PO41, a snake venom metalloproteinase inhibitor isolated from Philander opossum serum

PO41 was isolated from Philander opossum serum by DEAE-Sephacel, Phenyl Superose and Superdex 200 chromatographies and showed a molecular mass of 41,330 Da by MALDI-TOF MS. Molecular masses of 81.5 and 84.5 kDa were obtained by size exclusion chromatography and dynamic laser light scattering, respectively, suggesting that PO41 is dimeric. Its isoelectric point was estimated to be lower than 3.5. PO41 presented similar amino terminal sequence to those of DM40 and DM43, two antihaemorrhagins previously isolated from Didelphis marsupialis serum and was recognized by polyclonal antibodies raised against D. marsupialis antibothropic fraction. To study the inhibitory properties of this protein, the metalloproteinases bothrolysin and jararhagin were isolated from Bothrops jararaca venom by chromatographies on Superdex 200 and Phenyl Superose. Jararhagin was further submitted to a Mono Q column. The proteolytic and haemorrhagic effects of these haemorrhagins were neutralized by PO41. Both snake venom metalloproteinases formed stable complexes with PO41. The stoichiometry of the complex PO41-jararhagin was one inhibitor subunit to one molecule of the enzyme. These results show that PO41 has physicochemical, structural, immunoreactive and biological properties similar to other metalloproteinase inhibitors belonging to the supergene family of immunoglobulins.