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Characterization of inhibition of xanthine on Candida species uricase by integrated method
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 19
- DOI:
- 10.3321/j.issn:1000-2790.2005.19.001
- Key Word:
- 积分法;双倒数分析法;尿酸酶;黄嘌呤;米氏常数;抑制常数
Abstract： 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(apparent Michaelis-Menten constant, Kmapp)和表观最大反应速度(apparent maximal reaction rate, Vmapp). 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(inhibition 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<1/6 Kmapp而起始底物足够高,此积分法测定Kmapp的偏差和变异低于20%而Vmapp精度更高. 用积分法所得Kmapp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(Michaelis-Menten constant, Km)4倍以上的起始底物浓度并固定终点底物浓度<1/6 Km,此积分法能用于筛选特殊类型的抑制剂.
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