用积分法考察黄嘌呤对Candida species尿酸酶的抑制作用

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

目的:以Candida species尿酸酶为模型,考察用积分法筛选抑制剂的条件.方法:用293 nm吸收记录尿酸酶反应.用反应时间为自变量的积分速度方程拟合反应曲线确定表观米氏常数(apparentM ichaelis-M enten constant,Km app)和表观最大反应速度(apparentm axim al reaction rate,Vm app).据上述参数随抑制剂浓度的变化确定抑制类型和抑制常数(inh i-b ition constant,Ki).结果:被分析数据中的畸异值显著降低结果可靠性.通常终点底物<1/6Km app而起始底物足够高,此积分法测定Km app的偏差和变异低于20%而Vm app精度更高.用积分法所得Km app与双倒数法一致,且和黄嘌呤浓度成正比,对应Ki为(5.4±0.8)μmol/L(n=3),也与双倒数法一致.结论:被分析数据精度足够高,用无抑制剂时米氏常数(M ichaelis-M enten constant,Km)4倍以上的起始底物浓度并固定终点底物浓度<1/6Km,此积分法能用于筛选特殊类型的抑制剂.

AIM ： To investigate grated method for characterizing the prerequisite of the inteinhibitors 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 Michaelis-Menten constant, Kmapp, and apparent maximal reaction rate, Vmapp） were estimated by nonlinear fitting reaction curve to the integrated Michaelis-Menten rate equation with the predictor variable of reaction time. The inhibition type and inhibition constant （ K ） 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 one-sixth 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 Lineweaver-Burk plot and the response of Kmapp to xanthine concentrations gave inhibition constant of （5.4 ±0.8） μmol/L （n = 3）, also consistent to that by Lineweaver-Burk plot. CONCLUSION. This integrated method is feasible for fast screening of special inhibitors with data of higher precision and initial substrate above fourfold of Michaelis-Menten constant （Km ） in the absence of inhibitor and residual substrate below one-sixth of this Km for analysis.