Modulations in the self-assembly of bovine serum albumin by enhanced depolymerisation and condensation induced upon stirring

Abstract

An unusual phenomenon in the aggregation profile of BSA in the presence of CTAB, brought about by stirring, is reported here. Stirring the solution resulted in an apparent modification of the monophasic aggregation pathway into a biphasic one, and consequently delayed the formation of aggregates, while the extent of aggregation still remained the same. Later, it was found that the second growth phase was a consequence of the spontaneous ceasing of bead stirring due to the formation of larger aggregates; thus leading to the conclusion that stirring hindered the growth of aggregates. Interestingly, the monomers in solution were depleted only at very long times, much after the turbidity measurements showed saturation. Thus, the aggregation did not really come to a halt; rather, a temporary equilibrium was followed by a slower monomer depletion, leading to the formation of aggregates which could not be detected by turbidimetric studies. We conclude that the aggregation of BSA in the presence of CTAB proceeds via a series of oligomerization, and the growth in turbidity is detected once the oligomers reach a certain size, large enough to become insoluble. The temporary halt is actually brought about by the condensation of aggregates predominating the aggregation pathway beyond a certain aggregate concentration. This study shows that stirring does not affect the lag phase of the aggregation, as is found for many proteins, but affects the aggregation by promoting the condensation of larger aggregates. The rapid approach to the condensation process ultimately leads to lesser incorporation of the monomers into the aggregates, thus reducing the aggregation. The enhanced condensation in the stirred reaction presents a temporary halt in the aggregation much before that in the unstirred reaction. Once the factor of enhanced diffusion brought about by stirring is eliminated, the aggregation resumes, although much more sluggishly than before the halt, and again reaches a saturation once a certain concentration of aggregates are formed.