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

Abstract

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.

Observing Single Enzyme Molecules Interconvert between Activity States upon Heating

“…These enzymes are irreversibly denatured and contribute to the decrease in average activity over time. The denaturation temperature of β-galactosidase was calculated to be 55°C by CD experiments and confirmed by kinetic measurements (Figure S3A, B in File S1), and agrees well with denaturation temperatures reported in the literature [15], [24]–[26]. In the present experiments, denaturation of the enzymes was minimized by heating only to 47°C, which is below this denaturation temperature.…”

“…Previous experiments indicate that enzymes can also exhibit long-lived activity differences at room temperature resulting in a distribution of activities between ostensibly identical molecules, termed “static heterogeneity” [2], [3], [12]. This static heterogeneity arises either from different conformations of the enzyme and/or different primary sequences between molecules, with the latter possibility resulting from errors in transcription and translation [11], [13]–[15]. When a nascent protein folds, it is possible that each molecule becomes trapped in a local energy minimum and many different local minima, i.e.…”

Production, purification, characterization, immobilization, and application of β‐galactosidase: a review

“…[99] Moreover, it was found that electrophoretic mobility and catalytic activity of individual molecules of β-galactosidase synthesized by E. coli were different, although they had potentiality to act on the same substrate molecule. [100]…”

Single Molecule Assay ofEscherichia coliβ-Galactosidase Using Two Competing Substrates Simultaneously, DDAO-β-D-Galactoside and Resorufin-β-D-Galactoside

“…This observed heterogeneity is not limited to catalytic rate. Activation energy of catalysis (Craig et al 1996;Craig and Nichols 2008), K m (Craig et al 2010), V max (Craig et al 1996), electrophoretic mobility Craig and Nichols 2009), and dependence upon metal ions to maintain stability (Craig, Hall, and Goltz 2000) have also been found to vary amongst individual enzyme molecules. In addition, catalytic rate has been found to vary for a given enzyme molecule over time (Lu, Sun, and Xie 1998;Edman et al 1999;English et al 2006;Craig and Nichols 2008).…”