Intramolecular perturbation of tryptophans induced by the protonation of ionizable groups in goat alpha-lactalbumin.

Goat alpha-lactalbumin shows two septral changes at acid pH. One of these corresponds to the acid denaturation, while the other has been regarded as a result of the intramolecular perturbation of tryptophans induced by the protonation of ionizable groups in the native molecule. In order to distinguish between these spectral changes and to investigate the perturbation caused by the ionizable groups, the absorption changes with a change in pH have been followed by the stopped-flow pH-jump method. Only the acid denaturation can be observed kinetically while the perturbation is too rapid to follow by the kinetic method. Extrapolations of the time-dependent absorption changes to zero time in the unfolding and refolding pH jumps give the apparent titration curves of the ionizable groups that participate in the perturbations in the native and in the acid-denatured molecule. The perturbation of the native protein is induced by the protonation of at least two ionizable groups, while the acid-denatured molecule does not show the perturbation because of the loss of the unique spatial arrangement of the ionizable groups around tryptophans. The results of circular dichroism measurements and the comparison with known results of the charge perturbation of lysozyme are also discussed.