Accessible Requires Authentication Published by De Gruyter June 1, 2005

Fast Homogeneous Assay for Plasma Procarboxypeptidase U

Katinka A. Schatteman, Filip J. Goossens, Judith Leurs, Yasushi Kasahara, Simon S. Scharpé and Dirk F. Hendriks
From the journal

Abstract

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.

:
Published Online: 2005-06-01
Published in Print: 2001-09-07

Copyright © 2001 by Walter de Gruyter GmbH & Co. KG