Physicochemical Properties and States of Sulfhydryl Groups of Uricase from Candida utilis

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Abstract
Highly purified uricase [urate: oxygen oxidoreductase, EC 1.7.3.3] was obtained from Candida utilis by affinity chromatography with xanthine-agarose conjugate followed by chromatography with Sephadex G-200 in the presence of dithiothreitol. The uricase molecule had a molecular weight of 120,000 and was composed of four identical subunits with a molecular weight of 30,000. The amino acid composition was determined and the N-terminal amino acid was identified as methionine. Other physicochemical properties obtained were as follows; isoelectric point (5.6), α-helix content (12%; b0=−76± 15°), specific activity (25.8 units/mg protein) and Km value (1.0×10−5 M) for uric acid at pH 8.5 and 25°C. The uricase preparation did not contain detectable amounts of the following metals; Fe, Cu, Mg, Mn, Co, Ni, Zn, and Cd. There were three cysteine residues in the subunit molecule and these were classified into two types by a chemical modification study; one is accessible to a reagent and the remaining two become accessible only after denaturation. The former may be located in the vicinity of the active center of the molecule, although it does not participate directly in the catalysis.