elimination can occur after ortho cleavage of chlorocatechols. Some compounds, such as 3-chlorobenzoate, may only be dehalogenated after ring fission (Dorn and Knackmuss, 1978b).Chloride appears to be eliminated spontaneously after the carbon-halogen bond has been labilized through isomerases or reductases (Reineke and Knackmuss, 1988). Studies on the cyclosiomerase enzyme in the metabolic pathway of 3-chlorobenzoate indicated that dehalogenation was a secondary reaction of the cycloisomerization of halomuconic acid (Schmidt and Knackmuss, 1980).

C. RINGCLEAVAGE The aromatic rings of catechol and protocatechuate are cleaved via the reactions of the ortho- or meta-cleavage pathways illustrated in Figs. 1 2 and 13. The reactions of the catechol and protocatechuate branch are catalyzed by different enzymes. There is, for example, a catechol-1,2oxygenase and a protocatechuate-4,5-oxygenase. The products of either cleavage, cis, cis-muconate and 3-carboxy-cis,cis-muconate, yield in two subsequent reactions the first common intermediate, 4-oxoadipate enol lactone. This compound is degraded further to yield succinate and acetyl-CoA. The channeling of diverse compounds into a few central pathways benefits the microbe by simplifying regulatory circuits, genetic control, and reducing energy requirements (Harayama et al., 1987). The meta-cleavage pathway uses catechol-2,3-dioxygenaseto open the ring adjacent to the hydroxyl groups. Further metabolism leads to the formation of pyruvate, formate, and acetaldehyde. The catabolism of catechol produced during the metabolism of naphthalene by PseudoI I I O ~ Q Sspp. has been shown to involve the meta pathway in which the first reaction was catalyzed by catechol-2,3-dioxygenase (Barnsley, 1976). Toluene and substituted toluenes have also been shown to be degraded via the meta pathway (Chatfield and Williams, 1986). Oxygenase enzymes are responsible for the incorporation of the oxygen molecule directly into the organic substrate to yield hydroxyl groups. Oxygenases are classified as either dioxygenases or as monooxygenases. The monooxygenases are also referred to as hydroxylases or mixed-function oxidases. The dioxygenases catalyze incorporation of two atoms of oxygen, and the monooxygenases incorporate only one atom. Most dioxygenase enzymes characterized contain Fe(I1) but have no labile sulfur. Several dioxygenase enzymes that have been crystallized are nonheme iron proteins and others, such as tryptophan dioxygenase,

27


protocatechuate

catechol

4

COOH COOH

D -carboxy

cis, cis-muconate

c o=c HooC o ,

c=o

Y-carboxy-

muconolactone

4-oxoadipate

en%

,

;~~~3c;~;~

lactone

CoA

succinate

succinyl-CoA

FIG.1 2 . Reactions of the ortho-cleavage pathway. 1, Catechol 12-oxygenase; 2, muconate-lactonizing enzyme; 3, muconolactone isomerase; 4 , protocatechuate 3,4oxygenase; 5, carboxymuconate-lactonizingenzyme; 6, carboxymuconolactone decarboxylase; 7,oxoadipate enol lactone hydrolase; 8, oxoadipate succinyl-CoA transferase; 9,oxoadipyl-CoA thiolase (Gottschalk, 1979).

are heme enzymes (Metzler, 1977).The dioxygenase enzyme is typically made up of four subunits with a molecular weight of about 40,000 each (Crawford et al.,1975;Que et al.,1981).Dioxygenase enzymes are active when the iron is in the ferrous form. The formation of a Fe(II)-02 complex may be an essential first step. A proposed mechanism is as follows: Fe(I1)-0, + Fe(III)+-02 + Fe(II1) (ferriheme) (oxygenated complex) 02- (attacking reagent)

28


catechol

protocatechuate

noH COOH

\CHO

2-hydroxymuconic

H o o c ~ ~ ~ o H CHO

2-hydroxy semialdehyde

6

oxopent-4-enoate

carboxvpentenoate

HOOC HCOH"~ E'O

CH3

COOH

oxovalerate

+

\

rCH. COH

r

1

4& pyruvate

MCOOH

CH3

c=o

I COOH

oxovalerate

acetaldehyde

2 pyruvate FIG. 13. Dissimilation of cathechol and protocatechuate by the pathways involving meta cleavage. 1, Catechol 2,3-oxygenase; 2, muconic semialdehyde hydrolase; 3, 2oxopent-4-enoic acid hydrolase; 4, oxovalerate aldolase; 5, protocatechuate 4,soxygenase; 6, carboxymuconic semialdehyde hydrolase; 7, 2-oxo-4-carboxypent-4-enoic acid hydrolase; 8, oxovalerate aldolase (Gottschalk, 1979).

Monooxygenase enzymes are classified as external or internal. Monooxygenases that require a cosubstrate in addition to the substrate being hydroxylated are known as external monooxygenases. If the substrate being hydroxylated also serves as the cosubstrate, then the monooxygenase is classified as internal. Most internal monooxygenases contain flavin cofactors and are devoid of metal (Metzler, 1977). A well-


29

characterized monooxygenase is salicylate hydroxylase. Salicylate hydroxylase is a flavoprotein that catalyzes the hydroxylation and simultaneous decarboxylation of salicylate (White-Stevens and Kamin, 1972). The enzyme contains one FAD and one polypeptide chain per 57,200 molecular weight, and exhibits a strong specificity for substrates bearing hydroxyl and carboxyl substituents at the ortho position.

D. CHLOROCATECHOL METABOLISM Chlorocatechols are generally degraded by an ortho fission pathway. Meta cleavage of chlorocatechols may result in toxic or dead-end intermediates (Reineke et al., 1982). Normal dioxygenase and cycloisomerase enzymes exhibit low activity for halogenated substrates (Schmidt and Knackmuss, 1980). However, dioxygenase and cycloisomerase enzymes that have a high affinity for chloroaromatic substrates have been identified (Dorn and Knackmuss, 1978b). In Pseudomonas sp. strain B13, catechol and chlorocatechols were assimilated via two separate othro-cleavage pathways (Reineke and Knackmus, 1988). Correspondingly, two types of isofunctional enzymes for ring fission were found. Pyrocatechase I was present in cells grown on benzoate and was highly specific for catechol. Pyrocatechase I1 was induced when S-chlorobenzoate was the growth substrate. Pyrocatechase I1 had a high activity for the chlorosubstituted benzene. Two types of isofunctional enzymes were also found for cycloisomerization of cis,cis-muconate and cis,cischloromuconate. Cycloisomerase I was highly specific for cis,cismuconate whereas cycloisomerase I1 had high activity for 2chloromuconate and 3-chloromuconate. The proposed metabolisms of 3-chlorobenzoic acid and 4-chlorocatechol are shown in Fig. 14. A number of microbes have been isolated and identified that can metabolize 2,4-D (Bollag et al., 1968; Fisher et al., 1978; Sandmann and Loos, 1988; Don and Pemberton, 1981).A proposed metabolic pathway for the degradation of 2,4-D is shown in Fig. 15. The ether linkage of 2,4-D is cleaved by enzymes present in various soil bacteria (Bollag et al., 1968). The resulting 2,4-dichlorophenol is converted to chlorocatechol by a phenol hydroxylase. VIII. Cornetabolism

The term cometabolism implies the concomitant but incomplete oxidation of a nongrowth substrate during the growth of a microorganism on a utilizable carbon and energy source. Characteristics of cometabolism are that (1)the energy derived from oxidation of the cometabolite

30


HO

COOH

J

6.” c1

5.

HO COOH

c1

QooH

4

3

COOH

HC

c1 HOOC HOOC

c1-

J

5

Rc

COOH (COOS 6 CH2

, c=o /COO13

I1 HC

,/ C H 2 C=O

TCA cycle

H0OC-CH2-C0-C~-~~-~00~

FIG. 14. Proposed catabolic route of 3-chlorobenzoic acid to maleoyl acetic acid and proposed pathway for the metabolism of 4-chlorocatechol (Schmidt and Knackmuss, 1980; Chatterjee et al., 1981).

alone does not support microbial growth, (2) transformations of the cometabolite involve advantageous utilization of existing nonspecific enzyme systems, (3) utilization of the cometabolite is associated with increased oxygen consumption, and (4) production of waste products is stochiometrically related to the disappearance of the cometabolite (Hul-


31

2.44

ac
200°C 1.57 3.41 x mm Hg 1.16 X lo-' 1.94 0.1 150°C Stable Stable Resistant Resistant 6.5 360 480

46


CI CH,

METOLACHLOR

CGN

l

2,4-D

CH,OCH,NCCH,CI

I

OH

BROMOXYNIL

ALACHLOR

FIG.24. Structures of some important herbicides.

identification of biological factors involved in the metabolism of dicamba has not been accomplished. There are no previous reports in the literature on the metabolism of dicamba by a pure culture of microorganisms. Increased bacterial growth and increased O2 consumption have been reported when soil organisms capable of growth on o-anisate were grown on dicamba in the presence of this compound (Ferrer et a]., 1985). However, no analytical data on the disappearance of dicamba were presented. The biological degradation of dicamba has been investigated and documented by Krueger et al. (1989, 1990, 1991; Krueger, 1989). As mentioned previously, this chloroaromatic compound affects the growth of annual and perennial broadleaf weeds, as well as several grassy weeds. Unfortunately, dicamba affects the growth of certain commerical crops, namely, soybean. Using I4C-labeled dicamba, Krueger demonstrated the ability of Pseudomonas species to completely miner-

47


6+ @ 6 3,4-dichlorophenol

CI

CI

distilled off

-

CI

pressure

CI

pressure OH', MeOH

Q: J COOH

3.6-dichlorosalicylate

CI

COOH

dicamba

cl&~H~=-~ OCH3

CI

FIG.25. Chemical synthesis of dicamba.

alize dicamba to ' * C 0 2 . Through thin-layer chromatography and highpressure liquid chromatography, Krueger and Cork proposed a pathway for microbe-mediated dicamba degradation (Fig. 26). By the structure of the intermediates and the absence of anaerobic metabolism of dicamba, the mechanism of dicamba degradation is thought to be similar to those previously discussed.

48

DOUGLAS J, CORK AND JAMES P. KRUEGER

co <x)(IH

+ g . - - + c Q 2

OH

0

cl

FIG.26. Proposed pathway for the degradation of dicamba (Krueger, 1989).

Bacteria capable of utilizing the herbicide dicamba as a sole carbon source were obtained by enrichment from soil and water with a long history of dicamba exposure. Three strains that grew rapidly in liquid medium containing dicamba were identified by biochemical tests and by GC analysis of whole cell fatty acid profiles as follows: strain DI-6, Pseudomonas sp.; strain DI-7, Moraxella sp.; and strain DI-8, Pseudomoms sp. The organisms isolated that degrade dicamba represent genera that are commonly found in water, soil, and sewage and are often responsible for the decomposition of man-made chemicals in the environment. Members of the genus Pseudomonas are known to possess a wide range of degradative activities and have the ability to develop new degradative activities. Long-term intermittent exposure (approximately 25 years) of microbes to dicamba in the storm water retention ponds at the dicamba manufacturing facility appears to have provided the selective conditions necessary to develop dicamba-degrading capabilities. An excellent example of the development and spread of herbicide-degrading activity has been demonstrated for phenoxyacetic acid herbicides. A large number of the different genera that have been isolated are capable of degrading these herbicides (Helling et al., 1981).


49

XIII. Growth Kinetics in Liquid Culture

When dicamba-degrading organisms (strains DI-6, DI-7, and DI-8) were grown in liquid media containing 1000 pg/ml dicamba, an average of 97% of the chloride was released after 30 hours (Table XIV). The absorbance at 274 nm (wavelength of maximum absorbance for dicamba) decreased to zero. Chloride release and the decrease in absorbance at 274 nm indicate complete removal of dicamba. Radioassay of the media indicates that up to 80% of the substrate carbon is mineralized to COz (Fig. 27). The substrate carbon remaining in the media was probably incorporated into cell biomass because the cell number increased (Table XVI). Initial reactions in the metabolism of dicamba by microorganisms and subsequent increases in biomass require an expenditure of energy by the microbe. The evolution of l4COZfrom phenyl-labeled [14C]dicambasuggests that dicamba is fragmented to compounds that are channeled into oxidative cycles (the Krebs cycle) from which the organism can derive useful energy. Mineralization of 2,4-D to COz, with a corresponding increase in biomass, has also been demonstrated. Up to 40% of the substrate radiocarbon from 2,4-D was mineralized to COz in 20 hours. (Amy et a]., 1985). 80'

70

..

60. m

250. C

8

a 30.

....QY, ,.,-0. .........

2010.

6

.... ........... ........ ............... Control .................. 5....... ...A/

.'

..........

20 30 Time (Hours) FIG.27. Removal of soluble 14C from liquid medium containing 1000 pg/ml (14C]dicamba by three dicamba-degradingstrains and an uninoculated control. 0

10


50

TABLE XVI GROWTH KINETICSOF DICAMBA-DEGRADING ORGANISMS I N LIQUIDCULTURE CONTAINING 1000 pg/ml OF (14C]DICAMBA

Strain DI-6

DI-7

DI-8

Time (hours) 0 6 24 30 0 6 24 30 0 6 24 30

Viable cells perm1

2.1 x 7.5 x 1.3 x 6.8 X 7.8 x 8.5 X 9.8 X 1.0x

lo8 108 109

lo9 107

lo8 10’ 109

1.ox lo8

1.4 X lo8 1.1x 109 1.0x 109

Absorbance (274 nm)

Dicamba (pg/ml)”

2.36 2.38 NDc ND 2.38 2.56 ND ND 2.43 2.56

818 828 c = c\

A/

\

/c-c\

FIG.8. Epoxidation by cytochromes P-450(Guengerich and Macdonald, 1984).

R1\

,c=c\

/ R2

-\ /R' / c - C\-R2

FIG. 9. Oxidation group migration by cytochromes P-450 (Miller and Guengerich, 1982).

CH-CHC=C FIG. 10. Dehydrogenation by cytochromes P-450(Ortiz de Montellano, 1989).

+ leC- R c. + R FIG.11. Reduction by cytochromes P-450(Pohl et al., 1984).

-

XRR'COOH XRCO + R'H FIG. 12. Reductive cleavage of hydroperoxides by cytochromes P-450(Coon and Vaz, 1988).

ill. Microbial Cytochromes P-450

A. PROCARYOTIC CYTOCHROMES P-450

Procaryotic P-450 enzymes are generally multicomponent soluble systems that fall within the Type I1 category (Fig. 1) with respect to the organization of their electron transfer components. These P-450 systems were extensively reviewed by Sligar and Murray (1986). The majority of these enzymes are either extremely substrate specific or act only on a limited number of structurally related substrates. However, isolated examples of procaryotic P-450 enzymes with a different redox component organization or with versatile catalytic activities have also been

140

F. SIMA SARIASLANI

reported. Various procaryotic P-450 systems, which are involved in xenobiotic oxidation, are reviewed below: 1. Pseudomonas putida

Cytochrome P-450 systems have been identified in various strains of P. putida (Unger et al., 1986a). a. Pseudomonas putida (ATCC 17453). The cytochrome P-450 (P450,,,) system of P. putida involved in stereo- and regiospecific hydroxylation of camphor at the 5-eXO position (Fig. 13A) has been studied

(A)

P. pulida (Wild type) v

9

&.-

5-Exo ( 100%)

Camphor (B) P. pulida (Y96 mutant)

-

5-Exo (92%)

6-Ex0 (4%) 3-EXo (4%)

5-Exo (82%)

6-Ex0 (15%)

3-Exo (2.5%)

5-Exo ( 6 3 % )

6-Ex0 (31%)

347x0 (6%)

5-Exo (45%)

6-Ex0 (47%)

3-Exo (2.5%)

9-Hydroxy (0.1%)

(A) P. pufida (Wild type)

-

9-Hydroxy (0.5%)

l-CH3-norcamphor

(B) P. pufida (Y96 mutant)

-

(A) P. pulida (Wild type)

Norcamphor

(B) P. pufida (Y96 mutant)

-

$-Ex0 (36%)

6-Exa (41%)

3-Exo (23%)

FIG.13. (A] Oxidation of camphor, l-CH,-norcamphor, and norcamphor by P-450,., of Pseudornonas putida. (B) Oxidation of camphor, l-CH3-norcamphor, and norcamphor by P. putida (Y96 mutant) (Atkins and Sligar, 1988,1989).

MICROBIAL CYTOCHROME P-450

141

in great detail. Comprehensive reviews discussing this cytochrome P-450 system and its crystal structure have previously been published (Sligar and Murray, 1986; Poulos, 1986). Herein, the most recent information on this P-450 system will be reviewed. This multicomponent system, which was initially identified and characterized by Gunsalus and co-workers in the 1960s (Hedegaard and Gunsalus, 1965), is currently being studied in several laboratories. The P-45OC,, system is composed of a 45,000-Da cytochrome P-450 [P450cam), a 11,726-Da [2Fe-2S] ferredoxin (putidaredoxin), and a 48,000Da FAD-containing NADH-specific ferredoxin reductase (putidaredoxin reductase). Each of these components has been studied in great detail (Unger et a]., 1986b). The crystal structure of P-45OC,, has been resolved with highresolution X-ray crystallography (Poulos, 1986). Elucidation of the P-45OC,, structure has made considerable contributions to the advancement of our understanding of the mechanism of action of P-450 enzymes. These studies have shown that P-45OC,, is an asymmetrical, 30-kthick triangular prism-shaped protein with 1 2 helical segments (A-L), which contribute to 45% of its structure, and four antiparallel p pairs. The heme, which is completely surrounded by the protein, is positioned between helix L [proximal helix) and helix I (distal helix). The substrate is buried in an internal pocket above the heme distal surface adjacent to the oxygen-binding site. The orientation of camphor in the active site allows for contact between its C5 and the activated iron-bound oxygen atom for the production of the 5-eXOhydroxycamphor. The structures of both camphor-bound and camphorfree forms of P-45OC,, have been studied in detail (Poulos, 1988). In the camphor-free P-45OC,,, the substrate pocket of the enzyme is filled with five water molecules. In addition, a sixth water molecule forms a ligand with the heme iron at L6. No significant conformational changes are observed upon binding of the substrate to the enzyme. Raag and Poulos (1989) studied the X-ray structure of the ternary complex between CO, camphor, and P-45OC,, and showed that whereas camphor maintains its nonbonded contact with CO, it moves about 0.8 A, allowing the oxygen atom of the CO molecule to nestle in the enzyme’s groove. Crystallography studies have also indicated that the high stereo- and regiospecificity of P-45OC,, are achieved through specific interaction of several amino acid residues, with the substrate, at the active site of the enzyme (Poulos et al., 1987).In particular, studies with camphor-bound P-450 have indicated the presence of a hydrogen bond between the carbonyl moiety of camphor and the active site residue tyrosine-96. In

142

F. SIMA SARIASLANI

addition to X-ray crystallography, site-directed mutagenesis has been used to study the mechanism of camphor hydroxylation by P-45Ocam. Recently, Atkins and Sligar (1988, 1989) described the influence of the site-specific mutation of three active site residues-valine-295, valine247, and tyrosine-96-on the regiospecificity of P-45OCamduring oxidation of camphor and two of its analogs, 1-CH3-norcamphorand norcamphor (Fig. 13B). Replacement of tyrosine-96 by phenylalanine (mutant Y96F), through site-directed mutagenesis, destroys the strict regiospecificity observed in camphor oxidation (Fig. 13B). In addition to 5-exohydroxycamphor, 6-exo-, 3-eXO-, and 9-hydroxycamphors were also formed during camphor oxidation by this mutant. On the other hand, oxidation of norcamphor and l-CH,-norcarnphor by the wild-type P . putida usually results in the formation of several hydroxylated metabolites. Hydroxylation of norcamphor by the Y96F mutant generated similar metabolites, except that hydroxylation at the 3-position was significantly increased (from 2.5 to 23%). A similar effect was observed when 1-CH3-norcamphor was used as substrate for this mutant. These results underline the directive role played by the putative hydrogen bond between the substrate and tyrosinel96 of the active site in the regiospecificity of P-450,,,-catalyzed reactions. Atkins and Sligar (1989) have also studied the effect of hydrophobic interactions between substrate and the active site of P-45OCa, by changing valine-295 to isoleucine (mutant V295I) and valine-247 to alanine (mutant V247A). When camphor binds the enzyme, these residues are in contact with its 8,9-gem-dimethyl groups and 6- and 10-carbon atoms. It was concluded that these hydrophobic interactions had negligible effects on the control of regiospecificity of camphor hydroxylation by P-45OCa,. On the other hand, because hydroxylation of camphor analogs (1-CH3-norcamphor and norcamphor) was influenced by these mutations, Atkins and Sligar (1989) proposed that the steric bulk of the side chain of alanine-247 affects substrate orientation in these reactions. Recently Imai et al. (1989) showed that threonine-252 plays an important role in camphor hydroxylation by P-450,,, . When threonine-252 was replaced by alanine or valine, the reaction was uncoupled and HzOz was produced from Oz. As a result, the amount of 5-exohydroxycamphor produced was appreciably decreased. On the other hand, when threonine-252 was replaced by serine, the monooxygenase activity was not impaired. Based on these results, Imai et al. proposed that uncoupling of oxygen and subsequent HzOz production is due to the lack of a hydroxyl side chain in alanine and valine in the mutant enzyme.


143

As mentioned above, P-45OCa, is a multicomponent system that, in addition to the P-450 component, requires participation of NADH, putidaredoxin, and putidaredoxin reductase for substrate turnover. In an interesting study, Smith Eble and Dawson (1984) substituted putidaredoxin reductase and putidaredoxin with an artificial electron mediator such as phenazine methosulfate (PMS) and studied the hydroxylation of camphor by this system. In this reaction, in which transfer of electrons from NADH to P-45OC,, was mediated by PMS, 5-exohydroxycamphor was formed as the reaction product. Addition of 2,3dimercaptopropanol or putidaredoxin to the NADH/PMS/P-45OCa, system was accompanied by a 4- or a 20-fold increase in product formation, respectively. These effects were enhanced dramatically when O2was constantly bubbled through the system. These studies provided the first example of an oxygen-dependent P-450-catalyzed reaction in which an artificial electron mediator replaced the indigenous redox proteins. Molecular biology studies have revealed that the CAM plasmid in P. putida PpGl encodes for the enzymes involved in the early steps of camphor oxidation (Koga et al., 1985, 1989; Unger et al., 1986b). These include 5-exo-hydroxycamphor dehydrogenase (camD gene), and the three P-45OCa, components, putidaredoxin reductase (camA), putidaredoxin (camB), and P-450,,, (camC). The genes form the camDCAB operon and are under negative control by the camR gene located immediately upstream from the camD gene (Koga et al., 1986). In an independent study, Peterson et al. (1990) reported that a 2.2 x lo3 base pair BamHI-StuI fragment from camphor-grown P. putida cells contained two open reading frames that encode for putidaredoxin and putidaredoxin reductase. A potential transcription termination site was found 3' to the putidaredoxin-coding region. The sequence coding for the FAD-binding site in the reductase is close to the N-terminus (residues 6-24), whereas the NADH-binding site lies between residues 151 and 169. Both genes were subcloned and were independently expressed in Escherichia coli. When the rare start codon of GTG of putidaredoxin reductase was changed to ATG, an 18-fold increase in the level of expression of this protein was observed (Peterson et al., 1990).This is a significant finding because it allows isolation of a large amount of protein for detailed biochemical studies. In addition to 5-ex0 hydroxylation of camphor, P-45OC,, has been used for other types of reactions. Reductive dehalogenation of chloropicrin, polyhalomethanes, and chloronitromethanes was accomplished by whole cells of P. putida containing P-45OCa, (Castro et al., 1983, 1985). The final product of chloropicrin degradation was nitromethane

144

-

F. SIMA SARIASLANI

-

C13CNOz CIZCHNOZ CICHzNOzCH3NOz FIG.14. Oxidation of chloropicrin to nitromethane by Pseudomonas putida containing P-450 cam (Castro et al., 1983, 1985).

(Fig. 14). Trichloronitromethane and chloroform were the fastest and the slowest reacting substrates, respectively. Castro et al. (1988) later compared the stoichiometries and kinetics of reduction of trichloromethane, bromotrichloromethane, carbon tetrachloride, ethylene dibromide, and 1,2-dibromo-3-chloropropaneby iron(I1) deuteroporphyrin IX, rat liver P-450PB,P-45OCam,and whole cells of P. putida. Polyhalomethanes underwent reductive hydrogenolysis in all systems studied. Quantitative conversion of the vicinal halides by all the P-450 systems studied resulted in the formation of olefins (Fig. 15). These studies indicated that halides that undergo a quick bond cleavage step exhibit a steric inhibition effect on P-45OCam,whereas the mammalian P - 4 5 0 ~ ~ remains unaffected. Castro et al. concluded that the heme moiety in P-450~ could ~ be more accessible than the heme in P-45Oca,. b. Pseudomonas putida (PpG777). A second cytochrome P-450 system, which is induced by linalool and performs the first two steps of linalool oxidation (Fig. 16), has been identified in P. putida (incognita, strain PpG777) (Ullah et al., 1990). This three-component system consists of a 43,700-Da FAD-containing reductase (reductaseu,), a 12,800Da ferredoxin with a [2Fe-2S] cluster (ferredoxinli,), and a 44,800-Da P-450 component (P-4501~~). In reconstituted systems in which putidaredoxin reductase was used with P-45Oljn and ferredoxinli,, hydroxylation of linalool occurred at less than 10% of the rate observed with the native component. The second product of the linalool hydroxylation reaction is 8-oxolinalool, which is also mediated by the linalool monooxygenase system. In spite of certain similarities, P-450cam and P-45O1in possess distinct physical properties. Hoa et al. (1989)investigated the pressure-induced spectral changes in these cytochrome P-450 systems. P-4501~~ is much more stable under increasing pressure compared to P-45Ocam. On the other hand, although formation of P-42Olin was accompanied with precipitation of the protein and its unfolding, interconversion of P-45Ocam to P-42OCa, occurred without loss of its tertiary structure. Hoa et al.

FIG.15. Conversion of vicinal halides to olefins by various P-450 systems (Castro et al., 1988).

145


Linalool

8-Hydroxy lindool

8-Oxolinalool

FIG.16. Oxidation of linalool by P-4501i, of Pseudomonas putida (incognita) (Ullah et al., 1990).

(1989) proposed that p-4501in possesses a rigid structure but that P450,,, is more elastic. 2. Bacillus megaterium

Independent studies in two laboratories with two separate strains of B. megaterium have resulted in identification of two vastly different cytochrome P-450 systems in this organism. a. Bacillus megaterium (ATCC 14581). Extensive studies performed in Fulco’s laboratory at the University of California in Los Angeles have led to the characterization of a unique cytochrome P-450 from B. megaterium (ATCC 14581) (Miura and Fulco, 1975; Matson and Fulco, 1981) that hydroxylates both long-chain fatty acids and their corresponding amides and alcohols. The position of the hydroxyl group on the substrate molecule regulates its oxidation by this P-450 enzyme. The 0-1,w-2, and w-3 monohydroxy products of the unsubstituted and saturated fatty acids cannot act as substrates for the enzyme. Among the saturated fatty acids, the order of activity for hydroxylation of the substrates was c15 > c16 > c14 > C17 > c13 > C18 = C12. For amides this order was C14 > Clz > C15 > c16 and for alcohols it was c 1 4>c 1 3 = c15 > C12 > CIS. Kim and Fulco (1983) investigated the effect of 19 barbiturates on P-450 induction in B.megaterium and found that 13 of these chemicals induced the enzyme. Among the substrates tested, secobarbital, thiamylal, and methohexital were very good inducers. Kim and Fulco reported that the enzyme induction was higher with lipophilic substrates. At least three distinct P-450 enzymes are induced by phenobarbital in B. megaterium. One of these P-450 enzymes, P-45OBM-1,which is a 47,439-Da enzyme with 410 amino acids (Schwalb et a]., 1985),has recently been cloned (He eta]., 1989).P-450BM-1exhibits 27% sequence homology to P-450,,, but is four amino acids smaller. Fulco and co-workers concluded that P - 4 5 0 ~belongs ~ - ~ to a new gene family. This enzyme is distinct from P-45oBM-3,which will be discussed

146

F. SIMA SARIASLANI

later. The catalytic function of P-450BM-1is still unknown; however, this P-450 exhibits fatty acid monooxygenase activity in the presence of iodosylbenzene diacetate. Recent studies in Fulco’s laboratory have concentrated on the third enzyme of this series, designated P-450BM-3. P - 4 5 0 ~ ~is3a unique single-component enzyme system that performs the NADPH-dependent oxygenation of a variety of fatty acids in the absence of other electron transport proteins. Due to the presence of noncovalently bound FAD and FMN in its structure, P-450BM-3 possesses a distinctive absorption spectrum in the 450- to 475-nm region. This enzyme contains both a P-450 and a NADPH:P-450 reductase component in a single protein. Phenobarbital induces cytochromes P-450 in other strains of B. megaterium that cross-react with P-45oBM-3 antibodies. Nahri and Fulco (1987) showed that when the substrate-bound P-45oBM-3 is subjected to limited trypsin digest, two domains (polypeptides) with molecular weights of 66,000 and 55,000 are formed. The 66,000-Da domain, which contains the FAD and the FMN moieties, performs the NADPH-dependent reduction of cytochrome c and is derived from the C-terminus of P-450BM-3. Three heme-containing peptides (TI, TII, and TIII) constitute the 55,000Da domain. All these peptides exhibit the characteristic spectral properties of P-450 in the presence of CO and dithionite. The 55,000-Da TI component, which binds substrate, contains the N-terminus of P-450BM-3. The TI1 and TI11 components do not bind substrate and lack the first 9 and 15 amino acids of the N-terminus of P-450BM-31respectively. When P-~~CIBM-~ was digested in the absence of substrate, only the 54,000-Da TI1 and the 53,500-Da TI11 peptides were generated, indicating that perhaps one or more residues of the first nine N-terminus amino acids of P-45oBM-3 are involved in substrate binding. Following cloning and sequencing of the self-sufficient P-450BM-3, Wen and Fulco (1987) succeeded in expressing this gene constitutively in E. coli. Because this recombinant constitutive protein could not be induced by phenobarbital, they introduced the cloned gene back into B. megaterium and observed that although its expression was constitutively repressed, it was induced by phenobarbital. Based on these results Wen and Fulco proposed that perhaps interaction of phenobarbital with a repressor-type molecule, which is absent in E. coli, is a prerequisite for P-450BM-3 induction. After sequencing a 5-kb DNA fragment that contained the P-45oBM-3 gene, Ruettinger et al. (1989) estimated a molecular weight of 117,641 for the self-sufficient protein. The P-450 domain of P-450BM-3 exhibits homology (25%) with the fatty acid o-hydroxylase of P-450 family IV. On the other hand, the reductase domain of this enzyme exhibits 33%


147

sequence homology with the NADPH:reductases of mammalian liver. Although both P-450 and the reductase domains of P-450BM-3 define new gene families, they contain highly conserved regions that exhibit up to 50% sequence homology with their mammalian counterparts. S1 mapping of the mRNA for P-45oBM-3 indicated a nucleotide length of 3339 2 10 bases for this molecule. Wen et al. (1989) recently examined the effects on gene expression of 5' and 3' deletion derivatives obtained from a 1.6-kb DNA fragment that includes the regulatory region and 88 bases of the N-terminus of P-450BM-3. They concluded that the P-450BM-3 gene, which is under positive control, requires binding with at least one trans-acting factor, probably a protein, to activate transcription from the P-450BM-3promoter. Barbiturates might facilitate binding of this protein with the gene to exert their inducer effect. It is also possible that barbiturates either increase the rate of the protein synthesis or retard its degradation. Additionally, barbiturates might also facilitate the release of the protein from the cell membrane and thus make it accessible to plasmid or chromosomal DNA in the cytoplasm. Detectable expression of P-45oBM-3 is achieved only when a minimum of 0.6-0.7 kb of chromosomal DNA, immediately upstream from the translation start site of the BM-3 gene, is present. Boddupalli et a]. (1990),who have recently overexpressed P-45oBM-3 in E. coli, have shown the hydroxylation of palmitic acid by this recombinant P - ~ ~ O BatMa- rate ~ of 1600 mol/min/mol of heme. Lauric and myristic acids were hydroxylated, regardless of their initial concentrations, into two metabolites. A 1:l stoichiometry of oxygen or NADPH consumed per molecule of substrate oxidized was observed with these substrates. However, when palmitic acid was used as substrate, the ratio of oxygen or NADPH consumed per molecule of substrate oxidized was dependent on the concentration of the substrate used. At high concentrations (>zoo p M ) ,monohydroxylation of palmitic acid resulted in the formation of three metabolites and a stoichiometry of 1:l was observed for oxygen and palmitic acid consumption. When lower concentrations (5 pm) 2. Galactose 3. Melibiose 4. Conjugation 5. Raffinose 6. Raffinose 7. Trehalose 8. Maltose 9. Pseudohyphae 10. Growth at 37°C 11. Xylose 12. Growth without vitamins 13. Trehalose

2

3 Saccharomyces pastorianus 5 Zygosaccharomyces microellipsoideus 7 8

Saccharomyces kluyveri 10 11

Pichia fermentans Issatchenkia orientalis Candida glabrata

4 Saccharomyces bayanus Saccharomyces cerevisiae 6

Zygosaccharomyces bisporus 9

Candida stellata Saccharomyces exiguus 13 Pichia membranaefaciens 12

Issatchenkia terricola Candida inconspicua

See text for explanation of key. Characters include assimilation of substrates (cellobiose, erythritol, galactose, inositol, maltose, mannitol, melibiose, nitrate, raffinose, trehalose, and xylose); urease +, hydrolysis of urea. a

I

h X

>

3 a m

+ c

rn a 0

e

-E .-

24

.-I

M

A

W

u

U

-0

m

n

I

242

Debaromyces polymorphus Dekkera anomala Dekkera intermedia Endomyces fibuliger Geotrichum candidum Hanseniaspora occidentalis Hanseniaspora osmophila Hanseniaspora uvarum Hyphopichia burtonii Issatchenkia orientalis Issatchenkia terricola Kluyveromyces lactis Kluyveromyces marxianus Kluyveromyces thermotolerans Lodderomyces elongisporus Metschnikowia pulcherrima Pichia anomala Pichia canadensis Pichia etchellsii Pichia farinosa Pichia fermentans Pichia guilliermondii Pichia jadinii Pichia membranaefaciens Pichia ohmeri Pichia subpelliculosa Rhodosporidium infirmominiatum Rhodotorula glutinis Rhodotorula minuta Rhodotorula mucilaginosa Saccharomyces cerevisiae

Acetate, filament Acetate Spores hat shaped

- - - -

- - - -

+ +++++ + +-+++

- - - - - - -

- - - - - - - - - - - - - v -

_ _ _ ++ ++++-

-++++ + + + +

-

+ + v + + v - v v - v v

- - - -

+ + v + v + + v + +

+++++

v

v - - v + v v + v + + v v + v v

+ - - v +

+- ++-++

- - - - -

- - v v v

+--++ +--++

- - - v - v v + + - v - - - - v + - - v v v v - v - + - - v - v - - - + - - v - v v - - - - - + - v v v

- -

v - -

- -

+--

- -

++-

- - + + - + - t v - v v - +

+ + - +

- - - -

- - - - - - -

v - -

Apiculate Apiculate Apiculate Spores hat shaped Spores round Spores round Spores round, esters Spores round, esters Spores round Spores round Spores needle shaped Spores hat shaped Spores hat shaped Spores round Spores round Spores hat shaped Spores hat shaped Spores hat shaped Spores roundihat shaped Spores round/hat shaped Spores hat shaped Amylo + , slimy Amylo -, slimy Amylo Amylo -, slimy Spores round, lysine (continued)

TABLE I11 (Continued) Properties" Species

Saccharomyces bayanus Saccharomyces pastorionus Saccharomyces exiguus Saccharornyces kluyveri Saccharomycodes ludwigii Schizosaccharomyces octosporus Schizosaccharomyces pombe Sporobolomyces roseus Torulaspora delbrueckii Trichosporon cutaneum Trichosporon pullulans Wickerhamiella domercqiae Yorrowia lipolytica Zygooscus hellenicus Zygosaccharomyces bailii Zygosacchoromyces bisporus Zygosaccharomyces fermentati Zygosaccharomyces microellipsoides Zygosaccharomyces rouxii

a b c d e

-_--

v v

--__

f g h i j

+ v - + v + + + + v

k l m n o

- - - - -

+----

- - - - - - + + + - - - - - -

_ - -

v v

++++- +----

p q r s t

+ + + + + +

u v w x y

Spores round, lysine Spores round, lysine Spores round, lysine Spores round, lysine + Apiculate Spores round Spores round Amylo Spores round

- v v v - v v v v - - - v + v - - v - - v +

+--++

+ - - + v

+ + v v + v v v + v + v + v v

- - + - +

_ _

+ - + v +

- - _ -

+ +-+++ +++++

_ - - -

+ --_ ++ - - - - + - _ _ - +

v - -

- - v - - - - - - + - v v + v + v v + - v - v v + v v v + _ - v - - - - - -

- - - - -

-_--

+

Spores round Spores roundthat shaped

- + - v v + + - v v + - - v + - - v v + + + + v - + + v v - v v -

- - - - -

- - - - - - - - -

+----

+---+ ++-++ + - - - v + - V V +

Other

_-

v - -

Spores round Spores round Spores round Spores round Spores round

a a, Urease; b, erythritol; c, nitrate; d, cellobiose; e, mannitol; f , maltose; g, raffinose; h, galactose; i, trehalose; j, melezitose; k, melibiose; 1, lactose; m, rhamnose; n, xylose; 0,inositol; p. fermented glucose; q, cycloheximide;r, without vitamin; s, growth at 3%; t, 50% glucose; u, arthroconidia; v, true hyphae; w, pseudohyphae; x, pellicle; y, red colony. Notation in the table body has the following meaning: +, positive; -, negative; v, variable reaction; Amylo: starch production.

FOODBORNE YEASTS

245

C. DESCRIPTION OF MAINGROUPS OF FOODBORNE YEASTS The groups are described in the order of the groups of the key in Table 11. 1. Urease-Positive Yeasts

The first group includes urease-positive yeasts, among them all basidiomycetous species considered. As interesting as they are from a taxonomical standpoint, the small groups of basidiomycetous yeasts have little practical importance to the food industry. The basidiomycetous character can be indicated by the formation of dicaryotic hyphae with clamp connections and more unequivocally by the development of basidia or thick-walled teliospores. Most basidiomycetous yeasts are, however, heterothallic and the different mating types develop separate colonies. Hence, sexual reproduction can be observed only rarely. Nevertheless, the DBB color reaction and the urease test have proved to be a useful way of revealing the basidiomycetous character of anamorphic forms (Hagler and Ahearn, 1981). The positive color reactions often coincide with the formation of carotenoid pigments, mucoid colonies, or ballistoconidia, and with the lack of fermentation. Based upon these traits, the basidiomycetous nature of an anamorphic yeast can easily be recognized even in the absence of a known teleomorphic (perfect) state (Weijman et al., 1988). According to the rules of yeast nomenclature, a species has to bear the name of the teleomorph, whenever it is known. Accordingly the basidiomycetous species may belong to the genera Sporidiobolus, Rhodosporidium, Leucosporidium, Filobasidium, and others (van der Walt, 1987). However, as a rule, they are isolated from natural sources in their anamorphic forms, whose names are better known, such as Sporobolomyces, Rhodotorula, and Cryptococcus. Proposals have been made that the genus Candida be restricted only to ascomycetous anamorphs, and those former Candida species possessing basidiomycetous characters have been transferred to the genus Rhodotorula or Cryptococcus (van der Walt, 1987; Weijman et al., 1988).Of the foodborne yeasts, only about 15% are of basidiomycetous species. They are mostly associated with plant materials and occur infrequently; however, a few species are rather common and widespread in various foods. The red-colored and often mucoid colonies of Rhodotorula species easily catch the eye. The most common species is R. glutinis, which has been reported from fruits, vegetables, grains (Spicher and Mellenthin, 1983; Rale and Vakil, 1984; Messini et al., 1985; Parish and Caroll, 1985;

246

T. DEAK

DeAk and Beuchat, 1988), alcoholic and nonalcoholic beverages (Put et al., 1976; Lafon-Lafourcade, 1983; Ruiz et al., 1986; Back, 1987), milk, dairy products (Suarez and Inigo, 1982; Fleet and Mian, 1987),fresh and chilled meat, poultry, fish, seafoods (Comi and Cantoni, 1985; Banks and Board, 1987; Jay, 1987), and chilled salads (Kobatake and Kurata, 1980b). The somewhat less frequently observed species R. mucilaginosa (R. rubra) and R. minuta, as well as the similarly red-pigmented S. roseus, are mainly found on fruits and in fruit products (Buhagiar and Barnett, 1971; Rale and Vakil, 1984; De6k and Beuchat, 1988),as well as on meat and in meat products (Johannsen et al., 1984; Comi and Cantoni, 1985; Jay, 1987). The most important physiological property of these species is their capability of growth at low temperatures (Davenport, 1980a). In turn, their heat resistance is low (for R. mucilaginosa the D value at 51°C was in the range of 30 minutes) (Beuchat, 1983a). Although some Rhodotorula strains are able to grow at a 10% NaCl concentration and possess lipolytic activity (Comi and Cantoni, 1985),they are not among the determining spoilage yeasts in foods. The Cryptococcus species are characterized by the assimilation of inositol and glucuronate (Barnett et al., 1983). This holds true for R. infirmo-miniatum, which has a Cryptococcus anamorph. These yeasts share the food habitats with the rhodotorulas, and are mostly found in fruits, juices, must, vegetables (Buhagiar and Barnett, 1971; Parish and Caroll, 1985; Deak and Beuchat, 1988), grains, flour (Kurtzman et al., 1970; Spicher and Mellenthin, 1983), meat, poultry, fish (Dalton et al., 1984; Lowry and Gill, 1984; Jay, 1987), and sometimes in cheese and chilled salads (Banks and Board, 1987; Kobatake and Kurata, 1980a). The most frequenty observed species are C. albidus and C. laurentii. Among cryptococci there are a number of psychrotrophic and even psychrophilic strains growing in the temperature range from 0 to 5°C (Kobatake and Kurata, 1980b). Cryptococcus humicolus has been transferred from the genus Candida and develops both true hypha and pseudohypha. The genus Trichosporon is characterized by true hyphae breaking into arthroconidia as well as by a budding yeast phase. The morphologically similar genus, Geotrichum, is distinguished by its negative urease reaction and lack of budding cells (Weijman, 1979). Two species of Trichosporon, cutaneum and pullulans, are fairly widespread in various foods (Buck et al., 1977; Spicher and Mellenthin, 1983; Suresh et al., 1982; Sandhu and Waraich, 1984; Johannsen et a]., 1984; Ravelomanana et al., 1985). They possess extracellular enzymes, proteases, lipases, and pec-

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tinases, and often contribute to spoilage (Kobatake and Kurata, 1983b; Comi and Cantoni, 1983). In addition to the basidiomycetous yeasts, two endomycetous genera are exceptional in that they also give positive urease reactions (but are negative in the DBB test). These genera are Yarrowia and Schizosaccharomyces, which are also peculiar in other respects. The yeast Y. lipolytica has long been known for its ability to split fats. It occurs frequently on meat and meat products, butter, mayonnaise, salad dressings, and cheese (Muys, 1971; Kobatake and Kurata, 1980a,b; Hsieh and Jay, 1984; Brocklehurst and Lund, 1985; Banks and Board, 1987), and is also often isolated from fruits and soft drinks (Sand, 1974; Put et al., 1976). Since the discovery of its mating types, the species has been classified in various perfect genera such as Endomycopsis, Saccharomycopsis, and eventually Yarrowia (van der Walt and von Arx, 1980). The yeast Y. lipolitica forms budding cells, pseudohyphae, and septate hyphae, is nonfermentative, and assimilates only erythritol and mannitol but none of the common mono- and disaccharides (Barnett et a]., 1983). The genus Schizosaccharomyces belongs to the group of vigorously fermenting classical yeasts, from which it differs by the mode of vegetative reproduction called fission. Budding never occurs. The cell wall composition of these species also differs from the rest of the yeasts, and studies on 5 S ribosomal RNA have revealed that this genus represents a separate phylogenetic branch among yeasts (Walker, 1985). It has been proposed to split the four recognized species into three separate genera (Schizosaccharomyces, Octosporomyces, and Hasegawaea; von Arx and van der Walt, 1987), but for convenience the single former name is retained here. Schizosaccharomyces species are characterized by strong fermentation of sugars, They require vitamins for growth and often develop poorly on many media and in assimilation tests. Spores are formed after conjugation of cells and this is accompanied by the synthesis of starchlike compounds whose presence in the mature sporangia can easily be demonstrated using iodine solution. Schizosaccharomyces species develop well at 37°C and slightly above. They are xerotolerant, particularly S . octosporus [Octosporomyces octosporus), whose occurrence is mainly confined to high-sugar-containing products (Tokouka et al., 1985; Poncini and Wimmer, 1986).More widespread and frequent is S. pombe, which often occurs in must and wine and is able to convert malic acid to ethanol and C02 (Delfini, 1985). It is rather resistant to sulfur dioxide and preservatives (Warth, 1985).

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2. Erythritol-Assimilating Yeasts

The second group is split from the rest of the yeasts by the assimilation of erythritol, which is a fairly stable property. Of the yeasts grouped here, D. hansenii is variable in this trait, and, if negative, it keys out in group 4. Debaromyces hansenii is one of the most common foodborne yeasts. It is characterized by small spherical cells in which a single spore may develop. Hence, it is not easily observable under the microscope; however, colonies become brown when spores develop abundantly. The anamorphic state, Candida famata (Torulopsis candida), is frequently found. Debaromyces hansenii ferments poorly if at all, whereas a similar but less frequent species. Debaromyces polymorphus shows rather strong fermentation. Debaromyces hansenii typically occurs in salt-containing foods such as cured meats, ham, sausages (Comi and Cantoni, 1980a, 1983; Dalton et al., 1984), fermented olives, brined vegetables (Ravelomanana et al., 1985; Garrido Fernandez et al., 1985), and soy sauce (Mizunoma, 1984). It grows in 21% (w/w) NaCl or 50% (w/w) glucose concentrations and its minimum a, value for growth is 0.65 (Tilbury, 1980b). The species is equally common in cheeses and other dairy products (Schmidt and Lenoir, 1980; Fleet and Mian, 1987), fish, shellfish (Jay, 1987), high-sugar products (Tokouka et al., 1985), fruit juices, must, wine, and beer (Suresh et al., 1982; Dragoni and Comi, 1985; Back, 1987). It is rather sensitive to heat; Beuchat (1981a) measured a D value of 1 2 minutes at 48°C. Debaromyces polymorphus has been found in ham and sausage (Leistner and Bem, 1970), wine (Lafon-Lafourcade, 1983), and dough (Barber et al., 1983). Two yeasts in this group, P. anomala and P. subpelliculosa, are better known as Hansenula species; however, Kurtzman (1984) proposed to merge the two genera. Both species strongly assimilate nitrate. Pichia anomala can be distinguished by its ability to grow without added vitamins. It primarily assimilates galactose, whereas P. subpelliculosa does not; in turn, the latter produces more abundant pseudohyphae and often also true hyphae. Spores, when they develop, are hat-shaped. The two species share similar habitats, such as fruits, must, wine, and fermented and pickled vegetables (Etchells et al., 1975; Rale and Vakil, 1984; Dragoni and Comi, 1985; Ravelomanana et al., 1985; Brackett, 1987). Of the two, P. anomala is found more frequently. Both are moderately xerotolerant (minimum a, for growth is 0.75; Tilbury, 1980b) and may occur in raw sugar, molasses, and confectionery products (Tokouka et al., 1985). Pichia subpelliculosa has been found in dough (Barber et

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al., 1983), and P. anomala has been found in grains, flour (Spicher and Mellenthin, 1983), poultry, beer, and soft drinks (Put et al., 1976; Hardwick, 1983;Jay, 1987).It possesses a moderate tolerance to preservatives (Warth, 1985). Another nitrate-assimilating yeast in this group is C. boidinii. Its additional distinguishing property is the long, cylindrical shape of its cells, forming well-developed pseudohyphae. Candida boidinii occurs in soft drinks, wine, and beer (Sand et al., 1976; Kunkee and Goswell, 1977; Back, 1987), and also in fermented olives (Garrido Fernandez et al., 1985) and dairy products (Tilbury et al., 1974). Candida diddensiae is characterized by its irregular cell shape and pseudohyphae. It has been recovered from meat, fish, and shellfish (Buck et al., 1977; Hsieh and Jay, 1984; Jay, 1987), as well as olives and soft drinks (Sand, 1974; Garrido Fernandez et al., 1985). Candida canterellii and P. farinosa do not produce pseudohyphae and have rather narrow assimilation spectra. Both can be found in must and wine (Kunkee and Goswell, 1977),the former in jams, dairy products, and shellfish (Tilbury et al., 1974; Tilbury, 1976; Buck et a]., 1977), the latter in soy sauce, fruits, and fermenting cocoa (Noda et al., 1982; Pignal et al., 1985; Ravelomanana et a]., 1985). Hyphopichia burtonii and E. fibuliger are characterized by producing true hyphae. In their filamentous form they are typical representatives of the so-called yeastlike organisms. Both are frequent in dough and bread (Spicher, 1986), as well as in certain oriental fermented foods (Cronk et al., 1977; Sakai et al., 1983). They also occur on fruits and in beverages (Rale and Vakil, 1984; Dragoni and Comi, 1985). Endomyces fibuliger is strongly lipolytic and may cause spoilage of cooking oil (Spicher, 1984). 3. Nitrate-Assimilating Yeasts

Assimilation of nitrate is a very stable property among yeasts and is used for distinguishing the third group in the simplified identification system. Nevertheless, the peculiar group of Dekkera (anamorph Brettanomyces) species is variable in this property and a positive reaction may be masked by acid production when tested on an indicatorcontaining medium, according to Pincus et al. (1988). A remarkable property of the yeasts in the genus Dekkera is their acetic acid production, which lends an easily recognizable odor to their cultures. These species ferment glucose aerobically and grow very slowly in all media. They develop only tiny colonies that are short-lived due to acid production. All species are confined to beverages, especially

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beer and soft drinks (Back, 1987; Verachtert and Dawoud, 1984; Ison and Gutteridge, 1987). Perhaps the most frequently seen species is D. intermedia, and of the several other species with rather similar characteristics, D. anomala is recognizable by its branching filamentous cells. Each Dekkera species has its anamorph counterpart in the genus Brettanomyces (Jong et al., 1985). Of the nitrate-positive yeasts considered in group 3, two species, W. domercqiae and P. canadensis do not ferment sugars. Cells of the former are very small, only 2-3 pm in diameter. Spore formation is rare; the anamorph is known as C. domercqiae. The species occurs infrequently; nevertheless it has been found in various foods, including dairy products (Tilbury et al., 1974), meat (Dalton et al., 1984), and wine (Kunkee and Goswell, 1977). Pichia canadensis ,and the fermenting P. jadinii were previously classified as Hansenula. Both are more frequent in their anamorphic states, Candida melini and C. utilis, respectively. They can be found in various foods, for example, wine, fermented vegetables, and dairy products, though neither is common (Tibury et al., 1974; Lafon-Lafourcade, 1983; Comi et al., 1981a). Candida utilis is a well-known fodder yeast produced in large scale on molasses and agricultural wastes (Berry et al., 1987).

A number of common foodborne Candida species belong to this group. Most of them were previously described as Torulopsis because they do not form pseudohyphae. This was considered an unstable property, and the former Torulopsis genus was merged with Candida (Yarrow and Meyer, 1978), with the consequence that the latter became very large and heterogeneous. Further proposals have been made that the genus Candida should retain only endomycetous anamorphs (Weijman et a]., 1988). Of the species considered here only one, Candida glabrata, is known in teleomorphic form: Citeromyces matritensis. This and the other Candida species (versatilis, lactis-condensi, etchellsii, and magnoliae), with the exception of C. norvegica, possess a certain degree of xerotolerance. They grow in 11% (w/w) NaC1; C. etchellsii grows even in 21% NaCl concentration, and C. lactis-condensi and C. matritensis grow in 57% (w/w) glucose (a, 0.865) concentration; in sucrose-glycerol syrup the minimum a, for growth was found to be 0.7 (Tilbury, 1980b). These species can often be found in foods with high sugar and salt concentrations (brines, concentrates, soy sauce, dried fruits) (Etchells et a]., 1975; Tilbury, 1976; Madan and Gulati, 1980; Noda et al., 1982),but they also frequently occur in fruit juices, wine, and dairy products (Goto, 1980; Suriyarachchi and Fleet, 1981; Lafon-Lafourcade, 1983).

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Candida versatilis, C. magnoliae, and C. norvegica has been found in meat and shellfish (Buck et al., 1977; Dalton et al., 1984; Comi and Cantoni, 1985). 4. Cellobiose-Assimilating Yeasts

The fourth group of the simplified identification system comprises yeasts that are negative in the previous tests (urease hydrolysis and erythritol and nitrate assimilation), but do assimilate cellobiose. Only one species, C. sake, is variable in this respect, and, if negative, it falls into the next group. In turn, three yeasts discussed previously may be considered here, too, i.e., D. hansenii, if it fails to assimilate erythritol, and D. anomala and D. intermedia, if their nitrate assimilation is negative. The characteristic cell morphology (apiculate, lemon-shaped cells) easily distinguishes the yeasts that bud in a bipolar configuration. The unusually large cells (over 10 pm in diameter) set S . ludwigii apart from the rest of the apiculate yeasts belonging to the genus Hanseniaspora. Slight differences can be found among Hanseniaspora species, and all have an anamorphic phase called Kloeckera. Saccharomycodes ludwigii and the Hanseniaspora species ferment strongly. The former possesses a high resistance to sulfur dioxide, but has low ethanol tolerance (Minarik and Navara, 1977; Goto, 1980). Hanseniaspora species also tolerate less than 6% (v/v) ethanol and are most frequently found at the start of the spontaneous fermentation of grape must (Parish and Caroll, 1985). The most common species, H. uvarum (K. apiculata), also occurs in beer, soft drinks, fruit juices, and on fresh fruits (Stollarova, 1976; Suresh et al., 1982; Hardwick, 1983; Dragoni and Comi, 1985). The habitat of S . ludwigii is confined exclusively to beverages, particularly wine and cider (Carr, 1984; Heard and Fleet, 1986a; Back, 1987). The ability to assimilate raffinose divides into two groups the rest of the yeasts considered in the fourth group of the key. Vigor of fermentation and formation of pseudohyphae and pellicle are the useful distinguishing characteristics for the identification of species. For example, D. hansenii ferments weakly or not at all, and usually forms a thick pellicle on the surface of liquid media, although it does not develop pseudohyphae. The species in the genera Kluyveromyces and Zygosaccharomyces strongly ferment glucose. In the genus Kluyveromyces, spores are mostly bean-shaped and liberate easily from the sporangium. However, heterothallism often precludes spore formation and the anamorphs are well known as Candida species. A number of previously described

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Kluyveromyces species were lumped into a single one, K. marxianus, based on sexual hybridization and DNA homology studies. Due to this, however, most characteristics of the species become variable and the identification is difficult. Recently, some species have been reinstated, and both K. marxianus (synonyms K. fragilis and K. bulgaricus; anamorphs C. kefir and C. pseudotropicalis) and K. lactis (C. sphaerica) are recognized. Both species are capable of utilizing lactose; hence they are most often found in cheese and other dairy products (Schmidt and Lenoir, 1980; Engel et al., 1986; Fleet and Mian, 1987), Kluyveromyces marxianus, which is moderately xerotolerant and rather heat resistant (D, 60°C, for ascospores is 30 minutes in 10% sucrose solution; Put and De Jong, 1982b), may occur in molasses and sugar cane (Barwald and Hamad, 1984) as well as in must and wine (Kunkee and Goswell, 1977). A third species, Kluyveromyces thermotolerans, keys out in the next group because it does not utilize cellobiose, whereas Z. fermentati is a unique species among Zygosaccharomyces in that it assimilates cellobiose. Conjugating cells are often observed and this enhances identification. The species is uncommon and mostly found in must, wine, and soft drinks (Sand et al., 1976; Khayyat et al., 1982; Ruiz et al., 1986). Well-developed pseudohyphae are one of the distinguishing characteristics for four species treated here. Pichia ohmeri and C. intermedia also produce pellicles, but P. guilliermondii and C. steatolytica do not. The two Pichia species are usually heterothallic and are often found as nonsporing anamorphs, C. guilliermondii var. guilliermondii and C. guilliermondii var. membranaefaciens, respectively. The latter and C. steatolytica also produce true hyphae. The teleomorph of C. steatolytica has been recently described as Zygoascus hellenicus (Smith, 1986), but no teleomorph is yet known for C. intermedia. Pichia guilliermondii is the most frequently found member of this group in foods; it occurs in fermenting vegetables, soft drinks, wine, seafoods, syrups, and cane juice (Torok and DeAk, 1974; Buck et a]., 1977; Tilbury, 1976; Garrido Fernandez et al., 1985). Pichia ohmeri has been found in similar products (Goto and Yokotsuka, 1977; Tokouka et al., 1985; Atputharjah et al., 1986). Candida intermedia occurs frequently on meat and meat products, as well as in dairy products and beverages (Tilbury et al., 1974; Sand et al., 1976; Comi and Cantoni, 1985; Jay, 1987). Zygoascus hellenicus has been found in grapes and wine, as well as beef and shellfish (Buck et al., 1977; Goto, 1980; Hsieh and Jay, 1984; Guerzoni and Marchetti, 1987). Another four species occurring frequently in foods assimilate cellobiose but not raffinose. Candida tropicalis produces true hyphae, P. etchellsii and C. sake produce pseudohyphae, and M. pulcherrima does

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neither; on the other hand, its colonies turn reddish-brown when lipidcontaining chlamydospores developed. The commonest species of this group is C. tropicalis, which occurs in fresh and fermented fruits and vegetables (Suresh et al., 1982; Rale and Vakil, 1984), beverages (Put et al., 1976), and meat and dairy products (Zein et al., 1983; Jay, 1987). Candida sake is mostly found in soft drinks, wine, fermented foods (Sand, 1974; Sandhu and Waraich, 1984; Parish and Caroll, 1985; DeAk and Beuchat, 1988), and meat (Johannsen et al., 1984). Metschnikowia pulcherrima can be nearly always isolated from grape and must, and other fruits and fruit juices (Rosini et al., 1982; Suresh et al., 1982; Heard and Fleet, 1986a). Pichia etchellsii occurs less frequently in beverages, black olives, and beef, and is rather xerotolerant (Dalton et al., 1984; Dragoni and Comi, 1985; Garrido Fernandez et al., 1985). 5. Mannitol-Assimilating Yeast

The fifth group consists of yeasts assimilating only mannitol of the basic substrates included in the master key (Table 11).In this group, two genera of classical, strongly fermenting yeasts, Zygosaccharomyces and Torulaspora, are of particular importance in foods. Both were at one time considered to be Saccharomyces, but were split from this genus on the basis of a primarily haploid life cycle, whereas the genus Saccharomyces retained the primarily diploid species. The mode of sexual reproduction varies. In Zygosaccharomyces, conjugation between independent haploid cells usually precedes spore formation, whereas in Torulospora, conjugation occurs between mother cell and bud. In Saccharomyces, the diploid cells directly transform into sporangia. These features are not always easily observed and the identification of species by physiological criteria is also difficult because most of these criteria become variable after amalgamating a number of species on grounds of DNA homology, especially in S. cerevisiae and T. delbrueckii. In addition to the traditional identification tests, other criteria, not included in the simplified key, may greatly help identification. These are growth on lysine and ethylamine as well as in the presence of cycloheximide and 50% glucose. Some selective media applied in the brewery and in wine microbiology for distinguishing wild yeasts are based on these and similar criteria, and can be equally useful in identification. The Zygosaccharomyces species all grow on lysine and ethylamine and in the presence of 50% glucose. Zygosaccharomyces bailii and Z. bisporus are highly resistant to acetic acid and grow in a medium containing 1%acetate, whereas Z. rouxii does not. The delimitation of these species on the basis of utilization of sugars is uncertain. Zygosac-

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charomyces bailii is very resistant to preservatives (Thomas and Davenport, 1985; Warth, 1986); it grows on media containing 400 mglliter benzoic acid or 300 mg/liter sorbic acid, whereas Z. bisporus does not. Cells of Z. bailii are fairly large (diameter over 5 pm) compared to other Zygosaccharomyces species. Zygosaccharomyces bisporus and Z. microellipsoides are far less common in foods than are Z. bailii and Z. rouxii. The highly xerotolerant Z. bisporus is mostly confined to high-sugar products (Comi and Cantoni, 1984; Tilbury, 1976), but can be also found in beverages (Put et al., 1976), and Z. microellipsoides mostly occurs in soft drinks and wine (Sand et al., 1976; Kunkee and Goswell, 1977). With both species the assimilation of mannitol is variable and they may show up in the next group. Zygosaccharomyces rouxii is undoubtedly the most important and most frequent xerotolerant yeast; some strains are even xerophilic (Tokouka et al., 1985; Jermini and Schmidt-Lorenz, 1987a). It is often the sole yeast isolated from high-sugar products such as honey, syrups, dried fruits, and others (Tilbury, 1976; Comi and Cantoni, 1984; Tokouka et al., 1985; Jermini et al., 1987), and also from high-saltcontaining foods such as soy sauce (Noda et al., 1982; Tokouka et a]., 1985). Nevertheless, it often occurs in foods with a higher a,, for example, cheese and meats (Barnett et al., 1983; Comi and Cantoni, 1985).

The outstanding property of Z. bailii is its high tolerance and even adaptation to preservatives, which makes this organism a notorious spoilage agent in soft drinks and wine (Minarik, 1980; Sand, 1980; Warth, 1986) and in chemically preserved foods such as mayonnaise and salad dressing (Put et al., 1976; Smittle and Flowers, 1982; Baumgart et al., 1983). It is less xerotolerant than other Zygosaccharomyces species, although it frequently occurs in concentrates (Tilbury, 1976; MinArik and Hanicova, 1982). Other foods in which it has been found include bread, fermented cocoa, and fresh fruits (Ravelomanana et al., 1985; Vojtekova and Minarik, 1985; Spicher, 1986). Torulaspora delbrueckii is the only species of the genus that is widespread in foods. It commonly occurs in fruits, grapes, must, wine, soft drinks, beer, fermented vegetables, and sometimes high-sugar products and cheese (Sand et al., 1976; Tilbury, 1976; Suarez and Inigo, 1982; Ravelomana et al., 1985; Heard and Fleet, 1986a; Back, 1987; Jermini et al., 1987). Its cells are fairly small, especially compared to those of S . cerevisiae. Pseudohyphae are not produced. The species grows well on lysine, hardly or not at all on ethylamine, and also grows well in and even in 75% (w/w) glucose; the minimum a, for growth is 0.865

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(Tilbury, 1980b) and the temperature range is 5-40°C (Spicher, 1984). Its anamorph is known as Candida colliculosa. In its physiological properties and food niches occupied, K. thermotolerans is rather similar to T. delbrueckii. The two species differ in the mode of sexual reproduction, which is, however, not always observable. Kluyveromyces thermotolerans assimilates lysine and also ethylamine, and its anamorph is Candida dattila. In addition to the foods listed for T. delbrueckii, K. thermotolerans can be found in meat and shellfish (Bucket al., 1977; Lafon-Lafourcade, 1983;Comi and Cantoni, 1985; Put et al., 1976; Tokouka et al., 1985). A number of common Candida species belong to the fifth group. With the exception of C. apicola (formerly Torulopis apicola), each species produces pseudohyphae; C. albicans sometimes produces true hyphae. Fermentation is poor or lacking, although C. albicans and C. sake ferment strongly. The assimilation spectrum of a species is rather narrow. Perhaps the most frequently found species is C. parapsilosis, which has been found in all types of foods (Kobatake and Kurata, 1980a,b; LafonLafourcade, 1983; Johannsen et al., 1984; Spicher, 1986; Deak and Beuchat, 1988). Its relationship to the teleomorph Lodderomyces elongisporus has long been debated, but Hamajima et al. (1987) found convincing evidence that one form of C. parapsilosis corresponds to the anamorphic state of L. elongisporus, but other strains do not. Candida zeylanoides, C. catenulata, and C. rugosa are rather common in meat and meat products and also in wine (Kunkee and Goswell, 1977; Dalton et al., 1984; Banks and Board, 1987). Candida rugosa can be found in dairy products (Suarez and Inigo, 1982);C. vini and C. apicola occur in fruits and beverages (Suresh et al., 1982; Lafon-Lafourcade, 1983), and the latter is also found in high-sugar products (Tilbury, 1976). Candida albicans is the commonest human pathogenic yeast. It can easily be diagnosed by the development of germ tubes and chlamydospores. As the species now includes the former species C. claussenii and C. stellatoidea, which are mainly saprophytes, the frequent occurrence on foods of a yeast now called Ca. albicans is not surprising. It has been found in grape must, wine, soft drinks, cheese, beef, and shellfish (Sand, 1974; Buck et al., 1977; El-Bassiony et al., 1980; Dalton et al., 1984; Parish and Caroll, 1985). An easily distinguishable species in this group is a yeast-like organism, G. candidum, which produces true hyphae that break into arthroconidia, but never budding cells. In this and the negative urease reaction it sharply differs from Trichosporon. It occurs frequently in various foods, mostly in vegetables and dairy products and sometimes

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in meat products (Comi and Cantoni, 1983;Engel, 1986a;Guerzoni and Marchetti, 1987;DeAk and Beuchat, 1988). 6. Mannitol- and Cellobiose-Negative Yeasts

Yeasts falling into the sixth group are negative in all characteristics used in the master key and some of them also in most conventional physiological tests, which renders their identification rather difficult. The most important yeasts of all, the Saccharomyces species, key out in this group. Their main distinguishing characteristic is vigorous fermentation. Three species are common in foods: S. cerevisiae, S. exiguus, and S. kluyveri. The latter two species can be easily identified by their specific assimilation pattern. Unfortunately, this does not hold for S. cerevisiae, which includes some 20 former Saccharomyces species, and consequently most of its physiological properties are equivocal. The former species were distinguished primarily by sugar fermentation properties, which, however, proved to be inconsistent (Lodder, 1970). On the other hand, based on DNA homology, they appeared very closely related (Yarrow and Nakase, 1975). The present species, S. cerevisiae, appears to be a mixture of a number of natural biotypes and even hybrids. In contrast to wild strains, those adapted to, selected for, and exploited in industrial processes can be considered biotypes with definitive physiological properties. In the winery and brewery, the former species names Saccharomyces chevalieri (does not ferment maltose), S. bayanus (does not ferment galactose), S. cerevisiae (ferments only the fructose part of raffinose), and S. carlsbergensis (ferments raffinose and melibiose completely) have clear meaning. Recently, also on grounds of DNA reassociation studies, VaughanMartini and Martini (1987)newly delimited the species S. cerevisiae and S. bayanus and considered Saccharomyces pastorianus (synonym S. carlsbergensis) a natural hybrid of the two species. Unfortunately, the fermentative and assimilative properties are not consistent with the DNA/DNA homologies, and these species cannot be differentiated by physiological properties reliably. Nevertheless, in the key, traditional distinguishing characters are included for the provisional differentiation of these Saccharomyces species. Saccharomyces cerevisiae (and the similar species) can often be recognized by the large cell shape (6 x 13 pm). Neither S.cerevisiae nor S. exiguus develops on lysine and ethylamine, but S . kluyveri does. Saccharomyces cerevisiae is an indispensable organism for the production of a wide variety of alcoholic beverages, leavened bakery goods, and fermented foods (Berry et al., 1987).In many other cases it is a spoilage

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organism with a rather high heat resistance conferred by spores (D, 60°C, is 42 minutes; Put and De Jong, 1980), and a low pH tolerance conferred by vegetative cells (growth at pH 1.4; Pitt, 1974). Wine strains possess high resistance to SO2 (minimum inhibitory concentration 1.2 mM; Warth, 1985). The minimum a, for growth was stated to be 0.917 (55% w/w sucrose; Jermini and Schmidt-Lorenz, 1987a). It most frequently causes spoilage problems in soft drinks, fruit juices, bottled wine and beer, yoghurt, and cheese (Schmidt and Lenior, 1980; Suriyarachchi and Fleet, 1981; Put and De Jong, 1982a; Minarik et al., 1983; Back, 1987); sometimes it causes spoilage in bakery and meat products (Jay, 1987; Spicher, 1986). Saccharomyces exiguus is a special yeast used in sour doughs (Spicher, 1983),kefir (Engel et al., 1986), and certain types of beer (Novellie and Schaepdrijver, 1986); it causes spoilage in soft drinks, wine, and delicatessen salads (Put et al., 1976; Lafon-Lafourcade, 1983; Brocklehurst et al., 1983; Baumgart et al., 1983). It is often found on meat and fish (Buck et al., 1977; Comi and Cantoni, 1985). Saccharomyces kluyveri is less frequently found and its occurrence is mostly confined to soft drinks and wine (Torokand DeBk, 1974; Sand et al., 1976;Kunkee and Goswell, 1977). The rest of the yeasts grouped here are characterized by very restricted assimilation spectra and fermentative capabilities. Candida stellata is most active, strongly fermenting glucose and assimilating raffinose. It occurs commonly in fruits, must, and wine (Goto, 1980; Put et al., 1976; Heard and Fleet, 1986a), as well as on beef and shellfish (Buck et al., 1977; Comi and Cantoni, 1985).Neither this species nor C. inconspicua and C. glabrata produce pseudohyphae, and formerly they were considered Torulopsis species. Candida glabrata possesses some degree of xerotolerance and may occur in molasses and concentrated fruit juice (Kreger-van Rij, 1984),and is also frequently found on fish and shellfish (Bucket al., 1977; Jay, 1987). Candida inconspicua has also been found on meat (Dalton et al., 1984) and in various beverages (Hardwick, 1983; Lafon-Lafourcade, 1983; Muzikar, 1984). Both pseudohyphae and pellicles are produced by the two Pichia and two Issatchenkia species included this group. Although they can be differentiated by the mode of formation of sexual spores, this is not easily observed and anamorphic states are usually found. Pichia membranaefaciens (C, valida) ferments glucose very weakly. It is widespread in nearly all kinds of food, especially in fermented vegetables, alcoholic and nonalcoholic beverages, cheese, meat products, and delicatessen salads (Fleet et al., 1984; Brocklehurst and Lund, 1985;Garrido Fernandez et al., 1985; Back, 1987; Ravelomana et al., 1985; Dalton et

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al., 1984; Engel, 1986a, Messini et al., 1985). Pichia fermentans (C. lambica) is less common. It has been found in fresh meat (Hsieh and Jay, 1984), fermented cocoa (Ravelomanana et al., 1985), and more often in beverages (Torok and DeBk, 1974; Lafon-Lafourcade, 1983; Hardwick, 1983).

Issatchenkia orientalis [C. krusei) is very common in foods. Its only distinguishing property is that it grows well without added vitamins, unlike I. terricola. Though the latter species has been found in fruits and wine (Goto, 1980; Suresh et al., 1982), the former always occurs in fermented vegetables, fruits, fruit juices, wine, dairy products, and less frequently in dough and meat products (Suresh et al., 1982; Spicher, 1986; Fleet et al., 1984; Muzikar, 1984; Ravelomana et al., 1985; Jay, 1987). REFERENCES Alexander, N. J. (1986).Genetic manipulation of yeasts for ethanol production from xylose. Food Technol. 40(10),99-103. Anand, J.C., and Brown, A. D. (1968).Growth rate patterns of the so-called osmophilic and non-osmophilic yeasts in solutions of polyethylene glycol. J. Gen. Microbiol. 52, 205-212.

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High-Resolution Electrophoretic Purification and Structural Microanalysis of Peptides and Proteins ERIKP. LILLEHOJ* AND VEDPALS. MALIK+ *Cambridge Biotech Corporation Rockville, Maryland 20850 'Philip Morris Research Center Richmond, Virginia 23261 I. Introduction 11. Polyacrylamide Gel Electrophoresis

A. Theory and Development B. SDS-PAGE C. Two-Dimensional Polyacrylamide Gel Electrophoresis 111. Structural Analysis of Proteins Directly Eluted from One- and Two-Dimensional Polyacrylamide Gels A. Peptide Mapping, Epitope Mapping B. Protein Detection C. Photoaffinity Labeling D. Recovery of Proteins E. The Edman Degradation Cycle F. Automated Amino Acid Sequenators G. Radiochemical Peptide Mapping and Sequence Analysis H. Interference with Sequence Analysis IV. Structural Analysis of Proteins Electroblotted from One- and Two-Dimensional Polyacrylamide Gels A. Protein Electroblotting B. Microanalysis of Electroblotted Proteins C. Microsequence Analysis with Glass Fiber D. Microsequence Analysis with PVDF V. Electrophoretic Micropreparative Procedures as Part of a Comprehensive Purification Strategy A. One-Dimensional SDS-PAGE B. Two-Dimensional SDS-PAGE VI. Applications of Microsequence Analysis of Electrophoretically Purified Proteins A. Oligonucleotide Probes and Gene Cloning B. Polypeptide Processing, Homology Searches C. Peptide Antisera VII. Quality Control of Recombinant Proteins A. Expression Systems B. Protein Denaturation-Renaturation C. NH,-Terminal Methionine D. Posttranslational Modifications E. Fidelity of Translation VIII. Prospective Directions References

279 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 36 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ERIK P. LILLEHOJAND VEDPAL S.MALIK

1. Introduction

Substantial technical advance has occurred in the past 30 years toward the purification and structural analysis of proteins. Traditional methods of protein purification have relied upon the physicochemical properties of the protein of interest, often taking advantage of one or more unique characteristics that could be exploited to remove unwanted contaminants. Thus, a general feature emerged whereby each protein, or class of proteins, required its own particular purification scheme. It is now apparent that this strategy cannot be systematically applied to the study of the majority of proteins, particularly those of great biological significance that are present in only trace amounts. Rather, what is needed are generic procedures enabling the protein chemist to isolate a multitude of different proteins in a form amenable to subsequent structural analyses. In this regard, several techniques are now routinely being applied to achieve this goal, in particular the practice of protein purification by high-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE), electrotransfer to a chemically inert membrane, and high-sensitivity amino acid sequence determination. Two-dimensional PAGE in the presence of the anionic detergent sodium dodecyl sulfate (SDS) is presently the most powerful analytical technique available to examine the dynamics of protein expression during complex biological processes such as differentiation, development, and neoplastic transformation. Until recently, manipulation of the small amounts of proteins resolved on two-dimensional gels without serious losses restrained this procedure as a suitable alternative to existing purification techniques. New methods of amino acid microsequencing as a result of instrument miniaturization and enhanced detection of phenylthiohydantoin (PTH) amino acids now enable the sensitivity of sequencing (10pmol) to approach that of protein detection on two-dimensional polyacrylamide gels (1-10 ng by silver staining, equivalent to 0.05-0.5 pmol of a 50,000-Da protein). The purpose of this review is to provide historical perspectives of the developments of SDS-PAGE, protein electroblotting, and direct amino acid microsequencing, with special emphasis on those qualities that led to the evolution of a unified procedure for routine isolation and primary structural determination of proteins at the subnanomole level. We have directed the content herein primarily toward those individuals from other disciplines who require a concise reference source for application to their particular interests, yet who may be unfamiliar with state-of-theart protein purfication and analytical technologies. This topic has also

PEPTIDE AND PROTEIN PURIFICATION AND ANALYSIS

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been reviewed by Kent et al. (1987), Wilson (1988), and Simpson et aJ. (1989). Other sources that may be consulted are exclusively concerned with modern techniques of two-dimensional SDS-PAGE (Young and Anderson, 1982; Klose, 1983; Pearson and Anderson, 1983; Celis and Bravo, 1984; Dunbar, 1987a,b; Dunn, 1987; Endler et al., 1987), protein electroblotting (Gershoni and Palade, 1983; Towbin and Gordon, 1984; Bers and Garfin, 1985; Beisiegel, 1986), and/or amino acid microsequence analysis (Tschesche, 1983; Bhown, 1983; Shively, 1986a; Wittmann-Liebold et aJ., 1986; Walsh, 1987; Matsudaira, 1989). II. Polyacrylamide Gel Electrophoresis

A. THEORY AND DEVELOPMENT Electrophoresis has historically been the standard method of choice to analyze the homogeneity of a peptide or protein mixture. The technique is founded upon the observation that proteins possess a charge and will therefore move directionally under the influence of an electric field when placed in a solution with a suitable matrix to provide a nonreactive support and minimize convective forces. Although initial matrices used for this purpose consisted of paper, starch, or agarose, the unique porosity and rigidity of polyacrylamide has led to its almost universal use as the material of choice for electrophoretic purposes. Polyacrylamide is formed by the copolymerization of acrylamide and a cross-linking agent such as N,N’-methylene bisacrylamide (bis). Ammonium persulfate or riboflavin is used as a source of free radicals, with N,N,N’,N’-tetramethylethylenediamine(TEMED) or 3dimethylaminopropionitrile as a catalyst. Gel formation occurs by a two-step process. First, ammonium persulfate in aqueous solution yields a persulfate free radical that transfers an unpaired electron to TEMED, which in turn acts as an electron carrier to activate acrylamide monomers to a free-radical state (Bio-Rad, 1984). These acrylamide monomers polymerize into long chains that randomly incorporate the cross-linking bis monomer to produce a matrix containing pores of defined sizes. By adjusting either the concentration of acrylamide and/ or the degree of cross-linking, the effective pore size can be controlled. Gels with acrylamide concentrations between 3.5 and 40% can be prepared to separate most proteins (Chrambach and Rodbard, 1971). Crosslinking with high concentrations of bisacrylamide, however, is counterproductive and alternative agents, e.g., N,N’-diallyltartardiamide, are recommeded (Chrambach and Rodbard, 1981). When PAGE is used in a strictly zonal electrophoretic manner, proteins are

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ERIK P. LILLEHOJ AND VEDPAL S. MALIK

separated by their native conformation on the basis of not only their charge and molecular weight, but also structural interaction with the gel itself. PAGE has been used in both continuous and discontinuous (disc) buffer systems. The former uses a uniform buffer solution of constant pH throughout the gel and buffer reservoirs. The pH is chosen to be within the normal limits imposed by pH-dependent protein precipitation (generally, pH 3-10), yet near the PI values of the proteins present in the sample to maximize the relative charge differences and hence the resolution between them. The major disadvantage of continous PAGE systems is the poor resolution obtained with relatively large sample volumes. This problem was solved with the advent of disc electrophoresis, which utilizes a two-gel/two-buffer system to effectively reduce the volume and concentrate the sample as it migrates. The two-gel system consists of an upper (stacking) gel containing relatively large pores and a lower (resolving) gel with smaller pores. The two-buffer system is formulated so the pH of the buffer in the upper gel is less than that in the lower gel and running buffer. The pH values of the sample buffer and upper gel are equivalent. The running buffer also contains a weak acid with a pK, at or near the pH of the upper gel. In the disc system of Orenstein (1964) and David (1964), the buffer is tris(hydroxymethy1) aminomethane (TRIS) adjusted with HC1 to pH 6.7 in both the upper gel and sample buffer, and to pH 8.9 in the lower gel; glycine (pKa 9.6) serves as the weak acid (Fig. 1). In the presence of an electric field, the mobility of the glycinate ions is dictated by an equilibrium that exists between the poorly dissociated, low-mobility form at pH 6.7 and the anionic form with greater mobility at the higher pH. As the chloride and glycinate ions enter the sample buffer from the upper gel, a localized voltage gradient develops between the highly mobile leading chloride ions and the trailing glycinate ions. As this moving boundary sweeps past the proteins in the sample buffer, they acquire a mobility intermediate between that of the chloride and glycinate ions and consequently become stacked one above the other at the glycine/chloride boundary. Upon reaching the high-pH conditions of the lower gel, the mobility of the glycine ions surpasses that of the proteins such that the latter become unstacked and migrate in a uniform voltage gradient according to their charge and molecular weight. The basic principles of this discontinuous gel system have been experimentally adapted to other buffer systems covering the range of pH values between 3.5 and 9.5 (Reisfeld et a]., 1962; Williams and Reisfeld, 1964; Hedrick and Smith, 1968; Paterson and Strohman, 1970; Gabriel, 1971; Rodbard and Chrambach, 1971) and were used to develop a computer program to generate over 4000 different discontinuous buffer systems (Jovin, 1973a,b,c).

PEPTIDE AND PROTEIN PURIFICATION AND ANALYSIS Reservoir buffer h e . pH 8.3)

283

0

Stacking gel In Tris-HCl.pH 6.7

Proteins stacked Proteins fractionating in the resolving gal

Reservoir buffer (Tris- lycine. pH 8 . f )

6

GlycineI chloride boundary

(a) ( b) (C) FIG.1. Theory of discontinuous polyacrylamide gel electrophoresis before application of the electric field [a), during sample migration through the stacking gel [b), and during sample migration through the separating gel (c). Reproduced from Hames and Rickwood (1981),with permission.

B. SDS-PAGE

Because the migration of proteins in continuous or discontinuous polyacrylamide gels is influenced by both their molecular weight and charge, two methods have been developed to analyze protein migration solely under the influence of molecular weight. The first utilizes a technique developed by Ferguson (1964)whereby the relative mobility (Rf) of the protein of interest is determined on gels of different acrylamide concentrations. The Ferguson plot is a graphic analysis of protein Rf versus acrylamide concentration. A linear relationship exists between the slope of the Ferguson plot (KR)and the molecular weight of the protein in its native conformation (Hedrick and Smith, 1968). In most circumstances, however, it is necessary to perform electrophoresis after the proteins have been exposed to a denaturation agent to (1)obtain accurate estimation of molecular weight and (2) dissociate oligomeric protein complexes into their constituent subunits. SDS was used for this purpose initially by Shapiro et al. (1967) and subsequent studies by Weber and Osborn (1969), utilizing a phosphate (continous) buffer system, demonstrated, within molecular sieving limits of the gel, an inverse, linear relationship between the Rf values of a set of proteins and the logarithms of their molecular weight (Fig. 2). Although the exact mechanisms producing this relationship to molecular weight are unclear, SDS binds to proteins at a constant weight ratio (1.4 grams of SDS per gram of protein), inducing a conformational change to random coil, rod-shaped structures with nearly equal charge densities. A reducing

ERIK P. LILLEHOJ AND VEDPAL S.MALIK

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buffer front

200

I00 80

60 40

20 .-

E : 10

c

J

0

8

0

2

4

6 8 10 I2 14 Distance of migration (cm)

16

FIG. 2. Calibration curves of protein molecular weight plotted on a logarithmic scale on the ordinate versus distance of migration on the abcissa using 5, 10, or 15% total acrylamide concentrations and the SDS-containing discontinuous buffer system of Laemmli (1970). Reproduced from Hames and Rickwood (19811,with permission.

agent (P-mercaptoethanol or dithiothreitol), added to reduce intra- and interchain disulfide bonds, and heat are used to promote complete denaturation. The molecular weight of an unknown protein can therefore be deduced by coelectrophoresis of a series of protein standards with known molecular weights. A comprehensive list of commercially available protein standards has been published (Lillehoj and Malik, 1989). In actuality, this represents the protein’s apparent molecular weight, because its mobility is influenced by additional factors, such as the extent of glycosylation (see below). The most popular discontinuous buffers containing SDS are those of Laemmli (1970),utilizing the TRIS-glycine system described above,


285

and of Neville (1971),based on a TRIS-borate buffer. Under ideal conditions, these disc systems are capable of resolving proteins over a broad molecular weight range that differ in size by less than 2% (Rogers et a]., 1986). Figure 3 illustrates a separation of 37,500- and 38,000-Da proteins on a disc SDS-PAGE gel. However, disc polyacrylamide gels are handicapped in their ability to resolve polypeptides less than 15,000-20,000 Da. As pointed out by Wyckoff and co-workers (1977), SDS itself is stacked during electrophoresis in discontinuous buffers and the sizes of SDS micelles approach those of small peptide-SDS complexes, causing protein streaking at high acrylamide concentration (Schagger and von Jagow, 1987). Alternative PAGE systems have thus been developed to analyze small-molecular-weight polypeptides of biological interest either naturally occurring or generated, for example, by proteolytic cleavage for peptide mapping (see below). A modified Laemmli procedure incorporating 8 M urea and a high ratio of bisacrylamide to acrylamide monomer was used to resolve peptides from 20 to 150 residues in length (2500 to 17,000 Da) (Swank and Munkres, 1971). Other gel systems utilizing urea and/or increased cross-linking concentrations have been similarly described (Campbell et al., 1983; Hashimot0 et a]., 1983). The effect of urea is to decrease the gel pore size, probably as a consequence of interference with hydrogen bond formation between acrylamide monomers during polymerization. DeWald et al. (1986) described a continuous buffer gel system, devoid of urea, capable of resolving polypeptides in the range of 2000-200,000 Da. Increasing the mobility of glycine by replacement of TRIS with am-

FIG. 3. Separation of proteins of similar molecular weight by discontinuous SDSPAGE. [35S]Methionine-labeledmouse major histocompatibility complex class I proteins were immunoprecipitated and analyzed on an 11% SDS-PAGE gel according to the system described by Laemmli (1970).Three individual proteins were resolved. Their apparent molecular weights are indicated and were determined from a calibration curve prepared by coelectrophoresis of prestained protein standards. The maximum discernable resolution was achieved between the 37.5-and 38-kDa proteins, which differ in molecular weight by 1.3%. Reproduced from Rogers et al. (1986),with permission.

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mediol was used to enhance the separation of polypeptides down to 6000 Da (Wyckoff et al., 1977) and down to 1500 Da by further refinement using a linear 10-30% gradient acrylamide gel (Bothe et al., 1985).

Others have replaced glycine with alternative trailing ions, for example, 2-morphineethanesulfonic acid (Kyte and Rodriguez, 1983). Anderson and co-workers (1983) used a temporary stacking step provided by acetate ions interspersed between leading sulfate and trailing chloride ions to separate effectively polypeptides between 2500 and 90,000 Da. The use of Tricine instead of glycine allowed resolution of proteins in the range of 1000-100,000 Da using 10% total acrylamide plus 3% cross-linker (Schagger and von Jagow, 1987). Furthermore, the absence of urea and glycine was argued to make this procedure ideally suited for preparative purification of proteins in a form amenable to microsequence analysis. Tsugita et al. (1982) described a continuous PAGE system capable of separating peptides and proteins in the range of 200-100,OOO Da using a volatile buffer, triethylamine/formic acid at pH 11.7. Covalent modification of protein amino groups with trisulfonylpyrene isothiocyanate conferred a strongly negative charge, effectively replacing SDS and allowing proteins to be separated according to their molecular weight. Additionally, this charge modification was claimed to increase the extraction of proteins from the gel matrix, allowing them to be easily concentrated and desalted by evaporation in a form suitable for amino acid composition and NH2-terminal amino acid sequence analyses. A second commonly recognized limitation of SDS-PAGE is its inability to predict accurately the molecular weight on proteins containing various posttranslational modifications, particularly gly cosylation. Glycoproteins consistently exhibit reduced mobility compared to nonglycosylated proteins of closely similar molecular weights, probably as a consequence of their diminished binding to SDS resulting in reduced net charge on a weight basis. Because the difference between observed and actual molecular weight values decreases with increasing acrylamide pore size (Segrest et al., 1971; Leach et al., 1980), it has been proposed that more accurate molecular weight values of some glycoproteins can be estimated by plotting their apparent molecular weight at various acrylamide concentrations and extrapolating to the asymptotic minimal value (Segrest et al., 1971). More accurate estimation of glycoprotein molecular weight has also been reported using SDS gradient acrylamide gels in lieu of fixed acrylamide concentration gels (Lambin and Fine, 1979). Leach et al. (1980),however, were unable to arrive at a similar conclusion. Rather, the results of their studies demonstrated that


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gel filtration chromatography in the presence of denaturants (i.e., 6 M guanidinum chloride) was the most reliable method to predict the molecular weight of a glycoprotein. C. TWO-DIMENSIONAL POLYACRYLAMIDE GELELECTROPHORESIS Most proteins are present in complex mixtures that contain hundreds or thousands of individual components. For example, individual cells have been estimated to contain 10,000 different proteins ranging in abundance from a few hundred to over lo9 molecules per cell (Bravo and Celis, 1982; Pollard, 1984), although others have placed this upper estimate one order of magnitude lower (Duncan and McConkey, 1982). Electrophoresis on a one-dimensional SDS-PAGE gel is normally capable of resolving approximately 100 individual bands (Patton et al., 1990). Most of these bands, however, contain multiple protein species. Many techniques have been developed to increase the capacity of protein separation on a single slab gel by performing two different electrophoretic separations in tandem, with the second being performed perpendicularly to the first. The initial method described by Smithies and Poulik (1956) utilized two-zone electrophoretic processes in starch gel that were similar in nature and thus only able to moderately increase the resolution obtained with a single-dimension gel. The development of electrophoretic procedures capable of independently separating proteins on the basis of charge or size alone led several investigators to design two-dimensional protocols combining a charge-based separation in cylindrical gels in the first dimension, followed by either PAGE (Kenrick and Margolis, 1970) or SDS-PAGE (MacGillivray and Rickwood, 1975) on slab gels. Iborra and Buhler (1976) subsequently devised a two-dimensional system whereby proteins were initially separated on the basis of charge by isoelectric focusing (IEF) on polyacrylamide slab gels in the presence of urea, followed by discontinous SDS-PAGE using the Laemmli gel system. It was O’Farrell (1975), however, who introduced a high-resolution two-dimensional technique that today remains one of the most powerful tools for analytical and preparative separations of proteins from complex biological mixtures. Such two-dimensional gels can identify 100200 different proteins by Coomassie blue staining, 500-1000 proteins by silver staining, or 1000-2000 proteins by autoradiography (Bravo and Celis, 1982; Dunbar, 1987a; Patton et a]., 1990; Krauss et a]., 1990). Figure 4 illustrates a typical two-dimensional gel pattern of a complex protein mixture. Gel systems utilizing larger size formats and multiple gels with overlapping charge and molecular weight fractionation ranges

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ERIK P. LILLEHOJAND VEDPAL S.MALIK

E F -I

92.5

69

55

43

2 X

30

13

FIG. 4. Two-dimensional gel electrophoresis of 14C-labeled HeLa cell proteins. Proteins were separated by IEF in the horizontal direction and SDS-PAGE in the vertical direction. The migration of molecular weight standards is indicated on the right in kilodaltons. Reproduced from Bravo and Celis (1982),with permission.

have reportedly given resolution of up to 10,000 different proteins (Krauss et al., 1990). The sensitivity of this procedure is remarkable. Polypeptides derived from rare mRNAs and comprising as little as O.OOOO~-O.OOO~%of the total cellular protein can be detected using autoradiographic techniques (O’Farrell, 1975; Garrels, 1979; Bravo and Celis, 1982). The two-dimensional procedure developed by O’Farrell combines protein separation on the basis of charge by IEF in a cylindrical polyacrylamide gel in the presence of urea in the first dimension, followed by SDS-PAGE using a gradient polyacrylamide slab gel with the Laemmli system in the second dimension. IEF is normally performed in a nonrestrictive matrix, commonly agarose or large-pore polyacrylamide,


289

containing highly mobile carrier ampholytes consisting of a group of small-molecular-weight, zwitterionic, aliphatic polyamino/polycarboxylic acids with varying isoelectric points (Vesterberg, 1976).Under the influence of a potential difference, they migrate to a position in the gel where their net charge is zero, thus generating a pH gradient, with the most acidic ampholytes located near the anode and the most basic near the cathode. Similarly, because the charge of a protein is determined by the sum of the charges of the individual amino acid side chains (in addition to chemical groups added posttranslationally, e.g., phosphate and carbohydrate), the protein will also migrate to a position in the pH gradient where it has no net charge. The pH at this position is opertionally defined as the pl of the protein. Although IEF is theoretically an equilibrium procedure that reaches completion when protein migration ceases, it is practically difficult to achieve zero mobility because the protein’s velocity asymptotically decreases as it approaches its pl (An der Lan and Chrambach, 1981). It is therefore more appropriate to refer to the apparent PI of a given polypeptide under defined conditions of ampholytes, ionic strength, temperature, and time. Carrier ampholytes are available from a variety of commercial sources or can be synthesized in the laboratory to cover a broad pH range (Vinogradov et a]., 1973), generally between 2 and 11. In practice, however, the distribution of polypeptides across the pH gradient is not linear, because most proteins possess pl values in the range of 5-7 (Patton et al., 1990).Proteins outside of this range, particularly those with a basic pl, can be resolved by nonequilibrium pH gel electrophoresis (O’Farrell et a]., 1977;Willard et al., 1979).Another difficulty of two-dimensional electrophoresis is the variability associated with different sources of ampholytes, which hinders the ability to assign accurately PI values to individual proteins. Such problems include: (1)discontinuities in the pH gradient, (2) electrofocusing drift, (3) nonreproducible precast gels, and (4) difficulty in stabilization of widerange pH gradients (An der Lan and Chrambach, 1981;Hanash and Strahler, 1989).Internal charge standards prepared by progressive carbamylation of a purified protein, e.g., creatine phosphokinase (Anderson and Hickman, 1979), to produce a uniform series of charge isomers, provide a means to determine the relative pl of a particular protein. The advent of polyacrylamide gels containing immobilized pH gradients (Hanash and Strahler, 1989)has also alleviated some of these problems. Patton et al. (1990) have identified seven critical factors necessary for obtaining highly reproducible, high-resolution twodimensional gel patterns: (1)incorporation of a thread into the IEF gel, (2)use of large-format (22 x 22 cm) gels, (3) use of an ampholyte mixture, which broadens the pH 5-7 range, (4) shortening the second-

290


dimension separation time, (5) use of high-quality reagents, (6) maintaining constant temperature (20-23OC) during the second-dimension separation, and (7) use of a sensitive protein detection method. Other problems arise when two-dimensional gel electrophoresis, using either equilibrium or nonequilibrium electrofocusing in the first dimension, is incapable of resolving certain types of proteins. In these special circumstances, alternative gel systems have been developed. Traditionally, ribosomal proteins have been separated in a manner involving discontinuous gel electrophoresis at pH 8.6 in the first dimension, followed by continuous gel electrophoresis at pH 4.5 (Kaltschmidt and Wittmann, 1979; Howard and Traut, 1973). Datta et al. (1988) have designed an acid-urea gel for the first dimension and SDS-PAGE in the second as an aid to the identification of Escherichia coli ribosomal proteins. Histones are a class of very basic proteins that have also been difficult to separate by the classical two-dimensional procedure of O’Farrell. Hoffmann and Chalkley (1976) described an acid-urea gel system incorporating detergent in the second dimension, and Sinclair and Rickwood (1981) reported an acid-urea gel followed by an SDS separation at high pH to successfully separate histones. Other types of two-dimensional gels have been devised to analyze nuclear proteins (Orrick et al., 1973).

Ill. Structural Analysis of Proteins Directly Eluted from One- and Two-Dimensional Polyacrylamide Gels

PAGE systems have been used for several years for preparative isolation of proteins for primary structural analysis. The earliest of these investigations utilized polypeptides purified by one-dimensional SDSPAGE for subsequent peptide and epitope mapping studies. Later, as the sensitivity of amino acid detection methods increased, proteins were recovered by diffusion, electrodialysis, or electroelution in sufficient quantities to permit amino acid composition and NH2-terminal sequence analysis by Edman degradation. Further enhancements in the detectability of amino acids using radiolabeled proteins spawned the development of radiochemical methods of primary structural analysis. This section will briefly outline the historical developments of these primary structural techniques as they were applied to proteins isolated from one-dimensional polyacrylamide gels. Following this, recent advances in protein recovery from one- and two-dimensional polyacrylamide gels using novel electroblotting and microsequencing techniques will be summarized.


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A. PEPTIDE MAPPING, EPITOPEMAPPING Peptide mapping is a technique to analyze peptide fragments generated from an intact protein for the purpose of comparing the amino acid sequences of different gene products. High-resolution separation techniques, such as reversed-phase high-performance liquid chromatography (HPLC),are capable of resolving peptides with a single amino acid substitution. Epitope mapping is an extension of this method used to analyze particular antibody-binding sites on a protein antigen. Peptide fragments can be generated using a variety of different chemical or enzymatic procedures as listed in Table I. Judicious choice of the proper protein cleavage agent will expedite and facilitate peptide/epitope mapping studies, because the frequency at which a particular peptide bond occurs in the polypeptide chain dictates the sizes of the peptides generated. For example, cleavage with formic acid tends to produce relatively large fragments because the aspartyl-proline bond is found at an average frequency of once per 400 residues (Sonderegger et al., 1982b). Proteolytic cleavage by trypsin, in contrast, often results in peptides less than 15-20 amino acids in length. Thus, an initial cleavage with formic acid, for instance, can be used to preliminarily localize a particular TABLE I

REAGENTSTO PRODUCEPEPTIDEFRAGMENTS FROM INTACT PROTEINS~ Type of reagent Chemical

Enzyme

a

Peptide bond cleavedb

Reagent Cyanogen bromide (CNBr) 2-(2-Nitrophenylsulfony1)-3’-methyl3’-bromindolenine skatole (BNPSskatole), N-chlorosuccinimide/urea, iodobenzoic acid Nitrothiocyanobenzoic acid Hydroxylamine Formic acid

Methionyl-X Tryptophanyl-X

Trypsin Staphylococcus aureus VB Endoproteinase Arg-C Endoproteinase Asp-C Endoproteinase Lys-C Chymotrypsin, themolysin, pepsin, papain, clostripain, thrombin

Arginyl-X, lysyl-X Glutamyl-X Arginyl-X Aspartyl-X Lysyl-x Nonspecific

Adapted from Spande et 01. (1970),Han et X denotes any amino acid.

01.

X-Cysteiny1 Asparaginy1-glycine Aspartyl-proline

(1983),and Kessey (1987)

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ERIK P. LILLEHOJAND VEDPAL S. MALIK

peptide or epitope to a general region of the protein as well as to produce a substrate for further cleavage by typsin, to give a less complicated array of peptides than would be generated by direct trypsin cleavage of the intact protein. Once peptides have been produced, they are separated in a manner that allows their further characterization. Fractionation by one- or twodimensional gel electrophoresis has been a popular method of choice for several reasons. First, as indicated above, recent technical advances have permitted the resolution of small peptides on polyacrylamide gels. This is particularly useful for epitope mapping because antigenic determinants tend to occur once every 50-100 amino acid residues along the polypeptide chain (Luzio and Jackson, 1986]. Second, the separated peptide fragments can be accurately characterized according to their molecular weight and PI. If the amino acid sequence of the intact protein is known (e.g., those generated by recombinant DNA procedures), one may be able to predict the region of the protein from which the peptide was derived based on the specificity of the cleavage agent employed. Finally, the peptides can be recovered by electroelution or, more efficiently, by electrotransfer to a suitable matrix for antigenic or structural characterization. As will be discussed below, amino acid composition and NH2-terminal amino acid microsequence analysis have been successfully performed on proteins purified by such means. Historically, the earliest forms of structural characterization of proteins purified by polyacrylamide gel electrophoresis were peptide mapping techniques using either chemical- or protease-based cleavage protocols. In both cases, the proteins being analyzed were either directly electroeluted from the gel matrix or treated in situ and reelectrophoresed on a second gel at a right angle relative to the first separation. Typical examples of the latter using chemical reagents are cyanogen bromide (CNBr) (Lam and Kasper, 1979; Nikodem and Fresco, 1979), hydroxylamine (Lam and Kasper, 1979; Saris et al., 1983), formic acid (Wacker et a]., 1981; Sonderegger et a]., 1982a,b; Rittenhouse and Marcus, 19841, N-chlorosuccinimide (NCS)/urea (Lischwe and Ochs, 1982), and 2-(2-nitrophenylsulfonyl)-3’-methyl-3’-bromindolenine (BNPS)-skatole (Detke and Keller, 1982). Cleveland et al. (19771 produced SDS-PAGE peptide maps of the major bacteriophage T4 head protein following electroelution of [ 14C]leucine-labeledprotein and digestion with Staphylococcus aureus V8 protease. A similar procedure was used by Tijssen and Kurstak (1979) employing dansylated structural proteins of densonucleosis virus. Peptide mapping by in situ protease digestions has been described by Cleveland et al. (1977), Bordier and Crettol-Jarvinen (1979),Lam and Kasper (1980a,b),and Tijssen


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and Kurstak (1983). In these methods, the protein of interest was first separated by SDS-PAGE and the region of the gel containing the protein was excised and layered over a second SDS-PAGE gel. A solution of the particular proteolytic enzyme was then layered over this gel slice and coelectrophoresed with substrate into the stacking gel, where digestion was allowed to proceed and the products separated in the resolving gel of the second dimension. The utility of this procedure is made feasible by the fact that many proteolytic enzymes are active in the presence of SDS (Gooderham, 1984) and, in fact, some degree of substrate denaturation enhances the accessibility of peptide bonds to the enzyme. B. PROTEIN DETECTION Comprehensive bibliographies of polypeptide detection methods have been compiled (Hames and Rickwood, 1981; Kodak, 1985) and will be briefly summarized here. The most common method of visualizing protein in polyacrylamide gels is staining and the most popular protein stain is Coomassie blue G-250, an organic dye that binds to polypeptides by hydrophobic interactions. Coomassie blue staining is sensitive to about 1 pg/cm2. Other protein stains are amido black 10B, fast green FCF, nigrosine, uniblue A, and Ponceau S. Staining methods using these dyes are all based upon direct physical adsorption to the protein. An alternative principle utilizes photographic principles with metal stains such as silver (Oakley et al., 1980; Merril et al., 1981; Morrissey, 1981) and nickel (Yudelson, 1984).The advantage offered by these techniques is that they are much more sensitive than dye adsorption methods, routinely detecting as little as 5-10 ng/cm2. In addition to these general staining reagents, specific procedures have been developed to detect special types of proteins, including (1) glycoproteins (Zacharius et al., 1969; Wardi and Michos, 19721, (2) phosphoproteins (Cutting and Roth, 19731, (3) lipoproteins (Raymond et al., 1966; Naito et al., 19731, (4) nucleoproteins (Dahlberg et a]., 19691, (5) proteins with accessible sulfhydryl groups (Yamamoto et al., 1978; Zelazowski, 1980),and (6) collagens (McCormick et a]., 1979). Ultrasensitive visualization of proteins in polyacrylamide gels has also been achieved using fluorescent staining techniques, and a number of fluorophores have been employed for labeling either prior to (Ragland et al., 1974; Stephens, 1975; Barger et a]., 1976; Chen-Kiang et al., 1979; Tijssen and Kurstak, 1979) or following (Hartman and Udenfriend, 1969; Carson, 1977; Jackowski and Liew, 1980) electrophoresis. Autoradiography and fluorography of radiolabeled proteins separated chiefly on slab gels provide additional sensitive detection methods (see below).

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C. PHOTAFFINITY LABELING The utility of two-dimensional gel electrophoresis can be significantly enhanced if the protein of interest can be specifically identified on the gel. Antibody or chromogenic substrates can be used for this purpose. Photoaffinity labeling of certain proteins in a mixture before their resolution may also overcome the obstacle of identification. Purification of the photoaffinity-labeled glucagon receptor by gel electrophoresis from rat liver plasma membrane has been described (Horuk et al., 1984).Photoaffinity-labeled human P-adrenergic receptor migrated on SDS-PAGE at a mass equivalent to 68 kDa (Fraser et a]., 1987).Using photoaffinity labeling with [32P]8-azido-cAMPand two-dimensional gel analysis, 26 electrophoretic variants of CAMP-binding proteins were identified in six different tissues of the marine mollusk Aplaysia californica (Palazzollo et al., 1989).Fluorography of SDS-PAGE gels on which photoaffinity-labeled human testosterone-binding globulin was analyzed showed two subunits, 52.2 and 48.6kDa (Danzo et al., 1989). The level of charge heterogeneity in the aryl hydrocarbon receptor was analyzed by two-dimensional gel electrophoresis after photoaffinity la(Perdew beling with 2-azido-3-[1251]iodo-7,8-dibromodibenzo-p-dioxin and Hollenback, 1990). Direct photoaffinity-labeling techniques do cross-link nucleotides to many nucleotide-binding proteins ( Jansson and Eriksson, 1990).S-Adenosylmethionine has been used to photoaffinity label several methyltransferases (Billich and Zoecher, 1987;Som and Friedman, 1990). Many proteins are modified in vivo by covalent attachment of certain coenzymes. Prosthetic groups such as biotin, lipoic acid, and 4’phosphopantheine are linked to very few proteins. The ligases that covalently attach these coenzymes to proteins recognize specific amino acid sequences of the target protein. Protein segments recognized by a coenzyme ligase can be fused to a protein of interest and the fusion protein can be specifically labeled by growth of cell cultures in the presence of labeled coenzyme. The fusion protein can then be detected and/or purified by specific binding to the appropriate ligand. Biotin has been used for posttranslational labeling of proteins. The carboxyl-terminal protein segments are conserved in those proteins biotinylated by biotin ligase. Proteins from translational fusions of heterologous genes to the biotinylation-specific carboxyl-terminal sequences yield a molecule that is biotinylated in vivo (Cronan, 1990). The biotinylation sequence is 75 amino acids long and functions in both Saccharomyces and E. coli. Fusion proteins can be labeled with [3H]biotin in vivo and purified by binding to immobilized monomeric avidin followed by elution with buffers containing free biotin. The labeled protein can also be located on two-dimensional gels by fluorography.


295

D. RECOVERY OF PROTEINS

Purification of proteins by SDS-PAGE in a form suitable for amino acid analysis has been successfully performed for several years. The most common procedures for recovering proteins from gels include passive diffusion or electroelution. In the first case, the gel segment containing the protein of interest is immersed in a suitable solvent, for example, formic acid (Tsugita et al., 1987), acetic acid (Veronese et al., 1987), sodium hydroxidehhiodiglycol (Manabe et al., 1982), or triethylamine (Shoji et a]., 1986), and the protein is extracted by diffusion. A less time-consuming practice has been to place the gel segment in electrophoresis buffer, extract the protein with an applied potential difference,and collect it on a dialysis membrane (Mardian and Isenberg, 1978; Hanaoka et al., 1979; Bhown et al., 1980; Otto and Snejdarkova, 1981; Mendel-Hartvig, 1982; Walker et al., 1982; Hunkapiller et al., 1983,1984a;Hunkapiller and Lujan, 1986) or a protein-binding gel such as hydroxylapatite (Ziola and Scraba, 1976; Guevara et al., 1982) Figure 5 illustrates the hydroxylapatite/polyacrylamideslab gel used by Guevara et al. (1982) to recover electrophoretically separated proteins. Akaiwa (1982) has cited over 30 literature references pertaining to these

spacer

1

( 1 5 mm thick)

5.6 om

I1

upper phase

middlephase

lower phase

e.[Cm

15.0 em

b

Bottom (Anode end

FIG.5. Diagrammatic illustration of the hydroxylapatite-polyacrylamide slab gel used to isolate proteins. The upper phase consists of powdered polyacrylamide containing the protein of interest previously resolved on preparative two-dimensional gels. The middle phase consists of hydroxylapatite. The bottom phase consists of SDS-polyacrylamide. The protein migrates from the upper phase into the middle phase and is bound by the hydroxylapatite, from which it is recovered by elution with sodium phosphate in a glass column. Reproduced from Guevara et al. (1982),with permission.

296


procedures. As popular as they have been, however, they suffer from the drawback of being unsuitable for use with a large number of different proteins and of low recoveries, Kelly et al. (1983)have determined that the major cause of low protein yields during electrophoretic elution is depletion of SDS from the buffer and have thus recommended preincubation of the gel slice in 5% SDS prior to elution. Alternatively, polyacrylamide gel systems have been devised containing unique cross-linking agents that allow the gel matrix to be solubilized under appropriate conditions. Gels prepared with N’,Nbisacrylcystamine (Hansen, 1977) are dissolved with reducing agents such as P-mercaptoethanol or dithiothreitol. Alkaline conditions have been used to dissolve gels cross-linked with ethylene diacrylate (Choules and Zimm, 1965). Periodic acid will solubilize polyacrylamide cross-linked with N’,N-diallytartardiamide (Anker, 1979;Tas et al., 1979),and either periodic acid (O’Connell and Brady, 1977;Tas et al., 1979) or alkali (Mendel-Hartvig, 1982) will solubilize N‘-Ndihydroxyethylenebisacrylamide gels. Although proteins in SDSPAGE gels can easily be recovered using these reagents, they have not generally been applied to protein purification for primary structural studies due to the high concentration of acrylamide monomer that must be removed, amino acid modifications, and introduction of contaminants that interfere with the Edman chemistry and/or high-performance liquid chromatography (HPLC) identification of the released amino acids (see below). Polypeptides resolved by one-dimensional SDS-PAGE have also been purified using special electrophoretic instrumentation capable of continuous collection at the bottom of the gel. A variety of such devices have been described (see, e.g., Jovin et al., 1964;Ryan et al., 1976; Koziarz et al., 1978;Akaiwa, 1982;Hediger, 1986). Many of these suffered from problems related to inadequate heat dissipation, gel deformation, and low protein resolution. A commercially manufactured instrument utilizing short, small-diameter polyacrylamide gels and capable of continuous, microflow elution is available that circumvents many of these difficulties (HPEC; Applied Biosystems, Foster City, California). Sheer et al. (1990)have demonstrated the feasibility of this apparatus for micropreparative electrophoretic isolation of proteins suitable for peptide mapping and NH2-terminal amino acid sequence analyses.

E. THEEDMANDEGRADATION CYCLE A variety of procedures have been developed for the purpose of obtaining an amino acid sequence, but it was Edman (1950)who introduced a set of chemical reactions for the sequential removal of amino


297

acids from the NHz-terminus of a polypeptide chain; this procedure remains the most popular today. The Edman degradation cycle consists of three steps: (1)initial coupling of phenylisothiocyanate (PITC)to the a-amino group under basic conditions, (2) cleavage of the NHz-terminal amino acid via cyclization in acidic conditions, and (3) conversion of the anilinothiazolinone (ATZ) amino acid derivative to the more stable PTH derivative. These reactions are illustrated in Fig. 6. Due to a variety of factors such as physical protein loss, chemical side reactions, differential chemical reaction rates, NH2-terminal blockage, acid-catalyzed proteolysis, and incomplete coupling and cleavage, the yield of individual PTH amino acids from the polypeptide chain is always less than complete. Initial yield refers to the yield of PTH amino acid at cycle number 1compared to the total amount of protein applied to the reaction. Repetitive yield (RY) refers to the cumulative yields at successive degradation cycles. For a given pair of the same amino acid, RY is mathematically described by the following equation (Applied Biosystems, 1986):

RY = looyo x

e(ln Yj-ln Yi)

0 - i1-I

where e is the natural logarithm base and In Yi and Yj are the natural logarithms of PTH amino acid peaks at cycles i and j, respectively (with j > i). The average RY of the sequence analysis is the arithmetic average of the individual amino acid RY values. Because RY is always less than 100% as the degradation process proceeds, a gradual increase occurs in the fraction of the preceding residue, overlapping with the released PTH amino acid. In practical terms, this restricts the Edman degradation to a maximum of approximately 70 residues of continuous sequence (Allen, 1981).

F. AUTOMATED AMINOACIDSEQUENATORS Edman and Begg (1967) developed an instrument capable of automated Edman degradation of a protein sample in a solvent film on the inner walls of a rotating vessel. This spinning-cup sequencer was capable of handling nanomole quantities of protein but suffered from problems related to sample loss during washings and extractions, particularly with small peptides. Polybrene (1,5-dimethyl-1,5diazaundecamethylene polymethobromide) was introduced to reduce this problem (Tarr et a]., 1978; Klapper et a]., 1978). Further improvements in instrument design, HPLC analysis of the released PTH amino acids, and use of high-quality reagents and solvents enabled the spinning-cup sequencer to produce sequence data on subnanomole

298 A.


coupling

--

+ Hp-CH-CO-NH-CH-CO-NH-CH-CO-

Ph-N=C=B

I

I

R1

I

R2

->

R3

8

II

Ph-NH-C-MI-CH-CO-NH-CH-CO-NH-CH-CO-

I

R1

B.

I

I

R2

--

R3

Cleavage 8

II Ph-NH-C-NH-CH-CO-NH-CH-CO-NH-CH-CO-

I

I

R1

I

R2

+

Oh-NH-C=N-CH-CO

--

R3

--

*H~N-CH-CO-~-CH-CO-

I

I

I

R2

R3

R1

C.

H+

-

conversion

r8 i+

Ph-NH-C=N-CH-CO

8

HzO

H+

->

II

Ph-NH-C-NH-CH-COOH

I

I

->

R1

R1 8

II

rci

Ph-N-CO-CH-NH

I

+ HzO

R1 FIG. 6. Reactions of the Edman degradation cycle. (A) In the coupling step, phenylisothiocyanate (Ph-N+S) reacts with the a-amino group of the NH2-terminal amino acid. (B) In the cleavage step, the ATZ derivative is formed by cyclization and release of the NH,-terminal amino acid. (C) In the conversion step, the ATZ derivative is converted to the more stable PTH derivative via the intermediate phenylthiocarbamyl amino acid. R1,R2, and R3 indicate different amino acid side chains.


299

quantities of protein (Wittmann-Liebold, 1973; Hunkapiller and Hood, 1978,1980; Shively, 1981). Concurrent with the development of the spinning-cup sequenator, other procedures were developed for covalent attachment of polypeptides to solid supports for sequence analysis. These solid-phase sequencing techniques utilized the Edman reaction chemistry on proteins attached though their NH,-terminal, COOH-terminal, or internal amino acid residues. Specific coupling procedures (Laursen, 1977) and the current status of automated solid-phase sequencing (Machleidt, 1983; L'Italien, 1986) have been reviewed. However, it was not until the introduction of the gas-liquid solid-phase protein sequencer (commonly referred to as the gas-phase sequencer) by Hewick et al. (1981; Hunkapiller et al., 1986; Hunkapiller, 1988) that useful sequence information on as little as 5 pmol of protein was obtained. This enabled for the first time direct microsequence analysis of peptides and proteins purified by two-dimensional gel electrophoresis and electroblotted onto appropriate supports. A variety of amino acid microsequenators utilizing either gas- or liquid-phase solvent delivery systems are commercially available (Table 11). The gas-phase sequenator replaced the spinning cup of the EdmanBegg instrument with a cartridge containing a reaction chamber with a glass-fiber disk, in which the protein is embedded and through which flow liquid and vapor-phase reagents of the Edman chemistry. A similar instrument with minor modifications was described by Sively and coworkers (Hawke et al., 1985; Shively, 1986b). Improved gas-phase miTABLE I1 COMMERCIALLY AVAILABLE AMINO ACIDMICROSEQUENATORS~ Manufacturer

Sample attachment

Solvent delivery

Applied Biosystems, Foster City, California Chelsea Instruments, London, England Herbet Knauer GmbH, Berlin, Germany MilliGedBiosearch, Burlington, Massachusetts Porton Instruments, Tarzana, California, and London, England

Noncovalent

Liquid or gas phase

Noncovalent

Gas phaseb

Noncovalent

Liquid and gas phases"

Covalent

Liquid phase

Noncovalent

Gas phase

'Adapted from Knight (1989). Can be modified for liquid delivery. Solvents are delivered as liquids and converted to gas phase in the reaction chamber.

300

ERIK P. LILLEHOJ AND VEDPAL S . MALIK

crosequence analysis of low-picomole quantities of proteins was achieved by replacing the conventional cartridge with a continuousflow reactor containing Polybrene-coated porous glass beads (Shively et al., 1987). One problem with all of these sequencers is that they are mutually incompatible, thus limiting selection of the appropriate sequencing method to the particular instrument available. In an effort to circumvent this obstacle, Wittmann-Liebold and co-workers (1986; Wittmann-Liebold, 1983, 1986) designed a multipurpose sequencer combining a different version of automated degradation in a modular fashion, allowing interchange of cup, column, cartridge, or other reaction chambers for liquid-, solid-, or gas-phase sequence analysis. Further advancements in sequenator design and improvements in the sensitivity of PTH amino acid analysis should permit sequence determination in the low-femtomole range (Kent et al., 1987). PEPTIDE MAPPING AND SEQUENCE ANALYSIS G. RADIOCHEMICAL Difficulties in obtaining sufficient quantities of biologically important protein present in trace amounts have provoked technological advance in the development of high-sensitivity microanalytic methods of protein analysis. Initially, radiochemical microsequencing techniques were developed using existing instrumentation applied to nonradioactive proteins. They were particularly well adapted to primary structural characterization of proteins purified by SDS-PAGE. In this manner, amino acid sequence information was obtained from a variety of different proteins, most notably those of immunologic interest that are located on the surface of eucaryotic cells. However, the utility of this procedure was limited in its application to other types of proteins. Later, as a new generation of microsequencers and chromatographic instruments was developed, interest in radiosequence methodologies waned. Two different approaches to radiochemical amino acid sequence analysis have been undertaken. The first utilized 35S-labeledPITC during Edman degradation, followed by chromatography in the presence of unlabeled PTH amino acids, and liquid scintillation counting to identify the radioactive residue (Jacobs and Niall, 1975).Detection of radiolabeled PTH amino acid derivatives produced by this method was approximately 100-fold more sensitive than conventional methods available at the time. However, this method has not proved to be more sensitive than conventional sequence analysis using the gas-phase sequenator (Beyreuther et al., 1983). Alternatively, radiochemical sequence determination based upon in vitro biosynthetic incorporation of 35S-labeledand/or 3H-labeled amino


301

acids into polypeptides, electrophoretic purification of the labeled proteins, and subsequent Edman degradation in the spinning-cup sequencer has been more useful. Specific details of the methodology have been extensively reviewed (Coligan and Kindt, 1981;Coligan et al., 1983).Briefly, eucaryotic cells maintained in culture are exposed to a medium containing a single or few selected radioactive amino acids for a period of several hours, allowing uptake into intracellular amino acid pools and incorporation into newly synthesized proteins. Following cell disruption, the radioactive proteins are purified from other radiolabeled components and are subjected to primary structural analysis in the presence of unlabeled carrier proteins. With polypeptides containing a single labeled amino acid, determination of the positions of that particular residue in the primary sequence is accomplished by simply quantitating the amount of radioactive ATZ derivative released at each cycle of the Edman degradation, without the need for PTH amino acid identification. The sequence data obtained in this manner, however, are limited to the extent at which that particular amino acid occurs in the NH2 terminus of the polypeptide. Conversely, use of multiple amino acids as a group in the labeling process increases the amount of sequence information obtained in a single sequence determination but necessitates identification of the released radioactive PTH amino acid residues. Radiosequence analysis has also proved useful in the identification of posttranslational modifications, for example, by using [32P]orthophosphateto delineate sites of tyrosine phosphorylation (Patschinsky et al., 1982). H. INTERFERENCE WITH SEQUENCE ANALYSIS Two major problems that hamper amino acid sequence analysis are (1)introduction of contaminants during protein purification, interfering

with the Edman chemistry and/or subsequent PTH amino acid analysis, and (2) protein modification. For instance, degradation products from Coosmasie blue (Shoji et al., 1986;Wilson, 1988)may cause artifactual HPLC peaks in the first several Edman degradation cycles. High concentrations of glycine used in the Laemmli gel system may also interfere with PTH amino acid identification, and other buffer systems, for example, 10 mM 3-(cyclohexylamino)-l-propanesulfonic acid (CAPS) (Matsudaira, 1987),have been used to avoid this problem. Some of these contaminants are removed during the destaining process or can be extracted by additionally rinsing the gel in several changes of deionized, high-purity water (Shoji et a]., 1986).The period of staining and destaining, however, should be minimized in consideration of the acid lability

302

ERIK P. LILLEHOJ AND VEDPAL S.MALIK

of the aspartyl-proline peptide bond. Alternatively, selective protein precipitation with organic solvents, such as 90% methanol or ethanol at -20°C for 4-18 hours, has been used (Hunkapiller and Lujan, 1986; Ratajczak et al., 1988). The drawbacks of this method include: (1)a minimum of at least 1 pg of protein, (2) a requirement for SDScontaining buffers to achieve resolubility of some proteins, (3) substantial losses, and (4) variable recoveries. Pearson et al. (1987; Pearson, 1986) and Simpson et al. (1987,1989) have investigated reverse-phase HPLC techniques to further purify and consistently recover proteins from SDS-PAGE gel electroeluates in high yield. The most refractory of modifications is NH2-terminal blockage, rendering the protein inaccessible to Edman degradation. The NH2terminal blockage may either occur naturally or be generated during purification. For example, high concentrations of urea at elevated pH and temperature conditions result in carbamylation of primary amino groups by cyanate ions. A variety of nonurea polyacrylamide gel systems to purify proteins over a broad molecular weight range prior to sequencing have been developed (Tsugita et al., 1982; Anderson et a]., 1983; DeWald et al., 1986).Other chemical modifications that have been observed are cyclization of NHz-terminal glutamine residues, or oxidation, and formation of lactones (Allen, 1981).Residual-free radicals and oxidants present in the gel after acrylamide polymerization will destroy tryptophan, histidine, and methionine (Hunkapiller and Lujan, 1986). This effect can be minimized by letting the gels set overnight, prerunning the gels, degassing gel solutions to reduce the amount of catalyst needed for polymerization, or adding an antioxidant such as sodium thioglycolate (Hunkapiller and Lujan, 1986; Simpson et al., 1989).Other recommendations include (1)using a high proteidgel ratio that will not sacrifice resolution and (2) storage of gel solutions with mixed-bed ion exchangers to remove unwanted ions and avoid premature polymerization. Hunkapillar and co-workers (1983, 1986) have emphasized the need for careful attention to the source and quality of chemicals to minimize protein modification during electrophoresis. Specific recommendations regarding the preparation and purification of reagents and solvents for protein microsequencing have been published (Hunkapiller and Kim, 1986). In spite of attempts to minimize NH2-terminal modification during purification, most proteins exist as NHz-terminally blocked molecules in their native biological state (Benjamin et a]., 1989).Examples of these types of alterations include acetyl, formyl, pyroglutamyl, and pyrrolidone carboxylic acid groups (Allen, 1981). Chemical and enzymatic procedures exist to remove selectively some of these groups without


303

peptide bond cleavage, but these are not universally applicable to all proteins. Alternatively, gas chromatography, mass spectrometry, or nuclear magnetic resonance spectrometry can be used to identify the blocking groups. Another type of natural posttranslational modification that requires chemical derivatization prior to sequence analysis is amino acid cross-linking. The most common of such cross-links is disulfide bond formation between cysteine residues. These are generally broken by reduction and alkylation or oxidation. Cross-links between lysine and lysine, lysine and glutamic acid, and tyrosine and tyrosine have also been observed (Allen, 1981).

IV. Structural Analysis of Proteins Electroblotted from One- and Two-Dimensional Polyacrylamide Gels

The major limitation that initially hindered the use of twodimensional polyacrylamide gels for purification of proteins in a form suitable for primary structural analysis was the upper limit on the quantity of protein that could be isolated. With one-dimensional SDSPAGE, preparative-scale isolations were readily obtained, as with the apparatus described by Akaiwa (1982). By the very nature of their design, however, two-dimensional gel systems have proved less amenable to scale-up strategies. Typical protein loads accommodated in this case are on the order of 0.1 mg if maximum resolution is to be maintained (Benjamin et a ] . , 1989). Under these conditions, the problems inherent to electroelution (i.e., protein modification and sample loss) are magnified to a degree that limits the utility of the gel extraction techniques discussed above. Moreover, charge microheterogeneity introduced by posttranslational glycosylation (Fig. 7) reduces the proteinto-gel ratio, further complicating electroelution procedures. Two technologies have now matured sufficiently so as to have achieved solutions to these problems. First, recent developments in electroblotting techniques have introduced new protein binding matrices capable of withstanding the harsh chemical reagents used in the Edman degradation procedure. Second, a new generation of protein microsequenators has been introduced based upon the Edman chemistry, yet these are sufficiently sensitive to obtain sequences at the picomole range. Proteins purified electrophoretically and blotted to inert membranes can be directly submitted to microsequence analysis. The remainder of this review will examine these technological developments and their applications and consider what future prospects hold for further advancements.

304 a

ERIK P. LILLEHOJAND VEDPAL S. MALIK E Y

FIG.7. Protein microheterogeneity produced by variations in glycosylation. A rabbit antiserum against a synthetic peptide corresponding to the COOH terminus of the human T cell leukemia virus type I outer surface membrane glycoprotein gp46 was used to detect this molecule on immunoblots prepared from viral proteins electroblotted from one- or two-dimensional SDS-PAGE gels. (A) Immunoblot of a one-dimensional gel. A major immunoreactive protein at 46 kDa was detected. (B) Immunoblot of two-dimensional gel. IEF was performed in the horizontal direction and SDS-PAGE in the vertical direction. Nine immunoreactive spots were detected at 46 kDa varying in PI from 5.5 to 7.0. The migration of molecular weight standards is indicated on the right. This charge heterogeneity was due to variations in carbohydrate because deglycosylation with endoglycosidase F produced a single immunoreactive spot.

A. PROTEIN ELECTROBLOTTING

Although polyacrylamide is a superior support for resolving complex protein mixtures, it is often unsuitable for subsequent characterization of the separated proteins. Many investigators have thus developed methods for vectoral transfer of polypeptides from SDS-PAGE gels onto appropriate solid matrices in a form that facilitates not only their visualization but also immunological and physicochemical evaluation. Nitrocellulose was the first such support utilized for these purposes (Towbin et al., 1979; Renart et al., 1979). Protein binding to this membrane is mediated by hydrophobic (Schneider, 1980)and/or electrostatic (Farrah et al., 1981) forces, although not all polypeptides bind equally well. In


305

particular, low-molecular-weight polypeptides (less than 20 kDa) were found to be poorly retained on nitrocellulose (Burnette, 1981; Lin and Kasamatsu, 1983) using electrotransfer conditions appropriate for blotting of higher molecular weight proteins. Addition of methanol to the transfer buffer increases its capacity for low-molecular-weight proteins (Gershoni and Palade, 1982). Otter et al. (1987) developed a two-stage electroblotting procedure that efficiently transfers both highand low-molecular-weight components. Additional determinants (e.g., glycosylation and/or disulfide bond reduction, membrane pore size, and presence of organic solvents) may also influence the extent of protein immobilization (Miribel and Arnaud, 1988). A variety of other membrane supports have been introduced subsequently. Chargemodified nylon has been shown to possess a higher binding capacity and to retain some polypeptides that do not bind to nitrocellulose (Gershoni and Palade, 1982; Miribel and Arnaud, 1988), although other workers have disputed these claims (Pluskal et al., 1986). In addition to its greater mechanical strength compared to nitrocellulose, protein binding to nylon occurs mainly through ionic interactions and permits electrotransfers in the absence of methanol. Proteins immobilized on transfer membranes may be visualized with any of a number of staining reagents. A summary of protein staining techniques on nitrocellulose, nylon, and polyvinylidene difluoride (PVDF) membranes has been published (Pluskal et al., 1986). Glass fiber, nitrocellulose, and PVDF are compatible with classic stains such as Coomassie blue, amido black, India ink, Ponceau S , and silver particles. Nylon membranes, on the contrary, produce extremely high background staining using amido black or colloidal gold particles. Staining with colloidal metal particles is more sensitive than dye-binding procedures: about 1 ng/mm2for iron and less than 1 ng/mm2for gold (Moeremans et al., 1985a,b). Segers and Rabaey (1985) reported the latter was capable of detecting more protein spots than was silver staining on nitrocellulose electroblots of two-dimensional polyacrylamide gels. All of these visualization methods are compatible with amino acid compositional and sequence analyses (see below). In other cases, wherein staining dyes may interfere with other techniques (such as tryptic peptide mapping), reversible staining with Ponceau S (Salinovich and Montelaro, 1986; Montelaro, 1987) or direct visualization of unstained proteins by transillumination (Reig and Klein, 1988) may prove beneficial. Recovery of electroblotted proteins from membranes after separation by SDS-PAGE has been accomplished by two methods. In the first, the membrane is dissolved in a suitable organic solvent and the protein is

306


recovered in a form suitable for immunization (Knudsen, 1985) or primary structure determinations (Anderson, 1985).Alternatively, electrotransferred proteins can be eluted off the membrane under conditions that maintain the integrity of the support. Parekh et al. (1985) obtained the highest elution efficiencies from nitrocellulose using nonpolar solvents (50% pyridine or 40% acetonitrile). Yuen et al. (1989) reported extraction of 60% of p-lactoglobulin A from PVDF using 70% isopropanol/5% trifluoroacetic acid. Szyewczyk and Summers (1988) reported optimum elution of proteins electroblotted to PVDF using a detergent mixture consisting of 2% SDS/lYoTriton X-100. Polypeptides bound to poly(4-vinyl-N-methylpyridiniumiodide) (P4MVP)-coated glass-fiber paper were recovered using a solution of 80% formic acid (Bauw et al., 1988, 1989). A systemic survey of protein purification by preparative electroblotting onto a variety of membranes revealed a most efficient elution from nitrocellulose, nylon, or PVDF with acetonitrile and from glass-fiber membranes with formic acid (Montelaro, 1987). These procedures are useful for generating enzymatic or chemical cleavage fragments for peptide mapping and/or internal amino acid sequence studies. Judd (1987) obtained analytical fingerprints of peptides following direct radioiodination and cleavage on nitrocellulose. Scott and co-workers (1988) recovered peptide fragments generated by in situ CNBr digestion of PVDF-electroblotted immunoglobulins that were subsequently separated by reelectrophoresis and were electroblotted and analyzed by NHz-terminal microsequencing. Other workers have described a similar strategy to procure primary structural information of NHz-terminally blocked proteins after in situ protease digestion and recovery of peptides from the membrane (see below). B , MICROANALYSIS OF ELECTROBLOTTED PROTEINS

The ability to electroblot proteins from one- or two-dimensional polyacrylamide gels to chemically inert membranes permits established procedures of primary structural analysis to be performed at picomole levels. Both amino acid composition and NH2-terminal sequence information are essential parameters in the characterization of an unknown protein. Compositional data also provide an estimation of protein quantity. Microdetermination of amino acid composition has now been accomplished on PVDF (LeGendre and Matsudaira, 1988; Nakagawa and Fukuda, 1989; Plough et al., 1989; Santucci et al., 1989; Tous et al., 1989; Yuen et al., 1989) and glass-fiber (Vandekerckhove et al., 1985; Bauw et al.,1987; Bergman and Jornvall, 1987a; Eckerskorn et al.,1988) supports. Furthermore, the same protein-containing bot that was ini-


307

tially used for sequence analysis can be hydrolyzed in situ and the amino acid composition of the remaining unsequenced protein can be determined (Hildebrandt and Fried, 1989). Theoretically, other types of posttranslational modifications, for example, glycosylation and acylation, should be identifiable and characterizable by a similar method. C. MICROSEQUENCE ANALYSIS WITH GLASSFIBER

The greatest application of two-dimensional polyacrylamide gel electrophoesis and electroblotting to chemically inert membranes is the direct amino acid microsequence. Figure 8 diagrams the principles of this process. Amino acid sequencing of proteins electroblotted to nitrocellulose or nylon membranes is unavailable because these membranes are incompatible with the chemical reagents and solvents used in the

n/

BLOT TIN G

'

""'

E)

I

MIGRATION OF PROTEIN

4

STAINING

4 4

APPLICATION IN SEQUENATOR

SAMPLE IN CARTRIDGE

FIG. 8. Diagrammatic illustration of the two-dimensional gel/electroblotting/microsequencing process. The protein sample is resolved by IEF in the first dimension, SDSPAGE in the second dimension, and electrotransferred to an inert support (activated paper shown here) as indicated on the right side of the figure. As shown on the left, proteins migrate out of the polyacrylamide gel and are retained on the support. Their position is revealed by staining; the appropriate segment of the support is excised and placed in the reaction cartridge of a gas-phase sequenator. Reproduced from Aebersold et al. (1986a), with permission.

308


Edman degradation chemistry. Originally, the glass disk in the reaction cartridge of the gas-phase sequencer was coated with Polybrene, and proteins in solution were directly applied to it, then dried and subjected to sequence analysis. Vandekerckhove et al. (1985) were the first to obtain sequence data from a protein electroblotted onto glass-fiber sheets in the cartridge. Sperm whale myoglobin separated by SDSPAGE was transferred to Polybrene-coated glass fiber and was successfully sequenced through the first 18 NH2-terminal residues. Internal amino acid sequences of actin were also obtained from peptides after S. aureus V8 protease digestion, separation on one-dimensional polyacrylamide gels, and electroblotting to the Polybrene-coated glass fiber. Because Polybrene leads to a high level of background staining with Coomassie blue, these peptides were visualized by either Coomassie blue staining of the gel before transfer or fluorescamine staining of the electroblotted glass membrane. Bergman and Jornvall (1987b) visualized proteins in the gel with 1 M KC1 and electroblotted only those regions containing the protein of interest, prior to sequence analysis. Walsh et al. (1988) determined the NH2-terminal sequences of several Halobacterium marismortui proteins isolated by two-dimensional polyacrylamide gel electrophoresis, transferred to Polybrene-coated glass fiber, and directly visualized on the membrane with the fluorescent stain 33'-dipentyloxacarbocyanine iodide (DPOCC). Andrews and Dixon (1987)reported a procedure for in situ alkylation of cysteine residues on Polybrene-coated glass fiber for direct microsequence analysis. A variety of other modifications of glass fiber have been developed to increase protein-binding capacity and allow direct protein staining on the membrane. Table I11 lists the different electroblotting and polypeptide detection techniques that have been reported and compares the initial and repetitive yields of the sequences obtained. Bauw et al. (1987) and Vandekerckhove et al. (1987) replaced Polybrene with poly(4-vinyl-N-methylpyridiniumiodide) to improve membrane binding and changed the electrotransfer buffer to enhance protein gel elution. In this manner, Nicotiana plumbaginifolia proteins separated by two-dimensional SDS-PAGE and visualized by fluorescamine staining were partially sequenced. Eckerskorn et al. (1988) prepared a silylated glass fiber with blotting and staining characteristics superior to those of polybase-coated glass. The protein-binding capacity of this membrane was reported to be approximately 70 pg/cm2compared to 20-40 pg/cm2 for Polybrene- or P4VMP-modified glass. Furthermore, silylated glass fiber could be directly stained with Coomassie blue for detection of electroblotted proteins. Aebersold et al. (1986a) and subsequently Moos et al. (1988) immobilized SDS-PAGE-separated proteins on glass fiber


309

that had been activated by acid etching alone or subsequently modified for ionic interaction with either 3-aminopropyltriethoxy silane (APglass) or N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (QA-glass). Bound proteins were detected on the membranes by Coomassie blue or DPOCC staining. Fifteeen different peptides or proteins isolated on one- or two-dimensional gels in amounts between 5 and 140 pmol were sequenced for 9-21 amino acids (Aebersold et a]., 1986a). Figure 9 illustrates the electroblotted profile of a mixture of standard proteins from this study and subsequent NH2-terminal sequence analysis through the first three cycles of one of the proteins (soybean trypsin inhibitor). Aebersold et al. (1986a, 1988) also introduced covalent attachment of proteins electroblotted from polyacrylamide gels to 1,4-diphenylenediisothiocyanate (D1TC)-modifiedglass fiber. This concept was based upon previous techniques for covalent coupling of proteins to supports for solid-phase sequencing, but which were unsuitable for handling small quantities of proteins. Proteins electroblotted directly onto DITC glass through primary amino groups were detected with the fluorescent stain 7-(diethylamino)-3-[4-[(iodoacetyl)amino]phenyl]-4-methylcoumarin (DCIA). Using this technique, NH,-terminal sequences of 10 different proteins in the range of 5-60 pmol were reported. With the advent of solid-phase sequence analysis coupled with micropreparative isolation techniques, it is now possible to begin investigating alternative reaction conditions of the standard Edman chemistry (Kent et al., 1987). For example, more stringent washing solvents (e.g., neat trifluoroacetic acid) and higher flow rates that would normally be incompatible with noncovalently bound proteins can be employed to remove more effectively sample contaminants and shorten the cycle time. D. MICROSEQUENCE ANALYSIS WITH PVDF

In 1986, a new hydrophobic PVDF-based transfer membrane (Immobilon-P, Millipore, Bedford, Massachusetts) with superior protein-binding capacity, chemical resistance, and mechanical rigidity was described (Pluskal et al., 1986). This membrane was also compatible with the conventional protein-staining reagents (Coomassie blue, amido black, India ink), making it ideally suited as an electroblotting support. Matsudaira (1987) was the first to apply this support to obtain NH2-terminal amino acid sequences of low-picomole amounts of electroblotted proteins. The average initial and repetitive yields ranged from 30 to 100% and 88 to 93% respectively for myoglobin and p-lactoglobulin. Xu and Shively (1988) modified the procedure of Matsudaira to

TABLE I11 CHARACTERISTICS OF ELECTROBLOTTING MEMBRANES IN DECT AMINOACIDMICROSEQUENCING

Electrotransfer conditions Matrixa Activated GF

Reference Walsh et al. (1988)

Aebersold et al. (1986a)

Derivatized GF, Polybrene

Bergman and Jornvall

Buffer

Protein detection

Time (volts)

Sequencingb IY

RY

NR

25 mM TRIS, pH 8.4, 0.5 mM DTT 1-5% acetic acid, 0.5% NP-40 40 mM NH,HCO,, pH 8.8

1 hour (150-200 mA),4 hours (650 mA) 3-12 hours (70 V)

Coomassie blue

NR

Coomassie blue

61-74%

92-96%

6 hours (3 W)

KCI

8-48%

94-98%

25 mM TRIS, pH 8.4, 0.5 mM DTT 50 mM borate, pH 8.0

1 hour (150-200 mA), 4 hours (650 mA) 20 hours (3 V/cm)

50 mM TRIS, 50 mM

8 hours (35 V)

Fluorescamine, DPOCC Fluorescamine, Coomassie blue Fluorescamine

50%

91-96%

2 hours (50 V)

DPOCC

61-76%

93-94%

10-90 minutes (50 or

Coomassie blue

40-70 minutes (50 V)

Autoradiography

38-78Yo

92-93Yo

2-5 hours (mA/cmZ)

Coomassie blue, amido black, Ponceau S

50%

93-96Vo

(1987a)

Walsh et al. (1988)

cr W

Vandekerckhove et al. (1985)

0

P4VMP

Bauw et al. (1987)

AP, QA

Aebersold et al. (1986a)

Moos et al. (1988)

Xu and Shively (1988)

Silicone

Eckerskorn et al. (1988)

borate 25 mM TRIS, pH 8.3, 10 mM glycine, 0.5 mM DTT 25 mM TRIS, 10 mM glycine, 0.5 mh4 DTT, or 10 mM CAPS, 0.5 mM DTT 25 mM TRIS, pH 8.3 192 mM glycine 50 mM borate, pH 9.0

loo V)

1-48Y0,C 57-86%d

NR

DITC

Aebersold et al. (1988)

PVDF

Pluskal et al. (1986)

Matsudaira (1987)

LeGendre and Matsudaira (1988)

Walsh et al. (1988)

Xu and Shively W

Y CL

(1988)

Nokihara et al. (1988)

Benjamin et al. (1989)

24 mM N-

ethylmorpholine, pH 8.3 25 mM TRIS, 192 mM glycine, 15-20% methanol 10 mM CAPS, pH l l . O , l O Y o methanol 25 mM TRIS, pH 8.4, 192 mM glycine, 15% methanol 25 mM TRIS, pH 8.4, 0.5 mM DTT

25 mM TRIS, pH 8.4, 192 mM glycine 25 mM TRIS, pH 8.3, 192 mM glycine, 15% methanol 10 mM CAPS, pH 11.0, 10%

2 hours (50 V)

DCIA

1-2 hours (70 V)

10-30 minutes (500 mA)

Coomassie blue, amido black, Ponceau S Coomassie blue

30-100%

84-98Yo

NR

Coomassie blue

70-8Ovo

NR

1hour (150-200 mA), 4 hours (650 mA)

NR

90-94%

1.7 hours (25-30 V)

Fluorescamine, Coomassie blue, DPOCC, amido black Coomassie blue

20-6Oyo

92-94yo

2 hours (7 V/cm)

DHOC

10-30 minutes (90 V)

Coomassie blue, Ponceau S

50-6OYo

83-96Yo

1 6 hours (70 V)

Coomassie blue

80-85%

88-94%

20-4Oyo

NR

NR

92-94yo

NR

NR

methanol Yuen et al. (1989)

10 mM CAPS, pH 11.0

a Abbreviations: P4VMP. poly(4-vinyl-N-methylpyridinium iodide: AP, aminopropyl; QA, N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride; DITC, 1,4-diphenylenediesotbiocyanate; PVDF, polyvinylidene difluoride; DTT, dithiothreitol; CAPS, 3-(cyclohexylamino)-l-propanesulfonic acid; DPOCC, 3,3'-dipentyloxacarbocyanine iodide; DCIA, 7-(diethylamino)-3-[4-[(iodoacetyl)amino]phenyl]-4-methylcoumarin: DHOC, 3,3'-dihexyloxacarbocyanine iodide. IY, Initial yield: RY, repetitive yield; NR, not reported. Laemmli (1970),gel electrophoresis system. Jovin (1973a,b,c), gel electrophoresis systems.

0.00

3 !

a

0.OC

J b

a

0" 0

0.oc C

c

2

15

10

Minutes


313

achieve higher transfer yields by precoating PVDF with Polybrene prior to transfer and optimizing the electroblotting conditions. Considering the amounts of proteins resolved on SDS-PAGE gels, electroblotted to PVDF, and the initial sequencing yields, these authors reported overall yields of 20-30% for soybean trypsin inhibitor and bovine serum albumin and 50-60% for P-lactoglobulin. Compared to glass-fiber membranes, PVDF offered the advantages of (1)no preactivation or chemical modification, (2) lack of band spreading during the transfer step, (3) the ability to visualize immobilized proteins with staining reagents, and (4) lower backgrounds during PTH amino acid analysis. A multitude of other recent literature reports have described NHzterminal sequence determinations of proteins separated by polyacrylamide gel electrophoresis and electroblotted to PVDF membranes (LeGendre and Matusdaira, 1988; Nokihara et al., 1988; Reig and Klein, 1988; Scott et al., 1988; Szyewczyk and Summers, 1988; Walsh et al., 1988; Waters et al., 1988; Xu and Shively, 1988; Benjamin et al., 1989; Plough et al., 1989;Simpson et al., 1989; Yuen et al., 1989).As listed in Table 111, a variety of protein electrotransfer and detection conditions have been used with this support. Blotting buffers have consisted of CAPS or TRIS either containing or devoid of glycine. Blotting times have generally been on the order of one to several hours, and currents in the range of 200-600 mA, although shorter transfer times have also been successful (Matsudaira, 1987; Yuen et al., 1989). The list of protein detection reagents employed with PVDF includes Coomassie blue, amido black, Ponceau S , fluorescamine, DPOCC, and 3,3’-dihexyloxycarbocyanine iodide. Depending on the particular protein and electroblotting conditions, amino acid sequencing data reported by these investigations have varied between 20 and 100% initial yields and 83 and 98% repetitive yields. As indicated above, in some cases, low initial yield values may reflect partial NHz-terminalblockage incurred during purification, and specific

FIG.9. Electroblotting and microsequencing analysis after two-dimensional gel electrophoresis. (A) A mixture of E. coli p-galactosidase (a),bovine serum albumin (b),bovine carbonic anhydrase (c), soybean trypsin inhibitor (d), sperm whale myoglobin (e),and bovine a-lactalbumin (f) was resolved on a two-dimensional gel, electroblotted to an activated glass-fiber support, and stained with Coomasie blue. The region containing soybean trypsin inhibitor was excised and subjected to microsequencing in the gas-phase sequencer. (B) HPLC analysis through the first three degradation cycles. Background peaks are indicated by numbers. Released amino acids (a, b, and c) are identified by the singleletter code (D, Asp; F, Phe; V, Val). The identified sequence was Asp-Phe-Val. Reproduced from Abersold et al. (1986a). with permission.

314


procedures have been employed to minimize this effect. Most proteins, however, exist as NH,-terminally blocked molecules in their native biological state. In these instances, three approaches have been taken to obtain internal amino acid sequence information. In the first, proteins resolved by preparative two-dimensional polyacrylamide gels were cut out of the gel, digested in the gel slice with trypsin with the resulting peptides separated on a second single-dimension SDS-PAGE gel, electroblotted to PVDF, and sequenced (Kennedy et al., 1988a,b).An example of this approach used to obtain internal amino acid sequence data from actin is shown in Fig. 10. The other two methods have utilized on-membrane chemical or enzymatic cleavage followed by either direct, in situ microsequence analysis of the unseparated peptides or fragment

B 45

92-

66-

45 -

-

C

-

31 -

21 -

-(

NHz 6)VAALVODNGSGMXKAGFA

-(6) -(6)

VAALVh VAALVODNGSGMXKA

-(226)MiTAASSSSLEKSYELPDGQ

14 31-

-(95)

LRVAPEEHPVLLTEA

(318)ITALAPPTMKIKI IAPP

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