Electrophoresis of Enzymes: Laboratory Methods

311 Pages·1994·41.587 MB·English

by  Gunter M. Rothe

SPRINGER
LAB MANUAL
G.M. Rothe
Electrophoresis
of Enzymes
Laboratory Methods
With 59 Figures
Springer Verlag
Berlin Heidelberg New York
London Paris Tokyo
Hong Kong Barcelona Budapest
Professor Dr. GUNTER M. ROTHE
Johannes Gutenberg-Universitat
Institut fUr Allgemeine Botanik
Fachbereich Biologie
D-55099 Mainz
lSBN-13:978-3-642-79071-3 e-lSBN-13:978-3-642-79069-0
DOl: 10.1007/978-3-642-79069-0
Library of Congress Cataloging-in-Publication Data
Rothe, G. (Gunter)
Electrophoresis of enzymes: laboratory methods I Gunter M. Rothe.
(Springer laboratory)
Includes bibliographical references.
ISBN-13:978-3-642-79071-3
1. Enzymes -Separation -Laboratory manuals. 2. Electrophoresis -Laboratory manuals.
I. Title. II. Series.
QP601.R786 1994
574.19'25 -delo
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To my parents
Preface
This book is addressed to both new and experienced workers interested in the
properties of enzymes or using enzymes as gene markers in areas such as biochemi
stry, clinical pathology, zoology, botany, systematics, ecology, population genetics or
natural resources management.
The first chapter introduces into the classification, structure and size of enzymes
and isozymes. The second chapter provides protocols to extract (iso)enzymes from
microbial, plant, animal and human tissue and cells-respectively. Methods to extract
enzymes from cell-organelles of plant and animal tissues are also presented. The third
chapter is dedicated to reliable electrophoretic procedures being in use to separate
native enzymes. Some quantitative electrophoretic methods are also given to deter
mine the size and net charge of (iso)enzymes. Chapter four summarizes methods to
renature enzymes after sodium dodecylsulphate electrophoresis. Chapter five gives
an overview on the various "histochemical" reactions being in use to visualize
enzymes following electrophoresis. Specific staining protocols together with suitable
electrophoretic systems for more than 100 different enzymes are listed in chapter six.
Finally, methods of data acquisition from enzyme patterns produced by electro
phoresis are presented and various examples given to calculate populational genetic
statistics.
Acknowledgement
I am very grateful to Prof. Dr. Bertold J. Radola who encouraged me to write this book.
I am also much indebted to all my students but especially to Renate Bohrmann,
Ute Does, Dr. Werner D. Maurer, Brigitte Niethard, Monika Noll, Dr. Irene Novotny,
Ina Pappe, Dr. Huschang Pukhanbaba, Anke Richert and Beate Schellenberg. I thank
PO Dr. Klaus Honomichl and Hans Weidmann for their help in establishing the
computer program MOL-MASS and I wish to thank Peter Enders and the staff of
Springer-Verlag for their assistance and patience.
Mainz, March 1994 Gunter M. Rothe
Abbreviations
ADP: Adenosine-5'-diphosphate
AOL: Agar overlay
ATP: Adenosine-5'-triphosphate
BIS: N, N' -methylenebisacrylamide
CG: Cellogel
DNA: Deoxyribonucleic acid
EDTA: Ethylenediaminetetraacetic acid
FAD: Flavin adenine dinucleotide
FM: Flow method
IEF: Isoelectric focussing
MOL: Membrane overlay
M,: molecular mass [glmol]
MTT: 3-( 4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoli urn bromide
NAD: f3-Nicotinamide adenine dinucleotide
NADH: f3-Nicotinamide adenine dinucleotide, reduced form
NADP: f3-Nicotinamide adenine dinucleotide phosphate
NADPH: f3-Nicotinamide adenine dinucleotide phosphate, reduced form
PAA: Polyacrylamide
PAGE: Polyacrylamide gel electrophoresis
Pi: Orthophosphate
PMS: Phenazine methosulphate
POL: Paper overlay
SDS: Sodium dodecylsulphate
SGE: starch gel electrophoresis
% T: (g Acrylamide + g BIS)/Ioo ml
Temed: N,N ,N', N'- Tetramethylethylenediamine
Tris: Tris (hydroxymethyl) aminomethane
U: Units
UTL: Ultrathin layer
Contents
Introduction . . . . . . . . . .
1.1 Enzyme Classification . . . . .
1.2 Enzyme Subunit Composition . 3
1.3 Isozyme Classification . 14
1.4 References . . . . . . . . . . . 18
2 Extraction of Enzymes from Tissues, Cells and Cell-Organelles . 19
2.1 Methods to Extract Enzymes from Microorganisms . . . . . 19
2.2 Methods to Extract Enzymes from Animal Soft Tissue . . . . 22
2.3 Differential Extraction of Cytosolic and Mitochondrial Enzymes
from Animal Soft Tissue . . 26
2.4 Extraction of Insects . . . . . . . . . . . . 28
2.5 Extraction of Plant Tissues ....... . 29
2.6 Concentration of Diluted Enzyme Extracts 31
2.7 Mammalian Blood . . . . . . . . . 34
2.8 PancreaticIslets . . . . . . . . . . . . . . 42
2.9 Isolation of Subcellular Organelles .... 43
2.10 Catalytic Activities of Enzymes Forming a Common Metabolic
Sequence.
2n ~&reoc9 . . . . . . . . . . . . . . .
3 Methods for Separating Native Enzymes 71
J.1 General Considerations . . . . . 71
3.2 Cellulose Acetate Electrophoresis 72
3.3 Starch Gel Electrophoresis . . . . 80
3.4 References . . . . . . . . . . . . 88
3.5 Analytical Polyacrylamide Gel Electrophoresis for Separating
Native Enzymes . . . . . . . . . . . . . . . . . . . . . 89
3.6 2-D-Electrophoresis under Non-Denaturing Conditions 120
3.7 References . . . . . . . . . . . . . . . . 124
4 Sodium Dodecylsulphate Electrophoresis . 127
4.1 Homogeneous Buffer Systems . 127
4.2 Discontinuous Buffer Systems . 128
4.3 Gradient Gel Systems ..... 130
XII Contents
4.4 Enzyme Visualization Following SDS-Electrophoresis 134
4.5 References . . . . . . . . . . . . . 138
5 Chemistry of Enzyme Visualization. 141
5-1 Methods for Visualizing Oxidative Enzymes 142
5.2 Methods for Visualizing Transferases 151
5.3 Methods for Visualizing Hydrolases 155
5.4 Methods for Visualizing Lyases . . . 167
5.5 Methods for Localizing Isomerases and Ligases 168
5.6 Visualization of Enzymes by Different Methods 168
5.7 References . . . . . . . . . . . . . . . . . . . . 176
6 A Compilation of Protocols to Visualize Enzymes. 181
6.1 Index to Enzyme Recipes and Separation Systems 181
6.2 Protocols to Visualize Enzymes Following Electrophoretic Separation. 186
6.3 References . . . . . . . . . . . . . . . . . . . . . . 268
7 Data Evaluation in Population Genetics and Evolution 273
7.1 Allozymes as Gene Markers . 273
7-2 Population Genetic Measures 277
7.3 Calculation of Dendrograms 295
7.4 References 300
Subject Index . 303
1 Introduction
Enzymes are protein molecules with catalytic abilities. To date about 1500-2000
enzymes have been described and catalogued [1].
Bacteria such as Escherichia coli are estimated to possess about 3000 different
proteins per cell while higher eukaryote cells may synthesize about 50 000 proteins of
which the majority are enzymes.
1.1 Enzyme Classification
1.1.1 The EC Nomenclature
The presently accepted nomenclature of enzymes is that recommended by the En
zyme Commission (set up in 1955 by the International Union of Biochemistry in con
sultation with the International Union of Pure and Applied Chemistry) [1]. This
system is based on the specific chemical reaction catalyzed by an enzyme. All en
zymes known so far can be grouped into six main groups (with several sub-groups
specifying the reaction more precisely).
The system for classification of enzymes also serves as a basis for assigning code
numbers to them. The code numbers are prefixed by EC (Enzyme Commission) and
contain four numbers separated by points, with the following meaning [1]:
(a) the first number shows to which of the six main classes an enzyme belongs to;
(b) the second figure indicates the sub-class;
(c) the third figure gives the sub-sub-class; and
(d) the fourth figure is the serial number of the enzyme in its sub-sub-class:
1.1.1.1 Oxidoreductases
To this group belong all enzymes catalyzing oxidation-reduction reactions. The sub
strate (AH,) that is oxidized is regarded as a hydrogen donor (AH, + B = A + BH,).
The recommended name is dehydrogenase but, as an alternative, reductase is used.
The name oxidase is restricted to enzymes which exclusively use 0, as the hydrogen
acceptor. The second figure in the code number of oxidoreductases indicates the
group in the hydrogen donor which undergoes oxidation (e. g. CH -OH, CHO, CH -CH,
G. M. Rothe, Electrophoresis of Enzymes
© Springer-Verlag Berlin Heidelberg 1994