Immunochemical studies on cobrotoxin.

Xxomnuoehemical Studies on the Tryptophan-modified 'ýobrotoxin The two-dimensional structure of cobrotoxin has recently been established and permits a study of structure-activi~ty i-elatioziships. Preceding studies on the chemical modification of c:Dbrotoxin nuggested that either the intact Tyr-25, Lys-47, or* G'>1-2*: is essential for full activity of the toxin. The importance -,f the sin~le Trpresidue at position 29 for the lethality of cobrv'.oz:-. ut: ic.as several neuratoxins isolated from the venoms of sea szia.-e:: been reported# The immnmwodiffusion of these Trp-.nodif71,cT' t c-i r.! showed similar procipitin lines to those of native toxins~. . ever, an for inducing the production of antibodiez in aijý<. n immunization with these modified derivatives and thedci.i..,nchemical studies on these products have not yet been undertaken. 4 ~In this study, the single Trp-rosidue in cobrotoxin ha" been converted into NO-formyl kynurenine by ozonization, oxidized to ozindole derivative with N-bromosuccinimide, and also modified by reactions with 2-hydroxy-5-nitrobenzyl (RIND) bromide and 2-nitrophenyloulfenyl chloride. An important feature of the investigation is to determine whether the chemical modification would affect on the antigenic specificit:of the toxin. Each modified derivativc gave a s.Lngle fused precipitini line with cobrotoxin on ir~nmunodiffusion against either anti-cobrotoxin Or anti-HNE-cobrotoxin sera. In heterologous precipitin reactions, no significant ducreases in antigenic activity of the modified derivatives were ob:;,.-rvad when they reacted with anti-cobrotexin sera* In the production of antibodies in rabbits by immunization with these modified derivatives (except for ozonized toxin), we have obtained 1.3 times more potent antisera in oni:, one-half the time by using HNB-cobrotoxin instead of native toxin. Therefore, W4B-cobrotoxin is superior to native toxin for the production of antibody in animals. These results suggest that although the Trp-residue in cobrotoxin in essential for lethal toxicity, it may not be involved in ýhe antigenic specificity of the toxin.