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Assay of uricase activity by integrated method at substrate concentration higher than Michaelis-Menten constant

AIM: To test the reliability of the integrated method for the assay of enzyme activity at substrate concentration above the Michaelis Menten constant ( K m ). METHODS: Uricase reaction curve was monitored by absorbance at 293 nm, and it was fit to the integrated Michaelis Menten rate equation with the predictor variable of reaction time for irreversible, single substrate reaction with the background absorbance as a nonlinear parameter while K m as a preset constant. RESULTS: V m was sensitive to residual substrate concentration while it was resistant to the background absorbance. With residual uric acid below 2.5 μmol/L, there were no differences among V m at initial substrate concentrations from 10 to 70 μmol/L. There were linear responses of both V m and initial rates to the amount of uricase with initial substrate concentration at 25 or 70 μmol/L if the residual substrate was below 2.5 μmol/L. The lower limits of V m 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 V m 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 K m .