Characterization of inhibition of xanthine on Candida species uricase by integrated method

    ZHAO Liª²Na, LIAO Fei, ZHAO Yunª²Sheng

    Department of Biochemistry, Chongqing University of Medical Science, Chongqing 400016, China

    ¡¾Abstract¡¿ AIM: To investigate the prerequisite of the integrated method for characterizing inhibitors with the inhibition of xanthine on Candida species uricase as the model. METHODS: Uricase reaction curve was monitored by absorbance at 293 nm. The apparent kinetic parameters (apparent Michaelisª²Menten constant, Kmapp, and apparent maximal reaction rate, Vmapp) were estimated by nonlinear fitting reaction curve to the integrated Michaelisª²Menten rate equation with the predictor variable of reaction time. The inhibition type and inhibition constant (Ki) were determined based on the responses of apparent kinetic parameters to xanthine concentrations. RESULTS: Outliers in reaction curve greatly reduced the reliability of parameters obtained by this integrated method. With residual substrate below oneª²sixth of Kmapp and initial substrate high enough, this integrated method usually showed deviation below 20% for Kmapp and smaller deviation for Vmapp. By this integrated method with the same range of data for analysis, Kmapp was consistent to that by Lineweaverª²Burk plot and the response of Kmapp to xanthine concentrations gave inhibition constant of (5.4¡À0.8) ¦Ìmol/L (n=3), also consistent to that by Lineweaverª²Burk plot. CONCLUSION: This integrated method is feasible for fast screening of special inhibitors with data of higher precision and initial substrate above fourª²fold of Michaelisª²Menten constant (Km) in the absence of inhibitor and residual substrate below oneª²sixth of this Km for analysis. ......