Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, 2610 Antwerp, Belgium.
Anal Biochem. 2013 Dec 15;443(2):232-9. doi: 10.1016/j.ab.2013.09.002. Epub 2013 Sep 10.
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.
脯氨酰羧肽酶（PRCP，EC 3.4.16.2）是一种溶酶体羧肽酶，于 45 年前被发现。然而，由于缺乏测量生物样本中低活性所需的经过充分验证的检测方法，研究一直受到阻碍。本文优化并验证了两种用于定量粗匀浆和血浆样品中 PRCP 活性的反相高效液相色谱（RP-HPLC）方法。PRCP 活性通过测量 N-苄氧羰基-L-脯氨酸（Z-Pro）-Phe 的水解来确定。在不同的 HPLC 条件下，分别独立测量酶促形成的 Z-Pro 和 Phe。内部方法显示出良好的精密度、线性、准确性和特异性。基于米氏常数，选择 Z-Pro-Phe 作为首选底物，而非 Z-Pro-Ala。与二肽基肽酶（DPPs）2、4 和 9 以及脯氨酰寡肽酶（PREP）的交叉反应研究证实了 PRCP 活性测定的特异性。在 32 名健康个体的血浆和血清中，平均 PRCP 活性分别为 0.65±0.02 和 0.72±0.03 U/L。这两种方法都可用于特异性测量不同生物样本中的 PRCP 活性，非常适合评估 PRCP 抑制剂。这些经过充分验证的方法是研究 PRCP 在心血管疾病、中风、炎症和代谢综合征中的作用的有价值工具。
