Elsevier

Journal of Hazardous Materials

Volume 192, Issue 3, 15 September 2011, Pages 1291-1298
Journal of Hazardous Materials

Study on the interaction of phthalate esters to human serum albumin by steady-state and time-resolved fluorescence and circular dichroism spectroscopy

https://doi.org/10.1016/j.jhazmat.2011.06.038Get rights and content

Abstract

Phthalate esters (PAEs) are globally pervasive contaminants that are considered to be endocrine disruptor chemicals and toxic environmental priority pollutants. In this paper, the interactions between PAEs and human serum albumin (HSA) were examined by molecular modelling, steady state and time-resolved fluorescence, ultraviolet–visible spectroscopy (UV–vis) and circular dichroism spectroscopy (CD). The association constants between PAEs and HSA were determined using the Stern–Volmer and Scatchard equations. The binding of dimethyl phthalate (DMP) to HSA has a single class of binding site and its binding constants (K) are 4.08 × 103, 3.97 × 103, 3.45 × 103, and 3.20 × 103 L mol−1 at 289, 296, 303, and 310 K, respectively. The Stern–Volmer and Scatchard plots both had two regression curves for HSA–butylbenzyl phthalate (BBP) and HSA–di-2-ethylhexyl phthalate (DEHP), which indicated that these bindings were via two types of binding sites: the numbers of binding site for the first type were lower than for the second type. The binding constants of the first type binding site were higher than those of the second type binding site at corresponding temperatures, the results suggesting that the first type of binding site had high affinity and the second binding site involved other sites with lower binding affinity and selectivity. The thermodynamic parameters of the binding reactions (ΔG°, ΔH° and ΔS°) were measured, and they indicated the presences of hydrophobic forces and hydrogen interactions in the PAEs–HSA interactions, which agreed well with the results from molecular modelling. The alterations of protein secondary structure in the presence of PAEs were confirmed by UV–vis and CD spectroscopy. The time-resolved fluorescence study showed that the lifetime of Trp residue of HSA decreased after the addition of PAEs, which implied that the Trp residue of HSA was the main binding site.

Highlights

• Molecular docking revealed PAEs to be located in the hydrophobic pocket of HSA. • HSA–DMP had one class of binding sites while HSA–BBP and HSA–DEHP had two types. • Hydrophobic and hydrogen interactions dominated in the association of HSA–PAEs. • The lifetime of Trp residue of HSA decreased after the addition of PAEs. • The presences of PAEs could alter the second structure of HSA.

Introduction

Phthalate esters (PAEs) are widely used as plasticizers in industrial processes, and as non-plasticizers in consumer products [1], [2]. Due to their wide applications and growing demand, PAEs have become one of the highest yielding chemicals in the world. Nowadays their worldwide production including manufacturing of household goods amount to 6 million tones per year [3], [4]. With the widespread manufacture and application of PAEs, they are inevitably discharged into environment and also have been detected in human serum and plasma [5], [6], [7], [8]. In recent years, PAEs have attracted the interests of an increasing number of scientists since they are known with their carcinogenic, endocrine disrupting, and toxic effects on the environment and humans. In light of this negative health potential, environmental regulations have been brought to prevent the human intake of PAEs. These developments have led to an increasing interest and research effort on the effects and control of PAEs [9], [10], [11]. The affinity between PAEs and serum albumin is an important factor to understand the pharmacokinetics and pharmacodynamic properties of PAEs as it strongly influences PAEs distribution and determines the free fraction that is available for subsequent interactions with targeted receptors [12].

Human serum albumin (HSA) is the most abundant protein in plasma, which functions in the maintenance of colloid osmotic blood pressure and in the binding and transportation of various ligands such as fatty acids, hormones, and drugs, then transports them between tissues and organs [13]. It has been shown that the distribution, free concentration, and metabolism of various ligands can be significantly altered as a result of their binding to HSA [14]. Ligand interactions at the protein binding level will in most cases significantly affect the apparent distribution volume of the ligands and also affect the elimination rate of ligands. Therefore, investigating the interactions of PAEs and HSA are significant for knowing their transports and distributions in the body and clarifying their action mechanisms and pharmaceutical dynamics. As of yet, however, no work has been reported for the mechanism of these interactions and the detailed physicochemical characterizations of PAEs binding to HSA.

In this paper, we present a spectroscopic analysis of the interaction of HSA with three PAEs (structure shown in Fig. 1) such as dimethyl phthalate (DMP), butylbenzyl phthalate (BBP), and di-2-ethylhexyl phthalate (DEHP) in aqueous solution at physiological conditions, using constant protein concentration and various PAEs compositions. Structural information regarding PAEs binding mode and the effects of PAEs–HSA complexation on the protein stability and secondary structure are reported here.

Section snippets

Materials

HSA (fatty acid content <0.05%) was purchased from the Sigma Chemical Company and used without further purification. Its molecular weight was assumed to be 66,478 in calculating molar concentration. HSA was dissolved under simulated physiological conditions (pH 7.40), and this stock solution (3.0 × 10−5 M) was kept in the dark at 277 K. PAEs (≥95%) were obtained from the Dr. Ehrenstorfer GmbH (Germany). Stock solutions of DMP, BBP, and DEHP were prepared at a concentration of 5.0 × 10−2, 3.0 × 10−3,

Molecular modelling study of the interaction between PAEs and HSA

Molecular modelling was carried out using the Sybyl 6.9 software to investigate whether PAEs bind to HSA. Descriptions of 3D structure of crystalline albumin have revealed that human serum albumin comprises of three homologous α-helical domains (I–III): I (residues 1–195), II (196–383), III (384–585), that assemble to form a heart-shaped molecule. Each domain has two subdomains (A and B), which are six (A) and four (B) α-helices, respectively [20].

Aromatic and heterocyclic ligands have been

Conclusions

The interactions between PAEs and HSA have been investigated by molecular modelling, steady state and time-resolved fluorescence, UV and CD spectroscopy. The linearity of modified Stern–Volmer and Scatchard plots indicated that DMP could bind to one class of sites on HSA, which was in agreement with the number of binding site n in some degree; While the modified Stern–Volmer and Scatchard plots for the HSA–BBP and HSA–DEHP showed that these bindings were via two types of binding sites: one with

Acknowledgement

This work was kindly supported by the National Natural Science Foundation of China (No. 20875040, J0730425).

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