Caracterização funcional e estrutural de novas proteases isoladas da peçonha de Bothrops alternatus e do látex de Euphorbia milii var. hislopii

Abstract

CHAPTER II: A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatography 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 29814 kDa in MALDITOF/ 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 Bß-chain. Its fibrinogenolytic activity is inhibited by b-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. CHAPTER III: A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatography 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 29814 kDa in MALDITOF/ TOF mass spectrometry. Eumiliin has caseinolytic and fibrinogenolytic activities, but no hemorrhagic or defibrinating activities. The enzyme readily hydrolyzes the A-chain of fibrinogen and, more slowly, the B-chain. Its fibrinogenolytic activity is inhibited by b-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. CHAPTER IV: Bhalternin and Eumiliin are proteases extracted from the venom of Bothrops alternatus and latex of Euphorbia milli var. hislopii, respectively. The stabilities of Bhalternin and Eumiliin against denaturation by heat and urea were determined and compared. The plant protease proved to be tougher when heated at high temperatures and also when subjected to the action of urea as denaturant. Further studies are needed to better characterize the conditions for stabilization of these enzymes, especially those related to the strategies needed to protect the transport and storage in the commercial, therapeutic and biotechnological processes.