Serum albumin ligand binding volumes using high pressure denaturation

The pressure shift assay (PSA, also termed either PressureFluor or differential pressure fluorimetry) was used to study the thermodynamics of decanoate and dodecanoate lipid binding to human serum albumin (HSA) in the temperature range from 25 degrees C to 80 degrees C and the pressure range from 0.1 MPa to 400 MPa. The ligands stabilized HSA against both pressure and temperature denaturation. The P-T phase diagram for HSA bound to saturated fatty acids is shown. Pressure induced HSA denaturation reversibility is demonstrated via either intrinsic tryptophan or extrinsic probe 1,8-anilinonaphthalene sulfonate (ANS) fluorescence. The effect of guanidinium in a PSA was studied. PSA provides information on ligand binding volumes. The volume changes from protein-ligand binding are thermodynamically important and could be used in designing compounds with specific volumetric binding properties. (C) 2011 Elsevier Ltd. All rights reserved.