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PubMed1986

Immobilization of uricase on protamine bound to glass beads and its application to determination of uric acid.

Uricase was found to be stabilized by protamine from salmon testis. Protamine was then bound to controlled-pore glass beads aminohexyl CPG 500 using glutaraldehyde. Microbial uricase was readily immobilized on the protamine bound to glass beads. The immobilized uricase proved to be stable even at 70 degrees C, whereas free uricase was inactivated at 45 degrees C and showed activity over a broader pH range than free uricase. Automated analysis of uric acid was facilitated using the immobilized uricase. The standard curve for uric acid was linear in the range of 2 to 10 micrograms/sample and passed through the origin. This automated procedure was also applicable to the determination of uric acid in human serum. Protamine bound to glass beads is expected to be useful for the simple immobilization and stabilization of enzymes.

Animals↗
PubMed1987

Determination of uric acid by an anion-exchange column chromatography.

A column chromatography using a conventional anion-exchange resin for the separation of uric acid from other purine metabolites is described. It uses a HCl gradient, and the amount of uric acid is quantified directly by monitoring the absorbance of the effluent at 285 nm. The linear range of response is 0.5 to 100 nmol. The method was applied to the analysis of uric acid in urine and serum. Urine was injected directly into the system, while serum required removal of an interfering substance which absorbs the light and coelutes with uric acid. However, this substance was simply removed by heat coagulation of serum by heating in a boiling water bath for 2 min.

Anion Exchange Resins↗
PubMed1983

Uricase from soybean root nodules: purification, properties, and comparison with the enzyme from cowpea.

A 45-fold purification of uricase (urate:O2 oxidoreductase, EC 1.7.3.3) from soybean root nodules by ammonium sulfate fractionation, gel filtration, and affinity chromatography is described. Electrophoresis on nondenaturing gels using an activity stain or on sodium dodecyl sulfate (SDS) gels demonstrated that the enzyme obtained was nearly homogeneous. The subunit molecular weight of uricase estimated from SDS gels was 32,000 +/- 3000. Gel-filtration studies indicated that the native enzyme is a monomer at pH 7.5 which associates to form a dimer at pH 8.8. Enzyme activity was stabilized by the addition of dithiothreitol. The pH dependence of the enzyme showed an optimum of 9.5. Initial rate kinetics showed Km values of 10 and 31 microM for uric acid and oxygen, respectively, with an intersecting pattern of substrate dependence. Uricase activity was inhibited strongly by xanthine, which was competitive with respect to uric acid (Ki = 10 microM). No significant inhibition was observed in the presence of a variety of amino acids, ammonium, adenine, or allopurinol, in contrast with results reported for the cowpea enzyme. Gel-filtration chromatography and SDS-gel electrophoresis of uricase purified by the same method from cowpea nodules indicated that the native enzyme exists as a monomer of Mr 50,000 at pH 7.5.

Hydrogen-Ion Concentration↗
PubMed1978

Uricase of Bacillus fastidiosus. Properties and regulation of synthesis.

Uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) of Bacillus fastidiosus was purified to homogeneity in a two-step procedure and was crystallized. The native molecule had a molecular weight of 145 000--150 000 and was composed of subunits of two kinds (Mr = 36 000 and 39 000) in a 1 : 1 ratio. The quaternary structure of the enzyme was reversibly altered, with concomitant loss of activity, at temperatures between 40 and 60 degrees C. No evidence was found for the involvement of metal ions or coenzymes in the uricase reaction. The enzyme was inhibited by various metal ions and by cyanide. The isoelectric point of the enzyme was 4.3, and pH optimum 9.5 and the optimal temperature 30--35 degrees C. Only uric acid was oxidized by the enzyme and 9-methyluric acid, xanthine, 8-azaxanthine and oxonic acid were competitive inhibitors. Uricase synthesis was repressed by allantoin and allantoate, even in the presence of uric acid, which induced synthesis of the enzyme. Molecular oxygen was an important environmental factor in the control of uricase synthesis, probably due to its effect, as cosubstrate in the uricase reaction, in assessing the cytoplasmic concentration of allantoin. The highest amounts of uricase, up to half of the intracellular soluble protein content, was found in cells growing under limited oxygen supply in media containing uric acid as the main substrate.

Allantoin↗