Binding of Perfluorooctanoic Acid to Rat Liver‐form and Kidney‐form α2u‐Globulins

Abstract

Perfluorooctanoic acid (PFOA) is an organic fluorochemical and is reported to have a long half-life in human blood. Its urinary elimination in rats is markedly sex-dependent, and characterized by significantly longer plasma half-life of PFOA in male rats than in females. It has been postulated that male-specific PFOA binding protein(s) is responsible for the long half-life of PFOA in male rats. In this paper, two male rat specific proteins, liver- and kidney-form alpha2u-globulins (A2U(L) and A2U(K)), were purified from male rat urine and kidney, respectively. The binding of these two nroteins to PFOA was investigated using ligand blotting, electrospray ionization mass spectrometry and fluorescence competitive binding assay. The results revealed that both A2U(L) and A2U(K) were able to bind PFOA in vitro under physiological conditions, and that PFOA and a fluorescent-labeled fatty acid shared the same binding site on both A2U(L) and A2U(K). The binding affinities, however, are relatively weak. The estimated dissociation constants are in the 10(-3) M range, indicating that bindings of PFOA to either A2U(L) or A2U(K) cannot adequately explain the sex-dependent elimination of PFOA in rats, and it is unlikely that PFOA-A2U(K) binding would induce A2U nephropathy as seen with, for example, 1,4-dichlorobenzene.

Experimental characterization of the mechanism of perfluorocarboxylic acids' liver protein bioaccumulation: The key role of the neutral species

“…Thus, it is concluded that while it is the neutral, undissociated PFOA species that binds to L-FABP, it does so for only a limited range (7þ to 10þ) of protein charge states. This is consistent with the observed binding of PFOA to rat serum albumin [9] and rat liver a2m globulin [39], where the dominant charge state was 18þ.…”

“…Electrospray ionization/mass spectrometry was employed to confirm the stoichiometry of PFOA binding. This technique has been used to observe noncovalent protein-ligand complexes in the gas phase [25,37,38], including the binding of PFOA to human serum albumin [39]. Figure 5A shows a mass spectrum of L-FABP over the entire scanned mass range and, in the inset, a magnification of the dominant charge state, 9þ.…”

Strong associations of short‐chain perfluoroalkyl acids with serum albumin and investigation of binding mechanisms

“…NanoESI-MS, employing a soft ionization technique, is used here to confirm direct binding of PFCAs (C 2 -C 9 ) and PFSAs (C 4 -C 8 ) to BSA and determine stoichiometries of binding at 1:1 PFAA:albumin mole ratios. Nanoelectrospray ionization mass spectrometry has been widely used to study proteins in their native conformation and noncovalent interactions of proteinligand complexes preserved in the gas phase [33], although to our knowledge this is the first reported detection of short-chain PFCA and PFSA complexes with BSA by nanoESI-MS. Several studies have analyzed intact PFOA-and PFNA-protein complexes via ESI-MS to elucidate the stoichiometry of binding [12,23,26,28,34]. Although caution must be taken when quantifying gas-phase affinities using this method [23] for comparison with solution-based results, the technique has been increasingly used to determine stoichiometries of specific binding for protein-ligand complexes.…”

Interaction of perfluorooctanoic acid with human serum albumin

“…arazine [ 16 ], ochratoxin [ 17 ], methyl parathion [ 18 ] and arsenic [ 19 ]. Bindings of PFOA to biomacromolecular such as rat and Human plasma proteins [ 20 ], rat liver-form and kidney-form alpha 2u-globulins [ 21 ], have been investigated at room temperature. The interaction of organic contaminants and HSA is always affected by various environmental conditions such as pH, strength and temperature [ 22 ].…”