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Volume 272, Number 1, Issue of January 3, 1997 pp. 69-75
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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A Two-Stage Mechanism for the Reductive Unfolding of Disulfide-containing Proteins

(Received for publication, July 17, 1996, and in revised form, September 16, 1996)

From the Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel CH-4002, Switzerland

Reductive unfolding of disulfide-containing proteins can be experimentally dissected into two distinct stages. In the presence of denaturant and thiol catalyst, native proteins unfold by reshuffling their native disulfides and convert to a mixture of scrambled structures. Subsequent reduction of the disulfide bonds of scrambled proteins requires only mild concentration of reductant (0.2-0.5 mM reduced dithiothreitol) and undergoes intermediates that consist of highly heterogeneous disulfide isomers. These properties have been characterized with three cystine-containing proteins, namely hirudin, tick anticoagulant peptide (TAP), and bovine ribonuclease A. In the cases of hirudin and TAP, most intermediates observed during the oxidative folding were found to exist along the pathway of reductive unfolding as well.

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