Protein Sequence Determination, a Sourcebook of Methods and Techniques

1 General Considerations.- 2 Physical Characterization of the Protein Molecule.- I. Introduction to the Problem.- II. The Accessible Physical Parameters.- III. Sedimentation Methods.- A. Sedimentation Velocity.- B. Sedimentation Equilibrium.- C. Density Gradient Techniques.- IV. Gel Permeation Methods.- V. Electrophoresis.- A. Moving Boundary Electrophoresis.- B. Zonal Electrophoresis on Supporting Media.- C. Isoelectric Focusing.- D. SDS Gel Electrophoresis.- VI. Protocol for the Characterization of a Typical Protein, in Preparation for Sequencing.- A. Tests for Homogeneity of the Native Protein.- B. Detection and Enumeration of Subunits.- C. Preparative Chain Separation.- 3 End Group Determination.- I. Introduction.- II. N-Terminal Group Determination.- A. Dinitrofluorobenzene Method.- B. Dansyl Chloride Method.- C. Cyanate Method.- D. Other Methods.- Chemical Methods.- Enzymatic Methods.- III. C-Terminal Group Determination.- A. Hydrazinolysis Method.- B. Tritium-Labeling Method.- C. Carboxypeptidases.- D. Other Methods.- IV. Masked Terminal Groups.- A. N-Acylated Terminal Group.- Acetylated Terminus.- Formylated Terminus.- Pyroglutamyl Terminus.- B. C-Amidated Terminal Group.- 4 Improved Tritium-Labeling for Quantitative C-Terminal Analysis.- I. Improved Reaction Conditions for Tritium-Labeling.- II. Structural Effect on Tritium Incorporation.- III. Approach to Quantitative Analysis of the C-Terminal Residues (Internal Standard Method).- IV. Improvement in the Characterization Procedure of the Tri- tiated C-Terminus.- V. Further Problems.- 5 Fragmentation of Proteins for Sequence Studies and Separation of Peptide Mixtures.- I. Introduction.- II. Non-Enzymic Cleavage of Peptide Bonds.- A. Cyanogen Bromide.- B. Partial Acid Hydrolysis.- III. Enzymic Degradation.- A. General Considerations.- B. Preparation of a Protein for Enzymic Digestion.- C. Methods of Measuring Hydrolysis.- D. Trypsin.- Specificity.- Chemical Modifications Altering Trypsin Specificity.- Method of Hydrolysis.- E. Chymotrypsin.- Specificity.- Method of Hydrolysis.- F. Pepsin.- Specificity.- Method of Hydrolysis.- G. Thermolysin.- Properties and Specificity.- Method of Hydrolysis.- IV. Fractionation of Peptides.- A. Ion-Exchange Chromatography.- B. Gel Filtration.- C. Preparative Paper Electrophoresis and Chromatography.- 6 Identification of Specific Amino Acid Residues.- I. Introduction.- II. Presentation of the Results.- A. Quantitative Expression.- B. Protein Determination.- III. Estimation of Tryptophan.- A. Acid Hydrolysis.- B. Spectrophotometry.- C. Colorimetry.- D. N-Bromosuccinimide.- E. Other Methods.- IV. Estimation of Sulfhydryl Groups.- A. Mercaptide Formation.- B. Alkylating Agents.- C. Colorimetry.- D. Comments.- V. Estimation of Disulfides.- A. Reaction with Sulfite.- B. Reductive Cleavage.- C. Oxidative Cleavage.- VI. Estimation of Other Amino Acids.- VII. Estimation of Amino and Amide Groups.- A. Amino Groups.- B. Amide Groups.- VIII. Detection of Amino Acids and Peptides in Paper Chromatograms.- A. Non-Specific Reagents.- B. Specific Reagents.- 7 Amino Acid Composition by Column Chromatography.- I. Introduction.- II. Principles.- A. Resolution, Speed, and Sensitivity in Column Chromatography.- B. Resin Effects.- C. Buffers and Temperature Effects.- D. Column and Extra Column Effects.- E. Sensitivity - Column Effects.- F. Sensitivity - Detectors.- G. Sensitivity of Fluorescence Systems.- H. Sensitivity of Systems Other than Ion Exchange.- III. Instrumentation.- A. Commercial Amino Acid Analyzers.- B. Modification of Standard Amino Acid Analyzer Equipment.- C. Construction of Simplified Instrumentation for Amino Acid Analysis.- IV. Procedures and Techniques.- A. Preparation.- B. Contamination Problems.- C. Preparation of Reagents.- D. Preparation of Buffers and Reagents.- E. Sample Preparation.- F. Racemization and the Determination of D and L Amino Acids.- G. Amino Acid Analysis and Sequencing.- V. Conclusion.- 8 Sequence Determination.- I. Introduction.- II. Isothiocyanate Degradation.- A. Reaction Mechanism.- B. Preparation of Phenylthiohydantoins.- C. Properties of Phenylthiohydantoins.- D. Identification of Amino Acids.- Paper Chromatographic Methods.- Gas-Liquid Chromatography.- Mass Spectrometry.- Hydrolysis.- General Comments.- E. Sequential Degradation.- F. Related Procedures.- III. Other Chemical Degradation Procedures.- A. From N-Terminus.- B. From C-Terminus.- IV. Enzymatic Degradation Procedures.- A. From N-Terminus.- B. From C-Terminus.- V. Conclusion.- 9 Analysis of Amino Acid Phenylthiohydantoins by Gas Chromatography and High Performance Liquid Chromatography.- I. General Methods for PTH Identification.- II. Gas Chromatography.- A. Equipment.- B. Materials.- C. Preparation of the Support.- D. Preparation of Columns and Chromatographic Conditions.- E. Standard Solutions.- F. Silylation of Phenylthiohydantoins.- G. Chromatography.- H. Methylthiohydantoins (MTHs).- I. Applications.- III. High Performance Liquid Chromatography (HPLC).- A. Instrumentation.- B. Columns and Reagents.- C. Comments on HPLC.- D. Quantitation.- IV. Alternate Methods.- A. Thin-Layer Chromatography (TLC).- B. Mass Spectrometry (MS).- 10 Reconstruction of the Primary Sequence of a Protein from Peptides of Known Sequence.- I. Introduction.- II. Determination of the Amino Terminal Peptide.- III. Determination of the Carboxyl Terminal Peptide.- IV. Alignment of Peptides by Analogy.- V. Alignment of Peptides by Peptide Overlap.- A. Digestion of the Protein with Two Enzymes of Different Specificity.- B. Hydrolysis of the Protein with a Single Agent Having High Degree of Limited and Absolute Specificity.- C. Reconstruction of the Protein Sequence by Manual Operation.- VI. Qualities of Computer Programs.- 11 Peptide Synthesis.- I. Introduction.- II. Protecting Groups.- A. Amino-Protecting Groups.- Urethane-Type Protecting Groups.- Alkyl-Type Protecting Groups.- Acyl-Type Protecting Groups.- B. Carboxyl-Protecting Groups.- Ester Groups.- Amides and Substituted Hydrazides.- Protection by Salt Formation.- C. Sulfur-Protecting Groups.- D. Hydroxyl-Protecting Groups.- III. Peptide Bond Formation.- A. Acid Chloride Method.- B. The Azide Procedure.- C. Mixed Anhydride Method.- D. The Carbodiimide Method.- E. Isoxazolium Salts.- F. N, N-Carbonyldiimidazole.- G. Active Ester Method.- H. Coupling via Oxidation.- I. Leuch's Anhydride Method.- J. Enzymatic Synthesis.- IV. Merrified Solid Phase Method.- References.