title: Validation of a specific prolylcarboxypeptidase activity assay and its suitability for plasma and serum measurements.
abstract: Prolylcarboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, was discovered 45 years ago. However, research has been hampered by a lack of well-validated assays that are needed to measure low activities in biological samples. Two reversed-phase high-performance liquid chromatography (RP-HPLC) methods for quantifying PRCP activity in crude homogenates and plasma samples were optimized and validated. PRCP activity was determined by measuring the hydrolysis of N-benzyloxycarbonyl-l-proline (Z-Pro)-Phe. The enzymatically formed Z-Pro and Phe were measured independently under different HPLC conditions. The in-house methods showed good precision, linearity, accuracy, and specificity. Based on Michaelis-Menten constants, Z-Pro-Phe was chosen over Z-Pro-Ala as the substrate of preference. Cross-reactivity studies with dipeptidyl peptidases (DPPs) 2, 4, and 9 and prolyl oligopeptidase (PREP) confirmed the specificity of the PRCP activity assay. The average PRCP activity in plasma and serum of 32 healthy individuals was found to be 0.65 ± 0.02 and 0.72 ± 0.03 U/L, respectively. Both methods can be used to measure PRCP activity specifically in different biological samples and are well suited to evaluate PRCP inhibitors. These well-validated methods are valuable tools for studying PRCP's role in cardiovascular diseases, stroke, inflammation, and metabolic syndrome.
authors.affiliation: Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, 2610 Antwerp, Belgium.
authors.forename: Kaat
authors.initials: K
authors.lastname: Kehoe
authors.forename: Robert
authors.initials: R
authors.lastname: Verkerk
authors.forename: Yani
authors.initials: Y
authors.lastname: Sim
authors.forename: Yannick
authors.initials: Y
authors.lastname: Waumans
authors.forename: Pieter
authors.initials: P
authors.lastname: Van der Veken
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Lambeir
authors.forename: Ingrid
authors.initials: I
authors.lastname: De Meester
mesh_terms: D000818:Animals; D002268:Carboxypeptidases / Q000097:blood* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D011817:Rabbits; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
chemical_list: D002268:Carboxypeptidases; C022448:lysosomal Pro-X carboxypeptidase
keywords: Activity assays; Method validation; PRCP; Substrate affinity