Kinetic study of gamma-glutamyltransferase activity by electrophoretically mediated microanalysis combined with micellar electrokinetic capillary chromatography.
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.
Laboratorium voor Farmaceutische Chemie en Analyse van Geneesmiddelen, Katholieke Universiteit Leuven, Leuven, Belgium.
Sigrid
S
Van Dyck
Ann
A
Van Schepdael
Jos
J
Hoogmartens
D000818:Animals; D002417:Cattle; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods; D005973:Glutamine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008832:Microchemistry; D012015:Reference Standards; D013379:Substrate Specificity; D013696:Temperature; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D005973:Glutamine; C001300:gamma-glutamine-4-nitroanilide; D005723:gamma-Glutamyltransferase