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<sup>-1</sup>, a Km of 1 X 10<sup>-5</sup>M and a molecular weight of 13 x 10<sup>4</sup>. Each enzyme molecule comprises 4 subunits of molecular weight 3 32-34 X 10<sup>3</sup> 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<sup>-1</sup>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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:751152 |
| Date | January 1978 |
| Creators | bin Salleh, Abu Bakar |
| Contributors | Hornby, W. E. ; Ledingham, W. M. |
| Publisher | University of St Andrews |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10023/14387 |
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