Capture Reagent and Strategy for Retrieving Albumin-Bound Ligands from Plasma

A capture strategy is described and demonstrated for retrieving ligand entities in plasma that bind Human Serum Albumin. The method has applications for both exogenous and endogenous ligands. Exogenous ligands include drug candidates, performance enhancing drugs and toxic nerve agents that also interact quite strongly with HSA. Endogenous ligands are natural circulating compounds whose abundance corresponds to normal hemostasis or elevated levels that could be disease-specific molecular biomarkers. Melting curves of plasma solutions measured by differential scanning calorimetry produce “so-called” plasma thermograms that are physical signatures of the plasma solution. Patterns displayed by thermograms can be sensitive indicators of the presence of abnormal levels of exogenous and endogenous ligand components. Effects of ligand interactions on thermodynamic stability of proteins in plasma that they bind, primarily HSA, manifest on the plasma thermogram. The capture strategy is demonstrated for HSA binding in plasma of four “ideal” ligands of different types. The particular ligands were naproxen, bromocresol green, short double stranded and single strand DNA. Thermogram shapes and features were sensitive to the presence of ligands as thermograms of mixtures of plasma and HSA with these ligands were significantly different than thermograms of plasma or HSA alone. These results demonstrated directly that significant perturbations of plasma thermograms corresponded to ligand interactions with HSA in plasma.

Thermodynamic contributions of 5’- and 3’-single strand dangling-ends to the stability of short duplex DNAs

Differential scanning calorimetry (DSC) melting analysis was performed on 27 short double stranded DNA duplexes containing 15 to 25 base pairs. Experimental duplexes were divided into two categories containing either two 5’ dangling-ends or one 5’ and one 3’ dangling-end. Duplex regions were incrementally reduced from 25 to 15 base pairs with a concurrent increase in length of dangling-ends from 1 to 10 bases. Blunt-ended duplexes from 15 to 25 base pairs served as controls. An additional set of molecules containing 21 base pair duplexes and a single four base dangling-end were also examined. DSC melting curves were measured in varying concentrations of sodium ion (Na+). From these measurements, thermodynamic parameters for 5’ and 3’ dangling-ends were evaluated as a function of dangling end length. 5’ ends were found to be slightly stabilizing but essentially constant while the 3’ ends were destabilizing with increasing length of the dangling-end. 3’ ends also display a stronger dependence on Na+ concentration. In lower Na+ environment, the 3’ ends were more destabilizing than in higher salt environment suggesting a more significant electrostatic component of the destabilizing interactions. Analysis of thermodynamic parameters of dangling ended duplexes as a function of Na+ concentration indicated the 3' dangling ends behave differently than 5' dangling ended and blunt-ended duplexes. Molecules with one 5' and one 3' dangling end showed variation in excess specific heat capacity (ΔCp) when compared to the blunt-ended molecule, while the molecules with two 5’ ends had ΔCp values that were essentially the same as blunt-ended duplexes. These observations suggested differences exist in duplexes with 3’ and 5’ dangling ends, which are interpreted in terms of composite differences in interactions with Na+, solvent, and terminal base pairs of the duplex.

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

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.