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Title: Purification and immobilisation of uricase for use in automated analysis
Author: bin Salleh, Abu Bakar
Awarding Body: University of St Andrews
Current Institution: University of St Andrews
Date of Award: 1978
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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, a Km of 1 X 10-5M and a molecular weight of 13 x 104. Each enzyme molecule comprises 4 subunits of molecular weight 3 32-34 X 103 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-1the 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.
Supervisor: Hornby, W. E. ; Ledingham, W. M. Sponsor: Universiti Pertanian Malaysia
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP603.U7S2 ; Urate oxidase