Fast homogeneous assay for plasma procarboxypeptidase U.
Carboxypeptidase U (EC 3.4.17.20, CPU, TAFIa) is a novel determinant of the fibrinolytic rate. It circulates as an inactive zymogen, procarboxypeptidase U, which becomes active during the process of coagulation. We developed a high throughput method on microtiter plates for the determination of the procarboxypeptidase U concentration in human plasma samples. Following activation of procarboxypeptidase U by thrombin-thrombomodulin, the resulting enzyme activity cleaves p-OH-Hip-Arg and the generated p-OH-hippuric acid is converted by hippuricase to p-hydroxybenzoic acid and glycine. Finally, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine by NaIO4 forms the quinoneimine dye. The absorbance of the latter dye is determined at 506 nm in a microtiter plate reader. A mean value of 620 U/l was found, with a CV of 3.0% within-run and 4.3% between-run. The assay showed a good correlation with the activities observed using a HPLC assay as reference method (n = 25, r = 0.979). The presented method enables the routine analysis of large sample pools in clinical setting.
Schatteman
K A
KA
Department of Medical Biochemistry, University of Antwerp, Wilrijk, Belgium.
Goossens
F J
FJ
Leurs
J
J
Kasahara
Y
Y
Scharpé
S S
SS
Hendriks
D F
DF
D000328:Adult; D000368:Aged; D000581:Amidohydrolases / Q000378:metabolism; D025901:Carboxypeptidase B2 / Q000097:blood*; D004789:Enzyme Activation; D005260:Female; D005342:Fibrinolysis; D006626:Hippurates / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013917:Thrombin; D018180:Thrombomodulin
D006626:Hippurates; D018180:Thrombomodulin; D025901:Carboxypeptidase B2; D013917:Thrombin; D000581:Amidohydrolases; C019672:aminoacylase I; C030514:hippuric acid