ERIC Number: EJ1062262
Record Type: Journal
Publication Date: 2015-Feb
Abstractor: As Provided
Reference Count: 21
A Sensitive and Robust Enzyme Kinetic Experiment Using Microplates and Fluorogenic Ester Substrates
Johnson, R. Jeremy; Hoops, Geoffrey C.; Savas, Christopher J.; Kartje, Zachary; Lavis, Luke D.
Journal of Chemical Education, v92 n2 p385-388 Feb 2015
Enzyme kinetics measurements are a standard component of undergraduate biochemistry laboratories. The combination of serine hydrolases and fluorogenic enzyme substrates provides a rapid, sensitive, and general method for measuring enzyme kinetics in an undergraduate biochemistry laboratory. In this method, the kinetic activity of multiple protein variants is determined in parallel using a microplate reader, multichannel pipets, serial dilutions, and fluorogenic ester substrates. The utility of this methodology is illustrated by the measurement of differential enzyme activity in microplate volumes in triplicate with small protein samples and low activity enzyme variants. Enzyme kinetic measurements using fluorogenic substrates are, thus, adaptable for use with student-purified enzyme variants and for comparative enzyme kinetics studies. The rapid setup and analysis of these kinetic experiments not only provides advanced undergraduates with experience in a fundamental biochemical technique, but also provides the adaptability for use in inquiry-based laboratories.
Descriptors: Science Instruction, Science Experiments, College Science, Undergraduate Study, Biochemistry, Kinetics, Spectroscopy, Light, Science Laboratories, Laboratory Experiments, Molecular Structure, Inquiry, Teaching Methods
Division of Chemical Education, Inc and ACS Publications Division of the American Chemical Society. 1155 Sixteenth Street NW, Washington, DC 20036. Tel: 800-227-5558; Tel: 202-872-4600; e-mail: firstname.lastname@example.org; Web site: http://pubs.acs.org/jchemeduc
Publication Type: Journal Articles; Reports - Research
Education Level: Higher Education; Postsecondary Education
Sponsor: National Science Foundation
Authoring Institution: N/A
IES Grant or Contract Numbers: NSF DUE-1140526