Human serum albumin (HSA), the most abundant plasma protein, plays a vital role in transporting endogenous and exogenous substances. While the interaction of drug molecules with albumin has been extensively studied, the complex interplay between drug formulation components and HSA remains largely unknown. As a result, many binding sites are still uncharacterized and competitive binding of endogenous ligands and excipients is still poorly understood. Gaining deeper insights into these interactions is crucial for comprehensively understanding the fate of innovative drug delivery systems. Protein binding in the context of drug-target interactions is often highly specific. Binding to HSA by contrast is largely driven by hydrophobic interactions. Molecules with limited aqueous solubility—including lipids, fatty acids, and detergents—readily bind to HSA. As multiple molecules can occupy different binding sites simultaneously, interpreting experimental data is challenging. To address this, we established an isothermal titration calorimetry (ITC) method to characterize the parameters that govern binding to HSA at different temperatures. This approach enables the differentiation of binding sites or states, as well as additional processes such as surfactant demicellization upon dilution, based on their characteristic enthalpic contributions. Due to the strong temperature dependence of hydrophobic interactions—manifested as a large negative heat capacity change (Δ C p )—certain binding processes are detectable at one temperature but effectively “calorimetrically silent” (isoenthalpic) at another. A fit using four parameters—stoichiometry ( n ), binding constant ( K ), enthalpy (Δ H ), and heat capacity change (Δ C p )—greatly enhances the resolution of overlapping equilibria and provides a robust framework for investigating pharmaceutically relevant systems.
Hübschen et al. (Sun,) studied this question.