BmI, A SNAKE VENOM METALLOPROTEINASE INHIBITOR FROM Bothrops moojeni SERUM

Lopes, W.B.¹; Valente, R.H.¹; Neves-Ferreira, A.G.C.¹; Chapeaurouge, D.A.¹; Homsi-Brandeburgo, M.I.²; Hamaguchi, A.²; Domont, G.B.³; Perales, J.¹

1 Depto. de Fisiologia e Farmacodinâmica, IOC, FIOCRUZ; 2 INGEB, Lab. Química de Proteínas e Produtos Naturais, UFU; 3 Depto. de Bioquímica, IQ, UFRJ.

Many animals are known to possess natural resistance to the effects of snake venom metalloproteinases (SVMPs). This resistance can be attributed to neutralizing factors present in their blood sera. The aim of this work was to isolate a metalloproteinase inhibitor from Bothropsmoojeni serum. The serum was submitted to differential precipitation with ammonium sulphate (0-40; 40-60; 60-80 and >80% saturation). The fraction in the 40-60% saturation range was further fractionated using a hydrophobic interaction (phenyl) column and the peak of interest was applied to a reversed-phase (C4) high performance liquid chromatography (HPLC). Fraction II (named BMI) from HPLC run was submitted to a 12.5% SDS-PAGE under reducing conditions and a band with an approximate molecular mass of 55 kDa was stained. This band was excised and submitted to trypsin in gel digestion followed by analysis on a Voyager DE-PRO MALDI-TOF mass spectrometer (Applied Biosystems), a process called peptide mass fingerprinting. The peptide map obtained was submitted to a search on the latest NCBI database using the Protein Prospector software and a positive match was obtained, namely BJ46a (SVMP inhibitor isolated from Bothropsjararaca serum). In another experiment, the 55 kDa band was blotted to a PVDF membrane and submitted to Edman degradation automatic sequencer. The primary sequence identity (for the first 50 residues) was of 92 and 74% when compared to BJ46a and Habu Serum Factor (HSF- SVMP inhibitor from Trimeresurus flavoviridis serum), respectively. Hence, B. moojeni has a SVMP inhibitor (BMI) in its serum which, just like BJ46a and HSF, is presently classified as a member of the fetuin family, cystatin superfamily.

Supported by: CNPq, FAPERJ and PAPES-FIOCRUZ

Correspondence to: wbl@ioc.fiocruz.br