Coagulant thrombin-like enzymes from the venoms of Brazilian and Peruvian bushmaster (Lachesis muta muta) snakes.
Two isoforms of a thrombin-like enzyme designated TLE-B and TLE-P were purified from the venoms of Lachesis muta muta (bushmaster) snakes captured in two different geographical localities, Manaus (Brazil) and Pucallpa (Perú). TLE-B and TLE-P showed Mr values of 44000 and 43000 under reducing conditions on SDS-PAGE, which decreased to 27000 after deglycosylation with N-glycosidase F (PNGase F). The purified proteinases split off fibrinopeptide A rapidly from human fibrinogen and fibrinopeptide B more slowly. In addition, both enzymes released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Their specific clotting activities were equivalent to 1000 and 900 NIH thrombin units/mg on human fibrinogen and 526 and 606 NIH thrombin units/mg on bovine fibrinogen for TLE-B and TLE-P, respectively. Kinetic properties of these enzymes were determined using representative chromogenic substrates. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Purified rabbit IgG against TLE-B reacted with both enzymes forming a continuous precipitin line on immunodiffusion. Furthermore, Western blot and indirect ELISA were used to compare the antigenic cross-reactivity for both enzymes as well as the venoms of L. muta muta and Bothrops snakes. Incubation of human alpha2-macroglobulin (alpha2-M) with each enzyme at molar ratios of 1:1, 1:2 and 1:4 enzyme:inhibitor resulted in retarding their clotting activities by approximately 12 times, whereas their amidolytic activities were not affected. However, the Mr 180000 subunits of alpha2-M were not cleaved by these enzymes, suggesting that alpha2-M inhibits TLEs by steric hindrance. Similarly, inhibitions of their clotting activities were obtained using high concentrations of rabbit IgG (40 microg, corresponding to molar ratio enzyme:inhibitor of 1:2) against TLE-B.
Magalhaes
Arinos
A
Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG 30510-010, Brazil.
Ferreira
Rodrigo N
RN
Richardson
Michael
M
Gontijo
Silea
S
Yarleque
Armando
A
Magalhaes
Henrique P B
HP
Bloch
Carlos
C
Sanchez
Eladio F
EF
D000818:Animals; D001938:Brazil; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003429:Cross Reactions; D003435:Crotalid Venoms / Q000201:enzymology* / Q000276:immunology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010568:Peru; D011817:Rabbits; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D017819:Viperidae; D000511:alpha-Macroglobulins / Q000494:pharmacology
D003435:Crotalid Venoms; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C072644:Lachesis venom; D010446:Peptide Fragments; D000511:alpha-Macroglobulins; D005340:Fibrinogen; D014357:Trypsin; D013917:Thrombin; D008666:Metalloendopeptidases; C068795:hemorrhagic factor LHFII