Tetrodotoxin-binding proteins isolated from five species of toxic gastropods

Abstract

Toxic gastropods Polinices didamy, Natica lineata, Oliva miniacea, O. mustelina and O. hirasei are tetrodotoxin (TTX) bearing animals, which accumulate TTX in their muscle and digestive gland. Analysis by gel filtration on Sepharose CL-6B revealed that 0.05 M NaCl extracts of the muscle of five species of toxic gastropods contained TTX-binding high molecular weight substances (HMWS) (1500–2000 kDa). The TTX-binding capacities of those HMWS were 0.12, 0.62, 0.45, 0.28 and 0.35 MU/mg protein, respectively, but those HMWS had no neutralising effect on TTX or paralytic shellfish poison. The HMWS of the five toxic gastropods could be hydrolyzed with HCl and protease at 37 °C, pH 7.4, but not with ribonuclease T2, deoxyribonuclease I or α-amylase. After purifying the TTX-binding protein of N. lineata by Q Fast-Flow strong anion exchanger and then BioSep-SEC-S 2000, the TTX-binding capacity increased to 3.5 MU/mg and 4.2 MU/mg protein, respectively. The TTX-binding capacity of N. lineata HMWS had no obvious seasonal variation. The molecular weight of TTX-binding protein of N. lineata was estimated to be about 434 kDa, while it comprised two subunits with molecular weights of about 272 kDa and 47 kDa, respectively, under SDS–PAGE.