Single enzyme molecule assay with time resolution using capillary electrophoresis

Abstract

The continuous flow assay is a capillary electrophoresis-based methodology for the simultaneous measurement of the catalytic rate and electrophoretic mobility of individual molecules of the enzyme β-galactosidase. The method also provides time resolution. This method was used to measure the distribution of single-molecule activities and mobilities of a population of the wild-type Escherichia coli β-galactosidase. The catalytic rate was found to vary over time at an elevated temperature, suggesting a switching between different conformations. The successive incubation of individual molecules at 27°C, 45°C, and again at 27°C was found to convert molecules from one form with a stable catalytic rate to a different form with a different stable catalytic rate. Incubation at higher temperatures was found to cause a sudden and catastrophic loss in activity, which was consistent with denaturation. Increasing the incubation temperature over time was used to generate an Arrhenius plot for a single enzyme molecule. Finally, assaying the enzyme as the slow-release inhibitor d-galactal dissociated allowed for the measurement of the activity of individual subunits within a single molecule.

Keywords

• β-galactosidase
• capillary electrophoresis
• heterogeneity
• laser-induced fluorescence
• single molecule