Purification and partial characterization of a new proteolytic enzyme from the venom of Bothrops moojeni (CAISSACA).
A basic serine protease which is active on casein and fibrinogen was purified from Bothrops moojeni venom using a single step chromatography on a CM-Sepharose fast flow column. The enzyme, MOO3, was not hemorrhagic and presented only a trace of blood-clotting activity. Synthetic chromogenic substrates (azoacasein and azoalbumin) where not hydrolyzed by MOO3. Using polyacrylamide gel electrophoresis at pH 4.3, MOO3 showed as a single protein band. Using sodium dodecyl sulfate-polyacrylamide electrophoresis, MOO3 behaved as a single-chain protein with an approximate mol. weight of 27,000, both in the presence and absence of beta-mercaptoethanol. Its pI was 7.8 by electrofocusing. The enzyme did not contain neutral carbohydrates and its N-terminal amino acid was alanine. The amino acid composition showed 249 residues/mole, a high content of hydrophilic amino acids and 14 half-cystine residues, which should account for 7 disulfide bonds. The protease cleaved the A-alpha chain faster than the B-beta of bovine fibrinogen and showed no effect on the delta-chain. Specific esterolytic activity of MOO3 on alpha-N-tosyl-l-arginine methyl ester was 29.64 mumol min-1 x mg-1. MOO3 represented 1.42% (w/w) of the initial desiccated venom. Its proteolytic activity was inhibited by beta-mercaptoethanol, leupeptin, phenylmethylsulphonyl fluoride and ethylenediamine tetraacetate.
Oliveira
F
F
Departamento de Ciências, Faculdade de Filosofia, Ciências e Letras, FAFI, Araguari-MG, Brazil.
Rodrigues
V M
VM
Borges
M H
MH
Soares
A M
AM
Hamaguchi
A
A
Giglio
J R
JR
Homsi-Brandeburgo
M I
MI
D000818:Animals; D017837:Bothrops / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012910:Snake Venoms / Q000201:enzymology*; D013379:Substrate Specificity
D002364:Caseins; D015842:Serine Proteinase Inhibitors; D012910:Snake Venoms; D005340:Fibrinogen; C120988:MOO3 enzyme; D012697:Serine Endopeptidases