Laboratório de Química e Função de Proteínas Bioativas, Departamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brasil.
PLoS One. 2013 May 28;8(5):e63571. doi: 10.1371/journal.pone.0063571. Print 2013.
Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30-60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10(-8) mol.L(-1) and constant inhibition (Ki) of 1.0×10(-8) mol.L(-1), by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation.
从豆科植物种子中分离出的肽酶抑制剂因其药理学特性而受到研究。本研究集中于从 Erythrina velutina 种子中纯化、生化表征、评估一种新型 Kunitz 胰蛋白酶抑制剂（EvTI）的抗炎和抗凝作用。胰蛋白酶抑制剂通过硫酸铵（30-60%）沉淀、分级、胰蛋白酶-Sepharose 亲和层析和反相高效液相色谱进行纯化。纯化的抑制剂表现出 19210.48 Da 的分子量。此外，还观察到第二个同工型，分子量为 19228.16 Da。表现出最高胰蛋白酶特异性和提高回收率的抑制剂命名为 EvTI（P2），并选择用于进一步分析。由胰蛋白酶和胃蛋白酶消化生成的 EvTI 肽片段，通过 MALDI-ToF-ToF 质谱进一步分析，允许部分初步结构阐明。EvTI 对胰蛋白酶表现出抑制活性，IC50 为 2.2×10(-8) mol.L(-1)，抑制常数（Ki）为 1.0×10(-8) mol.L(-1)，为非竞争性机制。除了抑制胰蛋白酶活性外，EvTI 还抑制因子 Xa 和中性粒细胞弹性蛋白酶，但不抑制凝血酶、糜蛋白酶或肽酶 3。研究了 EvTI 的抗炎和抗凝特性。首先，EvTI 对人外周血单个核细胞没有细胞毒性作用。然而，抑制剂能够通过体外和体内模型以剂量依赖的方式延长凝血时间。由于 EvTI 的抗炎和抗凝作用，本文在此挑战了两种脓毒症模型。EvTI 抑制白细胞迁移，并且特别通过抑制 TNF-α释放和刺激 IFN-α和 IL-12 合成起作用。所呈现的数据清楚地有助于更好地理解 Kunitz 抑制剂在败血症中的应用，作为一种能够干扰凝血和炎症的生物活性物质。
