Kinetic study of γ-glutamyltransferase activity by electrophoretically mediated microanalysis combined with micellar electrokinetic capillary chromatography

Abstract

The use of capillary electrophoresis for the determination of gamma-glutamyltransferase (GGT) activity with gamma-glutamyl-p-nitroanilide (Glu-p-NA) as a substrate was investigated. The reaction velocity was quantified spectrophotometrically by the corrected peak area of the product p-nitroaniline (pNA) at 380 nm. Micelles composed of sodium deoxycholic acid were used in the background electrolyte in order to obtain a baseline separation between the substrate and the product. The presence of the micelles did not influence the enzymatic reaction. The electrophoretic system was used, not only for the separation and quantitation of the different reaction compounds but also for the in-capillary mixing of the enzyme and substrate plugs. This methodology is known as electrophoretically mediated microanalysis (EMMA). With the developed in-capillary activity assay an average Michaelis constant (K(M)) for GGT was calculated to be 2.09 mM (RSD = 7.3%, n = 3), a value consistent with previously reported values.

Microscale separation methods for enzyme kinetics assays

“…These are summarized in Table 1. Sodium dodecylsulfate (SDS) is a typically used micelle-forming agent, although cetyltrimethylammonium bromide (CTAB) [59][60][61][62], sodium cholate [24,63,64], and deoxycholate [56,65,66] have also been used. Use of other additives, for example sodium octanesulfonate [67], Tween 20 [26] or Brij 35 [68], has been reported occasionally.…”

“…Enzymatic reaction must, therefore, be performed in a different buffer and the reaction mixture cannot be in contact with the background electrolyte (BGE) until the reaction is complete. Occasionally the enzyme is compatible with MEKC separation buffer and classical EMMA formats could be used [24,26,65]. Van Dyck et al [23] solved the problem of incompatibility of amine oxidase with SDS by introducing the so called "partially filled capillary" concept.…”

Electrophoretically mediated microanalysis

“…In this variant, enzyme and substrate(s) are introduced into the capillary as distinct plugs, the first reactant injected being the one with the lower electrophoretic mobility [5,[26][27][28][29][30][31][32][33][34][35][36][37][38][39]. Upon the application of an electric field, the two zones interpenetrate due to differences in their electrophoretic mobilities.…”

Separation methods applicable to the evaluation of enzyme–inhibitor and enzyme–substrate interactions

“…Upon application of a current, the rapidly-migrating substrate zone enters the sluggish enzyme zone, which initiates the enzymatic reaction. The internal standard, enzyme, substrate, and newly formed product then migrate along the column (reproduced from [117]). toxins, produced by cyanobacteria, are considered a significant world wide public health threat [103].…”

“…Enzyme-substrate reactions are model systems for analysis using EMMA [109][110][111][112][113][114][115][116][117][118][119]. For example, the Glatz group has used EMMA to analyze the activity of rhodanese, the enzyme which catalyzes the reaction of thiosulfate with cyanide to form thiocyanate and sulfite [120][121][122].…”