Investigation of binding of fatty acids to serum albumin to determine free concentrations: Experimental and in-silico approaches

The binding characteristics of fatty acids (FA) to human serum albumin (HSA) have been garnering increased attention due to the importance of FAs in numerous in clinical and biological fields. In this study, we investigate the binding characteristics of two long-chain FAs — linoleic acid (LA; FA 18:2, ω-6) and alpha-linoleic acid (aLA; FA 18:3, ω-3) —studying their binding isotherms to HAS using solid-phase microextraction (SPME) probes. The binding isotherms showed that ligand saturation occurred at B ≈ 7 for both ligands, which indicates that there are at least seven specific binding sites available in HSA for FAs. The most notable feature of the binding isotherm graph was the presence of only one set of specific binding sites – we ignored nonspecific binding interaction because it is not important for these ligands. A Scatchard plot was employed to understand how these binding sites interacted with each other, both positively and negatively, and the apparent affinity of these ligands was estimated by their Hill's coefficients (KaLA = 7.7 × 105 L mol−1 and KLA = 2.1 × 106 L mol−1 for aLA and LA ligands, respectively) – with similar values as those values previously published in the literature, establishing the feasibility of SPME as an appropriate technique for binding studies. The experimental data is supported by mathematical models that were developed using the experimental data and COMSOL Multiphysics software. An in-silico comparison of the SPME extraction kinetics for aLA in an HSA binding matrix confirmed the suitability of mathematical models and provided insight into the microextraction mechanism in complex samples.