Amino acid sequence and chemical modification of a novel alpha-neurotoxin (Oh-5) from king cobra (Ophiophagus hannah) venom.
A novel alpha-neurotoxin, Oh-5, was isolated from king cobra (Ophiophagus hannah) venom and purified by successive SP-Sephadex C-25 column chromatography and reversed-phase HPLC. The complete sequence of Oh-5 was determined by Edman degradation of peptide fragments generated by endopeptidases, i.e., trypsin, Saccharomyces aureus V8 protease and lysyl endopeptidase. This novel toxin comprises 72 amino acid residues with 10 cysteines. The sequence shows 89% sequence homology with Oh-4, and 60% with Toxins a and b from the same venom. The tyrosine, tryptophan, lysine and arginine residues in Oh-5 were modified with tetranitromethane (TNM), 2-nitrophenylsulfenyl (NPS) chloride, trinitrobenzene sulfonate (TNBS), and p-hydroxyphenylglyoxal (HPG), respectively. Modification of Tyr-4 or Trp-27 did not affect the lethal toxicity at all, while the Tyr-4 and 23 nitrated derivative retained about 50% of the lethality of native toxin. Selective trinitrophenylation of Lys-51 or 69 resulted in a decrease in lethality by 29%, and 50% lethality was retained after modification of Lys-2, 51, and 69. A drastic decrease in lethality to 26% was observed when both Arg-35 and 37 were modified. The neurotoxicity was further decreased when Arg-9 was additionally modified. These results suggest that the aromatic residues, Tyr-4 and Trp-27, are not crucial for the neurotoxicity, whereas the cationic residues are involved in multipoint contact between the toxin molecule and the nicotinic acetylcholine receptor (nAChR). The residues Tyr-23 and Arg-35 and 37 in the central loop of Oh-5 seem to contribute greatly to the neurotoxicity.
Lin
S R
SR
School of Chemistry, Kaohsiung Medical College, Taiwan.
Leu
L F
LF
Chang
L S
LS
Chang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry* / Q000633:toxicity*; D007928:Lethal Dose 50; D051379:Mice; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D003039:Cobra Neurotoxin Proteins; C106861:Oh-5 alpha-neurotoxin; D001120:Arginine