Purification and biochemical characterization of Eumiliin from Euphorbia milii var. hislopii latex

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.