Physiological changes in the albumin-bound non-esterified free fatty acids critically influence heme/bilirubin binding properties of the protein: A comparative, in vitro, spectroscopic study using the endogenous biomolecules.

Heme and bilirubin (BR), as by-products of red blood cells (and hemoglobin) degradation, show increased plasma concentrations in some diseases. These two toxic hydrophobic molecules are mainly transported in the blood-stream by human serum albumin (HSA) that carries a wide variety of ligands. Under normal physiological conditions, ~3 fatty acid (FA) molecules are bound to each HSA; and its possible effect on BR/heme binding remains to be more clarified. In the present study, to provide deeper insight on this issue, we purified albumin from healthy individuals (as purified non-defatted albumin or PA) with normal plasma levels of FA, then defatted some of the purified protein (as defatted-HSA; or DA). In the next step, using various spectroscopic methods, their interactions with heme and BR were investigated. By 1: 1 binding of the ligands, quenching and thermodynamic analysis of parameters indicated that binding constants (K) values of bilirubin and heme for PA and DA are different. It could be perceived that the presence of FAs in high-affinity FA binding sites (FABSs) exerted considerable conformational changes in the structure followed by an improved BR binding while hindered heme interaction. The data was confirmed by determining surface hydrophobicity of the purified albumin (PA) and DA, and then supported by bioinformatics analyses. The physiological and clinical relevance of the observed dynamic interactions is also discussed. This study, also, re-confirmed that the primary BR binding site is subdomain IIA not subdomain IB.