Experiments in the Purification and Characterization of Enzymes. A Laboratory Manual

Table of Contents

Safety Guidelines for Biochemistry Laboratories General Guidelines for Handling Solutions of Protein Maintaining a Laboratory Notebook Introduction to Enzymes Catalyzing Oxidation-Reductions with the Coenzyme NAD(P) Computational Techniques for Biochemistry  Software for Analysis of Data Computational Methods and Bioinformatics for Studying the Structure and Function of Proteins Background: Software and Databases; 1. Retrieve Entries from a Database and Compare Sequences of Amino Acids; 2. Compare the Tertiary Structures of Polypeptides Section 1: FNR; Purification and Characterization of Ferredoxin-NADP+ Reductase from Chloroplasts of S. oleracea 1. Preparation of a Lysate of Chloroplasts and Sequestration of Proteins by an Anion-exchange Solid Phase; 2. Elution of Proteins from the Anion-exchange Solid Phase and Assays for the Concentration of Total Protein and FNR Activity; 3. Isolation of FNR by Adsorption and Elution 4. Electrophoresis of Proteins from the Chloroplasts and Purified FNR on Gels of Polyacrylamide Cast in Solutions of Dodecyl Sulfate; 5. Kinetic Assay for the Km of FNR for NADPH; 6. Estimation of the Size and Quaternary Structure of Native FNR Using Chromatography by Molecular Exclusion; 7. Detection of FNR in Extracts from Chloroplasts of Spinach by Immunoblotting Section 2: LuxG; Purification and Characterization of a Recombinant FMN Reductase from P. leiognathi 1. Expression of LuxG-hexahistidine in E. coli and Its Release from the Cells by Lysis; 2. Affinity Adsorption of LuxG-hexahistidine to a Solid Phase to which Ni2+ is Chelated; 3. Electrophoresis of the Polypeptides in the Lysate and Purified LuxG-hexahistidine; 4. Assay for the Flavin Reductase Activity of LuxG-hexahistidine; 5. Determination of the Michaelis Constant of LuxG-hexahistidine for NADH; 6. Determination of the Molar Concentration of LuxG-hexahistidine Spectrophotometrically Section 3: LDH; Purification and Characterization of Bovine LDH 1. Preparation of the Isoenzymes of LDH from Extracts of Various Tissues; 2. Spectrophotometric Assay of the Activity of Bovine LDH; 3. Separation and Visualization of the Isoenzymes of LDH by Electrophoresis on Cellulose Acetate; 4. Fractionation of Proteins in Bovine Heart by Precipitation with Sulfate; 5. Purification of LDH from Heart by Affinity Adsorption and Elution; 6. Concentration of Protein by the Bradford Stain and Absorbance at 280 nm; 7. Electrophoresis of Native Proteins from Bovine Heart and Purified LDH on a Gel of Polyacrylamide; 8. Electrophoresis of Unfolded Polypeptides from Bovine Heart and Purified LDH Saturated with Dodecyl Sulfate; 9. Determination of the Michaelis Constant, KmNADH, of the Isoenzymes of LDH for NADH; 10. Chromatography by Molecular Exclusion to Evaluate the Quaternary Structure of LDH; 11. Detection of LDH in Extracts of Bovine Tissue by Immunoblotting Section 4: Experimental Design; 1. Association of FNR with the Membranes of the Thylakoids in the Chloroplasts from Spinach; 2. Search for Multiple Flavin Reductases in P. leiognathi; 3. Assay for LDH Activity Specific to (R)-lactate in Bovine Mitochondria Appendices I. Measurement of Absorbance with Multiwell Plates and an Automated Plate Reader; II. Operation of the Spectronic 20D+ Spectrophotometer

Authors

Thomas E. Crowley Department of Mathematics and Natural Sciences, National University, La Jolla, CA, USA. Thomas Crowley studied biochemistry as an undergraduate at the University of Illinois Urbana-Champaign. He then pursued graduate studies in molecular biology in the Division of Biology at the California Institute of Technology. As a graduate student and later during postdoctoral studies he used biochemical and genetic methods to examine the regulation of gene expression and the intracellular localization of proteins during animal development. He has taught courses covering a wide range of chemical and biological topics such as general chemistry, biochemistry, microbiology, cell biology and developmental biology. These courses were taught at Montclair State University in New Jersey, Columbia University in New York City, the University of California San Diego and National University in La Jolla, California. He has authored articles derived from his research in a variety of journals and articles derived from his teaching in Biochemistry and Molecular Biology Education (BAMBED). He is currently an adjunct faculty member in the Department of Mathematics and Natural Sciences at National University and a member of the American Chemical Society. Jack Kyte Professor, Dept. of Chemistry & Biochemistry, UCSD, San Diego, USA. Jack Kyte graduated from Carleton College in 1967, completed his doctoral studies at Harvard University in the laboratory of Guido Guidotti in 1972, and was a postdoctoral fellow with S. Jonathan Singer at the University of California at San Diego. In 1974 he joined the faculty of the Department of Chemistry at the University of California at San Diego and retired from the department as Professor of Chemistry in 1999. In his research, he studied the structures of Na+/K+-exchanging ATPase and anion carrier using chemical labeling and site-directed immunochemistry. He also studied the mechanism of the activation of the receptor protein-tyrosine kinase for epidermal growth factor and the mechanism of the reaction catalyzed by ribonucleoside-diphosphate reductase. He has written two books, Structure in Protein Chemistry and Mechanism in Protein Chemistry.