Purification and immobilisation of uricase for use in automated analysis
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A procedure for the purification of uricase from porcine liver is described, utilising the technique of bioaffinity chromatography as a major purification step. Bioaffinity support is prepared by coupling of urate to bisoxirane-activated Sepharose 4B. Purified uricase shows a single protein band corresponding to the activity band, when applied to polyacrylamide disc-gel electrophoresis. A single protein band but no activity band is obtained by SDS-acrylamide disc-gel electrophoresis. The enzyme has a pH optimum in the range of 8.9-9.1, with a Vmax of 12.6 U.mg<super>-1</super>, a Km of 1 X 10<super>-5</super>M and a molecular weight of 13 x 10<super>4</super>. Each enzyme molecule comprises 4 subunits of molecular weight 3 32-34 X 10<super>3</super> each. Nylon tube is directly activated by alkaline glutaraldehyde solution to generate reactive centres for enzyme immobilisation. The optimal conditions for activation are studied. Purified uricase is immobilised to PEI-glutaraldehyde-nylon tube with about 20% activity retention. The derivatised enzyme has a pH optimum in the range of 9.0-9.2 and a Km of about 4 times that of the soluble enzyme. Immobilised uricase is incorporated into a continuous flow Auto-Analyser for use in the automated analysis of serum urate. For this purpose, the immobilised enzyme shows good storage and operational stability. Linear calibration plots can be obtained for a urate range of 2-20 mg.100ml<super>-1</super>the method exhibiting a high degree of precision and accuracy. The results obtained also compare favourably with an established method of urate assay which employs soluble uricase.
Thesis, PhD Doctor of Philosophy
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