Effects of Selective Biotinylation on the Thermodynamic Stability of Human Serum Albumin

Abstract

Thermal denaturation and stability of two commercially available preparations of Human Serum Albumin (HSA), differing in their advertised level of purity, were investigated by differential scanning calorimetry (DSC). These protein samples were 99% pure HSA (termed HSA99) and 96% pure HSA (termed HSA96). According to the supplier, the 3% difference in purity between HSA96 and HSA99 is primarily attributed to the presence of globulins and fatty acids. Our primary aim was to investigate the utility of DSC in discerning changes in HSA that occur when the protein is specifically adducted, and determine how adduct formation manifests itself in HSA denaturation curves, or thermograms, measured by DSC. Effects of site specific covalent attachment of biotin (the adduct) on the thermodynamic stability of HSA were investigated. Each of the HSA preparations was modified by biotinylation targeting a single site, or multiple sites on the protein structure. Thermograms of both modified and unmodified HSA samples successfully demonstrated the ability of DSC to clearly discern the two HSA preparations and the presence or absence of covalent modifications. DSC thermogram analysis also provided thermodynamic characterization of the different HSA samples of the study, which provided insight into how the two forms of HSA respond to covalent modification with biotin. Consistent with published studies [1] HSA96, the preparation with contaminants that contain globulins and fatty acids seems to be comprised of two forms, HSA96-L and HSA96-H, with HSA96-L more stable than HSA99. The effect of multisite biotinylation is to stabilize HSA96-L and destabilize HSA96-H. Thermodynamic analysis suggests that the binding of ligands comprising the fatty acid and globulin-like contaminant contributes approximately 6.7 kcal/mol to the stability HSA96-L.