Kinetics of disulfide reduction in α-lactalbumin by dithiothreitol are investigated by measuring time-dependent changes in absorption at 310 nm and in CD ellipticity at 270 nm (pH 8.5 or 7.0, and 25 °C). When the disulfide-intact protein is folded, the kinetics are biphasic. The disulfide bond between the half-cystines-6 and - 120 is reduced in the fast phase, and the other three disulfide bonds are reduced in the slow phase. The apparent rate constants of the two phases are both proportional to the concentration of dithiothreitol, indicating that both phases are expressed by bimolecular reactions. However, detailed molecular mechanisms that determine the reaction rates are markedly different between the two phases. The slow phase shows a sigmoidal increase in the reaction rate with increasing concentration of a denaturant, urea, and is also accelerated by destabilization of the native state on removal of the bound Ca2+ ion in the protein. The disulfide bonds are apparently protected against the reducing agent in the native structure. The fast phase reaction rate is, however, decreased with an increase in the concentration of urea, and the disulfide bond shows extraordinary superreactivity in native conditions. It is 140 times more reactive than normal disulfides in the fully accessible state, and three-disulfide α-lactalbumin produced by the fast phase assumes nativelike structure under a strongly native condition. As ionic strength does not affect the superreactivity of this disulfide bond, electrostatic contributions to the reactivity must be negligible. Inspection of the disulfide bond geometry based on the refined X-ray coordinates of baboon α-lactalbumin [Acharya et al. (1989) J. Mol. Biol. 208, 99-127] and comparison of the geometry with those in five other proteins clearly demonstrate that the superreactivity arises from the geometric strain imposed on this disulfide bond by the native structure folding. Relationships of the disulfide strain energy to the protein stability and the disulfide reactivity are discussed.
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