Substrate specificity of two thrombin like enzymes (elegaxobin, elegaxobin II) from the venom of Trimeresurus elegans (Sakishima-habu), using neutralizing antibody

We reported previously that elegaxobin and elegaxobin II cause the release of fibrinopeptide A alone from rabbit fibrinogen. Elegaxobin II is a thrombin-like enzyme that possesses kinin-releasing activity, and shows a characterization obviously different from elegaxobin. To study the substrate specificities of elegaxobin and elegaxobin II against fibrinogen, we investigated whether anti-elegaxobin or anti-elegaxobin II antibody counteracts those enzymatic activities. Anti-elegaxobin II antibody effectively counteracted the thrombin-like activity of elegaxobin and elegaxobin II in comparison to the anti-elegaxobin antibody. The anti-elegaxobin II antibody only counteracted the thrombin-like activity, whereas did not counteract TAME hydrolytic activity and weakly counteract the kinin-releasing activity. Furthermore, we investigated which part of elegaxobin II as an antigen this antibody connects with specifically, using M-peptides derived from digests with CNBr. As a result, anti-elegaxobin II antibody bound specifically to the M-peptide including C-terminal portion. In particular, the anti-elegaxobin II antibody connected to position 220–239 (AQPHEPGSYTNVFDHLDWIK), containing His223.