- Research article
- Open Access
Interaction of perfluorooctanoic acid with human serum albumin
© Wu et al; licensee BioMed Central Ltd. 2009
Received: 09 March 2009
Accepted: 14 May 2009
Published: 14 May 2009
Recently, perfluorooctanoic acid (PFOA) has become a significant issue in many aspects of environmental ecology, toxicology, pathology and life sciences because it may have serious effects on the endocrine, immune and nervous systems and can lead to embryonic deformities and other diseases. Human serum albumin (HSA) is the major protein component of blood plasma and is called a multifunctional plasma carrier protein because of its ability to bind an unusually broad spectrum of ligands.
The interaction of PFOA with HSA was investigated in the normal physiological condition by equilibrium dialysis, fluorospectrometry, isothermal titration calorimetry (ITC) and circular dichroism (CD). The non-covalent interaction is resulted from hydrogen bond, van der Waals force and hydrophobic stack. PFOA binding to HSA accorded with two-step binding model with the saturation binding numbers of PFOA, only 1 in the hydrophobic intracavity of HSA and 12 on the exposed outer surface. The interaction of PFOA with HSA is spontaneous and results in change of HSA conformation. The possible binding sites were speculated.
The present work suggested a characterization method for the intermolecular weak interaction. It is potentially useful for elucidating the toxigenicity of perfluorochemicals when combined with biomolecular function effect, transmembrane transport, toxicological testing and the other experiments.
It is well known that the Teflon event involving the Dupont Company of USA drew serious international attention to perfluorooctanoic acid (PFOA) ; PFOA is formed from the raw materials used in the production of Teflon-lined non-stick cooking appliances. Fluoropolymers such as Teflon have very good performances e.g. as fire retardants and for oil and fat resistance; their byproducts such as PFOA can be formed by cooking, burning and environmental degradation. PFOA is still widely used in basic processes in the aviation, automobile, building materials, chemicals, electronic, semiconductor and textile industries. It is persistent and non-biodegradable and becomes widely distributed in nature, e.g. water , biological bodies , human tissues  and foods . It can certainly enter the gastrointestinal tract via the intake of foods and water and then it is absorbed and permeates into the blood and various tissues. Sampling studies have revealed the presence of PFOA in the bloods of over 90% of US residents . It may have serious effects on the endocrine, immune and nervous systems and it can be delivered to the fetus through the umbilical cord and can accumulate . It can also cause cancers of the liver, testis, pancreatic and mammary glands , and can lead to embryonic deformities and other diseases [9, 10]. In recent years, PFOA has become a significant issue in many aspects of environmental ecology, toxicology, pathology and life sciences [11, 12].
Human serum albumin (HSA) is a major protein component of blood plasma but is also found in the interstitial fluid of body tissues. In mammals, albumin is synthesized by the liver and has a half-life of 19 days in the circulation . It is the major contributor to the oncotic pressure of the blood plasma . It is called a multifunctional plasma carrier protein because of its ability to bind an unusually broad spectrum of ligands e.g. inorganic ions, various drugs, amino acids, fatty acids, etc. Binding to HSA facilitates their transport throughout the circulation . Without doubt, interaction of any toxicant with HSA influences the transport of nutrients and drugs. Recently, studies have been conducted on the binding of organic contaminants or toxins to HSA e.g. arazine , ochratoxin , methyl parathion  and arsenic . Bindings of PFOA to biomacromolecular such as rat and Human plasma proteins , rat liver-form and kidney-form alpha 2u-globulins , 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 . In this work, we investigated the interaction of PFOA with HSA by equilibrium dialysis, fluorospectrometry, isothermal titration calorimetry (ITC) and circular dichroism (CD) under normal physiological condition, pH 7.40 and 0.15 M electrolyte and 37°C. The object is to analyze the interaction forces, sites and type and then further understand the toxigenicity of PFOA.
Results and discussion
Equilibrium dialysis of PFOA
Characterization of the interactions of PFOA with HSA
Both cL0 and cM0 are the initial mole concentrations of PFOA and HSA, cL is the equilibrium concentration of PFOA described above. N is the saturation binding number of PFOA. The effective fraction (f) of PFOA bound to HSA and its molar binding ratio (γ) are calculated by the relations: f = 1-cL/cL0 and γ = fcL0/cM0. The γ value will approach N with increasing PFOA.
Thermodynamic property of the HSA-PFOA interaction at pH 7.40 at 37°C
K b, i
ΔH i , (kcal/mol)
24.7 ± 6.5
0.55 ± 0.14
Besides a long carbon chain, PFOA has strong extensibility on interface of water – particles so that it may spread on the exposed outer surface of HSA. When cL0/cM0 is more than 2, PFOA began to bind on the hydrophilic surface of HSA till a saturation (N2 = 12) via the polar bonds e.g. ionic interaction, hydrogen bond and F⋯N and F⋯O halogen bonding (Fig. 3B). By comparison of N obtained by equilibrium dialysis (Fig. 2) and that (N = N1 + N2 = 13) obtained by ITC (Fig. 3B), two methods achieves the same result. From the higher ΔH2 value, the binding sites of PFOA may bridge between any two helixes all over the outer surface of HSA (Fig. 5). From ΔG2 value (Table 1), the PFOA-HSA reaction is spontaneous. In the 2nd step, the binding of PFOA to HSA caused an obvious entropy decreasing, i.e. more negative ΔS2 (Table 1), the HSA structure changed refolding.
The current work investigated the interaction of PFOA with HSA in the normal physiological acidities of blood and intestinal tract tissue where PFOA molecules may be present. The interaction of PFOA with HSA accorded with the Langmuir isothermal model in two-step sequence, in which only one PFOA molecule entered the hydrophobic intracavity in the first step and 12 PFOA molecules binding on the hydrophilic outer surface in the second step. The interaction of PFOA and HSA is spontaneous and the non-covalent bond results in change of HSA conformation. The possible binding sites were also speculated. The present work proposed a determination and characterization method for the intermolecular weak interaction. If combined further with the other experiments e.g. biomolecular function effect , cell membrane transport of contaminant  and toxicological testing , it is more helpful for elucidating the toxigenicity of perfluorochemicals.
Instruments and chemicals
Model S-4100 spectrophotometer (Sinco Co., Korea), which was computer-controlled by Labpro Plus firmware (Version 060105); Model MSC-ITC system (MicroCal Inc., USA) with measurement software; Model J-715 CD Spectropolarimeter (Jasco Instrum., Japan) with secondary structure Estimation-Standard Analysis Measurement software (715/#B014460524, JASCO); Model RC 30 – 5K semi-permeable membrane (Molecular Weight Cut Off 5 KDa, Shanghai Green Bird STD). Both 0.100 mM HSA (Sigma) was prepared and stored at 4°C. The other solutions were prepared: a standard PFOA solution (New Jersey, USA) (5.00 mM); Britton-Robinson (BR) buffers (pH 7.40); ECR (1.00 mM, A. R., Sigma) solution; CPC (1.00 mM, purity > 99%, Shanghai Reagents).
Determination of PFOA
A simple and rapid spectrophotometry method for determining PFOA was developed. The anionic color ligand eriochrome cyanine R (ECR) and cationic surfactants cetylpyridinium chloride (CPC) were employed to determine PFOA. The detailed analytical procedure was described by .
By means of a ITC device, PFOA solution (2.50 mM in pH 7.40 BR buffer) was injected about 40 times in 6-μl increments at 3-min intervals into an isothermal cell containing HSA (10.0 μM in pH 7.40 BR buffer). The cell temperature was maintained at 37°C and all the solutions contained 0.15 M NaCl. Heats of dilution of PFOA, obtained separately by injecting into the buffer, were used to correct the data. The corrected heats were divided by the number of moles injected and analyzed using the Origin software (version 7.0).
BR buffer (1.0 ml, pH 7.40) was mixed with 0.010 mM HSA in three flasks; or 1.00 mM PFOA was added and the solutions were diluted to 10.0 ml with distilled water. Simultaneously, a reagent blank without PFOA was prepared. Before measurement, all the solutions were diluted from 0.1 to 10.0 ml with 10% BR buffer. Each sample was allowed to equilibrate for 15 min, then injected into a 0.1-cm light path cell, and the mean residue ellipticity (MRE) of HSA was measured between 195 and 250 nm by spectropolarimetry.
This work was supported by the National Major Project of Science & Technology Ministry of China (Grant No.2008ZX07421-002) and the State Key Laboratory Foundation of Science and Technology Ministry of China (Grant No. PCRRK08003). We would like to thank Dr. Shao-Feng Luo of USTC for his help with ITC measurement and Dr. William Gelb and Dr. Xue-Ling Ao for their assistances in use of the computer programs and data analysis.
- Stokstad E: Environmental research – DuPont settlement to fund test of potential toxics. Science 2006, 311: 26–27. 10.1126/science.311.5757.26aView ArticlePubMedGoogle Scholar
- Wania FA: Global mass balance analysis of the source of perfluorocarboxylic acids in the Arctic ocean. Environ Sci Technol 2007, 41: 4529–4535. 10.1021/es070124cView ArticlePubMedGoogle Scholar
- Lau C, Butenhoff JL, Rogers JM: The developmental toxicity of perfluoroalkyl acids and their derivatives. Toxicol Appl Pharmacol 2004, 198(2):231–241. 10.1016/j.taap.2003.11.031View ArticlePubMedGoogle Scholar
- Maestri L, Negri S, Ferrari M, Ghittori S, Fabris F, Danesino P, Imbriani M: Determination of perfluorooctanoic acid and perfluorooctanesulfonate in human tissues by liquid chromatography/single quadrupole mass spectrometry. Rapid Commun Mass Spectrom 2006, 20: 2728–2734. 10.1002/rcm.2661View ArticlePubMedGoogle Scholar
- Karrman A, Ericson I, van BB, Darnerud PO, Aune M, Glynn A, Lignell S, Lindstrom G: Exposure of perfluorinated chemicals through lactation, levels of matched human milk and serum and a temporal trend, 1996–2004, in Sweden. Environ Health Perspect 2007, 115(2):226–230.PubMed CentralView ArticlePubMedGoogle Scholar
- Calafat AM, Kuklenyik Z, Reidy JA, Caudill SP, Tully J, S Needham LL: Serum concentrations of 11 polyfluoroalkyl compounds in the US population, Data from the National Health and Nutrition Examination Survey NHANES. Environ Sci Technol 2007, 41: 2237–2242. 10.1021/es062686mView ArticlePubMedGoogle Scholar
- Abbott BD, Wolf CJ, Schmid JE, Das KP, Zehr RD, Helfant L, Nakayama S, Lindstrom AB, Strynar MJ, Lau C: Perfluorooctanoic acid-induced developmental toxicity in the mouse is dependent on expression of peroxisome proliferator-activated receptor-alpha. Toxicol Sci 2007, 98: 571–581. 10.1093/toxsci/kfm110View ArticlePubMedGoogle Scholar
- Martin MT, Brennan RJ, Hu WY, Ayanoglu E, Lau C, Ren HZ, Wood CR, Corton JC, Kavlock RJ, Dix DJ: Toxicogenomic study of triazole fungicides and perfluoroalkyl acids in rat livers predicts toxicity and categorizes chemicals based on mechanisms of toxicity. Toxicol Sci 2007, 97: 595–613. 10.1093/toxsci/kfm065View ArticlePubMedGoogle Scholar
- Maras M, Vanparys C, Muylle F, Robbens J, Berger U, Barber JL, Blust R, Coen DW: Estrogen-like properties of fluorotelomer alcohols as revealed by MCF-7 breast cancer cell proliferation. Environ Health Persp 2006, 114: 100–105. 10.1289/ehp.8149View ArticleGoogle Scholar
- Yeung LWY, Guruge KS, Yamanaka N, Miyazaki S, Lam PKS: Differential expression of chicken hepatic genes responsive to PFOA and PFOS. Toxicology 2007, 237: 111–125. 10.1016/j.tox.2007.05.004View ArticlePubMedGoogle Scholar
- Service RF: Meeting – American Chemical Society: Safer alternative could replace widespread contaminant. Science 2005, 309: 1810. 10.1126/science.309.5742.1810aView ArticlePubMedGoogle Scholar
- Washburn ST, Bingman TS, Braithwaite SK, Buck RC, Buxton LW, Clewell HJ, Haroun LA, Kester JE, Rickard RW, Shipp AM: Exposure assessment and risk characterization for perfluorooctanoate in selected consumer articles. Environ Sci Technol 2005, 39: 3904–3910. 10.1021/es048353bView ArticlePubMedGoogle Scholar
- He XM, Carter DC: Atomic structure and chemistry of human serum albumin. Nature 1992, 358: 209–215. 10.1038/358209a0View ArticlePubMedGoogle Scholar
- Bhattacharya AA, Grune T, Curry S: Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin. J Mol Biol 2000, 303: 721–732. 10.1006/jmbi.2000.4158View ArticlePubMedGoogle Scholar
- Kragh-Hansen U: Molecular aspects of ligand binding to serum albumin. Pharmacol Rev 1981, 33: 17–53.PubMedGoogle Scholar
- Purcell M, Neault JF, Malonga H, Arakawa H, Carpentier R, Tajmir-Riahi HA: Interaction of atrazine and 2, 4-D with human serum albumin studied by gel and capillary electrophoresis FTIR spectroscopy. Biochim Biophys Acta 2001, 1548: 129–138.View ArticlePubMedGoogle Scholar
- Berger V, Gabriel AF, Sergent T, Trouet A, Larondelle Y, Schneider YJ: Interaction of ochratoxin A with human intestinal Caco-2 cells, possible implication of a multidrug resistance-associated protein MRP2. Toxicol Lett 2003, 140: 465–476. 10.1016/S0378-4274(03)00043-2View ArticlePubMedGoogle Scholar
- Silva D, Cortez CM, Cunhua-Bastos J, Louro SRW: Methyl parathion interaction with human and bovine serum albumin. Toxicol Lett 2004, 147: 53–61. 10.1016/j.toxlet.2003.10.014View ArticlePubMedGoogle Scholar
- Uddin SJ, Shilpi JA, Murshid GMM, Rahman AA, Sarder MM, Alam MA: Determination of the binding sites of arsenic on bovine serum albumin using warfarin site-I specific probe and diazepam site-II specific probe. J Biol Sci 2004, 4: 609–612. 10.3923/jbs.2004.609.612View ArticleGoogle Scholar
- Han X, Snow TA, Kemper RA, Jepson GW: Binding of Perfluorooctanoic Acid to Rat and Human Plasma Proteins. Chem Res Toxicol 2003, 16: 775–781. 10.1021/tx034005wView ArticlePubMedGoogle Scholar
- Han X, Hinderliter PM, Snow TA, Jepson GW: Binding of Perfluorooctanoic Acid to Rat Liver – form and Kidney – form α2u – Globulins. Drug Chem Toxicol 2004, 27: 341–360. 10.1081/DCT-200039725View ArticlePubMedGoogle Scholar
- Zhang X, Chen L, Fei XC, Ma YS, Gao HW: Binding of PFOS to serum albumin and DNA: insight into the molecular toxicity of perfluorochemicals. BMC Mol Biol 2009, 10: 16. 10.1186/1471-2199-10-16PubMed CentralView ArticlePubMedGoogle Scholar
- Gao HW, Xu Q, Chen L, Wang SL, Wang Y, Wu LL, Yuan Y: Potential protein toxicity of synthetic pigments, binding of poncean S to human serum albumin. Biophys J 2008, 94: 906–917. 10.1529/biophysj.107.120865PubMed CentralView ArticlePubMedGoogle Scholar
- Yang M: Molecular recognition of DNA targeting small molecule drugs. J Beijing Med Univ 1998, 30: 97–99.Google Scholar
- Cooper A, McAlpine A, Stockley PG: Calorimetric studies of the energetics of protein-DNA interactions in the E. coli methionine repressor MetJ system. FEBS Lett 1994, 348: 41–45. 10.1016/0014-5793(94)00579-6View ArticlePubMedGoogle Scholar
- Kamiya M, Torigoe H, Shindo H, Sarai A: Temperature dependence and sequence specificity of DNA triplex formation, an analysis using isothermal titration calorimetry. J Am Chem Soc 1996, 118: 4532–4538. 10.1021/ja952287jView ArticleGoogle Scholar
- Luque I, Todd MJ, Gomez J, Semo N, Freire E: Molecular basis of resistance to HIV-1 protease inhibition, a plausible hypothesis. Biochemistry 1998, 37: 5791–5797. 10.1021/bi9802521View ArticlePubMedGoogle Scholar
- Cabot R, Hunter CA: Non-covalent interactions between iodo-perfluorocarbons and hydrogen bond acceptors. Chem Comm 2009.Google Scholar
- Xu Z, Liu XW, Ma YS, Gao HW: Interactions of nano-TiO2 with lysozyme: insights into the enzyme toxicity of nanosized particles. Environ Sci Pollut Res Int 2009. 10.1007/s11356-009-0153-1Google Scholar
- Li L, Gao HW, Ren JR, Chen L, Li YC, Zhao JF, Zhao HP, Yuan Y: Binding of Sudan II and IV to lecithin liposomes and E. coli membranes: insights into the toxicity of hydrophobic azo dyes. BMC Struct Biol 2007, 7: 16. 10.1186/1472-6807-7-16PubMed CentralView ArticlePubMedGoogle Scholar
- Incardona JP, Collier TK, Scholz NL: Defects in cardiac function precede morphological abnormalities in fish embyos exposed to polycyclic aromatic hydrocarbon. Toxicol Appl Pharmacol 2004, 196: 191–205. 10.1016/j.taap.2003.11.026View ArticlePubMedGoogle Scholar
- Wu LL, Chen L, Song C, Liu XW, Deng HP, Gao NY, Gao HW: Potential enzyme toxicity of perfluorooctanoic acid. Amino Acids 2009. 10.1007/s00726-008-0217-4Google Scholar
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