Highly sensitive assay for gamma-glutamyltranspeptidase activity by high-performance liquid chromatography with electrochemical detection.
A highly sensitive assay for gamma-glutamyltranspeptidase activity involving high-performance liquid chromatography (HPLC) with electrochemical detection was devised. gamma-Glutamyl-DOPA, a new synthetic dipeptide, which consists of naturally occurring amino acids, was found to be a good substrate for gamma-glutamyltranspeptidase purified from Proteus mirabilis. Enzymatically formed DOPA was adsorbed on an aluminium oxide column, eluted with 0.5 M hydrochloric acid and determined by HPLC with electrochemical detection. The sensitivity limit of this method was 0.5 pmol of DOPA formed. Some properties of gamma-glutamyltranspeptidase purified from P. mirabilis were investigated using gamma-glutamyl-DOPA as a substrate. In the presence of 0.15 M glycylglycine, the KM value of the enzyme for gamma-glutamyl-DOPA was 0.013 mM, and the maximum velocity was 247 nmol/min per mg protein. This method was applied to the assay of the enzymatic activity in human serum.
Kiuchi
K
K
Kiuchi
K
K
Nagatsu
T
T
Togari
A
A
Kumagai
H
H
D002851:Chromatography, High Pressure Liquid; D004295:Dihydroxyphenylalanine / Q000032:analysis; D004563:Electrochemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D011513:Proteus mirabilis / Q000201:enzymology; D005723:gamma-Glutamyltransferase / Q000032:analysis*
D004295:Dihydroxyphenylalanine; D005723:gamma-Glutamyltransferase