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.

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.