Purification, characterization, and primary structure of four depressant insect-selective neurotoxin analogs from scorpion (Buthus sindicus) venom

Four depressant insect-selective neurotoxin analogs (termed Bs-dprIT1 to 4) from the venom of the scorpion Buthus sindicus were purified to homogeneit y in a single step using reverse-phase HPLC. The molecular masses of the pu rified toxins were 6820.9, 6892.4, 6714.7, and 6657.1 Da, respectively, as determined by mass spectrometry. These long-chain neurotoxins were potent a gainst insects with half lethal dose values of 67, 81, 103, and 78 ng/100 m g larva and 138, 160, 163, and 142 ng/100 mg cockroach, respectively, but w ere not lethal to mice even at the highest applied dose of 10 mug/20 g mous e. When injected into blowfly larvae (Sarcophoga falculata), Bs-dprIT1 to 4 induced classical manifestations of depressant toxins, i.e., a slow depres sant flaccid paralysis. The primary structures of Bs-dprIT 1 to 4 revealed high sequence homology (60-75%) with other depressant insect toxins isolate d from scorpion venoms. Despite the high sequence conservation, Bs-dprIT1 t o 4 showed some remarkable features such as (i) the presence of methionine (Met(6) in Bs-dprIT1 and Met(24) in Bs-dprIT2 to 4) and histidine (His(58) and His(57) in Bs-dprIT1) residues, i.e., amino acid residues that are unco mmon to this type of toxin; (ii) the substitution of two highly conserved t ryptophan residues (Trp43 --> Ala and Trp53 --> His) in the sequence of Bs- dprIT1; and (iii) the occurrence of more positively charged amino acid resi dues at the C-terminal end than in other depressant insect toxins. Multiple sequence alignment, sequence analysis, sequence-based structure prediction , and 3D homology modeling studies revealed a protein fold and secondary st ructural elements similar to those of other scorpion toxins affecting sodiu m channel activation. The electrostatic potential calculated on the surface of the predicted 3D model of Bs-dprIT1 revealed a significant positive pat ch in the region of the toxin that is supposed to bind to the sodium channe l. (C) 2001 Academic Press.