Human Serum Albumin (HSA) is the most abundant protein in body fluids, where it serves as a carrier and depot for many molecules that can interact with the multiple binding sites located in its three-domain structure. The protein is also known to undergo conformational transitions towards partially unfolded forms triggered by acidification below pH 4.5. However, the potential loss of affinity for the ligands related to such structural variations has not be thoroughly elucidated so far. We propose to use a time-dependent pH scanning protocol to monitor the process of acid unfolding of complexes of HSA with specific spectroscopically active ligands, to verify the possible release phenomena determined by the protein conformational variations. Clarifying the interplay between the protein conformational transitions in acid conditions and the ligand binding/release equilibria could pave the way to the use of albumin as a specific pH-dependent carrier in nanotechnological formulations.
Studies on albumin-drug complexes are very numerous in the literature, but the possible impact of the acid-induced conformational transition of this protein on the variation of the binding affinity is a scarcely investigated question. In particular, the time-dependent acidification method based on GdL hydrolysis kinetics [32] we propose to apply has a notable novelty content in the context of protein unfolding. It also allows for a rather simple experimental implementation which could be easily extended to several albumin-ligand systems.
The first objective is to provide a proof-of-principle for the direct monitoring of the ligand release upon the albumin transition towards the acid conformers. We expect that after clarification of the basic aspects of this phenomenon at the molecular level in solution conditions, the method could be applied to formulate pH-dependent drug encapsulation and release protocols using albumin as a safe and versatile macromolecular carrier.[33,34]
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