Thermal stabilization of human albumin by medium‐ and short‐chain n‐alkyl fatty acid anions

Abstract

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.

Stability of Protein Pharmaceuticals: An Update

“…Ribonuclease has also been shown to bind two moles of chloride ion, resulting in significant structural stabilization (∼2-3 kJ/mol) (431). Stabilization of HSA has been observed from binding chloride (432) and carboxylates, such as formate and acetate (433).…”

Unfolding properties of recombinant human serum albumin products are due to bioprocessing steps

“…An average value of the primary transition from pooled plasma HSA samples (I–1, J, N, and O, all with fatty acids) gives a of T m1 = 68.2°C. Shrake et al also measured the T m1 and Δ H m of defatted HSA as 64.7°C and 242.9 kcal mol −1 , (1016 kJ mol −1 ) respectively …”

“…Previous DSC measurement by Shrake et al have demonstrated that the although HSA has a propensity to aggregate on denaturation, the unfolding transition itself is microscopically reversible with respect to the individual protein macromolecules . To analyze the DSC data, we used the Origin software package from the manufacturer to model the behavior of the heat capacity curves.…”

“…In a review of the current literature, we have found that even when keeping the variables such as pH, scan rate, and the presence or absence of fatty acids constant, there is considerable variation in the reported melting temperatures T m and enthalpy values of HSA (Table ) …”

Albumin-based nanoparticles as magnetic resonance contrast agents: II. Physicochemical characterisation of purified and standardised nanoparticles

“…Cleaning and stabilisation of HSA Albumin was prepared by the method of Chen (1967) and then stabilised by addition of octanoate according to Shrake et al (2005): a solution containing 50 mg/ml of human serum albumin in distilled water was prepared at room temperature. Then charcoal powder was added under continuous stirring (HSA:charcoal = 2:1 w/w) and the pH was adjusted to 3 with 1 M HCl.…”