Conformational stability of α-lactalbumin missing a peptide bond between Asp⁶⁶ and Pro⁶⁷

Abstract

The peptide bond between Asp⁶⁶-Pro67 of α-lactalbumin was cleaved with formic acid (cleavedα-lactalbumin). Secondary structural changes of the cleavedα-lactalbumin, in which the two separated polypeptides were joined by disulfide bridges, were examined in solutions of sodium dodecyl sulfate (SDS), urea, and guanidine hydrochloride. The structural changes of the cleavedα-lactalbumin were compared with those of the intact protein. The relative proportions of secondary structures were determined by curve fitting of the circular dichroism spectrum. The cleavedα-lactalbumin contained 29%α-helical structure as against 34% for the intact protein. Some helices of the cleavedα-lactalbumin which had been disrupted by the cleavage appeared to be reformed upon the addition of SDS of very low concentration (0.5mM). In the SDS solution, the helicities of both the intact and cleaved proteins increased, attaining 44% at 4mM SDS. On the other hand, the helical structures of the cleavedα-lactalbumin began to be disrupted at low concentrations of guanidine hydrochloride and urea compared with that of the intact protein. However, no diffrence was observed in the thermal denaturations of the intact and cleaved proteins, except for the difference in the original helicities. The helicities of both proteins decreased with an increase of temperature up to 65°C and recovered upon cooling.