Purification and characterization of an anti-hemorrhagic protein from Naja naja (Indian cobra) venom. (15th December 2017)

• Record Type: Journal Article
• Authors: Suvilesh, K.N.; Yariswamy, M.; Savitha, M.N.; Joshi, Vikram; Nanjaraj Urs, A.N.; Urs, Amog P.; Choudhury, M.; Velmurugan, D.; Vishwanath, B.S.
• Is Part Of: Toxicon. Volume 140(2017)
• Page Start: 83
• Page End: 93
• Publication Date: 2017-12-15
• Subjects: Kunitz-type proteins -- Snake venom metalloproteases -- Extracellular matrix -- Hemorrhage -- Anti-hemorrhagic protein -- Peptide mass fingerprinting; SVMPs Snake venom metalloproteases -- ECM Extracellular matrix -- PMF peptide mass fingerprinting -- CK creatine kinase; Toxins -- Periodicals; Venom -- Periodicals; 615.9
• DOI: 10.1016/j.toxicon.2017.10.019
• Languages: English
• ISSNs: 0041-0101

Abstract

Abstract: Snake venom Kunitz-type proteins are well known to inhibit serine proteases but a few studies have also shown matrix metalloproteases (MMPs) inhibition. In view of the fact that MMPs and snake venom metalloproteases (SVMPs) have similar catalytic site, inhibition of SVMP activity by Kunitz-type proteins remains to be studied. Recent proteomic studies of Naja naja ( N. naja ) venom revealed the abundance of Kunitz-type proteins. In this regard, present study aimed at purification of a protease inhibitor from N. naja venom that inhibits the toxicity of SVMPs rich Echis carinatus ( E. carinatus ) venom. N. naja venom effectively inhibited E. carinatus venom-induced hemorrhage. Purification of the active principle responsible for anti-hemorrhagic effect was achieved by fractionation of N. naja venom in three successive chromatographic steps. SDS-PAGE revealed that purified anti-hemorrhagic protein (NNAh) has an apparent molecular mass of ∼44 kDa and single peak in RP-HPLC demonstrated its homogeneity. NNAh also inhibited myonecrosis induced by E. carinatus venom and reduced activity of creatine kinase in NNAh treated animal sera substantiated the anti-myonecrotic effect. Hemorrhage and myonecrosis inhibitory effects of NNAh were further supported by inhibition of E. carinatus venom-mediated gelatinolysis and collagenolysis. NNAh falls into the category of Kunitz-type serine protease inhibitor as determined by peptide mass fingerprinting and shown to be a strong inhibitor of chymotrypsin. Collectively our data signify that NNAh is a Kunitz-type chymotrypsin inhibitor which also inhibited metalloprotease activities of E. carinatus venom. In future, complete sequence of NNAh and peptide region(s) responsible for inhibition will assist to deduce the mechanism of action. Graphical abstract: Highlights: Echis carinatus venom-induces progressive tissue damage including hemorrhage and myonecrosis. Naja naja venom inhibited Echis carinatus venom-induced hemorrhage. Anti-hemorrhagic protein belongs to Kunitz-type protease inhibitor family and inhibited chymotrypsin activity. Anti-hemorrhagic protein also inhibited myonecrotic activity of Echis carinatus venom.