TRANSITION-STATE IN THE FOLDING OF ALPHA-LACTALBUMIN PROBED BY THE 6-120 DISULFIDE BOND

The guanidine hydrochloride concentration dependence of the folding an d unfolding rate constants of a derivative of alpha-lactalbumin, in wh ich the 6-120 disulfide bond is selectively reduced and S-carboxymethy lated, was measured and compared with that of disulfide-intact alpha-l actalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models , the intermediate-controlled folding model and the multiple-pathway f olding model, that we had proposed previously. All of the data support ed the multiple-pathway folding model. Therefore, the molten globule s tate that accumulates at an early stage of folding of alpha-lactalbumi n is not an obligatory intermediate. The cleavage of the 6-120 disulfi de bond resulted in acceleration of unfolding without changing the ref olding rate, indicating that the loop closed by the 6-120 disulfide bo nd is unfolded in the transition state. It is theoretically shown that the chain entropy gain on removing the cross-link from a random coil chain with helical stretches can be comparable to that from an entirel y random chain. Therefore, the present result is not inconsistent with the known structure in the molten globule intermediate. Based on this result and other knowledge obtained so far, the structure in the tran sition state of the folding reaction of alpha-lactalbumin is discussed .