Colorimetric coupled enzyme assay for gamma-glutamyltransferase activity using glutathione as substrate.
A colorimetric coupled enzyme assay for the determination of gamma-glutamyltransferase (GGT) activity using glutathione as substrate is described. The cysteine released from glutathione upon sequential action of GGT and leucine aminopeptidase is spectrophotometrically detected through its reaction with ninhydrin at 100 degrees C in acidic conditions. The method was applied to the determination of the activity of both bovine kidney and human serum GGT. In the described assay conditions with final GGT concentrations ranging from 0.18 to 4 mU/ml, a linear relationship between produced cysteine and incubation times up to 90 min was observed. When a standard chromogenic assay for GGT using L-gamma-glutamyl-3-carboxy-4-nitroanilide as substrate and the proposed assay were applied on the same serum sample a linear relationship between the two method was observed. Since the use of GSH as substrate, the proposed method can be usefully adopted for enzymological studies on GGT-related enzymes, a class of enzymes which is still waiting to be characterized.
Del Corso
Antonella
A
Dipartimento di Biologia, Sezione di Biochimica, Università di Pisa, via S. Zeno, 51, 56100 Pisa, Italy. delcorso@dfb.unipi.it
Cappiello
Mario
M
Buono
Francesca
F
Moschini
Roberta
R
Paolicchi
Aldo
A
Mura
Umberto
U
D000818:Animals; D002417:Cattle; D004796:Clinical Enzyme Tests / Q000379:methods*; D003124:Colorimetry / Q000379:methods*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D013223:Statistics as Topic; D005723:gamma-Glutamyltransferase / Q000032:analysis*
D005723:gamma-Glutamyltransferase; D005978:Glutathione