Subunit Structure of Glucose Oxidase from Penicillium amagasakiense

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Abstract
Glucose oxidase (β-D-glucose: O2 oxidoreductase, EC 1.1. 3. 4) was purified by using DEAE-cellulose. The purified enzyme was homogeneous with respect to ultracentrifugal and electrophoretical criteria. The submit structure of the glucose oxidase was investigated by the sedimentation velocity and sedimentation equilibrium analyses. The sedimentation coefficient and the molecular weight of the native enzyme were calculated to be 7.5S and 160, 000, respectively. In 6 M guanidine hydrochloride (pH 3.0), they were reduced to 3.3S and 81, 000, respectively. A sedimentation coefficient of 2.8S and a molecular weight of 45, 000 were obtained for the enzyme in 6 M guanidine hydrochloride (pH 8.0) containing 0.3 M mercaptoethanol. In 6 M guanidine hydrochloride (pH 3.0), the sulf-hydryl content of the glucose oxidase was estimated to be 1.8 moles per mole enzyme. The sulfhydryl content increased to 5.9 moles by treatment with 0.3 M mercaptoethanol in 6 M guanidine hydrochloride (pH 8.0), which indicated that the glucose oxidase had two sulfhydryl and two disulfide groups. From these results, it is concluded (1) that the glucose oxidase molecule consists of four polypeptide chains equal in molecular size, and (2) that two polypeptide chains are held together by a disulfide bond to form a dimer, and two dimeric units associate noncovalently to form a tetramer.

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