Bioanalytics, Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, D-40225 Düsseldorf, Germany.
Electrophoresis. 2010 Jan;31(4):634-40. doi: 10.1002/elps.200900514.
A new assay for protein kinase CK2 activity determination based on the quantification of a phosphorylated substrate was developed. The common CK2 substrate peptide RRRDDDSDDD, conjugated with the fluorophore 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid at the C-terminus served as the analyte. By means of CZE using 2 mol/L acetic acid as electrolyte and UV detection at 214 nm, the non-phosphorylated and the phosphorylated peptide variants could be resolved within 6 min from a complex assay mixture. By this means, activity of human CK2 could be monitored by a kinetic, as well as an endpoint, method. Inhibition of human recombinant CK2 holoenzyme by 6-methyl-1,3,8-trihydroxyanthraquinone and 4,5,6,7-tetrabromobenzotriazole resulted in IC(50) values of 1.33 and 0.27 microM, respectively, which were similar to those obtained with the standard radiometric assay. These results suggest that the CE/UV strategy described here is a straightforward assay for CK2 inhibitor testing.
基于对磷酸化底物进行定量的方法，开发了一种用于测定蛋白激酶 CK2 活性的新分析方法。常见的 CK2 底物肽 RRRDDDSDDD，在 C 末端与荧光团 5-[(2-氨乙基)氨基]萘-1-磺酸连接，作为分析物。通过使用 2 mol/L 乙酸作为电解质的 CZE 和在 214nm 处的 UV 检测，可以在 6 分钟内从复杂的分析混合物中分离出非磷酸化和磷酸化肽变体。通过这种方法，可以通过动力学和终点方法监测人 CK2 的活性。6-甲基-1,3,8-三羟基蒽醌和 4,5,6,7-四溴苯并三唑对人重组 CK2 全酶的抑制作用导致 IC50 值分别为 1.33 和 0.27 microM，与标准放射性分析方法获得的值相似。这些结果表明，这里描述的 CE/UV 策略是一种用于 CK2 抑制剂测试的直接分析方法。
