Biological and Biomedical Applications of Isoelectric Focusing

ISBN-13: 9781461341833 / Angielski / Miękka / 2011 / 352 str.

Nicholas Catsimpoolas

Opis

It has now been over a decade since isoelectric focusing became estab lished as a valuable addition to our arsenal of physical methods for separa tion of proteins and other amphoteric substances. The high resolving power of the technique and its unique ability to separate as well as concentrate have been amply demonstrated in several international symposia. The past few years have led to the wide acceptance of standardized methodologies for analytical and preparative purposes. These procedures afford rapid, reproducible separations that have led to a dramatic increase in our under standing of many areas of biological and biomedical research. Conse quently, we considered it desirable to review some of the important appli cations of the isoelectric focusing technique. To accomplish this, we have solicited the participation of noted authorities to highlight major progress in their fields of expertise. Thus, the material in this book will emphasize recent advances in knowledge rather than methodological aspects, except when special procedures are reviewed. Nicholas Catsimpoolas James W. Drysdale vii Contents Chapter J Isoelectric Focusing of Human Saliva, Cerebrospinal Fluid, and Urine Josie A. Beeley I. Introduction I II. Isoelectric Focusing of Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 A. Gel Rods and Thin Layer Gels . . . . . . . . . . . . . . . . . . . . . . . . 2 B. Liquid Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 C. a-Amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 D. Immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 E. Blood Group Specific Substances . . . . . . . . . . . . . . . . . . . . . 11 F. Vitamin B12 Binding Proteins (Cobalophilins) . . . . . . . . . . . 12 G. Other Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 H. Lysozyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 I. Specific Salivary Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 J. Dental Plaque . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 K. Other Proteins . . . . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . . . . 16 III. Isoelectric Focusing of Cerebrospinal Fluid . . . . . . . . . . . . . . . .

Spis treści

1 Isoelectric Focusing of Human Saliva, Cerebrospinal Fluid, and Urine.- I. Introduction.- II. Isoelectric Focusing of Saliva.- A. Gel Rods and Thin Layer Gels.- B. Liquid Columns.- C. ?-Amylases.- D. Immunoglobulins.- E. Blood Group Specific Substances.- F. Vitamin B12 Proteins Binding Proteins (Cobalophilins).- G. Other Glycoproteins.- H. Lymozyme.- I. Specific Salivary Proteins.- J. Dental Plaque.- K. Other Proteins.- III. Isoelectric Focusing of Cerebrospinal Fluid.- IV. Isoclectdc Focusing of Urine.- V. Conclusions.- VI.References.- 2 The Use of Gel in the Analysis of Hemoglobins.- I. Introduction.- II. Practical Applications.- A. Normal Human Hemoglobins.- B. Clinical Studies.- C. Identification of Hemoglobin Variants.- D. Subunit Identification.- E. Identification of Animal Hemoglobins.- III. Experimental Applications.- A. Preparation of Purified Hemoglobins.- B. Conformational Isomerization.- C. Asymmetrical Hybrids.- D. Valence Hybrids.- E. Chemical Modification.- IV. References.- 3 Isoelectric Focusing of Allergens.- I. Introduction.- II. Methods.- A. Isoelectric Focusing Method Used for the Characterization of Allergens.- B. Paper Disk Radio- Allergo-Sorbent Test (Paper Disk RAST) Used for the Detection of Allergens.- C. The Preparation of Agents for the Detection of Allergens.- D. The Effect of Freezing Allergens.- III. Isoelectric Focusing of Allergens.- A. Effect of Polyacrylamide Gel Concentration.- B. Effect of Different Times of Electrophoresis.- C. Reproducibility of Method.- D. Correlation of I soelectrophoretically Separated Allergen Components with Protein Patterns.- IV. Applications.- A. Allergen Dose-Response Relationship.- B. Variability in pl Component Spectrum of Allergens Determined with Several Allergen Sensitive Sera.- C. Differences in pl Component Spectra of Allergen Extracts Prepared by Different Producers.- D. Changes in pl Component Spectra of Allergen Extracts Resulting from Storage.- E. Changes in Dose-Response Relationships and in Isoelectric Focusing Patterns of Allergen Extracts Treated with Enzymes.- F. Detection of Allergens by Enzyme-Linked Allergo-Sorbent Test (ELAST).- G. Isoelectric Focusing Patterns of Some Purified Allergens.- V. General Comments.- VI. References.- 4 Isoelectric Focusing of Muscle Proteins.- I. Introduction.- II. Technical Considerations.- A. Preparative Procedures.- B. Analytical Procedures.- III. Specific Applications.- A. Preparations and Isolations of Muscle Proteins.- B. Analyses of Muscle Proteins.- C. Applications of Special Interest.- IV. Conclusions and Predictions.- V. References.- 5 Studies of the Structure and Synthesis of Immunoglobulins by Isoelectric Focusing.- I. Introduction.- II. Methods of Isoelectric Focusing of Immunoglobulins.- A. Liquid Density Gradient Stabilized Columns.- B. Acrylamide Gel Stabilized Methods.- C. Granular Gel Stabilized Methods.- D. The Use of Urea for Solubilization.- E. Isoelectric Focusing of Immunoglobulin Chains and Fragments.- III. Isoelectric Focusing of M-Proteins.- A. Focusing Patterns of Monoclonal Immunoglobulins.- B. The Nature of Immunoglobulin Microheterogeneity.- IV. Studies of Antibody Structure and Heterogeneity by Isoelectric Focusing.- A. Antihapten Antibodies.- B. Anticarbohydrate Antibodies.- C. Antiprotein Antibodies.- V. Studies of Antibody Synthesis.- A. In Vivo Cloning.- B. In Vitro Cloning (Monofocal Antibodies).- C. Estimates of Antibody Diversity in a Single Animal.- D. Studies of Antibody Production in Neonatal Animals.- E. Clonal Dominance.- F. Gene Linkage Studies.- VI. References.- 6 The Use of Isoelectric Focusing in the Separation and Characterization of Nuclear Nonhistone Proteins.- I. Introduction.- II. General Properties of NHP.- III. Isoelectric Focusing of NHP.- A. Separation of NHP from Histones.- B. Analyses of NHP Using Isoelectric Focusing.- C. Preparation of NHP Using lsoelectnc Focusing.- IV. Artifacts and the Heterogeneity of NHP.- A. General.- B. Nucleic Acids.- C. Modifications of NHP.- D. Effect of Denaturing Agents.- E. Binding of Ampholines.- F. Effects of Extremes of pH.- G. Effects of Proteolytic Activity.- V. Conclusion.- VI. References.- 7 Isoelectric Focusing of Membrane Components.- I. Introduction.- II. Methods.- A. Solubilization of Membrane Proteins.- B. Focusing Procedure.- C. Two-Dimensional Techniques.- III. Biological Applications.- A. Erythrocyte Membranes.- B. Plasma Membranes from Lymphoid Cells.- C. Bacterial Membranes.- D. Chloroplast Membranes.- E. Membrane Lipids in Isoelectric Focusing.- IV. References.- 8 Isoelectric Focusing of Seed Proteins.- I. Introduction.- II. Food Proteins.- A. Cereals.- B. Oilseeds.- C. Other Plant Proteins.- III. Enzymes.- A. Carbohydrases.- B. Proteases and Peptidases.- C. Lipoxygenases.- D. Peroxidases.- E. Esterases.- F. Urease.- G. Myrosinase.- IV. Protease Inhibitors.- A. Soybean.- B. Groundnut.- C. Barley.- D. Potato.- V. Lectins.- A. Nutritional Effects.- B. Isolectins.- C. Aggregation.- D. Sugar Binding.- E. Subunit Structure.- VI. Genetic Studies.- A. Location of Genes Controlling Protein Synthesis.- B. Phylogenetic Relationships.- C. Variety Identification.- D. Food Analysis.- VII. Conclusion.- VIII. References.- 9 Isoelectric Focusing of Microbial Proteins.- I. Introduction.- II. Materials.- A. Ampholytes.- B. Anticonvection Media.- C. The Sample.- III. Methods.- IV. Isoelectric Focusing of Microbial Proteins.- A. Viral Proteins.- B. Protozoan Proteins.- C. Fungal Proteins.- D. Bacterial Proteins.- V. Conclusions.- VI. References.- 10 Isoelectric Focusing of Serum Proteins.- I. Introduction.- II. Gel Isoelectric Focusing Alone.- A. Gel Isoelectric Focusing in One Dimension.- B. Gel Rod Electrofocusing of Whole Serum and Plasma Proteins.- III. Gel Electrofocusing Followed by Electrophoresis.- A. Identification of Human Serum Protein Zones.- B. Animal Sera.- IV Individual Types of Protein in Serum or Plasma.- A. Albumin.- B. ?1-Antitrypsin.- C. Coagulation and Fibrinolytic Factors.- D. ?1-Fetoprotein.- E. Glycoproteins.- F. Haptoglobins.- G. Hemopexin.- H. Immune-Response Proteins.- I. The Lipoproteins.- J. ?2-Macroglobulin.- K. Transport Proteins.- L. Hormones and Related Proteins.- M. Miscellaneous Observations.- V. Clinical Applications.- A. Australia Antigen.- B. Cystic Fibrosis.- C. Gammopathies.- D. Lipoprotein Disorders.- E. Miscellaneous.- VI. References.