Synthesis and disulfide structure determination of agelenin: identification of the carboxy-terminus as an amide form.

Agelenin, a blocker of the presynaptic calcium channel, is a 35-amino acid peptide having six cysteinyl residues recently isolated from the venom of the spider Agelena opulenta. However, it has not been confirmed whether the carboxy-terminus of this peptide is a free acid or amide. To elucidate this, we synthesized both peptides by a solid phase procedure, and compared their elution profiles with that of the natural product on RP-HPLC. The retention time on HPLC as well as the biological activity of the synthetic peptide-amide was found to be identical with those of natural agelenin, confirming that the carboxy-terminus of agelenin is amidated. The disulfide structure of agelenin was also determined to be linked between 3-19, 10-24 and 18-34, by comparing the tryptic peptide linked by two disulfide bonds with those synthesized by the selective formation of disulfide bonds.