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[74]Glycolic acid oxidase from pig liver
Book chapter   Peer reviewed

[74]Glycolic acid oxidase from pig liver

Marilyn Schuman Jorns
Methods in Enzymology, pp 337-343
1975
DOI: https://doi.org/10.1016/S0076-6879(75)41076-X
PMID: 236456

Abstract

This chapter describes the glycolic acid oxidase from pig liver, and focuses on its assay method, purification procedure, and properties. The oxidation of substrate (usually glycolic acid) is followed spectrophotometrically at 420 nm with potassium ferricyanide or at 600 nm with 2,6-dichlorophenolindophenol as electron acceptor. The potassium ferricyanide assay is performed at 25° in a cuvette with a 1-cm light path using a Gilford recording spectrophotometer, and the reaction is initiated by the addition of enzyme, with the exception of crude preparations. This assay is used to monitor the yield and purification during each stage of enzyme purification and for comparing the specific activity of samples isolated in successive preparations. 2,6-dichlorophenolindophenol (DCIP) assay is approximately 6 times more sensitive than the K3Fe(CN)6 assay, and it is used to assay dilute enzyme solutions obtained in column fractions. All steps of the purification are carried out at 2–4° in the presence of 0.3 mM EDTA. Glass-distilled water is used throughout the purification procedure. Glycolic acid oxidase is a fiavoprotein with a molecular weight of 100,000 as determined by molecular sieve chronlatography. The enzyme contains two chromophores: FMN and 6-hydroxyFMN. The oxidation of glycolic acid, coupled with the reduction of DCIP, shows converging-line steady-state kinetics, whereas the reaction with oxygen as the electron acceptor shows parallel-line kinetics.

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