Differential scanning fluorimetry to assess PFAS binding to bovine serum albumin protein.

The rapid screening of protein binding affinity for poly- and perfluoroalkyl substances (PFAS) benefits risk assessment and fate and transport modelling. PFAS are known to bioaccumulate in livestock through contaminated food and water. One excretion pathway is through milk, which may be facilitated by binding to milk proteins such as bovine serum albumin (BSA). We report a label-free differential scanning fluorimetry approach to determine PFAS-BSA binding over a broad temperature range. This method utilizes the tryptophan residue within the protein binding pocket as an intrinsic fluorophore, eliminating the need for fluorophore labels that may influence binding. BSA association constants were determined by (a) an equilibrium-based model at the melting temperature of BSA and (b) the Hill adsorption model to account for temperature dependent binding and binding cooperativity. Differences in binding between PFAS and fatty acid analogs revealed that a combination of size and hydrophobicity drives PFAS binding.

PFAS fluidize synthetic and bacterial lipid monolayers based on hydrophobicity and lipid charge.

Poly- and Perfluoroalkyl substances (PFASs) are pollutants of emerging concern that persist in nature and pose environmental health and safety risks. PFAS disrupt biological membranes resulting in cellular inhibition, but the mechanism of disruption and the role of lipid composition remain unclear. We examine the role of phospholipid saturation and headgroup charge on the interactions between PFASs and phospholipid monolayers comprised of synthetic phosphocholine (PC) and phosphoglycerol (PG) lipids and prepared from bacteria membrane extracts rich in PG lipids from an environmentally relevant marine bacterium Alcanivorax borkumensis. When deposited on a buffered subphase containing PFAS, PFAS mixed within and fluidized zwitterionic and net-anionic monolayers leading to increases in monolayer compressibility that were driven by a combination of PFAS hydrophobicity and monolayer charge density. Differences in the monolayer response using saturated or unsaturated lipids are attributed to the ability of the unsaturated lipids to accommodate PFAS within 'void space' arising from the bent lipid tails. Similar fluidization and compressibility behavior were also observed in A. borkumensis lipid monolayers. This work provides new insight into PFAS partitioning into bacterial membranes and the effect PFAS have on the physicomechanical properties of zwitterionic and charged lipid monolayers.

Replacement per- and polyfluoroalkyl substances (PFAS) are potent modulators of lipogenic and drug metabolizing gene expression signatures in primary human hepatocytes.

Per- and polyfluoroalkyl substances (PFAS) are a class of environmental toxicants, and some, such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), have been associated with hepatic steatosis in rodents and monkeys. It was hypothesized that perfluorosulfonic acids (C4, 6, 8), perfluorocarboxylic acids (C4-14), perfluoro(2-methyl-3-oxahexanoic) acid (HFPO-DA), 1H, 1H, 2H, 2H-perfluorooctanesulfonic acid (6:2 FTS) along with 3 PFOS precursors could induce expression of lipid metabolism genes and lipid deposition in human hepatocytes. Five-donor pooled cryopreserved human hepatocytes were cultured and treated with 0.1% DMSO vehicle or various PFAS (0.25 to 25 µM) in media. After a 48-h treatment, mRNA transcripts related to lipid transport, metabolism, and synthesis were measured using a Quantigene Plex assay. After 72-h treatments, hepatocytes were stained with Nile Red dye to quantify intracellular lipids. Overall, PFAS were transcriptionally active at 25 µM. In this model, lipid accumulation was not observed with C8-C12 treatments. Shorter chain PFAS (C4-C5), 6:2 FTS, and PFOS precursor, metFOSA, induced significant liver lipid accumulation, and gene activation at lower concentrations than legacy PFAS. In summary short chain PFAS and other alternative PFAS were more potent gene inducers, and potential health effects of replacement PFAS should be critically evaluated in humans.

Critical new insights into the binding of poly- and perfluoroalkyl substances (PFAS) to albumin protein.

With an increasing number of health-related impacts of per- and polyfluoroalkyl substances (PFAS) being reported, there is a pressing need to understand PFAS transport within both the human body and the environment. As proteins can serve as a primary transport mechanism for PFAS, understanding PFAS binding to proteins is essential for predictive physiological models where accurate values of protein binding constants are vital. In this work we present a critical analysis of three common models for analyzing PFAS binding to bovine serum albumin (BSA) based on fluorescence quenching: the Stern-Volmer model, the modified Stern-Volmer model, and the Hill equation. The PFAS examined include perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorobutanesulfonic acid (PFBS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanesulfonic acid (PFOS), and the replacement compound 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate (HFPO-DA or GenX). While all three models capture the general effects of hydrophobicity and steric limitations to PFAS binding, the Hill equation highlighted a unique relationship between binding cooperativity and the number of fluorinated carbons, with PFOA exhibiting the greatest binding cooperativity. The significance of steric limitations was confirmed by comparing results obtained by fluorescence quenching, which is an indirect method based on specific binding, to those obtained by equilibrium dialysis where PFAS binding directly correlated with traditional measures of hydrophobicity. Finally, the binding constants were correlated with PFAS physicochemical properties where van der Waals volume best described the steric limitations observed by fluorescence quenching.

Dominant entropic binding of perfluoroalkyl substances (PFASs) to albumin protein revealed by 19F NMR.

Mechanistic insight into protein binding by poly- and perfluoroalkyl substances (PFASs) is critical to understanding how PFASs distribute and accumulate within the body and to developing predictive models within and across classes of PFASs. Fluorine nuclear magnetic resonance spectroscopy (19F NMR) has proven to be a powerful, yet underutilized tool to study PFAS binding; chemical shifts of each fluorine group reflect the local environment along the length of the PFAS molecule. Using bovine serum albumin (BSA), we report dissociation constants, Kd, for four common PFASs well below reported critical micelle concentrations (CMCs) - perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexanesulfonic acid (PFHxS), and perfluorooctanesulfonic acid (PFOS) - as a function of temperature in phosphate buffered saline. Kd values were determined based on the difluoroethyl group adjacent to the anionic headgroups and the terminal trifluoromethyl groups. Our results indicate that the hydrophobic tails exhibit greater binding affinity relative to the headgroup, and that the binding affinities are generally consistent with previous results showing that greater PFAS hydrophobicity leads to greater protein binding. However, the binding mechanism was dominated by entropic hydrophobic interactions attributed to desolvation of the PFAS tails within the hydrophobic cavities of the protein and on the surface of the protein. In addition, PFNA appears to form hemimicelles on the protein surfaces below reported CMC values. This work provides a renewed approach to utilizing 19F NMR for PFAS-protein binding studies and a new perspective on the role of solvent entropy.