Methods of Protein Separation

This open-end treatise on methods concerning protein separation had its beginning in an American Chemical Society symposium entitled "Con- temporary Protein Separation Methods" which was held in Atlantic City, New Jersey in September 1974. The purpose of the symposium-and subse- quently of the present work-was to review the available modern techniques and underlying principles for achieving one of the very important tasks of experimental biology, namely the separation and characterization of proteins present in complex biological mixtures. Physicochemical characterization was covered only as related to the parent method of fractionation and there- fore involved mostly mass transport processes. Additionally, the presentation of methods for gaini. ng insight into complex interacting protein profiles was considered of paramount importance in the interpretation of separation patterns. Finally, specific categories of proteins (e. g. , chemically modified, deriving from a specific tissue, conjugated to different moieties, etc. ) require meticulous trial and selection andjor modification of existing methodology to carry out the desired separation. In such cases, the gained experience provides valuable guidelines for further experimentation. Although powerful techniques exist today for the separation and related physicochemical characterization of proteins, many biological fractionation problems require further innovations. It is hoped that the description in the present treatise of some of the available separation tools and their limitations will provide the necessary integrated background for new developments in this area.

Table of Contents:
1 Sedimentation and Gel-Permeation Chromatography of Associating-Dissociating Macromolecules: The Role of Ligand Mediation and Rates of Reaction.- I. Introduction.- II. Theoretical Formulation.- III. Results.- A. Ligand-Mediated Association-Dissociation Reactions.- B. Kinetically Controlled Nonmediated Interactions.- IV. Discussion.- V. References.- 2 Trans Electrophoresis.- I. Introduction.- A. Principle.- B. History of in Situ Electro-Optical Scanning.- C. Types of Electrophoresis and Supporting Media.- D. Anatomy of the TRANS Electrophoresis System.- II. Instrumental Aspects.- A. The Electro-Optical Unit.- B. The Scanning Stage Module.- C. The Scanner Control Servo Unit.- D. The Electrophoresis Cell Cassette.- E. The Filling/Purging/Cooling Module.- F. Other Components.- G. The Digital Data Acquisition Module.- H. General Operational Procedure.- I. Column-Coating Procedure.- III. Data Processing.- A. Slope Analysis.- B. Moment Analysis.- IV. TRANS-CZE and TRANS-MZE.- A. Preliminary Considerations.- B. Peak Velocity.- C. Kinetic Peak-Variance Measurements.- D. Resolution.- E. Boundary Displacement.- V. TRANS-IF.- A. Preliminary Considerations.- B. Minimal Focusing Time.- C. Segmental pH Gradient and Apparent Isoelectric Point.- D. Resolving Power and Resolution.- E. Retardation Coefficient.- F. Kinetics of Defocusing and Refocusing.- G. Nonideal Effects in TRANS-IF.- VI. Future Developments.- VII. References.- 3 Immunodiffusion.- I. Introduction.- II. Antigen-Antibody Reactions.- III. Immunodiffusion.- IV. Immunodiffusion Combined with Electrophoresis.- V. Helpful Hints.- VI. References.- 4 Isoelectric Focusing in Polyacrylamide Gel.- I. Introduction.- II. Background.- III. Methodology.- A. Apparatus.- B. Electrolyte Solutions.- C. Gel Composition.- D. Electrolysis Conditions.- E. Sample Application.- F. Sample Load.- G. Measurement of pH Gradients.- H. pH-Gradient Instability.- IV. Sample Detection.- A. Staining for Proteins.- B. Histochemical Staining.- C. Gel Fractionation.- D. Recovery of Focused Zones.- E. Markers.- V. Applications.- A. Ferritins.- B. Hemoglobins.- C. Interacting Systems.- D. Nucleic Acids.- E. Two-Dimensional Procedures.- VI. Discussion.- VII. References.- 5 Purification of Chemically Modified Proteins.- I. Introduction.- II. General Types and Purposes of Chemical Modification.- A. Modification to Change Biological Activity.- B. Modification to Change Physical Properties.- C. Modifications to Block Deteriorative Reactions.- D. Introduction of a Label.- E. Reversible Modifications.- III. Some Problems Encountered in Chemical Modification.- A. Denaturation.- B. Reversibility of Modification.- C. Heterogeneity of Products.- D. A Product with Properties Very Similar to Those of the Original Native Protein.- E. Side Reactions of a Chemical Nature Occurring During Modification.- IV. Analysis of Chemically Modified Proteins.- A. Analysis of Amino Acid Residues.- V. Typical Examples of the Purification of Chemically Modified Proteins.- A. Heterogeneity Commonly Encountered in the Introduction of Groups Causing Changes in Charges.- B. Modifications with No Change in Charge.- C. Some Special Procedures for Chromatography.- VI. The Use of Chemical Modification as a Tool for Purification of Proteins.- A. Purification by Reversible-Complex Formation.- B. Reversible Chemical Modification for Separating Hybrids of Variants of Oligomeric Proteins.- C. The Use of Chemical Modification to Separate Products with Different Biochemical Activities.- D. Reversible Modification to Allow for Another Modification.- E. The Use of Cross-Linking Agents to Determine Degree of Polymerization and Localization of Proteins in Tissues.- VII. References.- 6 Chromatographic Peak Shape Analysis.- I. Introduction.- II. Moment Analysis.- A. Definition.- B. Mass Balance and the Moments.- C. Other Uses of Moments Analysis.- III. Experimental Studies.- A. Verification of Skew and Excess Utility.- B. Peak Identification.- IV. Slope Analysis.- V. Conclusion.- VI. References.- 7 Sedimentation Equilibrium of Proteins in Density Gradients.- I. Introduction.- II. Theory.- A. Two-Component Theory.- B. Three-Component Theory.- C. Four-Component Theory.- D. Resolution.- III. Experimental.- A. Solution Preparation.- B. Run Conditions.- C. Data Analysis.- D. Computation.- E. Use of a Density Marker in the Determination of ?o.- IV. Results.- A. Proteins at the Isoelectric Point.- B. Buoyant Titrations of Proteins.- C. Buoyant Titrations of Synthetic Polypeptides.- D. Buoyant Titrations of Chemically Modified Proteins.- E. Related Studies.- V. References.- 8 Hollow-Fiber Separation Devices and Processes.- I. Introduction.- II. Hollow-Fiber Membranes.- III. Device Configurations.- IV. Dialysis.- V. Combined Dialysis and Concentration.- VI. Concentration.- VII. Drug-Binding Studies.- VIII. Enzyme Reactor.- IX. Conclusion.- X. Appendix.- 9 Affinity Chromatography, Principles and Applications.