Chemical synthesis of a neurotoxic polypeptide from the sea anemone Stichodactyla helianthus.
The sea anemone Stichodactyla helianthus neurotoxin I, a 48-residue polypeptide, was synthesized by automated solid phase methodology. The fully reduced polypeptide was subsequently refolded in the presence of a glutathione oxidoreduction buffer to the biologically active species containing three disulfide bonds. The overall yield after rigorous purification was 12.5%. The circular dichroism (CD), and proton nuclear magnetic resonance (1H NMR) spectra of the HPLC-purified synthetic toxin were indistinguishable from those obtained concurrently with the natural toxin. A subtilisin digest of the synthetic neurotoxin generated peptide fragments identical to that of a sample of the natural toxin subjected to the same treatment. The toxicity of the synthetic polypeptide was identical to that of the natural toxin (crab LD50, 3.1 micrograms/kg). The equilibrium dissociation constant (28 nM) for interaction of the synthetic toxin with crab axolemma vesicles was nearly identical to that of the natural toxin (25 nM).
Pennington
M W
MW
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville.
Kem
W R
WR
Norton
R S
RS
Dunn
B M
BM
D000595:Amino Acid Sequence; D000818:Animals; D003386:Brachyura; D002942:Circular Dichroism; D003064:Cnidarian Venoms / Q000138:chemical synthesis* / Q000633:toxicity; D004220:Disulfides / Q000032:analysis; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D009498:Neurotoxins / Q000138:chemical synthesis* / Q000633:toxicity; D010449:Peptide Mapping; D011487:Protein Conformation; D012615:Sea Anemones
D003064:Cnidarian Venoms; D004220:Disulfides; D007202:Indicators and Reagents; D009498:Neurotoxins; C059713:neurotoxin I, Stichodactyla helianthus