A simple and reliable method for determination of optimum pH in coupled enzyme assays

Determination of the optimum pH in a coupled enzyme assay poses significant challenges because altering the pH of the reaction mixture can affect the performance of both enzymes. Here, we demonstrate a simple and reliable method to determine the pH optimum for pyruvate kinase using the pyruvate kina...

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Bibliographic Details
Published in:	BioTechniques Vol. 68; no. 4; pp. 200 - 203
Main Authors:	Bowman, Lee, Motamed, Rachael, Lee, Paul, Aleem, Kadijah, Berawala, Astha S, Hayden, Katherine L, Bzik, David J, Chattopadhyay, Debasish
Format:	Journal Article
Language:	English
Published:	England Future Science Ltd 01-04-2020 Taylor & Francis Group
Subjects:	coupled enzyme assay Cryptosporidium parvum enzyme kinetics lactate dehydrogenase pH optimization Plasmodium falciparum pyruvate kinase Plasmodium falciparum Cryptosporidium parvum coupled enzyme assay pyruvate kinase lactate dehydrogenase pH optimization enzyme kinetics
Online Access:	Get full text
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Summary:	Determination of the optimum pH in a coupled enzyme assay poses significant challenges because altering the pH of the reaction mixture can affect the performance of both enzymes. Here, we demonstrate a simple and reliable method to determine the pH optimum for pyruvate kinase using the pyruvate kinase/lactate dehydrogenase coupled enzyme assay. This simple and reliable method can be broadly adapted to determine the pH optimum for various enzymes that are assayed using a coupled enzyme assay. This article describes a simple method to determine the optimum pH for the activity of one enzyme in a coupled enzyme assay. Decoupling of the two-step reaction is achieved by heat denaturing the enzyme catalyzing the first reaction at different pHs followed by the addition of the second enzyme at a constant pH. The velocity of the second enzymatic reaction is determined by the concentration of the product produced in the first reaction, therefore, allowing the measurement of the optimum pH of the first enzyme by monitoring the rate of the second reaction.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0736-6205 1940-9818
DOI:	10.2144/btn-2019-0126