Assay of uricase activity by integrated method at substrate concentration higher than Michaelisª²Menten constant

    LIAO Fei, ZHU Xiaoª²Yun, WANG Yongª²Mei, ZENG Zhaoª²Chun, ZUO Yuª²Ping

    Department of Biochemistry, Chongqing University of Medical Sciences, Chongqing 400016

    ¡¾Abstract¡¿ AIM: To test the reliability of the integrated method for the assay of enzyme activity at substrate concentration above the Michaelisª²Menten constant (Km). METHODS: Uricase reaction curve was monitored by absorbance at 293 nm, and it was fit to the integrated Michaelisª²Menten rate equation with the predictor variable of reaction time for irreversible, single substrate reaction with the background absorbance as a nonlinear parameter while Km as a preset constant. RESULTS: Vm was sensitive to residual substrate concentration while it was resistant to the background absorbance. With residual uric acid below 2.5 ¦Ìmol/L, there were no differences among Vm at initial substrate concentrations from 10 to 70 ¦Ìmol/L. There were linear responses of both Vm and initial rates to the amount of uricase with initial substrate concentration at 25 or 70 ¦Ìmol/L if the residual substrate was below 2.5 ¦Ìmol/L. The lower limits of Vm by this integrated method showed no difference at these two initial substrate concentrations and were comparable to those of initial rates. But the upper limit of Vm was twice above that of initial rate, and this difference was more pronounced at lower substrate concentrations. CONCLUSION: This integrated method with reaction time as the predictor variable in the integrated rate equation is reliable for enzyme activity assay at substrate concentration above the Km. ......