Structure of peptide fragments of a cross-linked complex of [Lys(Abz)26]neurotoxin II from Naja naja oxiana with the nicotinic acetylcholine receptor from Torpedo californica

After irradiation of a complex of the nicotinic acetylcholine receptor (AChR) with iodinated [Lys(Abz)26]neurotoxin II, the labeled δ-subunit of AChR was isolated, and it was cleaved with the aid of LysC endoproteinase, the hydrolysate being separated by rfHPLC. In a mass-spectrometric analysis of the radioactive fraction, the peptide of the δ-subunit (Mr 2593) was detected. By purification of the radioactive rfHPLC fraction with the aid of electrophoresis in tricine gel, three radioactive bands were obtained (M ∼ 16, 10, and 8 kDa). Edman degradation gave for all of them the sequence of a fragment of the δ-subunit beginning from Phe148. On further cleavage of the radioactive fraction within the gel by the action of AspN proteinase, followed by rfHPLC, the radioactive peak was eluted under conditions close to those for the elution of the single radioactive peptide 30-44 obtained by the successive cleavage of the [125I] neurotoxin II by LysC/AspN proteinases. This result shows the presence of the corresponding neurotoxin fragment in the sample in which the above-mentioned sequence of the receptor was detected. Since no sequences of the neurotoxin were detected in the radioactive products of the cross-linkages in model experiments at the picomolar level, neurotoxin II and its fragments were investigated by Edman degradation at the picomole level and so was the influence of the p-azidobenzyl group and its photoactivation on the degradation. On the whole, the sequencing of neurotoxin II and its fragments containing photolabeled and iodinated residues took place with extremely low initial yields; a further fall in the yields was observed on the degradation of irradiated Lys26-peptides. The results obtained explain the difficulties in the detection of the sequences of the neurotoxin in cross-linkage products available in amounts of only 10-20 pmole