Unfolding and refolding of the reduced constant fragment of the immunoglobulin light chain: Kinetic role of the intrachain disulfide bond

Abstract

The constant fragment of the immunoglobulin light chain whose intrachain disulfide bond is reduced (reduced C L fragment) assumes a conformation very similar to the intact C L fragment (Goto & Hamaguchi, 1979). The kinetics of reversible unfolding and refolding of the reduced C L fragment by guanidine hydrochloride at pH 7.5 and 25 °C were studied and were compared with those of the intact C L fragment described in the accompanying paper (Goto & Hamaguchi, 1982). Tryptophyl fluorescence, far-ultraviolet circular dichroism, and reactivity of the SH groups toward 5,5′-dithiobis-(2-nitrobenzoic acid) were used to follow the kinetics. The results obtained were thoroughly explained on the basis of the three-species mechanism, U 1 ▪ U 2 ▪ N, where U 1 and U 2 are slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The rate constants of interconversion between U 1 and U 2 and the rate constant for the process from N to U 2 were very similar to the respective values for the intact C L fragment. Only the rate constant for the process from U 2 to N was greatly different between the intact and reduced C L fragments; the rate constant for the reduced C L fragment was about 100 times smaller than that for the intact C L fragment. These results indicated that the slow isomerization of the unfolded molecule is independent of the presence of the disulfide bond and that the kinetic role of the intrachain disulfide bond is to accelerate the folding process. This kinetic role in the folding of the C L fragment was explainable only in terms of the decreased entropy in the unfolded state of the intact C L fragment due to the presence of the disulfide bond.