Resolution of isoforms of mutalysin II, the metalloproteinase from bushmaster snake venom.
Mutalysin II, a zinc endopeptidase possessing direct-acting fibrinolytic activity has been previously purified from bushmaster (Lachesis muta muta) snake venom. We now report a method to isolate two isoforms of natural mutalysin II (mut IIa and mut IIb) using chromatographies on Sephacryl S-200, CM Sepharose CL 6B and Sephadex G-50. The two proteins are monomeric non-glycosylated proteinases with similar molecular masses of 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Both isoforms showed high similarity in all enzymatic properties using fibrinogen, fibrin and dimethylcasein as substrates. Thus, the specific fibrinolytic activity was estimated as 12+/-1.04 and 11.5+/-1.02 U/microg for mut IIa and mut IIb, respectively. The antigenic cross-reactivity of both isoforms was examined using rabbit hyperimmune serum or immunoglobulin G anti-mut IIa assays on immunodiffusion microscope slides, indirect enzyme-linked immunoabsorbent assay and western blots. From these experiments it was concluded that the two metalloproteinases mut IIa and mut IIb share identical antigenic structures. Since the stability of mutalysin II is dependent upon the presence of zinc, we examined the EDTA sensitivity of the isoforms of mutalysin II. Thus, the IC(50) values (concentration of EDTA to produce 50% inhibition of dimethylcasein hydrolysis) for mut IIa is 180 microM and 165 microM for mut IIb.
Sanchez
Eladio F
EF
Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, 30510-010, Belo Horizonte, MG, Brazil. eladio@funed.mg.gov.br
Souza
Christiane T
CT
Bello
Cynthia A
CA
Richardson
Michael
M
Oliveira
Eduardo B
EB
Magalhaes
Arinos
A
D000818:Animals; D002364:Caseins / Q000187:drug effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005337:Fibrin / Q000187:drug effects / Q000378:metabolism; D005340:Fibrinogen / Q000187:drug effects / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D010449:Peptide Mapping; D011817:Rabbits; D012910:Snake Venoms / Q000737:chemistry / Q000201:enzymology / Q000302:isolation & purification* / Q000378:metabolism*; D013057:Spectrum Analysis; D017819:Viperidae / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D002364:Caseins; D006023:Glycoproteins; D007527:Isoenzymes; C002087:N,N-dimethylcasein; D012910:Snake Venoms; C478150:mut IIa protein, Lachesis muta muta; C478151:mut IIb protein, Lachesis muta muta; D005337:Fibrin; D005340:Fibrinogen; D010447:Peptide Hydrolases; D008666:Metalloendopeptidases; C068795:hemorrhagic factor LHFII; D015032:Zinc