title: Limited proteolysis of fibrinogen by fibrinogenase from Echis multisquamatis venom.
abstract: Previously we purified fibrinogenase from venom of Echis multisquamatis and showed that the enzyme predominantly cleaves BβArg42-Ala43 peptide bond of fibrinogen. A much slower hydrolysis of its Aα-chain was also shown. To evaluate the accessibility of the hydrolysis sites to fibrinogenase's hydrolytic action, the pathway of cleavage of Aα- and Bβ-chains of fibrinogen, monomeric and polymeric fibrin desA and desAB has been investigated using western blot with monoclonal antibodies to Bβ 26-42 and Aα 20-78 of fibrinogen. The data indicated that the BβArg42-Ala43 peptide bond is available for cleavage in all forms of fibrin(ogen) with the exception of polymerized fibrin desAB. This is direct evidence of BβN-domain involvement in formation of protofibrils that makes it inaccessible to protease. The Aα-chain of fibrinogen remained intact after 3 min of incubation with fibrinogenase. Further incubation resulted in cleaving of the fibrin(ogen) αC-regions with the formation of two kinds of degradation products (~30 and ~60 kDa). In the case of monomeric fibrin desA or desAB we observed simultaneous hydrolysis of Aα and Bβ-chains and the cleavage of Aα-chain was more apparent for both forms of polymeric fibrin.
authors.affiliation: Protein Structure and Functions Department, Palladin Institute of Biochemistry, 9, Leontovych Str., Kiev, 01601, Ukraine, bio.cherv@gmail.com.
authors.forename: V O
authors.initials: VO
authors.lastname: Chernyshenko
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000737:chemistry*; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry*; D059748:Proteolysis; D057057:Serine Proteases / Q000302:isolation & purification / Q000494:pharmacology*; D014757:Viper Venoms / Q000201:enzymology*; D017819:Viperidae
chemical_list: C115499:BBeta fibrinogen; D010446:Peptide Fragments; D014757:Viper Venoms; C464794:fibrinogen Aalpha; D005340:Fibrinogen; D057057:Serine Proteases