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Assay of uricase activity by integrated method at substrate concentration higher than Michaelis-Menten constant
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 17
- DOI:
- 10.3321/j.issn:1000-2790.2004.17.005
- Key Word:
- 积分法;初速度;尿酸酶;自变量;上限;底物浓度;酶活性测定
Abstract： 目的:以尿酸酶为模型,考察在底物浓度高于酶米氏常数(Km)时用积分法测定酶活性(Vm)的可靠性. 方法:用293 nm光吸收记录尿酸酶反应曲线. 用以反应时间为自变量的积分速度方程,以尿酸酶Km为常数而本底为非线性参数拟合尿酸酶反应曲线,搜寻对应最好拟合的Vm. 结果:此积分法测定Vm主要受剩余底物浓度影响,本底基本没有干扰. 剩余底物低于2.5 μmol/L时用10～70 μmol/L起始底物所得Vm无差异. 用25和70 μmol/L起始底物浓度时初速度和Vm都与酶量呈正比. 在此两种底物浓度下积分法的下限相同,且与初速度法下限无明显差异. 但用积分法测定酶活性的上限明显高于初速度法,而且起始底物浓度越低则差异越显著. 结论:在积分法中使用以反应时间为自变量的积分速度方程拟合酶反应曲线,在高于Km的底物浓度下能可靠测定酶活性.
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