Colorimetric determination of carboxypeptidase A activity in serum.
This simple, reproducible colorimetric method for determining the activity of carboxypeptidase A (EC 3.4.17.1) is based on measuring the absorbance at 505 nm of a quinoneimine dye produced from the action of this enzyme on the new substrate p-hydroxybenzoyl-glycyl-L-phenylalanine. The enzyme acts on the substrate to produce p-hydroxybenzoyl-glycine and L-phenylalanine. The former is then hydrolyzed by hippuricase (EC 3.5.1.14) to produce p-hydroxybenzoic acid and glycine. Finally, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine by sodium periodate forms a quinoneimine dye. The Km for the reaction with this substrate is 3.6 mmol/L; the optimum pH is 7.8. Our within-run and between-run CVs are 4.3% and 6.6%, respectively. The activity of carboxypeptidase A in serum correlates well with that of lipase (r = 0.96) and immunoreactive elastase-1 (r = 0.76).
Saruta
H
H
Ashihara
Y
Y
Sugiyama
M
M
Roth
M
M
Miyagawa
E
E
Kido
Y
Y
Kasahara
Y
Y
D000581:Amidohydrolases / Q000378:metabolism*; D002268:Carboxypeptidases / Q000097:blood*; D043422:Carboxypeptidases A; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D004151:Dipeptides / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D062385:Hydroxybenzoates / Q000378:metabolism; D007700:Kinetics; D008049:Lipase / Q000378:metabolism; D010226:Parabens
D004151:Dipeptides; D062385:Hydroxybenzoates; D010226:Parabens; C048692:4-hydroxybenzoylglycylphenylalanine; D008049:Lipase; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D000581:Amidohydrolases; C019672:aminoacylase I; C038193:4-hydroxybenzoic acid; D005998:Glycine