Development of a sensitive and selective assay for the determination of procarboxypeptidase U (thrombin-activatable fibrinolysis inhibitor) in plasma.
To date, several assays for procarboxypeptidase U (proCPU) determination exist, all having their own inherent disadvantages and advantages. A drawback of activity-based assays is the interference of the constitutively active carboxypeptidase N (CPN) in plasma. Recent screening of Bz-Xaa-Arg peptides with modified aromatic amino acids at the P1 position revealed a selective CPU substrate, N-benzoyl-ortho-cyano-phenylalanyl-arginine (Bz-o-cyano-Phe-Arg), which will allow straightforward determination of proCPU in plasma. Our assay shows an excellent linearity in the concentration range of 20-2600 U/L, with within- and between-run precision values of 2.7% and 4.6%, respectively. A good correlation with our high-performance liquid chromatography (HPLC)-assisted proCPU activity assay using hippuryl-l-arginine (HipArg) as substrate was found. Besides the major improvement regarding the selectivity, the assay is much easier to perform and far less time-consuming compared with the proCPU activity assay using HipArg as substrate.
Heylen
Evelien
E
Laboratory of Medical Biochemistry, University of Antwerp, B-2610 Wilrijk, Belgium.
Van Goethem
Sebastiaan
S
Willemse
Johan
J
Olsson
Thomas
T
Augustyns
Koen
K
Hendriks
Dirk
D
D002138:Calibration; D025901:Carboxypeptidase B2 / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D001121:Lysine Carboxypeptidase / Q000097:blood; D012015:Reference Standards; D013379:Substrate Specificity
D025901:Carboxypeptidase B2; D001121:Lysine Carboxypeptidase