Properties and cDNA cloning of an antihemorrhagic factor (HSF) purified from the serum of Trimeresurus flavoviridis

Habu serum factor (HSF) is a metalloproteinase inhibitor that is isolated from the serum of habu snake (Trimeresurus flavoviridis), and it can suppress snake venom-induced hemorrhage. In the present study, the inhibitory property and fundamental structure of HSF were analyzed in detail. HSF inhibited all the hemorrhagic and most of the non-hemorrhagic metalloproteinases tested from the venoms of T. flavoviridis and Gloydius halys brevicaudus. HSF was extremely stable in a broad range of temperature and pH, and the treatments with a temperature of 100 °C or pH ranging from 1 to 13 barely affects its reactivity against G. halys brevicaudus H6 protease. Gel filtration chromatography revealed that HSF binds to the H6 protease with a 1:1 molar ratio. A secondary structure profile of HSF that was monitored by circular dichroism spectrum remained unvaried up to 2 M urea. The activity of HSF was stoichiometrically abolished by chemical modification with 2,4,6-trinitrobenzene sulfonic acid and N-bromosuccinimide; this indicates that Lys and Trp residues in its sequence play a role in the inhibitory mechanism. In this study, the amino acid sequence of HSF that was obtained by cDNA cloning was identical to that reported previously, except for five substitutions. We concluded that these discrepancies reflect a difference in the places of capture of the snake specimens.