Characterization of tyrosine kinase and screening enzyme inhibitor by capillary electrophoresis with laser-induced fluoresce detector

An effective, rapid and reliable capillary electrophoresis-laser induced fluorescence (CE-LIF) procedure was built to study the characterization of tyrosine kinase (TK), which was a target for drug screening. In this procedure, CE separated the sample of the TK reaction and LIF selectively detected the fluorescence-labeled polypeptide substrate and product. The precise TK activity was quantitated by introducing the transformation ratio of the substrate (T%) to avoid the deviation resulted from the detection sensitivity and the injection amounts in different runs and different capillaries. By measuring the T%, the effects of various reaction conditions were optimized. Meanwhile, the progression of the enzyme reaction was monitored. The K-m and V-max were calculated for TK under the optimized experimental conditions. In addition, the inhibition effectiveness of two model inhibitors, Staurosporine and SU6656 were evaluated. The results indicated that the screening platform based on electrophoresis was suitable for TK analysis and laid a foundation for the HTS of TK inhibitors. (C) 2010 Elsevier B.V. All rights reserved.