Preface | p. v |
List of protocols | p. xv |
Abbreviations | p. xvii |
Proteolytic enzymes: nomenclature and classification | p. 1 |
Introduction | p. 1 |
Terminology and nomenclature | p. 1 |
Peptidase and related terms | p. 1 |
Specificity-subsite terminology | p. 3 |
Catalytic type | p. 3 |
Homology | p. 3 |
The EC classification of peptidases | p. 4 |
What is the EC system? | p. 4 |
What information does the EC list contain? | p. 6 |
When and how can a newly-discovered peptidase be added to the EC list? | p. 7 |
The MEROPS system for the classification of peptidases | p. 12 |
Families | p. 13 |
Clans | p. 17 |
Individual peptidases | p. 17 |
Uses of the MEROPS system | p. 18 |
Steps one might take on discovering a new peptidase | p. 19 |
Acknowledgement | p. 20 |
References | p. 20 |
Purification of proteolytic enzymes | p. 23 |
Introduction | p. 23 |
Prelude to purification | p. 24 |
Assay | p. 24 |
Initial considerations | p. 26 |
Source | p. 26 |
Buffer composition | p. 26 |
Membrane-bound or soluble? | p. 26 |
Membrane-bound enzymes | p. 27 |
Endogenous inhibitors and activators | p. 29 |
General scheme for purification of proteolytic enzymes | p. 30 |
Initial steps | p. 30 |
Intermediate and final steps | p. 32 |
Specialized techniques for proteolytic enzymes | p. 33 |
Peptidyl aldehyde affinity chromatography | p. 33 |
Affinity columns for cysteine proteinases | p. 34 |
Affinity columns for trypsin-like enzymes | p. 34 |
Affinity columns for metalloproteinases | p. 35 |
Other affinity columns | p. 35 |
Optimization of the purification protocol | p. 37 |
Determination of homogeneity | p. 38 |
Purification of the proteasome; EC 3.4.99.46 | p. 38 |
Conclusions: many roads lead to Rome | p. 42 |
References | p. 42 |
Protease assay methods | p. 45 |
Introduction | p. 45 |
Assays with natural substrates | p. 45 |
Endopeptidase assays | p. 45 |
Exopeptidase assays | p. 55 |
Assays with synthetic substrates | p. 55 |
Endopeptidase and aminopeptidase substrates | p. 55 |
Spectrophotometric assays | p. 56 |
Fluorimetric assays | p. 59 |
Miscellaneous fluorimetric methods | p. 62 |
Carboxypeptidase substrates | p. 64 |
Radiometric assays | p. 64 |
HPLC assays for peptidases | p. 65 |
Capillary electrophoretic analyses of proteases | p. 67 |
Solid-phase protease assays | p. 69 |
Gel electrophoretic methods | p. 70 |
Plate assays | p. 73 |
Miscellaneous solid-phase assays | p. 73 |
Assays for histochemical studies | p. 74 |
Acknowledgements | p. 75 |
References | p. 75 |
Determination of protease mechanism | p. 77 |
Introduction--the importance of mechanistic classification | p. 77 |
The serine peptidases | p. 78 |
The cysteine peptidases | p. 80 |
The aspartic peptidases | p. 81 |
The metallopeptidases | p. 81 |
Methods for determining the mechanistic class | p. 83 |
Classification based on 'standard' inhibitors | p. 83 |
Chemical modification/identification | p. 86 |
Site-directed mutagenesis | p. 87 |
Mechanistic distinctions--intermediates | p. 88 |
Kinetic studies to probe the mechanism in more detail | p. 91 |
Notes on 'ideal' assays | p. 91 |
Kinetic determination of K[subscript m] and k[subscript cat] | p. 92 |
pH dependence of the kinetic parameters | p. 95 |
Solvent deuterium isotope effects | p. 96 |
Transition state analogues and substrate alteration | p. 97 |
Determination of primary specificity of a protease | p. 97 |
Degradation of standard proteins and peptides | p. 98 |
Cleavage of homologous synthetic peptides | p. 99 |
References | p. 102 |
Inhibition of proteolytic enzymes | p. 105 |
Introduction: which inhibitors? | p. 105 |
Principles for using irreversible and reversible inhibitors | p. 106 |
General structure of synthetic inhibitors | p. 106 |
How to live with crossreactivity | p. 106 |
Practical use of inhibition constants | p. 107 |
Irreversible inhibitors | p. 107 |
Reversible inhibitors | p. 108 |
Non-specific inhibitors | p. 109 |
[alpha]-Macroglobulins | p. 109 |
Peptide aldehydes | p. 110 |
Peptide chloromethyl ketones | p. 110 |
Metal chelators | p. 111 |
Class-specific inhibitors | p. 112 |
Serine proteases | p. 112 |
Cysteine proteases | p. 116 |
Proteasome | p. 119 |
Metalloproteases | p. 120 |
Aspartic proteases | p. 122 |
Inhibitors as active-site titrants | p. 122 |
Cysteine proteases | p. 122 |
Serine proteases | p. 124 |
Protease inhibitors in cell culture | p. 125 |
Suppression of proteolysis | p. 126 |
Therapeutic value of protease inhibitors | p. 127 |
References | p. 128 |
Finding, purification and characterization of natural protease inhibitors | p. 131 |
Introduction | p. 131 |
The meaning of inhibition | p. 131 |
Finding protease inhibitors | p. 132 |
Screening of inhibitors from natural sources | p. 132 |
Finding inhibitors by reverse zymography | p. 133 |
Finding inhibitors from DNA sequences | p. 135 |
Combinatorial protease inhibitors | p. 137 |
Purification of natural protease inhibitors | p. 137 |
Use of the target enzyme as an affinity ligand | p. 137 |
Conventional purification | p. 137 |
Reverse zymography | p. 138 |
Characterization of inhibitors: inhibition kinetics | p. 138 |
The importance of kinetics | p. 138 |
IC[subscript 50] and percentage inhibition | p. 138 |
Practical inhibitor kinetics | p. 139 |
Reversible inhibitors | p. 140 |
Irreversible inhibitors | p. 143 |
Practical applications of protein inhibitors | p. 146 |
Acknowledgements | p. 146 |
References | p. 146 |
Mass spectrometry of proteolysis-derived peptides for protein identification | p. 149 |
Introduction | p. 149 |
Mass spectrometry | p. 150 |
Methods of ionization | p. 150 |
Mass analysis | p. 152 |
Tandem mass spectrometry | p. 153 |
Interrogation of sequence databases using mass spectrometry data | p. 155 |
Choice of protease | p. 155 |
Peptide mass mapping | p. 157 |
Database identification via tandem mass spectrometry | p. 159 |
Analytical strategy for the identification or cloning of proteins using mass spectrometry | p. 161 |
Low-level protein preparation for characterization by mass spectrometry | p. 161 |
Protein visualization | p. 163 |
Proteolytic cleavage of gel separated proteins | p. 165 |
Protein identification by MALDI peptide mass mapping | p. 167 |
Peptide sequencing by nanoelectrospray tandem mass spectrometry | p. 172 |
Towards cloning of proteins using mass spectrometry data | p. 177 |
Post-translationally modified proteins | p. 181 |
Concluding remarks | p. 183 |
Acknowledgements | p. 183 |
References | p. 183 |
Using proteinases for Edman sequence analysis and peptide marking | p. 187 |
Introduction | p. 187 |
The need for digesting proteins to peptides | p. 187 |
Substrate preparation | p. 188 |
Purification techniques | p. 188 |
Sample requirements | p. 189 |
Reduction and alkylation of proteins | p. 191 |
Digestion | p. 193 |
Choice of proteinase | p. 193 |
Conditions for proteolytic digestions | p. 196 |
Digestion of proteins isolated on polyacrylamide gels | p. 199 |
Monitoring a reaction | p. 200 |
Analysis of the proteolytic digestion | p. 200 |
Mass spectrometry | p. 200 |
Electrophoresis | p. 200 |
Ion exchange | p. 200 |
Reverse-phase chromatography | p. 201 |
Data interpretation | p. 209 |
Acknowledgments | p. 209 |
References | p. 209 |
Prevention of unwanted proteolysis | p. 211 |
Introduction | p. 211 |
Proteolytic susceptibility of native proteins | p. 213 |
Intrinsic factors determining the susceptibility of proteins to proteolysis | p. 213 |
The influence of other molecules on susceptibility to proteolysis | p. 213 |
Properties of endogenous proteinases | p. 214 |
Identification of proteolysis as a problem | p. 214 |
Changes in protein properties | p. 214 |
Mimicking an effect with added proteinases | p. 216 |
Checking samples for proteinase activity | p. 216 |
Inhibition of proteinases | p. 217 |
Outline of approaches for reducing proteinase activity | p. 217 |
Suppression of endogenous proteinase activity | p. 217 |
Preventing proteolysis by denaturation | p. 217 |
Use of proteinase inhibitors | p. 220 |
Removal of proteinases | p. 227 |
Choice of starting material | p. 227 |
Cell disruption and fractionation | p. 230 |
Selective removal of proteinases during purification | p. 231 |
Acknowledgements | p. 231 |
References | p. 231 |
Proteolysis of native proteins as a structural probe | p. 233 |
Introduction | p. 233 |
Factors influencing susceptibility | p. 235 |
Molecular recognition and limited proteolysis | p. 235 |
Prediction of nicksites | p. 236 |
A tool to aid in prediction of sites of limited proteolysis | p. 239 |
Experimental considerations | p. 240 |
Choice of proteinase | p. 242 |
Ratio of proteinase to substrate | p. 245 |
Solution conditions | p. 245 |
Determination of site of proteolysis | p. 246 |
Strategies for limited proteolysis experiments | p. 247 |
Analysis of limited proteolysis data and simulations | p. 249 |
Obtaining quantitative data | p. 250 |
Analysing the data by non-linear curve fitting | p. 254 |
Example reaction schemes | p. 257 |
Simulations and modelling | p. 258 |
References | p. 264 |
Proteases in peptide synthesis | p. 265 |
Introduction | p. 265 |
Enzyme properties influencing the product yield and steric purity in protease catalysed peptide synthesis | p. 267 |
Kinetically controlled synthesis | p. 267 |
Equilibrium-controlled peptide synthesis | p. 270 |
Selecting the optimal protease | p. 271 |
Purity of the protease | p. 271 |
P[subscript 1] and P'[subscript 1] specificity | p. 272 |
Factors controlling the yield and steric purity in the synthesis of a peptide bond with a given enzyme | p. 278 |
Protection of the P'[subscript 1] and activation of the P[subscript 1]-carboxyl group | p. 279 |
pH | p. 279 |
Temperature | p. 281 |
Ionic strength | p. 282 |
Solvent composition | p. 283 |
Peptide synthesis in suspensions with solid product or substrate | p. 284 |
Planning a protease-catalysed synthesis of a peptide bond | p. 286 |
What enzyme? | p. 286 |
Equilibrium-controlled or kinetically controlled synthesis? | p. 287 |
Free or immobilized enzyme? | p. 287 |
Experimental methods for protease-catalysed peptide synthesis | p. 288 |
Enzyme purity and purification | p. 288 |
Enzyme immobilization | p. 288 |
Substrates and buffers | p. 288 |
Monitoring the synthesis; purification of products | p. 289 |
Optimizing the yield | p. 289 |
Proteases in peptide synthesis: limitations and perspectives | p. 289 |
References | p. 291 |
The Schechter and Berger nomenclature for proteinase subsites | p. 293 |
Some commercially available proteases | p. 295 |
Commercially available proteinase inhibitors | p. 317 |
List of suppliers | p. 331 |
Index | p. 337 |
Table of Contents provided by Syndetics. All Rights Reserved. |