Protein Sequence Determination: A Sourcebook of Methods and Techniques (Paperback, 2, 1975. Softcover)

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.