底物浓度选择在酶Km值测定中的重要性

The Importance of Selecting a Right Substrate Concentration in the Km Assay of a Michaelis-Menten Enzyne

以胰蛋白酶对酪蛋白的Ｋｍ值测定为例，讨论了底物浓度选择在酶Ｋｍ值测定中的重要性。底物酪蛋白的浓度分为三组：高浓度（１～５ｍｇ／ｍｌ），低浓度（０．１～１ｍｇ／ｍｌ）和极低浓度（０．０２～０．０８ｍｇ／ｍｌ），高底物浓度（＞８Ｋｍ）的测定结果不能有效地区分多酶（作用于同一底物的多种酶）的存在，也不符合作图法要求的准确度，所得到的Ｋｍ值可能是不准确的或错误的。因此，测定Ｋｍ值时所用的底物浓度必须在０．２～５Ｋｍ范围内，必要时应包括更广泛的浓度范围（包括高底物浓度）。

The data of Km assay of casein hydrolyzed by trypsin was taken was taken as a example for discussing the importance of selecting a right substrate concentrations in the Km assay of a Michaelis-Menten enzyme.Casein(as substrate of trypsin) was divided to three levels of high concentrations(1-5mg/ml),low concentrations (0.1-1mg/ml) and very low concentrations(0.02-0.08mg/ml).High substrate concentrations (>8Km) is not enough to prove the exist of a multi-enzymes system,(two or more than two enzymes which catalyze the same substrate) and to come to the precision require by a plotting.It may result in a mistake or incorrect Km.The substrate concentrations used to assay Km must be in 0.2-5Km which is in the levels of low and very low concentrations,if necessaray,a larger range of substrate concentrations should be selected that include high level.