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The protein Human Serum Albumin (HSA) is known to undergo conformational transitions towards partially unfolded forms triggered by acidification below pH 4.5. The extent of Fatty Acids (FA) binding has been thought to have an impact on the conformational equilibrium between the native and acid forms and to be a possible explanation for the observation of more than one band in early electrophoretic migration experiments at pH 4. We compared the acid-induced unfolding processes of commercial FA-free HSA, commercial "fatted" HSA and FA-HSA complexes, prepared at FA:HSA molar ratios between 1 and 6 by simple mixing and equilibration. We used a method for continuous acidification based on the hydrolysis of glucono-δ-lactone from pH 7 to pH 2.5, and followed the average protein changes by the blue shift of the intrinsic fluorescence emission and by performing a small angle X-ray scattering analysis on selected samples. The method also allowed for continuous monitoring of the increase of turbidity and laser light scattering of the protein samples related to the release of the insoluble ligands with acidification. Our results showed that the presence of FA interacting with albumin, an aspect often neglected in biophysical studies, affects the conformational response of the protein to acidification, and slightly shifts the loss of the native shape from pH 4.2 to pH 3.6. This effect increased with the FA:HSA molar ratio so that with three molar equivalents a saturation was reached, in agreement with the number of high-affinity binding sites reported for the FA. These findings confirm that a non-uniform level of ligand binding in an albumin sample can be an explanation for the early-observed conformational heterogeneity at pH 4., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The most abundant plasma protein, human serum albumin (HSA), is known to undergo several conformational transitions in an acidic environment. 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. The progressive acidification, based on the hydrolysis of glucono-δ-lactone from pH 7 to pH 2.5, highlighted a multistep 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. The effect of acid unfolding at this intermediate stage was assigned to the rearrangement of the three albumin domains drifting apart toward a more elongated conformation, with a partial unfolding of one of the outer domains. To test the stabilizing effect of fatty acids, 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.