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Enzyme kinetics in the undergraduate biochemistry laboratory is often a frustrating experience because of the difficulty in obtaining precise measurements. This difficulty 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:
Beta-D-glucose + O sub 2 --> D-gluconolactone + H sub 2 O sub 2
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 sub 2 O sub 2 + Donor sub reduced --> Donor sub oxidized + 2 H sub 2 O.
This electron donor is usually a dye, which in our case Is 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS). This is a stable, nontoxic, water-soluble dye substrate that forms a brilliant blue-green color when oxidized (1-4). Because the oxidation of ABTS involves removal of one electron to generate the colored radical cation; the stoichiometry of the reaction is two molecules of ABTS oxidized for every molecule of glucose oxidized (1). The standard dye substrate for the glucose oxidase/HRP 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 an undergraduate biochemistry experiment utilizing the glucose oxidase/peroxidase system, they utilized o-dianisidine as a peroxidase dye substrate (6).
Experimental Procedure
Materials and Equipment
Glucose oxidase (catalog # G-6125), horseradish peroxidase (catalog # P-6140), ABTS (catalog # A-1888), glucose and other sugars were obtained from Sigma Chemical Company (St. Louis, MO). Sugar solutions were made up as 1 M stocks, the ABTS/HRP stock contained 50 mM ABTS and 25 units/mL HRP, and the glucose oxidase stock was 0.025 mg/mL (= 0.5 Sigma units/mL). An accurate measurement of glucose oxidase subunit concentration can be obtained from the flavin extinction coefficient at 450 nm of 1.4...