12‐Channel Peltier array temperature control unit for single molecule enzymology studies using capillary electrophoresis

Abstract

Capillary electrophoresis has been used to demonstrate that individual molecules of a given enzyme support different catalytic rates. In order to determine how rate varies with temperature, and determine activation energies for individual β-galactosidase molecules, a 12-channel Peltier array temperature control device was constructed where the temperature of each cell was separately controlled. This array was used to control the temperature of the central 30 cm of a 50 cm long capillary, producing a temperature gradient along its length. Continuous flow single β-galactosidase molecule assays were performed allowing measurement of the catalytic rates at different temperatures. Arrhenius plots were produced and the distribution of activation energies for individual β-galactosidase molecules was found to be 56 ± 10 kJ/mol with a range of 34-72 kJ/mol.

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…Under the experimental conditions applied, the product DDAO migrated faster than the enzyme so that a zone of product formed in front of the enzyme resulting in a box-shaped electropherogram [95]. The methods were applied to the determination of the catalytic rate of enzymes [96] including mutants [97] as well as the activation energy of individual enzyme [98][99][100]. This is consistent with conformational differences between molecules being an underlying basis for enzyme heterogeneity.…”

“…This may be due to the fact that this mode typically does not produce sharp substance peaks but rather broad zones. Nonetheless, Craig and coworkers published a series of applications of continuous CE assays for the measurement of individual molecules of β-galactosidase using 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7yl)-β-d-galactopyranoside (DDAO-gal) as substrate [95][96][97][98][99][100][101][102]. In the so-called double static incubation assay, 50 cm, 5 μm id, fused-silica capillaries were filled with buffer containing the substrate and highly diluted enzyme, so that on average only 5-10 enzyme molecules were distributed throughout the capillary.…”

“…A chiral ligand exchange-CE method based on the coordination effect of Zn(II)-l-Leu complexes and β-CD was developed for the separation of Dns-labeled amino acids [30]. Baseline separations of Phe, Tyr, Val, Thr, Met and Asn were obtained in a 60/45 cm, 75 μm id, fused-silica capillary using a pH 8.2 background electrolyte composed of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM Zn(II), 6.0 mM l-Leu and 4.0 mM β-CD. The method was used to screen for inhibitors of tyrosinase using l-Tyr as substrate which was derivatized with dansyl chloride prior to CE analysis.…”