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

Abstract

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.

Fungicide Tebuconazole Influences the Structure of Human Serum Albumin Molecule

“…Faroongsarng [30] studied assessment of the dissociation energies of diazepam and ibuprofen bound on HSA by DSC method and found that the denaturation of HSA incubated with the drugs was done at higher temperatures than HSA itself depending on drugs′ concentrations. Similar findings were demonstrated by other authors [31,32]. The thermograms of free HSA and HSA (fixed concentrations) in the presence of different TB concentrations are shown in Figure S1.…”

Thermal analysis of protein stability and ligand binding in complex media

“…Samples were prepared by adding specific reaction components to pre-prepared media. For DSC melting experiments, the sample heating rate was approximately 1 °C/min while monitoring changes in the excess heat (microwatts) of the sample versus temperature [6][7][8]. In their primary form, melting curves or thermograms are displayed as plots of changes in microwatts measured for the sample, versus temperature.…”

Structural stability of Human serum albumin is modified in rheumatoid arthritis

“…We found an increase in the melting temperature for a small fraction (~10%) of the HSA pool could explain the observed HDC shift (Supplemental Figure 3). Changes in HSA melting temperature could result from new ligand binding, protein interactors, or tertiary structure (48)(49)(50). Here, we tested for structural changes of HSA through analysis of covalently modified amino acid profiles between the RA and non-RA samples.…”