Inhibitory effects of xanthine on uricase analyzed by a linear kinetic method

    ZHAO Liª²Na, LIAO Fei, ZHAO Yunª²Sheng

    Department of Biochemistry, Chongqing Univeristy of Medical Sciences, Chongqing 400016, China

    ¡¾Abstract¡¿ AIM: To investigate the reliability of a linear kinetic method based on calibrated specific activities of enzyme at two substrate concentrations in estimating apparent kinetic parameters and characterizing inhibitors by using two uricases with different Km as models. METHODS: Uricase reaction was monitored by the absorbance at 293 nm and apparent kinetic parameters (apparent Michaelisª²Menten constant, Kmapp, and apparent maximal reaction rate, Vmapp) were estimated based on the calibrated specific activities of uricase at two substrate concentrations. The inhibition type and inhibition constant (Ki) were determined by the responses of apparent kinetic parameters to the xanthine concentrations. RESULTS: Km of Bacillus fastidiosus uricase and Candida species uricase were estimated with lower concentration of uric acid (SL) above fourª²fifth of Km and higher concentration of uric acid (SH) above 1.4ª²fold of SL in consistence to doubleª²reciprocal analysis. With SL at 3.8ª²fold Km and SH at 3ª²fold SL, there was linear response of Kmapp of Candida species uricase by this linear kinetic method to xanthine concentrations, and the inhibition constant was (4.8¡À0.5) ¦Ìmol/L, consistent to doubleª²reciprocal analysis. This linear kinetic method did not yield reliable inhibition constant of xanthine on Bacillus fastidiosus uricase of much higher Km. CONCLUSION: This linear kinetic method, reliable in estimating kinetic parameters and characterizing inhibitors of enzyme with lower Km by using two concentrations of the substrate above its Km, makes it possible to use routine technique for quantification. ......