Abstract
Measurement of urine and plasma oxalate with reusable strip of amaranthus leaf oxalate oxidase
Biochemistry Research Laboratory, Department of Biochemistry and Genetics, M. D. University, Rohtak - 124 001, India
Correspondence Address:
C S Pundir Biochemistry Research Laboratory, Department of Biochemistry and Genetics, M. D. University, Rohtak - 124 001 India E-mail: pundircs@rediffmail.com
A reusable strip of oxalate oxidase was prepared by immobilizing covalently partially purified amaranthus leaf oxalate oxidase on to alkyl amine glass beads affixed on a plastic strip. The immobilized enzyme gave a conjugation yield of 48 mg/g support with 87% retention of initial activity of free enzyme. The strip showed maximum activity at pH 3.5 when incubated at 40º for 15 min. A method for discrete analysis of oxalate in urine was developed employing this enzyme strip. The method is based on measurement of hydrogen peroxide generated from urinary/plasma oxalate by strip bound oxalate oxidase using a colour reaction consisting of 4-aminophenazone, phenol and horseradish peroxidase as chomogenic system. The minimum detection limit of the method was 0.1 mM. The recovery of added oxalate was 96.5% in plasma and 98% in urine. Within and between assay coefficient of variation were <6% and <5%, respectively in plasma and urine. The method provides enormous ease in handling of immobilized enzyme during its reuse and is unaffected by chloride ions found in biological fluids.
