Abstract
Echistatin is the smallest member of the disintegrin family of snake venom proteins, containing four disulfides in a peptide chain of 49 residues. Partial assignment of disulfides has been made previously by NMR and chemical approaches. A full assignment was made by a newly developed chemical approach, using partial reduction with trisβ(2βcarboxyethyl)βphosphine at acid pH. Reduction proceeded in a stepwise manner at pH 3, and the intermediates were isolated by high performance liquid chromatography. Alkylation of free thiols, followed by sequencer analysis, enabled all four bridges to be identified: (1) at 20 Β°C a single bridge linking Cys 2βCys 11 was broken, giving a relatively stable intermediate; (2) with further treatment at 41 Β°C the bridges Cys 7βCys 32 and Cys 8βCys 37 became accessible to the reagent and were reduced at approx. equal rates; (3) the two bicyclic peptides produced in this manner were less stable and could be reduced at 20 Β°C to a peptide that retains a single bridge linking Cys 20βCys 39; and (4) the monocyclic peptide can be reduced to the linear molecule at 20 Β°C. Some disulfide exchange occurred during alkylation of the bicyclic intermediates, but results unambiguously show the pattern to be [2β11; 7β32; 8β37; 20β39]. A comparison is made with kistrin, a longer disintegrin whose disulfide structure has been proposed from NMR analysis.