Whisnant A R, Johnston S E, Gilman S D
Department of Chemistry, The University of Tennessee, Knoxville 37996-1600, USA.
Electrophoresis. 2000 Apr;21(7):1341-8. doi: 10.1002/(SICI)1522-2683(20000401)21:7<1341::AID-ELPS1341>3.0.CO;2-9.
An analytical method for studying enzyme inhibition has been developed using capillary electrophoresis with laser-induced fluorescence detection. This technique is based on electrophoretic mixing of zones of enzyme and inhibitor in substrate-filled capillaries. Enzyme catalytic activity is measured by detecting the fluorescent reaction product as it migrates past the detector. Reversible enzyme inhibition is indicated by a transient decrease in product formation. The enzyme, alkaline phosphatase, has been studied using the fluorogenic substrate AttoPhos ([2,2'-bibenzothiazol]-6-hydroxy-benzthiazole phosphate). This assay has been used to quantify theophylline, a noncompetitive, reversible inhibitor of alkaline phosphatase. The detection limit for theophylline is estimated at 3 microM, and 8.6 amole of alkaline phosphatase are required for each assay. The calculated K(i) for theophylline is 90 microM for the capillary electrophoretic enzyme-inhibitor assays.
已开发出一种利用毛细管电泳结合激光诱导荧光检测来研究酶抑制作用的分析方法。该技术基于在充满底物的毛细管中酶和抑制剂区域的电泳混合。通过检测荧光反应产物迁移经过检测器时的情况来测定酶的催化活性。产物形成的短暂减少表明存在可逆酶抑制作用。已使用荧光底物AttoPhos（[2,2'-联苯并噻唑]-6-羟基-苯并噻唑磷酸酯）对碱性磷酸酶进行了研究。该测定法已用于定量茶碱，茶碱是碱性磷酸酶的一种非竞争性、可逆抑制剂。茶碱的检测限估计为3 microM，每次测定需要8.6 amole的碱性磷酸酶。毛细管电泳酶抑制剂测定中茶碱的计算解离常数（K(i)）为90 microM。
