Protease-induced aggregation of bovine α-lactalbumin: Identification of the primary associating fragment

Abstract

Details in the hydrolysis of α-lactalbumin known to result in formation of highly ordered nanotubules, was investigated by incubation of solutions with 10 g α-lactalbumin/l with a specific protease from Bacillus licheniformis (BLP). After 50 min of incubation, soluble aggregates were formed, the concentration of which increased until precipitation occurred after 200 min. The latter aggregates were dissolved in urea or at low pH, like the nanotubules characteristic of gels formed by the action of BLP on α-lactalbumin at 100 g/l. On the molecular level, α-lactalbumin was initially cleaved into two large hydrophobic fragments with masses of 11·6 and 11·3 kDa, which in turn were cleaved in a stepwise manner into the ultimate fragment of 8·8 kDa. This fragment was the predominating component in the insoluble aggregates, and was identified as the sequences 26–37 and 50–113 of α-lactalbumin linked together by a disulphide bond. Cleavage of α-lactalbumin into this fragment probably created new hydrophobic surfaces and new calcium binding sites allowing its association into ordered structures.