Two distinct phases characterize thermal aggregation of beta-lactoglobulin at neutral pH.

Bovine beta-lactoglobulin( BLG) has been abundantly studied as model-protein. Moreover, it is one of the major components of the whey of cow’s milk. Aggregation and gelation processes have been characterized in different experimental conditions. At neutral pH, the protein assumes a dimeric structure and is characterized by nine antiparallel beta-strands and one short and one long alfa-helix at the carboxyl terminus. It is well known that BLG thermal aggregation at neutral pH occurs by the formation of intermediate oligomers. We report a study of thermal (60 °C) aggregation of BLG at pH 7 in the absence and presence of copper and zinc ions. A joint application of different techniques was used. The growth of the aggregates was followed by using a particle sizer, whereas the changes in the secondary and tertiary structures were monitored by IR, Raman and fluorescence spectroscopies, as done for other proteins. Also AFM measurements were carried out in order to monitor the aggregates morphology. In this respect, the presence of oligomeric and fibrillar aggregates was revealed. Our work highlights the existence of two phases characterizing the aggregation pathway of BLG. The first phase is dependently on the aggregative state of the protein: its time increased at lower protein concentration. Moreover, it is characterized by the formation of intermolecular beta-sheets and by simultaneous occurring of H-D exchange, usually probe of the partial protein unfolding. In the second phase, when H-D exchange is ended, modifications in the protein tertiary structure take place, as revealed by the quenching and simultaneous increase of Trp and Tyr fluorescence, respectively. Our data support the idea that BLG aggregation during the first phase occurs with formation of stable and metastable oligomers. The latters may evolve towards fibril formation. This phenomenon is accompanied by a slow process of consolidation and tertiary structural arrangement. The presence of metals promotes the formation of fibrillar aggregates. On the contrary, they do not markedly affect the time of the distinct phases, but slightly modify their evolution.