Unfolding and refolding of a type .kappa. immunoglobulin light chain and its variable and constant fragments

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Abstract
By limited proteolysis of a type .kappa. immunoglobulin light chain (Oku) with clostripain, both the VL and CL fragments were obtained with a high yield. The unfolding and refolding by guanidine hydrochloride of light chain Oku and its VL and CL fragments were studied at pH 7.5 and 25.degree. C with circular dichroism and tryptophyl fluorescence. The concentration of guanidine hydrochloride at the midpoint of the unfolding curve was 1.2 M for the VL fragment and 0.9 M for the CL fragment. As in the case of the CL fragment of light chain Nag (type .lambda.) [Goto, Y., and Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910], the unfolding and refolding kinetics of the CL fragment could be explained in principle on the basis of the three-species mechanism U1 .dblarw. U2 .dblarw. N, where N is native protein and U1 and U2 are the slow-folding and fast-folding species, respectively, of unfolded protein. The unfolding and refolding kinetics of the VL(Oku) fragment were described by a single exponential term. Double-jump experiments, however, showed that two forms of the unfolding molecule exist. The equilibrium and kinetics of unfolding of light chain Oku were explained by the sum of those of the VL and CL fragments. On the other hand, the refolding kinetics of light chain Oku were greatly different from the sum of those of the VL and CL fragments. The amplitude of the slow phase of the light chain was greater than that expected from the refolding kinetics of the isolated domains, and an additional slow phase that did not exist in the isolated domains appeared. These results indicate that while the unfolding and refolding of the light chain were approximately explained in terms of the independent folding of the two domains, some interactions exist between the domains during the kinetically determined refolding process.