Covalent immobilization of uricase inside a plastic vial for uric acid determination in serum and urine.
Uricase from Candida species was immobilized covalently onto the inner wall of a plasticized polyvinyl chloride (PVC/plastic) vial through glutraldehyde coupling with a 65.23% retention of its initial activity and a conjugation yield of 0.37 mg/cm(2). The vial-bound enzyme showed the optimum activity at pH 7.2, when incubated at 45°C for 5 min. There was a linear relationship between the immobilized uricase activity and the uric acid concentration in the range of 0.01 to 1.2 mM with an apparent Km for uric acid of 0.17 mM. The vial-bound enzyme was employed for an enzymic colorimetric determination of uric acid in serum and urine. The minimum detection limit of the method was 0.01 mM. The analytical recoveries of added uric acid in serum (10 and 20 mM) were 98.0 and 96.5%, respectively. Within and between assays, the coefficients of variation (CVs) for urate in sera determinations were 5.6 and 4.7%, respectively. A good correlation (r = 0.997) was obtained between the serum uric acid values by the standard enzymic colorimetric method using free enzyme and the present method. The vial was reused 200-times over a period of 4 months, when stored at 4°C.
Department of Biochemistry, M. D. University.
Nidhi
N
Chauhan
Preeti
Pinky
C S
CS
Pundir
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D002175:Candida / Q000201:enzymology; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D004867:Equipment Design; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D011143:Polyvinyl Chloride / Q000737:chemistry; D011994:Recombinant Proteins; D014503:Urate Oxidase / Q000235:genetics / Q000378:metabolism*; D014527:Uric Acid / Q000097:blood* / Q000652:urine*
D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D014527:Uric Acid; D011143:Polyvinyl Chloride; D014503:Urate Oxidase