Experiments in the Purification and Characterization of Enzymes: A Laboratory Manual provides students with a working knowledge of the fundamental and advanced techniques of experimental biochemistry. Included are instructions and experiments that involve purification and characterization of enzymes from various source materials, giving students excellent experience in kinetics analysis and data analysis. Additionally, this lab manual covers how to evaluate and effectively use scientific data. By focusing on the relationship between structure and function in enzymes, Experiments in the Purification and Characterization of Enzymes: A Laboratory Manual provides a strong research foundation for students enrolled in a biochemistry lab course by outlining how to evaluate and effectively use scientific data in addition to offering students a more hands-on approach with exercises that encourage them to think deeply about the content and to design their own experiments. Instructors will find this book useful because the modular nature of the lab exercises allows them to apply the exercises to any set of proteins and incorporate the exercises into their courses as they see fit, allowing for greater flexibility in the use of the material.
Written in a logical, easy-to-understand manner, Experiments in the Purification and Characterization of Enzymes: A Laboratory Manual is an indispensable resource for both students and instructors in the fields of biochemistry, molecular biology, chemistry, pharmaceutical chemistry, and related molecular life sciences such as cell biology, neurosciences, and genetics.
Key Features
- Offers project lab formats for students that closely simulate original research projects
- Provides instructional guidance for students to design their own experiments
- Includes advanced analytical techniques
- Contains adaptable modular exercises that allow for the study proteins other than FNR, LuxG and LDH
- Includes access to a website with additional resources for instructors
Dedication
List of Figures
Preface
Acknowledgements
Notes Regarding Nomenclature
Abbreviations
Safety Guidelines for Biochemical Laboratories
General Guidelines for Handling Solutions of Protein
Maintaining a Laboratory Notebook
Introduction to Enzymes Catalyzing Oxidation-Reductions with the Coenzyme NAD(P)
Physiological Role of NAD(P)
Cosubstrates for NAD(P) in the Reactions Catalyzed By FNR, LuxG, and LDH
Does FNR Function as a Monomer or a Dimer?
Do FNR, LuxG, and LDH Have Different Michaelis Constants for NAD(P)?
Computational Techniques for Biochemistry
Software for Analysis of Data
Computational Methods and Bioinformatics for Studying the Structure and Function of Proteins
Computational Exercise 1
Computational Exercise 2
Section 1. Purification and Characterization of Ferredoxin-NADP+ Reductase from Chloroplasts of S. oleracea
Background: The Role of FERREDOXIN-NADP+ REDUCTASE in Linear and Cyclic Electron Transport in Photosynthesis
FNR Exercise 1
FNR Exercise 2
FNR Exercise 3
FNR Exercise 4
FNR Exercise 5
FNR Exercise 6
FNR Exercise 7
Section 2. Purification and Characterization of a Recombinant FMN Reductase from P. leiognathi
Background: Flavin Reductases, Bioluminescence and the lux Operon
LuxG Exercise 1
LuxG Exercise 2
LuxG Exercise 3
LuxG Exercise 4
LuxG Exercise 5
LuxG Exercise 6
Section 3. Purification and Characterization of Bovine l-Lactate Dehydrogenase
Background: Importance of l-Lactate Dehydrogenase in Bacterial and Eukaryotic Physiology
Summary of the Exercises with LDH
LDH Exercise 1
LDH Exercise 2
LDH Exercise 3
LDH Exercise 4
LDH Exercise 5
LDH Exercise 6
LDH Exercise 7
LDH Exercise 8
LDH Exercise 9
LDH Exercise 10
LDH Exercise 11
Section 4. Experimental Design
Background
Sources for Biochemical Methods
Experimental Design Exercise 1
Experimental Design Exercise 2
Experimental Design Exercise 3
Appendix I. Measurement of Absorbance with Multiwell Plates and an Automated Plate Reader
Appendix II. Operation of the Spectronic 20D+ Spectrophotometer
Bibliography
Index
