Crowley / Kyte Experiments in the Purification and Characterization of Enzymes

A Laboratory Manual
1. Auflage 2014
ISBN: 978-0-12-409593-9
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 266 Seiten

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. - 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

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.

Autoren/Hrsg.

Crowley, Thomas E.

Kyte, Jack

Inhaltsverzeichnis

1;Front Cover;1
2;Experiments in the Purification and Characterization of Enzymes: A Laboratory Manual;4
3;Copyright;5
4;Dedication;6
5;Contents;8
6;LIST OF FIGURES;12
6.1;COMPUTATIONAL TECHNIQUES FOR BIOCHEMISTRY;12
6.2;SECTION 1: FNR;12
6.3;SECTION 2: LUXG;13
6.4;SECTION 3: LDH;13
6.5;SECTION 4: EXPERIMENTAL DESIGN;14
7;Preface;16
8;Acknowledgements;18
9;Notes Regarding Nomenclature;20
10;ABBREVIATIONS;22
10.1;MASS;22
10.2;VOLUME;22
10.3;CONCENTRATION;22
10.4;MOLES;22
10.5;SPECTROPHOTOMETRY AND TIME;22
10.6;ELECTROPHORESIS;23
10.7;MISCELLANEOUS;23
11;Safety Guidelines for Biochemical Laboratories;24
11.1;ESSENTIAL PERSONAL PROTECTIVE EQUIPMENT FOR ALL PROCEDURES IN A LABORATORY;24
11.2;HEALTH HAZARDS FROM LABORATORY EQUIPMENT;25
11.3;HAZARDOUS CHEMICALS;26
11.4;HAZARDOUS WASTE DISPOSAL;27
11.5;EMERGENCY RESPONSE;27
12;General Guidelines for Handling Solutions of Protein;30
13;Maintaining a Laboratory Notebook;32
13.1;PERSONAL ELECTRONIC DEVICES;32
14;Introduction to Enzymes Catalyzing Oxidation-Reductions with the Coenzyme NAD(P);34
14.1;PHYSIOLOGICAL ROLE OF NAD(P);34
14.2;COSUBSTRATES FOR NAD(P) IN THE REACTIONS CATALYZED BY FNR, LUXG, AND LDH;35
14.3;DOES FNR FUNCTION AS A MONOMER OR A DIMER?;38
14.4;DO FNR, LUXG, AND LDH HAVE DIFFERENT MICHAELIS CONSTANTS FOR NAD(P)?;39
14.5;Source of Conventions for Discussion of Enzyme Kinetics;42
15;Computational Techniques for Biochemistry;44
15.1;SOFTWARE FOR ANALYSIS OF DATA;44
15.2;COMPUTATIONAL METHODS AND BIOINFORMATICS FOR STUDYING THE STRUCTURE AND FUNCTION OF PROTEINS;44
15.3;COMPUTATIONAL EXERCISE 1;48
15.4;COMPUTATIONAL EXERCISE 2;54
16;Section 1 - Purification and Characterization of Ferredoxin-NADP+ Reductase from Chloroplasts of S. oleracea;58
16.1;BACKGROUND: THE ROLE OF FERREDOXIN-NADP+ REDUCTASE IN LINEAR AND CYCLIC ELECTRON TRANSPORT IN PHOTOSYNTHESIS;58
16.2;FNR EXERCISE 1;64
16.3;FNR EXERCISE 2;70
16.4;FNR EXERCISE 3;82
16.5;FNR EXERCISE 4;92
16.6;FNR EXERCISE 5;104
16.7;FNR EXERCISE 6;108
16.8;FNR EXERCISE 7;124
17;Section 2 - Purification and Characterization of a Recombinant FMN Reductase from P. leiognathi;136
17.1;BACKGROUND: FLAVIN REDUCTASES, BIOLUMINESCENCE AND THE LUX OPERON;136
17.2;LUXG EXERCISE 1;140
17.3;LUXG EXERCISE 2;150
17.4;LUXG EXERCISE 3;156
17.5;LUXG EXERCISE 4;160
17.6;LUXG EXERCISE 5;166
17.7;LUXG EXERCISE 6;170
18;Section 3 - Purification and Characterization of Bovine l-Lactate Dehydrogenase;174
18.1;BACKGROUND: IMPORTANCE OF L-LACTATE DEHYDROGENASE IN BACTERIAL AND EUKARYOTIC PHYSIOLOGY;174
18.2;SUMMARY OF THE EXERCISES WITH LDH;180
18.3;LDH EXERCISE 1;182
18.4;LDH EXERCISE 2;186
18.5;LDH EXERCISE 3;190
18.6;LDH EXERCISE 4;200
18.7;LDH EXERCISE 5;208
18.8;LDH EXERCISE 6;218
18.9;LDH EXERCISE 7;222
18.10;LDH EXERCISE 8;230
18.11;LDH EXERCISE 9;234
18.12;LDH EXERCISE 10;238
18.13;LDH EXERCISE 11;240
19;Section 4 - Experimental Design;246
19.1;BACKGROUND;246
19.2;SOURCES FOR BIOCHEMICAL METHODS;246
19.3;EXPERIMENTAL DESIGN EXERCISE 1;248
19.4;EXPERIMENTAL DESIGN EXERCISE 2;250
19.5;EXPERIMENTAL DESIGN EXERCISE 3;252
20;APPENDIX I;256
20.1;MEASUREMENT OF ABSORBANCE WITH MULTIWELL PLATES AND AN AUTOMATED PLATE READER;256
20.2;CHOOSING THE APPROPRIATE TYPE OF PLATE;256
20.3;PREPARING SAMPLES OF PROTEIN AND OPERATING THE PLATE READER;256
21;APPENDIX II;258
21.1;OPERATION OF THE SPECTRONIC 20D+ SPECTROPHOTOMETER;258
21.2;PROPERTIES OF THE PHOTOTUBE;259
21.3;HANDLING THE TUBES FOR SAMPLES WITH THE SPECTRONIC 20D+;259
22;Bibliography;260
23;Index;266

Leseproben

Safety Guidelines for Biochemical Laboratories
No eating or drinking is allowed in any chemical laboratory. Essential Personal Protective Equipment for All Procedures in a Laboratory
Eye Protection
Everyone in the laboratory should be wearing eye protection whenever any work with chemicals is being done. For most of the procedures described in this book, plastic safety glasses are sufficient. Some procedures involve possible splashing of large volumes of hazardous liquids such as a mixture of isopropanol and dry ice, high-speed rotary devices, or high-frequency vibrational devices. For such procedures, safety goggles should be worn. Laboratory Coat
Everyone in the laboratory should be wearing a laboratory coat whenever any work with chemicals is being done. The sleeves should extend to the wrists. Gloves
Most of the procedures in this book may be performed safely without gloves. Wear gloves, however, when handling any hazardous chemicals (refer to Table s.1). The chemicals listed in this table are identified as hazardous within the appropriate exercises. If you are allergic to latex, wear nitrile or other hypoallergenic gloves. TABLE s.1 The Most Hazardous Chemicals to be Used in the Exercises in this Book Chemical Procedure(s) Hazarda Dithiothreitol Assay for LuxG-his6 activity Neurotoxin, respiratory tract irritant NiSO4 Purification of LuxG-his6 Carcinogen, teratogen Phenylmethanesulfonyl fluoride Lysis of spinach and bacteria Toxic to nerves, heart, blood, eyes Acrylamide Gel electrophoresis Neurotoxin, carcinogen, teratogen N,N,N´,N´-tetramethyl-ethylenediamine Polymerization of acrylamide Burns skin and eyes, flammable Ammonium persulfate Polymerization of acrylamide Burns skin and eyes 2-sulfanylethanol (2-mercaptoethanol) Denaturation of protein Skin and eye irritant, respiratory tract irritant, mutagen 5,5-diethylbarbituric acid (barbital) Electrophoresis of the isoenzymes of LDH Neurotoxin, carcinogen 5-methylphenazinium methyl sulfate Stain for activity of LDH Irritant to respiratory tract, eyes and skin Nitro Blue Tetrazolium Stain for activity of LDH Irritant to respiratory tract, eyes and skin Coomassie Brilliant Blue G-250 Bradford assay, detection of protein in gels Irritant to respiratory tract, eyes and skin Phosphoric acid Bradford assay Burns eyes, skin, and respiratory tract; toxic to liver, blood, and bone marrow aHealth hazard information from the Material Safety Data Sheet on the Sigma-Aldrich website: http://www.sigmaaldrich.com. Proper Footwear
Everyone in the laboratory must wear closed-top shoes that do not have high heels. Health Hazards from Laboratory Equipment
For all of the equipment described below, obtain instructions from the instructor or the teaching assistant before using the device. You should be supervised during your first use of the device. Motor-driven Homogenizers
Instructions and photos explaining proper use are in the experimental sections. To ensure your safety, be certain the pestle shaft is tightly secured within the chuck of the drive. During homogenization, the glass receptacle should be held near the top because it is more likely to shatter near the bottom than the top. Centrifuges
A two-pan balance must be used to bring samples that will be on opposite sides of the rotor to an equal mass before loading. Samples should be in a screw-cap tube or bottle that is designed for the amount of centrifugal force that will be applied (photo in the first exercise on ferredoxin-NADP+ reductase). The tubes or bottles will be open while balancing (to allow addition of more solution) but the caps should be on the balance trays. If rotor adaptors are to be used, they should also be on the trays. In most cases, the rotor is only secured to the centrifuge spindle if its cap has been screwed on tightly. After starting the centrifugation, stay near the centrifuge until it reaches the desired rotational speed. If severe vibration of the machine occurs, stop the centrifugation immediately! Devices for Electrophoresis
Instructions and photos on the proper use of these devices are provided in the experimental sections. Relatively high voltages of direct current are used, and the buffers are excellent conductors. The main concern for safety is that the operator might dip a finger into a buffer tank after the voltage has been applied, and if there is a conduction path through the operator, a severe electric shock might be experienced. The devices described in this manual have safety interlocks that are designed so that the electrical circuit is not completed until the lid is covering the buffer tanks. Most electrophoretic apparatuses have a similar design. In spite of this safety feature, have the power supply turned off when the lid is not on and the buffer tanks are not covered. Emitters of High-frequency Sound
The frequency emitted by a sonifier can damage the human ear. Instructions and photos regarding safe use of this device are provided in the first exercise for LuxG. In particular, the operator should wear ear muffs and eye protection. Gas Burners and Hot Plates
In preparing a boiling water bath for the denaturation of protein, boiling chips should be used and an electrical hot plate is preferred to a gas burner. When you are finished with the experiment, the hot plate should be turned off and unplugged. The valve at the counter top for any gas burner should be closed immediately after it has been used. Hazardous Chemicals
Definitions
Department of Environmental Health and Safety At most universities, this department establishes safety regulations for the use of all campus laboratories (it may have a different name in some cases). The staff of the department of environmental health and safety also enforce policies and provide services such as the certification of fume hoods, the disposal of hazardous waste, the maintenance of safety showers and stations for washing eyes, and the response to emergencies. Some colleges may not have a department of environmental health and safety, but all schools with chemistry laboratories will have a safety officer to handle these issues. Material Safety Data Sheet The Material Safety Data Sheet (MSDS) provides information on volatility, flammability, toxicity, corrosiveness, and other properties of a hazardous chemical. Carcinogen A chemical known to induce mutations that may lead to cancer in the individual that has been exposed. Concerns for Pregnant Women
Teratogen A chemical known to induce mutations that may lead to developmental abnormalities in a fetus. Fetotoxin A chemical that may be toxic to a fetus. Information Sources
The Web sites for chemical supply firms usually provide links to the Material Safety Data Sheets for their products. It is also helpful, however, to have access to open-source databases of Material Safety Data Sheets. Sigma-Aldrich Chemicals: http://www.sigmaaldrich.com (no subscription necessary). Where to Find a Material Safety Data Sheet on the Internet (by Interactive Learning Paradigms, Inc.): http://www.ilpi.com/msds/index.html. Chemwatch ChemFFX (from Global Chemical Data Inc.): mailto:[e-mail]. http://www.chemwatchna.com/OurServices/MSDSManagement.aspx. ChemQuik (from Actio Corp., Portsmouth, NH): mailto:[e-mail]. http://www.actio.net/default/index.cfm/products/chemquik/. Minimization of Exposure
Volatile Chemicals The laboratory should have a chemical fume hood that is certified at appropriate intervals by the staff of the department of environmental health and safety. Stock bottles and waste containers for volatile chemicals should be kept in the hood. Corrosive, Toxic or Carcinogenic Chemicals Wear disposable gloves when handling. If any are spilled on skin, rinse immediately with water. Hazardous Waste Disposal
Follow the instructions provided by the instructor or the teaching assistant for disposal procedures for sharp waste, hazardous chemicals, and biohazards (for example, material contaminated with recombinant bacteria). Emergency Response
The instructions below for providing immediate assistance are for the instructor, the teaching assistant, or the student. If it appears...