Elucidation of specific binding sites and extraction of toxic Gen X from HSA employing cyclodextrin

The presence of per and poly-fluoroalkyl substances (PFAS), commonly referred to as forever chemicals, in aquatic systems is a serious global health problem. While the remediation of PFAS from aqueous media has been extensively investigated, their interactions with and removal from biological systems have received far less attention. We report herein structural alterations to human serum albumin (HSA) upon addition of perfluoro(2-methyl-3-oxahexanoic) acid (Gen X) monitored by changes to the fluorescence and circular dichroism (CD) spectra of HSA. The equilibrium association constant for Gen X binding to HSA is 7( ± 1) × 10³ M^-1 determined from changes in HSA fluorescence emission data during titration. Site-specific HSA binding fluorophores, 8-anilinonaphthalene-1-sulfonic acid (1,8-ANS), warfarin and dansyl-L-proline were used to investigate the specific binding sites of Gen X on HSA. A competitive displacement study yields association constants for Gen X to HSA at the 1,8-ANS, warfarin, and dansyl-L-proline binding sites to be 6.25 ( ± 0.5) × 10⁴ M^-1, 1.1 × 10⁶ M^-1, and 2.5( ± 0.2) × 10⁹ M^-1 respectively. Addition of β-cyclodextrin (β-CD) and heptakis(6-deoxy-6-amino)-β-cyclodextrin heptahydrochloride to the HSA:Gen X complex leads to the effective extraction of Gen X from the complex with the return of HSA in its native form. Gen X also leads to displacement of site-specific binding fluorophores bound to HSA, while subsequent addition of β-CD extracts Gen X from HSA with the return of the characteristic fluorescence of the HSA bound site-specific agent. These results illustrate the strong and specific binding sites of Gen X on HSA and demonstrate the principles for the potential application of β-CD for the remediation of PFAS from biological systems.