Ohanin, a novel protein from king cobra venom, induces hypolocomotion and hyperalgesia in mice.
We have identified, purified, and determined the complete amino acid sequence of a novel protein, ohanin from Ophiophagus hannah (king cobra) venom. It is a small protein containing 107 amino acid residues with a molecular mass of 11951.47 +/- 0.67 Da as assessed by electrospray ionization-mass spectrometry. It does not show similarity to any known families of snake venom proteins and hence is the first member of a new family of snake venom proteins. It shows similarity to PRY and SPRY domain proteins. It is nontoxic up to 10 mg/kg when injected intraperitoneally in mice. Ohanin produced statistically significant and dose-dependent hypolocomotion in mice. In a pain threshold assay, it showed dose-dependent hyperalgesic effect. The ability of the protein to elicit a response at greatly reduced doses when injected intracerebroventricularly as compared with intraperitoneal administration in both the locomotion and hot plate experiments strongly suggests that ohanin acts on the central nervous system. Since the natural abundance of the protein in the venom is low (approximately 1 mg/g), a synthetic gene was constructed and expressed. The recombinant protein, which was obtained in the insoluble fraction in Escherichia coli, was purified under denaturing condition and was refolded. Recombinant ohanin is structurally and functionally similar to native protein as determined by circular dichroism and hot plate assay, suggesting that it will be useful in future structure-function relationship studies.
Pung
Yuh Fen
YF
Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117543.
Wong
Peter T H
PT
Kumar
Prakash P
PP
Hodgson
Wayne C
WC
Kini
R Manjunatha
RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002490:Central Nervous System / Q000187:drug effects*; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004546:Elapid Venoms / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006930:Hyperalgesia / Q000139:chemically induced*; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009068:Movement / Q000187:drug effects*; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D004546:Elapid Venoms; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C500360:ohanin protein, Ophiophagus hannah; D014357:Trypsin