Polyethylene terephthalate membrane as a support for covalent immobilization of uricase and its application in serum urate determination

摘要

A method is described for covalent immobilization of uricase onto polyethylene terephthalate (PET) membrane with a conjugation yield of 4.44 μg/cm~2 and 66.6% retention of initial activity of free enzyme. The enzyme exhibited an increase in optimum pH from pH 7.0 to 8.5 and K_m for uric acid from 0.075 mM to 0.13 mM but slight decrease in temp. for maximum activity from 37 °C to 35 °C after immobilization. A colorimetric method for determination of serum uric acid was developed using immobilized uricase, which is based on measurement of H2O2 by a color reaction consisting of 3,5-dichlorobenzene sulphonic acid (DHBS), 4-aminoantipyrine and peroxidase as chromogenic system. Minimum detection limit of the method was 0.05 mM. Analytical recovery of added uric acid (5 mg/dl and 10 mg/dl) was 94.3% and 89.8%, respectively. Within and between batch coefficient of variation (CV) were <3.2% and <4.3%, respectively. A good correlation (r=0.98) was found between uric acid values by standard enzymic colorimetric method and the present method. The immobilized uricase was reused 100 times during the span of 60 days without any considerable loss of activity, when stored in reaction buffer at 4°C The support chosen for the present study was biocompatible, antimicrobial, inert, impact resistant, light weight and had good shelf life.