Pace M, Mauri P, Pietta P, Agnellini D
Dipartimento di Scienze e Technologie Biomediche, University of Milano, Italy.
Anal Biochem. 1989 Feb 1;176(2):437-9. doi: 10.1016/0003-2697(89)90338-2.
Enzymes can be assayed by HPLC by calculating the amount of substrate(s) left over, or product formed, through the peak area ratios with a suitable internal standard. However, sometimes the substrates used are contaminated with small amounts of products and this can lead to errors in the determination of the enzyme activity. A method for a HPLC test of such enzymes, which prevents eventual errors, uses the ratio substrate/product at time zero as internal standard and the kinetics can be followed with the aid of a simple mathematical equation. This approach was applied to the determination of the activities of papain, urokinase, NAD glycohydrolase, and pyruvate kinase samples and it was compared with the data obtained by the internal standard method, giving reproducible results in all cases.
酶可以通过高效液相色谱法（HPLC）进行测定，方法是通过与合适的内标物的峰面积比来计算剩余底物的量或形成的产物的量。然而，有时所使用的底物会被少量产物污染，这可能会导致酶活性测定出现误差。一种用于此类酶的HPLC检测方法，可防止最终出现误差，该方法使用零时刻底物/产物的比率作为内标物，并且可以借助一个简单的数学方程式跟踪动力学过程。这种方法被应用于木瓜蛋白酶、尿激酶、NAD糖水解酶和丙酮酸激酶样品活性的测定，并与通过内标法获得的数据进行了比较，在所有情况下均给出了可重复的结果。
