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

• Published in: Analytical letters Vol. 44; no. 10; pp. 1835 - 1841
• Main Authors: Craig, Douglas B., Eggertson, Michael J., Chikamatsu, Miki, Horwood, Corie A.
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA Taylor & Francis Group 01.07.2011; Taylor & Francis
• Subjects: Analytical chemistry; Beta-galactosidase; Capillary electrophoresis; Chemistry; Chromatographic methods and physical methods associated with chromatography; Enzyme heterogeneity; Exact sciences and technology; Other chromatographic methods; Single molecule enzymology; Beta-galactosidase; Peak resolution; Single molecule enzymology; Chemical analysis; Enzyme heterogeneity; Enzyme; Capillary electrophoresis; Escherichia coli; Use; Product; Replica method; Glycosylases; Substrate; Heterogeneity; Molecules; Reporter gene; Glycosidases; Bacteria; Hydrolases; β-Galactosidase; Enterobacteriaceae
• ISSN: 0003-2719; 1532-236X
• DOI: 10.1080/00032719.2010.526264

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.