Time-dependent pH scanning of the acid-induced unfolding of Human Serum Albumin

The most abundant plasma protein, Human Serum Albumin (HSA), is known to undergo conformational transitions in acidic environment [1]. To avoid buffer effects and correlate global and local structural changes, we developed a continuous acidification method and simultaneously monitored the protein changes by both small-angle scattering (SAXS) and fluorescence [2], using a dedicated instrumental platform [3]. The progressive acidification, based on the hydrolysis of glucono--lactone from pH 7 to pH 2.5, highlighted a multi-step unfolding involving the putative F form (pH 4) and an extended and flexible conformation (pH < 3.5). The scattering profile of the F form was extracted by component analysis and further 3D modeled, suggesting the rearrangement of the three albumin domains in a more elongated conformation, with a partial unfolding of one of the outer domains at this intermediate stage. To test the stabilizing effect of fatty acids [4], here palmitic acid, we compared the acid unfolding process of albumin with and without ligand. We found that when binding the ligand the native conformation was favored up to lower pH values. Our approach solved the problem of realizing a continuous, homogeneous and tunable acidification with simultaneous characterization applicable to study processes triggered by a pH decrease.