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The cDNA encoding a long neurotoxin homolog was constructed from the cellular RNA isolated fom the venom glands of Naja naja atra (Taiwan cobra) by reverse transcription-polymerase chain reaction. BLAST searches for sequence similarity in the GenBank databases reveal that the cDNA sequence of the long neurotoxin homolog is not highly homologous with long and short neurotoxins. Although the long neurotoxin homolog exhibited an activity to inhibit acetylcholine-induced muscle contractions as Naja naja atra cobrotoxin, the degree of inhibition caused by the addition of long neurotoxin homolog was only approximately 35% of that observed with the addition of cobrotoxin. Moreover, the primary structure of the long neurotoxin homolog did not fulfill the characteristic features of long or short neurotoxins. Together with long neurotoxin homologs from other snake species, they probably represent an evolutionary divergence between long and short neurotoxins.

The effect of 2,2,2-Trifluoroethanol (TFE) on the structure of five all beta-sheet proteins, isolated from the venom of the Taiwan cobra (Naja naja atra), is studied. In all the toxins used, it is observed that significant amount of alpha-helix is induced at higher concentrations of TFE. In all these proteins, the induction of helical conformation and disruption of the tertiary structure seem to occur simultaneously. The structural transitions induced by TFE in reduced and denatured protein appear to be different from those observed in the native protein(s). In our opinion, the findings reported herein could have significant implications on research in the area of protein folding.

In order to identify the structural determinants of cobrotoxin that caused its anomalous mobility in sodium dodecyl sulfate (SDS) gel, comparative studies on the capacities of cobrotoxin and its modified derivatives for binding with SDS were made. Cobrotoxin migrates faster in SDS gel after modification of Lys residues or carboxylated groups. However, the capacities of modified derivatives for binding with SDS molecules did not show a positive correlation with their electrophoretic mobilities. Moreover, it was found that the segment at positions 22-38 had the greatest capacity to bind with SDS than other segments of cobrotoxin, but guanidination of Lys residues of this peptide fragment did not significantly change its migration. These results suggest that the SDS-binding capacity of cobrotoxin is not solely responsible for its anomalous mobility in SDS gel, and that the effects may arise from the electrostatic interactions between Lys residue and carboxylated groups.

In contrast to observations made with S. aureus V8 protease-digest hydrolysates, the antigenic structures of reduced and S-carboxymethylated (RCM)-cobrotixin were notably affected following hydrolysis of RCM-cobrotoxin with chymotrypsin. The peptide separated from the V8 protease-digest hydrolysates with a sequence at positions 22-38 of cobrotoxin exhibited a nearly equal reactivity toward the anti-RCM-cobrotoxin antibodies as RCM-cobrotoxin. Chymotryptic cleavage on this segment caused a precipitous drop in the antigenicity of RCM-cobrotoxin. Alternatively, the N-terminal and C-terminal regions of RCM-cobrotoxin encompassed other antigenic determinants which exhibited low reactivities toward anti-RCM-cobrotoxin antibodies. The epitope structures of RCM-cobrotoxin are in line with those predicted from the hydrophobicity profile of cobrotoxin, but the notably immunoreactive region in the C-terminal region of native toxin molecule (Ref. 1) cannot be predicted from analysis of its primary structure. Moreover, RCM-cobrotoxin had a superior reactivity toward anti-RCM-cobrotoxin antibodies than cobrotoxin did. These results indicate that the epitope structures in RCM-cobrotoxin and cobrotoxin are different.

2D NMR-derived spatial structures of neurotoxin II (NtII) and several homologous toxins in solution were assessed by comparison with their own amino acid sequences using a three-dimensional (3D) profile method. 3D profiles of all the toxin models match the sequences well and, therefore, the method of 3D profile was demonstrated to work correctly for these well-resolved NMR structures in aqueous solution. At the same time, the profile window plots reveal low scores in the bottom tip of loop II (residues 22-34 in NtII) and in beta-strand of loop III (residues 49-52). Some residues in the first poor-scoring region are of functional importance being involved in binding with nicotinic acetylcholine receptor (AChR). Furthermore, the second segment participates in intermolecular hydrogen bonding upon dimerization of postsynaptic neurotoxins in solution resulting in increasing of the 3D-1D score for residues at the interface between monomers. Therefore, the 3D profile method can be useful for detection functionally-important regions in well-resolved protein structures.

Antibodies which specifically reacted with reduced and S-carboxy-methylated(RCM) cobrotoxin were purified from anticobrotoxin antisera and anti-RCM-cobrotoxin antisera, respectively. Results using a competitive immunoassay revealed that the antibodies from anti-RCM-cobrotoxin antisera had a greater affinity for RCM-cobrotoxin than for cobrotoxin. Whereas the reactivity of the antibodies from the anticobrotoxin antisera toward cobrotoxin and RCM-cobrotoxin had a reversed order of binding. In contrast to observations made with S. aureus V8 protease-digest hydrolysates, the antigenic structures of RCM-cobrotoxin recognized by antibodies from anti-RCM-cobrotoxin antisera were notably affected following hydrolysis of RCM-cobrotoxin with chymotrypsin. Moreover, the chymotryptic hydrolysates showed a comparable reactivity as RCM-cobrotoxin toward the antibodies purified from anticobrotoxin antibodies, but a decrease in antigenicity with the V8 protease hydrolysates. These results reveal that the repertoire of antibodies against the unfolded cobrotoxin are not the same in anticobrotoxin and anti-RCM-cobrotoxin antisera. Moreover, it suggests that the repertoire of antibodies from the different sources against the same antigen can be differentiated by measurement of their reactivities with the proteolytic hydrolysates of the antigen.

The coating of the wells of microtiter plates with cobrotoxin in Tris (pH 9.8) or PBS (pH 7.2) buffer was assessed by enzyme-linked immunoassay (ELISA). It was found that the poor binding in neutral buffer was improved by adding glutaraldehyde (GA), and the bound amount reached the same extent as that measured with alkali buffer. The optimal concentration of GA was approx. 0.02%. A decrease in optical density was observed with GA concentration higher than 0.02%. This may result from the modification of cobrotoxin by GA, which induced a decrease in the antigenicity of the cobrotoxin as revealed by competitive immunoassay. This study implies that the coating of physiological samples with GA for ELISA may be carried out with physiological buffers without the need to change the buffer to one of alkaline pH.

The 3D solution structure of alpha-cobratoxin, a neurotoxin purified from the Naja naja siamensis snake venom, has been determined by Nuclear Magnetic Resonance spectroscopy, in conjunction with distance geometry and restrained molecular dynamics, at pH 7.5. A total of 490 distance restraints were obtained from NOE intensities and 25 phi dihedral angle restraints deduced from J-coupling data. The generated structures are well defined with root mean square deviations from a geometrical mean structure of 0.107 +/- 0.036 nm for the backbone atoms and 0.128 +/- 0.073 nm for the side-chain atoms (considering residues 1 to 66 minus 26 to 35). A comparison between the generated structures at pH 7.5 and the mean NMR solution structure at pH 3.2 revealed that the 3D structure of alpha-cobratoxin is more compact at neutral pH. This major difference is mainly due to the pH-dependent conformational variations of three residues His18, Thr44 and Thr59.

Acrylamide quenching studies indicated that the exposure degree of the Trp residue in cobrotoxin was higher than that in alpha-bungarotoxin. The Trp residue of cobrotoxin was in a positively charged environment as revealed by iodide quenching, while the Trp residue of alpha-bungarotoxin was not accessible for iodide. Analysis of hydrophilicity profile and local concentration of positively charged residues of toxin molecule also indicated that Trp in cobrotoxin was in a highly hydrophilic and positively charged environment. Measurement of Trp fluorescence with increasing temperature showed that the stability of environment of Trp in alpha-bungarotoxin was higher than in cobrotoxin. Result of competitive binding for nicotinic acetylcholine receptor (AchR) between cobrotoxin and alpha-bungarotoxin revealed that the molecular interaction of the two toxins with AchR was not the same. These, together with the fact that the cationic groups of the two toxins are involved in the binding with AchR, suggest that the observed different environment surrounded Trp residue and different AchR binding mechanism might fulfill a different requirement of the invariant Trp in the lethality of cobrotoxin and alpha-bungarotoxin.