Echistatin disulfide bridges: Selective reduction and linkage assignment

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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 tris-(2-carboxyethyl)-phosphine 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 °C a single bridge linking Cys 2-Cys 11 was broken, giving a relatively stable intermediate; (2) with further treatment at 41 °C the bridges Cys 7-Cys 32 and Cys 8-Cys 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 °C to a peptide that retains a single bridge linking Cys 20-Cys 39; and (4) the monocyclic peptide can be reduced to the linear molecule at 20 °C. Some disulfide exchange occurred during alkylation of the bicyclic intermediates, but results unambiguously show the pattern to be [2–11; 7–32; 8–37; 20–39]. A comparison is made with kistrin, a longer disintegrin whose disulfide structure has been proposed from NMR analysis.