Protein Sequence Determination

1 General Considerations.- 2 Physical Characterization of the Protein Molecule.- I. Introduction to the Problem.- II. The Accessible Physical Parameters.- III. Sedimentation Methods.- IV. Gel Permeation Methods.- V. Electrophoresis.- VI. Protocol for the Characterization of a Typical Protein, in Preparation for Sequencing.- 3 End Group Determination.- I. Introduction.- II. N-Terminal Group Determination.- III. C-Terminal Group Determination.- IV. Masked Terminal Groups.- 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.- III. Enzymic Degradation.- IV. Fractionation of Peptides.- 6 Identification of Specific Amino Acid Residues.- I. Introduction.- II. Presentation of the Results.- III. Estimation of Tryptophan.- IV. Estimation of Sulfhydryl Groups.- V. Estimation of Disulfides.- VI. Estimation of Other Amino Acids.- VII. Estimation of Amino and Amide Groups.- VIII. Detection of Amino Acids and Peptides in Paper Chromatograms.- 7 Amino Acid Composition by Column Chromatography.- I. Introduction.- II. Principles.- III. Instrumentation.- IV. Procedures and Techniques.- V. Conclusion.- 8 Sequence Determination.- I. Introduction.- II. Isothiocyanate Degradation.- III. Other Chemical Degradation Procedures.- IV. Enzymatic Degradation Procedures.- 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.- III. High Performance Liquid Chromatography (HPLC).- IV. Alternate Methods.- 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.- VI. Qualities of Computer Programs.- 11 Peptide Synthesis.- I. Introduction.- II. Protecting Groups.- III. Peptide Bond Formation.- IV. Merrified Solid Phase Method.- References.