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2748570 
Journal Article 
Homogentisic Acid Oxidase. II. Properties of the Crude Enzyme in Rat Liver 
Crandall, DI 
1955 
Yes 
Journal of Biological Chemistry
ISSN: 0021-9258
EISSN: 1083-351X 
NIOSH/00133386 
212 
2 
565-582 
The properties of crude homogentisic-acid-oxidase (HAO) prepared from rat liver was investigated. The crude enzyme was prepared by centrifuging homogenized rat liver. Compounds tested were bipyridyl (37275482), cyanide (74908), hydrogen-sulfide (7783064), cysteine (52904), diethyl-dithiocarbamate (147842), azide (14343692), mercuric-chloride (7487947), p-chloromercuribenzoate (59858) (PCMB), iodosobenzoate (27323359), iodoacetate (64697), and maleate. The optimal pH for HAO activity was 7.0. HAO was completely inhibited at pH 6 and this inhibition was reversible by a combination of ferrous ions and ascorbate ions and not by other metallic ions. Reversibility was obtained at very low concentrations of ferrous ions. HAO was inhibited by all test compounds except iodoacetate and maleate. The reversal of inhibition by PCMB required the presence of both reduced glutathione (70188) (GSH) and ferrous ions, neither of which was effective singly. The ferrous ion was not involved in the partial reversal of iodosobenzoate inhibition by ascorbate and GSH. Both ascorbate and GSH increased the activity of HAO at pH 7 but not at pH 6 in the absence of added ferrous ions. The enzyme was susceptible to irreversible inactivation by oxygen and hydrogen peroxide (7722841) and was not inactivated by dialysis at pH 7. HAO did not oxidize gentisic-hydroxyphenyl-acetic-acid or o-hydroxyphenyl-acetic-acid, indicating specificity for homogentisic-acid oxidation. HAO was strongly inhibited by benzoquinone-acetic-acid (10275077). The author concludes that ascorbate activates HAO by protecting ferrous but not sulfhydryl groups against oxidation.