SummaryThis paper describes a convenient oxidative refolding method that exploits the dual property of dimethylsulfoxide (DMSO) as an oxidant and a solvent 'chaperon' in assisting disulfide formation in peptides. Synthetic peptides with preferred secondary structures were used as models. For fl-sheet peptides, an active fragment of fibroblast growth factor containing the tetrapeptide Arg-Ser-Arg-Arg at a reverse turn was used. A disulfide scan consisting of 16 analogs is designed in which different pairs of amino acids on the antiparallel/3-sheets flanking the active determinants are replaced with cysteine. DMSO in aqueous buffer at pH 6 or 8 was found to minimize aggregation due to/3-sheet formation in all 16/3-stranded peptides and provided monocyclic disulfide peptides within 7 h. In contrast, air oxidation required a significantly longer duration for completion under similar conditions, and only 8 of 16 peptides formed intramolecular disulfides. Rate accelerations at pH 8 were found in exo-disulfide formation involving peptides with amino terminal cysteine, irrespective of oxidation conditions. The exo-disulfide effect in accelerating disulfide formation may be useful for regiospecific disulfide formation. For c~-helix peptides, the twodisulfide endothelin (ET) was used as a model. DMSO in combination with trifluoroethanol (TFE) was found to favor the desired bicyclic 1,4-disulfide bridged ET (1,4-ET) over the incorrectly folded 1,3-ET. Under aqueous conditions at pH 5-11, 1,4-ET to 1,3-ET was formed in the ratio of approximately 3:1, while the use of DMSO and TFE increased the ratio to 11:1. This solvent combination may stabilize an ol-helical stretch found in ET and contribute to enhanced selectivity. Thus, our results show that DMSO in disulfide formation in an aqueous or helix-promoting solution may serve as an oxidant and a 'chaperon' solvent system to provide regiospecificity for oxidative disulfide formation.

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