A CE-LIF detection-based assay was developed for the study of individual molecules of Escherichia coli beta-galactosidase. The assay allows for the simultaneous measurement of the electrophoretic mobility, catalytic activity and the variation in activity over time of individual enzyme molecules. In addition to showing the microheterogeneity of the enzyme molecules with respect to mobility and activity, it was demonstrated that at elevated temperatures the enzyme activity fluctuates over time. Incubation at varying temperatures showed that individual beta-galactosidase molecules exhibit differences in their change in activity upon a change in incubation temperature. Incubation at one temperature, followed by an incubation at an elevated temperature and subsequent incubation back at the initial lower temperature caused the molecules to have a different activity than they had initially. Additionally, thermal denaturation was found to cause a rapid and complete loss of activity.

The electrophoretic mobility and catalytic activity of individual molecules of Escherichia coli beta-galactosidase were measured using CE-LIF detection. Both the mobility and activity were reproducible for each molecule but differed between individual molecules. Assays were performed using uncoated capillaries and capillaries coated with different polymers, using enzymes from different sources and by three different experimental protocols. In all cases the observed ranges in electrophoretic mobilities were similar. The observed range in the electrophoretic mobility may be explained by structural microheterogeneity resulting in a gain or loss of up to 1.6 suppressed charge units. There was no observed relationship between the observed activities and electrophoretic mobilities. If the finding that individual beta-galactosidase molecules have heterogeneous electrophoretic mobility can be extended to other proteins, this may limit the resolution possible for capillary zone electrophoresis protein separations.

Teaching redox chemistry is an important facet of undergraduate biochemistry courses, particularly with respect to developing the students' understanding of the central metabolic processes such as the mitochondrial respiratory chain and photosynthesis. We have introduced an experiment for the undergraduate metabolism course involving the spectroscopic measurement of the redox potential of cytochrome c. The experiment permits a quantitative result within a single three-hour laboratory period and complements the basic redox concepts and calculations shown in class.

Assays were performed on individual Escherichia coli beta-galactosidase molecules at 2 different concentrations of the substrate DDAO-beta-D-galactoside using a free zone capillary electrophoresis-based protocol with an in-laboratory-constructed instrument utilizing laser-induced fluorescence detection. In a typical run, 2 enzyme molecules were injected into the capillary. They were separated from each other by a brief period of electrophoresis and incubated on the capillary in the presence of the substrate. They were then mobilized on the capillary into a zone of substrate at a different concentration, re-incubated, and the product peaks mobilized past the detector . The relative change in activity as the concentration was increased differed between molecules, suggesting differences in Km. In a different experiment, the capillary was filled with on average 13 enzyme molecules per run, incubated, and the activities of the individual molecules determined. The shapes of the distribution curves of single molecule activities obtained at different concentrations of the substrate resorufin-beta-D-galactoside were indistinguishable, suggesting a homogeneous Km. To explain why individual enzyme molecules behaved as if they were heterogeneous with respect to Km but the population behaved as if it were homogeneous, theoretical Michaelis-Menten curves were constructed. The curves for populations with heterogeneous Km values were found to be indistinguishable from that of a homogeneous population.

Beta-galactosidase was induced in E. coli wild type strains ATCC 8677 and 35321 in the presence of various protease inhibitors. Single enzyme molecule assays were performed using a capillary electrophoresis based protocol. The presence of the protease inhibitors had a minimal effect on the average and distribution of single molecule activities. Two novel capillary electrophoresis based single enzyme molecule assays for beta-galactosidase were developed using DDAO-beta-D-galactopyranoside and fluorescein-beta-D-digalactopyranoside as substrates. Double incubations were performed on individual enzyme molecules to demonstrate the reproducibility of the assays. Assays performed on beta-galactosidase from strains 8677 and 35321 demonstrated that the relative activities of the enzyme for the different substrates differed between the strains. Sequencing showed that these two strains differ in their primary sequence by a single amino acid substitution in position 280, which is in the region of the active site.