FABP3 versus FABP7: Quantitative evidence that heart-type FABP is a superior molecular selector for DHA over EPA

Fatty acid-binding proteins 3 and 7 (FABP3 and FABP7) shuttle long- chain fatty acids inside cells, yet their relative selectivity for the ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have not been defined quantitatively. Here we express human FABP3 and FABP7 in Escherichia coli and purify them to >95% homogeneity by Strep-tactin XT affinity and size-exclusion chromatography. Biolayer interferometry (BLI) shows that each isoform favors DHA over EPA by a factor of ~ 7.5, but with markedly different absolute affinities: FABP3 binds DHA and EPA with equilibrium dissociation constants (K d ) of 39.13 ± 3.83 μM and 300.0 ± 19.0 μM, respectively, whereas FABP7 binds the same ligands with K d values of 80.18 ± 3.71 μM and 600.2 ± 30.2 μM. Competitive capture experiments analyzed by GC-MS corroborate these kinetics. In equimolar DHA/EPA mixtures, immobilized FABP3 left an 80-fold excess of EPA over DHA in solution at sub-saturating protein load, whereas FABP7 achieved only a 2.5-fold excess. Thus, FABP3 is both the tighter binder and the far more discriminating selector for DHA. These quantitative benchmarks position FABP3 as a promising scaffold for engineering high-capacity, DHA-specific affinity media for nutritional and pharmaceutical purification of ω-3 lipids.