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从蚯蚓 Perionyx excavatus 中提取新型纤溶蛋白酶的纯化和特性分析及其作为潜在抗血栓药物的研究

Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavatus.

机构信息

Faculty of Food Processing Technology, Can Tho University of Technology, Can Tho, Vietnam.

出版信息

AMB Express. 2011 Sep 30;1(1):26. doi: 10.1186/2191-0855-1-26.

DOI:10.1186/2191-0855-1-26
PMID:21961566
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3210732/
Abstract

Six protease fractions, namely FI, FII, FIII-1, FIII-2, FIII-3 and FIV, were isolated from Perionyx excavatus earthworm biomass by acetone precipitation, followed by serial chromatography using anion exchange, hydrophobic interaction and size exclusion chromatography. All fractions exhibited strong hydrolytic activity towards casein. The activity of six fractions towards fibrin, determined by fibrin plate assay, ranged from 44 to 831 plasmin unit.mg-1 and ranked as FIII-3 > FIII-2 > FI > FIII-1 > FIV > FII. Casein degradation was optimal at pH 7 and 11, and at 45-60°C. All fractions were considerably stable at high temperature and wide pH range. They were completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The molecular weights (MW) and isoelectric points (pI) determined by 2D-electrophoresis were 27.5-34.5 kDa, and 4.3-5.2, respectively. Tandem mass spectrometry (MS) analysis was used to deduce the amino acid sequences of some peptides from FIII-1 and FIII-2. The sequences shared 16.9% and 13.2% similarity, respectively, with the fibrinolytic enzymes from two related earthworm species, Lumbricus rubellus and Eisenia fetida. The P. excavatus proteases were classified as serine proteases. They could perform rapid hydrolysis on both coagulated fibrous fibrin and soluble fibrinogen monomers without the presence of activators such as tPA or urokinase.

摘要

从环毛蚓（Perionyx excavatus）生物量中用丙酮沉淀分离出 6 种蛋白酶级分，即 FI、FII、FIII-1、FIII-2、FIII-3 和 FIV，然后通过阴离子交换、疏水相互作用和排阻色谱进行连续色谱分离。所有级分对酪蛋白均表现出很强的水解活性。通过纤维蛋白平板测定，6 个级分对纤维蛋白的活性范围为 44 至 831 纤溶单位.mg-1，活性顺序为 FIII-3>FIII-2>FI>FIII-1>FIV>FII。酪蛋白降解的最佳 pH 值为 7 和 11，最佳温度为 45-60°C。所有级分在高温和宽 pH 范围内都相当稳定。它们被苯甲基磺酰氟（PMSF）完全抑制。二维电泳法测定的分子量（MW）和等电点（pI）分别为 27.5-34.5 kDa 和 4.3-5.2。串联质谱（MS）分析用于从 FIII-1 和 FIII-2 推断一些肽的氨基酸序列。这两个序列分别与两种相关蚯蚓物种（Lumbricus rubellus 和 Eisenia fetida）的纤溶酶具有 16.9%和 13.2%的相似性。环毛蚓蛋白酶被归类为丝氨酸蛋白酶。它们可以在没有 tPA 或尿激酶等激活剂的情况下，快速水解凝固的纤维状纤维蛋白和可溶性纤维蛋白原单体。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aeb9/3210732/471ce5c67b9d/2191-0855-1-26-7.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aeb9/3210732/471ce5c67b9d/2191-0855-1-26-7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aeb9/3210732/f030705858cf/2191-0855-1-26-1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aeb9/3210732/24e448178d5d/2191-0855-1-26-2.jpg
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