Purification and biochemical characterization of Eumiliin from Euphorbia milii var. hislopii latex
Purification and biochemical characterization of Eumiliin from Euphorbia milii var. hislopii latex
复制标题
DOI：
10.1016/j.phytochem.2010.02.009
复制
发表时间：
2010-05-01
期刊：
影响因子：
3.8
通讯作者：
Oliveira, F.
中科院分区：
文献类型：
--
作者：
Fonseca, K. C.;Morais, N. C. G.;Oliveira, F.
本文报道了从小米大戟（Euphorbia milii var.）通过使用DEAE-Sephacel的离子交换色谱步骤和使用Sephadex G-75的凝胶过滤的组合来分离hislopii。Eumiliin是一种单体蛋白，通过还原条件下的SDS-PAGE测定其表观分子量为30 kDa，并在MALDI-TOF/TOF质谱中在29，814 KDa处产生一个主峰。Eumilin具有溶解酪蛋白和纤维蛋白原的活性，但没有出血或去凝血活性。该酶容易水解纤维蛋白原的A α链，而更慢地水解B β链。其纤维蛋白原溶解活性被β-巯基乙醇和亮抑酶肽抑制。EDTA和苯甲脒对Eumiliin的活性无明显影响。Eumiliin的酪蛋白分解活性的最适pH为8.0，并且在高达40 ℃的溶液中是稳定的;在>= 80 ℃时活性完全丧失。足底注射Eumiliin（1-25 μ g/爪）引起剂量和时间依赖性痛觉过敏，在酶注射后1-5小时达到峰值。足底内注射Eumiliin（1-25 μ g/爪）也引起致水肿反应，在1小时后达到最大。形态学分析表明，Eumiliin诱导了强烈的肌坏死，注射后24小时可见白细胞浸润和受损的肌细胞。(C)2010爱思唯尔有限公司版权所有。
A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatographic steps using DEAE-Sephacel and gel-filtration with Sephadex G-75. Eumiliin is a monomeric protein with an apparent molecular mass of 30 kDa by SDS-PAGE under reducing conditions and gave one main peak at 29,814 KDa in MALDI-TOF/TOF mass spectrometry. Eumiliin has caseinolytic and fibrinogenolytic activities, but no hemorrhagic or defibrinating activities. The enzyme readily hydrolyzes the A alpha-chain of fibrinogen and, more slowly, the B beta-chain. Its fibrinogenolytic activity is inhibited by beta-mercaptoethanol and leupeptin. In contrast, EDTA and benzamidine did not affect the activity of Eumiliin. The caseinolytic activity of Eumiliin had a pH optimum of 8.0 and was stable in solution at up to 40 degrees C; activity was completely lost at >= 80 degrees C. Intraplantar injection of Eumiliin (1-25 mu g/paw) caused a dose- and time-dependent hyperalgesia, which peaked 1-5 h after enzyme injection. Intraplantar injection of Eumiliin (1-25 mu g/paw) also caused an oedematogenic response that was maximal after 1 h. Morphological analyses indicated that Eumiliin induced an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 h after injection. (C) 2010 Elsevier Ltd. All rights reserved.