Partially oxidized active intermediates in refolding of reduced ribonuclease

Abstract

The refolding of reduced ribonuclease A has been studied by measurements of enzymatic activity under conditions where the oxidation of thiol groups into disulfide bonds is rather slow. The sensitivity to a treatment by N-ethylmaleimide has been used to distinguish between partially and totally oxidized active species. It is found that the first active protein molecules to be formed do not have all of their disulfide bonds. Because they are active, these partially oxidized intermediates probably have very close to native conformation, which they can reach without being trapped in a wrong structure by forming too many incorrect disulfide bonds. The significance of these intermediates to the refolding pathway of reduced ribonuclease is discussed.