Single Molecule Assay of Escherichia coli β-Galactosidase Using Two Competing Substrates Simultaneously, DDAO-β-D-Galactoside and Resorufin-β-D-Galactoside

Abstract

Single enzyme molecule assays were performed on E. coli β-galactosidase using a capillary electrophoresis-based protocol. Assays were performed using double incubations and two substrates, resorufin-β-D-galactoside and DDAO-β-D-galactoside, simultaneously. The variation between individual enzyme molecules in the ratio of product peak areas for the two different substrates used was indistinguishable from the variation in peak areas of the replicate incubations for a given enzyme molecule. This suggests that the enzyme is not heterogeneous with respect to its relative activity with the two different substrates used.

Keywords:

• Beta-galactosidase
• Capillary electrophoresis
• Enzyme heterogeneity
• Single molecule enzymology

Acknowledgments

This project was supported by a grant from the Natural Sciences and Engineering Research Council.