The Modern Student laboratory Using the Glucose Oxidase/Peroxidase System in Enzyme Kinetics Robert C. Bateman, ~ r and ? Jeffrey A. Evans Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, MS 39406
Enzyme kinetics in the undergraduate biochemistry laboratory is often a frustrating experience because of the difficulty in obtaining precise measurements. This dificulty can be due to nonlinearity in the assay itself, unstable substrates or enzymes, measurement of a product not easily visualized, etc. This paper describes the use of glucose oxidase coupled to horseradish peroxidase as a stable and easily measured enzyme system for demonstrating Michaelis-Menten kinetics. Glucose oxidase (EC 1.1.3.4) is an FAD-dependent oxidoreductase that catalyzes the following reaction: + D-gluconolactane+ HzO, p-D-glucose+ 0% To measure the reaction, the hydrogen peroxide produced is scavenged by horseradish peroxidase (HRP), which uses an electron donor to reduce further the hydrogen peroxide to water. H,Oz + Donorrhd + Donoro6dized + 2 HzO This electron donor is usually a dye, which in our case is
2.2'-azino-bis13-cthvlbenzthiazolinc-6-sulfonic acid -.(ABTS). This is a stable, nontoxic, water-soluble dye sub-
strate that forms a brilliant blue-green color when oxidized (14).Because the oxidation of ABTS involves removal of one electron to generate the colored radical cation, the stoichiometry of the reaction is two molecules ofABTS oxidized for every molecule of glucose oxidized (I).The standard dye substrate for the glucose oxidaseMRF' coupled reaction, o-dianisidine, is less soluble in water, carcinogenic and produces a pale brown color upon oxidation (5).Although Woolridge, Turchi, and Edwards have previously published a n undergraduate biochemistry experiment utilizing the glucose oxidaselperoxidase system, they utilized o-dianisidine as a peroxidase dye substrate (6). Exwrimental Procedure Materials and Equipment
Glucose oxidase (catalog # G6125), horseradish peroxidase (catalog # P-61401, ABTS (catalog # A-18881, glucose and other sugars were obtained from Sigma Chemical Company (St. Louis, MO). Sugar solutions were made up a s 1 M stocks, the ABTS/HRP stock contained 50 mM ABTS and 25 units/mL HRP, and the glucose oxidase stock was 0.025 mg1mL (= 0.5 Sigma unitslmL). An accurate measurement of glucose oxidase subunit concentration can be obtained from the flavin extinction coefficient at 450 nm of 1.4 x lo4 M-' em-' (8, 9). The buffer used throughout was 0.1 M sodium phosphate at pH 7.0. All solutions were stored at 4 "C and allowed to warm to room tem~erature prior to use. For the best results, solutions should be used within two to thrcedavs. The AI
