Fluorometric Assay of Serum γ-glutamyltransferase in Solution and on a Semi-Solid Surface

Abstract

We describe an enzymatic, fluorometric method for measuring the activity of serum γ-gIutamyItransferase (EC 2.3.2.2) in solution and on silicone rubber pads. N-γ-L-Glutamyl-α-naphthylamide in tris(hydroxymethyl)aminomethane buffer is used as substrate. In solution, measurements are performed at 37 °C with use of a reaction volume of 3.0 ml, in Pyrex cuvets. Measurements on silicone rubber pads are also made at 37 °C, after establishing a stable substrate film by lyophilizing the reagent for the γ-glutamyltransferase assay on the surface of the pads. Only 10-50 µl of buffered substrate and serum containing the enzyme to make a total volume of 60 µl are necessary for each assay. The rate of appearance of α-naphthylamine fluorescence (λex = 344 nm; λem = 445 nm), liberated from N-γ-L-glutamyl-α-naphthylamide by the enzymatic action of γ-glutamyltransferase, is measured and equated to its activity in serum. Calibration plots of the change in fluorescence per min vs. enzyme concentration for measurements in solution and on pads show a good proportionality in the range of 26.5-265 U/liter, and indicate the usefulness of these methods.