On the Reaction of the Glucose Oxidase from Aspergillus niger with Bisulfite

From the ¹ From the Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48104

Bisulfite reacts rapidly with glucose oxidase to produce characteristic changes in the absorbance and fluorescence of the enzyme. When the bisulfite-treated enzyme is dialyzed against buffer, the active native enzyme is regenerated.

The visible absorption spectrum of the glucose oxidase-bisulfite product is similar to but not identical with that of the reduced enzyme in which the bound flavin is in the fully reduced form (FADH₂). A major difference between reduced enzyme and the glucose oxidase-bisulfite product is the failure of the latter compound to react with electron acceptors such as oxygen, ferricyanide, or sulfate.

The formation of an addition compound between glucose oxidase and bisulfite has been postulated. The proportion of combined to uncombined enzyme was found to be dependent on the absolute concentration of the reactants (not their ratio), the temperature, and the pH. The presence of 4M urea depressed the amount of complex formed.

The complex has been shown to be enzymically inactive by the stopped flow method.

Bisulfite reacts with free flavins when very high concentrations are used. A dissociation constant of 4M has been calculated for the flavin mononucleotide-bisulfite complex.

The reaction of bisulfite with glucose oxidase brings about shifts and large increases in the blue fluorescence (emission peak, 450 mµ; excitation peaks, 360 and 290 mµ) and also the ultraviolet fluorescence (emission peak, 340 mµ; excitation peak, 290 mµ) of the enzyme. The bound FAD in glucose oxidase does not fluoresce. The blue fluorescence is a property of a component of the enzyme which has not yet been characterized, while the ultraviolet fluorescence is primarily a property of the aromatic amino acid residues of the protein.

The fluorescence and absorbance changes correlate during the titration of the enzyme with bisulfite, and are affected by the temperature, pH, and urea concentration in similar ways. It is proposed that both types of change are a function of the formation of an enzyme-bisulfite complex. Active site groups such as the bound FAD and possibly the blue fluorophore are involved. This conclusion is further substantiated by fluorometric and absorbance experiments in which glucose has been shown to displace bisulfite slowly from the complex.

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