Measurement of the Activity of Individual Subunits of Single Molecules of the Tetrameric Enzyme beta-Galactosidase

作者：Craig Douglas B^*; Morris Thomas T; Ong Justiniano Coleen Marie Q

来源：Analytical Chemistry, 2012, 84(10): 4598-4602.

DOI：10.1021/ac300777u

Escherichia coli beta-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) beta-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.

• 出版日期2012-5-15