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.
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