Thermal stabilization of human albumin by medium- and short-chain n-alkyl fatty acid anions.
A comprehensive study of the thermal stabilization of defatted human albumin monomer by n-alkyl fatty acid anions (FAAs), formate through n-decanoate, was carried out by differential scanning calorimetry (DSC). The concentration of each ligand affording maximum thermal stabilization was determined; n-nonanoate provides the greatest stabilization but is only marginally better than n-octanoate and n-decanoate. The use of reversible thermodynamics and a two-state denaturation model for albumin has been validated. Standard free energies of binding, calculated from increases in free energy of denaturation, for n-butanoate and longer FAAs, are linear with n-alkyl chain length whereas those for formate, acetate, and n-propionate deviate from linearity; those for acetate and n-propionate are even greater than that of n-butanoate, thereby suggesting, in addition to the common class of sites available to all such ligands, the presence of an additional class of lower affinity binding sites available only to these shortest ligands. Competition experiments involving acetate and n-octanoate and involving n-pentanoate and n-octanoate confirmed the binding of acetate to lower affinity sites unavailable to n-octanoate and n-pentanoate. Furthermore, an equation is provided, allowing computation of the transition temperature as a function of the free energy for any reversible process causing a change in thermal stability of a protein undergoing reversible, two-state denaturation. With this equation, modeling the competition experiments by using the binding parameters determined by DSC provides additional support for the class of lower affinity sites, which play a significant role in thermal stabilization of albumin at higher concentrations of these shortest FAAs.
Shrake
Andrew
A
Division of Hematology, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892, USA. shrake@cber.fda.gov
Frazier
Douglas
D
Schwarz
Frederick P
FP
D000838:Anions / Q000737:chemistry* / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D004355:Drug Stability; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D017510:Protein Folding; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
D000838:Anions; D005227:Fatty Acids; D012709:Serum Albumin