Measurement of the differences in electrophoretic mobilities of individual molecules of E. coli β‐galactosidase provides insight into structural differences which underlie enzyme microheterogeneity

Abstract

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.

Continuous flow assay for the simultaneous measurement of the electrophoretic mobility, catalytic activity and its variation over time of individual molecules of Escherichia coli β‐galactosidase

“…The catalytic rate of E. coli b-galactosidase has been found to vary with source [13] and these values fall within the normal range of the values observed. As has been noted previously [9], there was no observed relationship between electrophoretic mobility and catalytic activity.…”

“…Quadruple incubations of individual molecules of b-galactosidase, using incubation periods of 3 min, have shown reproducible activity [9]. This has also been shown with alkaline phosphatase [3] and lactate dehydrogenase [2], which suggests that time-average activities are consistent over the short term.…”

“…Differences between individual molecules in the change in activity upon incubation at different temperatures have been shown for bovine alkaline phosphatase [3]. Recently, individual molecules of E. coli b-galactosidase have been shown to exhibit differences in electrophoretic mobility [9], and these differences were used to make inferences regarding the structural basis of the observed enzyme microheterogeneity. …”

Comparison of the single molecule activity distributions of recombinant and non-recombinant bovine intestinal alkaline phosphatase

“…This heterogeneity has been found with respect to catalytic rate, in terms of both V max [5] and K m [6], activation energy of catalysis [5], electrophoretic mobility [7] and dependence upon exogenous metal ions to maintain activity [8]. Furthermore, catalytic rate has been found to vary for a given molecule over time [9].…”

Determination of the inhibitor dissociation constant of an individual unmodified enzyme molecule in free solution

“…Since the individual molecules have different electrophoretic mobilities [17], when more than one was injected the resultant electropherogram was comprised of sets of partially overlapping peaks, complicating the mathematical resolution and integration of the peaks. Since the individual molecules have different electrophoretic mobilities [17], when more than one was injected the resultant electropherogram was comprised of sets of partially overlapping peaks, complicating the mathematical resolution and integration of the peaks.…”