Chemistry Department, University of Winnipeg, Winnipeg, Manitoba, Canada.
Anal Chem. 2012 May 15;84(10):4598-602. doi: 10.1021/ac300777u. Epub 2012 Apr 26.
Escherichia coli β-galactosidase was incubated in the presence of the slow-release inhibitor D-galactal for 30 min at a concentration of 70 times its K(i). The sample was then diluted 20000-fold into buffer containing the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-on-7-yl) β-D-galactoside, reducing the inhibitor concentration to K(i)/280. The sample was subjected to a capillary electrophoresis continuous flow single enzyme molecule assay. As the inhibitor dissociated while the enzyme traveled the length of the capillary, a fraction of molecules showed stepwise increases in activity. This was due to the activation of individual subunits within single molecules. The changes in activity can be largely explained in terms of each molecule containing subunits of indistinguishable activity.
大肠杆菌β-半乳糖苷酶在浓度为其 K(i) 的 70 倍的慢释放抑制剂 D-半乳糖醛的存在下孵育 30 分钟。然后，将样品稀释 20000 倍到含有荧光底物 9H-(1,3-二氯-9,9-二甲基吖啶-2-酮-7-基)β-D-半乳糖苷的缓冲液中，将抑制剂浓度降低到 K(i)/280。将样品进行毛细管电泳连续流动单酶分子分析。由于抑制剂在酶穿过毛细管的过程中解离，因此一部分分子的活性呈逐步增加。这是由于单个分子内的各个亚基的激活。可以根据每个分子都包含无法区分的活性的亚基来解释大部分活性变化。
