Glycerol oxidase, a novel copper hemoprotein from Aspergillus japonicus. Molecular and catalytic properties of the enzyme and its application to the analysis of serum triglycerides.

T Uwajima,Y Shimizu,O Terada

doi:10.1016/s0021-9258(17)43209-1

Abstract

Glycerol oxidase purified from Aspergillus japonicus AT 008 had Mr = 400,000 and contained 1 mol of protoheme IX and 2 g atoms of copper/mol of enzyme protein. The absorption maxima of the oxidized form were found at 557, 530, 420, 280, and 238 nm, and those of the reduced form at 557 and 430 nm. Anaerobic addition of glycerol to the enzyme produced both a shift of the Soret band from 420 to 410 nm and bleaching of the alpha and beta bands at 557 and 530 nm. The ESR spectrum of glycerol oxidase showed three major signals at g = 1.99, g = 2.00, and g = 2.02. The signals at g = 1.99 and g = 2.02 were diminished by the anaerobic addition of glycerol, and the three signals completely disappeared after the addition of either dithionite or diethyldithiocarbamate. Exposure of glycerol oxidase to a borate buffer of pH 10.0 resulted in activation of the enzyme with concomitant enhancement of the ESR signals at g = 1.99 and g = 2.02. Since glycerol oxidase acts predominantly on glycerol, the enzyme can be employed in a specific colorimetric assay for serum triglycerides in combination with lipoprotein lipase.

Highlights

From the $Tok.yo Research Laboratory, Kyowa Hakko Kogyo Co., Machida-shi, Tokyo 194, Japan and the §Fuji Factory, Kyowa Medex Co., Nagaizumi-cho, Shizuoka-ken 411, Japan
The signals at g = 1.99 and g = 2.02 were diminished by the anaerobic addition of glycerol, and the three signals completely disappeared after the addition ofeither dithionite or diethyldithiocarbamate
Since glycerol oxidase acts predominantly on glycerol, the enzyme can be employed in a specific colorimetric assay for serum triglycerides in combination with lipoprotein lipase

Summary

RESULTS

Molecular Properties of Glycerol Oxidase densities of the a and /3 bands decreased, flattening into a Molecular Weight-The molecular weight of the enzyme was determined to be 400,000 bya sedimentation equilibrium method (3). The number of amino acid residues per mol of protein was 3,063, as calculated on the basis of M, = 400,000. Spectral Change of GlycerolOxidase Due to InhibitorsThe glycerol oxidase reaction was potently inhibited by potassium cyanide, hydroxylamine, and .sodium azide (3). T h e effect of these inhibitors on the absorption spectrum of glycerol oxidase was investigated by incubation of the enzyme with the inhibitors underanaerobic conditions (Fig. 2). Addition of hydroxylamine led to a drop in absorption at the Absorption Spectra of Glycerol Oxidase-The purified enzyme showed a typical absorption spectrumof a hemoprotein.

Procedure

Acetyketone Method

DISCUSSION