Protein Sequence Determination

In the few years since the first edition of this book made its appearance, under takings in the area of protein sequence determination have increased at an almost logarithmic pace. The logic governing such efforts has, predictably, retreated from a simple sense of curiosity in achieving something that had not previously been done, to be replaced by an absolute requirement for sequence information as the best means for understanding heretofore unattainable aspects of chemical, and structural and functional mechanistic phenomena. Witness, for example, recent volumes of the Annual Review of Biochemistry and other review journals, which treat the genetic control of mitochondrial proteins, mechanisms of enzyme action, the immunochemistry of collagen, and the role of lysosomal enzymes in the lipid storage diseases - all on the basis of known protein sequence information. One must note, too, the appearance of related works in the area of nucleic acid sequencing. The technologic advances in chemical sequencing procedures have proliferated at a remarkable rate. It is in the area of instrumentation, however, that the greatest advances have taken place. As a consequence of this, each chapter (with one single exception) in this book has been extensively revised and updated. Entire chapters have been replaced in several cases, without minimizing the value of their original content. In addition, a second volume will appear soon, dealing with subjects not covered in the present book.

Contenu
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.