Kinetic assay of gamma-glutamyltransferase with use of bilirubin oxidase as a coupled enzyme.

We studied the kinetic measurement of gamma-glutamyltransferase (EC.2.3.2.2), coupling the reaction with that catalyzed by bilirubin oxidase (EC 1.3.3.5), which oxidizes and combines a phenylenediamine derivative with an aniline derivative to produce a green pigment. We measured the formation of the pigment kinetically (at lambda max745 nm, epsilon = 75 000 L mol-1 cm-1), with L-gamma-glutamyl-N-hydroxyethylaminoanilide as substrate and N-ethyl-N-hydroxy-3-sulfopropyl)-m-toluidine as a the coupling derivative. The within-run CV for measuring this reaction in samples of normal sera was 2.4%. A calibration plot of the change in absorbance per minute vs enzyme activity concentration showed good proportionality in the range of 0-1300 U/L. The results of this assay correlated well (r = 0.995) with those of the Boehringer method, in which L-gamma-glutamyl-3-carboxy-4-nitroanilide is the substrate. This new, highly sensitive procedure may be adapted to other assays involving phenylenediamine derivates as synthetic substrates.