Purification and biochemical characterization of Eumiliin from Euphorbia milii var. hislopii latex |
| |
Authors: | KC Fonseca MR Queiroz MS Gomes CCN Mamede N Penha-Silva HAN Canabrava |
| |
Institution: | a Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Uberlândia, MG, Brazil b Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, MG, Brazil c Instituto Federal de Educação, Ciências e Tecnologia do Triângulo Mineiro, Campus Ituiutaba, MG, Brazil d Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil e Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica (N-Biofar), Brazil |
| |
Abstract: | 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 Aα-chain of fibrinogen and, more slowly, the Bβ-chain. Its fibrinogenolytic activity is inhibited by β-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 °C; activity was completely lost at ?80 °C. Intraplantar injection of Eumiliin (1-25 μg/paw) caused a dose- and time-dependent hyperalgesia, which peaked 1-5 h after enzyme injection. Intraplantar injection of Eumiliin (1-25 μg/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. |
| |
Keywords: | Euphorbia milii Crown-of-Thorns Euphorbiaceae Cysteine protease Eumiliin |
本文献已被 ScienceDirect 等数据库收录! |
|