The interaction of patulin with human serum albumin (HSA) was studied in vitro under normal physiological conditions. The study was performed using fluorescence, ultraviolet-visible spectroscopy (UV-Vis), circular dichroism (CD), atomic force microscopy (AFM), and molecular modeling techniques. The quenching mechanism was investigated using the association constants, the number of binding sites, and basic thermodynamic parameters. A dynamic quenching mechanism occurred between HSA and patulin, and the binding constants (
Patulin is a toxic secondary metabolite produced by a variety of food spoilage fungi, particularly by
Human serum albumin (HSA) containing 585 amino acid residues is the most abundant protein constituent of the circulatory system, contributing significantly to physiological functions as carrier proteins [
In this study, the interaction of patulin with HSA was investigated using steady-state and time-resolved fluorescence spectroscopy, UV-Vis spectroscopy, circular dichroism (CD) spectroscopy, atomic force microscopy (AFM), and molecular modeling.
HSA (fatty acid free < 0.05%) was purchased from Sigma Chemical Company (USA), and patulin (Figure
The chemical structure of patulin.
Steady-state fluorescence spectra were obtained using an F-4500 fluorescence spectrometer (Hitachi, Japan). The excitation and emission slit widths were 5 nm. An excitation wavelength of 295 nm and emission wavelengths between 300 and 500 nm were recorded.
The interactions between patulin and HSA were measured by the fluorescence method. The appropriate concentrations of HSA, NaCl, and patulin—15
Time-resolved fluorescence spectra were measured by steady-state spectroscopy (FLS-920) (Edinburgh Instruments) using a standard time-correlated and single-photon counting scheme. Samples were excited with a subnanosecond pulsed diode laser at a repetition rate of 10 MHz at 295 nm. Fluorescence spectra were detected three times at 345 nm and at 288 K. The decay of the fluorescence intensity was determined with the instrument-specific software based on tail fitting. The multiexponential values were assessed by considering the reduced chi-square value (
Absorption spectra were obtained with a UV-2450 UV-Vis spectrometer (Shimadzu, Japan) at 288 K in the range of 200–500 nm using a 1 cm quartz cell. The method employed was the same as that described in Section
CD measurements were collected with a JASCO J-810 circular dichroism spectrometer (Japan) at 288 K using a 0.1 cm quartz cell. The HSA concentration was 1.5
AFM was carried out with a MultiMode Nanoscope IIIa (USA) equipped with a normal NP probe. The spring constant of the cantilever was 0.32 N/m, and the typical imaging resonance frequency of the fluid was 7–9 kHz. All of the samples were imaged in fluid contact mode with an O-ring liquid cell. Samples were prepared as follows:
The 3D structure of the ligand was constructed with standard bond lengths and bond angles using the molecular modeling software program SYBYL8.0 (Tripos Inc., St. Louis, USA) for Linux. Geometry optimization was performed using the standard Tripos forcefield [
Molecular docking was implemented using MOE2009 for Windows (Chemical Computing Group Inc., Montreal, Canada). The available X-ray structure of HSA complexed with R-warfarin (PDB code: 1H9Z) was applied in this work as the receptor. Hydrogen atoms were added to the PDB file. Then, the 1H9Z complex was handled in LigX (a module of the MOE software) to meet the docking requirements. The conformer with the lowest
The intrinsic fluorescence of HSA is derived mainly from tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues. Phe residue fluorescence has a very low quantum yield, and Tyr residue fluorescence is nearly quenched when the residue is ionized or near an amino or carboxyl group of Trp. The only tryptophan residue (Trp-214) in HSA is located in domain II, Site I. The fluorescence of Trp-214 is sensitive to the ligand to which the residue binds. Therefore, Trp-214 is often used as a probe to investigate the interaction of small ligands with HSA. Figure
The fluorescence emission (a) and synchronous (b) spectra of the patulin-HSA system. The concentration of HSA was 1.5
Synchronous fluorescence is a very useful tool for investigating the microenvironments of fluorophore functional groups. According to Miller [
To further confirm the quenching mechanism, the fluorescence quenching data were analyzed with the Stern-Volmer equation:
Binding and thermodynamic parameters for the patulin-HSA interaction at different temperatures in Trisbuffer (pH = 7.4).
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Equation ( |
Equation ( |
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288 |
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0.9961 |
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0.9769 | 1.10 | −22.46 | ||
300 |
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0.9985 |
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0.9914 | 1.17 | 8.06 | 106.0 | −23.73 |
310 |
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0.9972 |
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0.9982 | 1.34 | −24.79 |
Time-resolved fluorescence spectroscopy is a tool that can be used to investigate the interaction between ligands and proteins. The fluorescence lifetime can be used to directly differentiate between dynamic and static quenching. The lifetime of static quenching does not depend on the quencher concentration (i.e.,
Fluorescence decay fitting parameters for the patulin-HSA system in Trisbuffer (pH = 7.4).
Substance |
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HSA | 3.06 | 0.64 | 7.80 | 0.36 | 1.007 | 5.37 |
Patulin-HAS (1 : 1) | 2.82 | 0.51 | 7.26 | 0.49 | 1.020 | 5.28 |
Patulin-HAS (2 : 1) | 2.55 | 0.47 | 6.19 | 0.53 | 1.024 | 5.11 |
The time-resolved fluorescence decay of the patulin-HSA system.
The apparent binding constant (
There are several types of noncovalent interaction modes between proteins and ligands, such as hydrogen bonds, van der Waals forces, hydrophobic interaction forces, and electrostatic forces [
The
The molecular distance
The overlap of (a) the absorption spectra of patulin and (b) the fluorescence emission spectrum of HSA.
Absorption at approximately 210 nm is indicative of the a-helix structure of HSA. Figure
UV absorption spectra of the patulin-HSA system. The concentration of HSA was 1.5
CD spectroscopy is a quantitative technique that is used to study the conformation of proteins in aqueous solutions. The CD spectra of HSA exhibited two negative bands at 208 and 219 nm, which characterize the
Secondary structure determination for free HSA and its drug complexes in Trisbuffer (pH = 7.4) at different molar concentration ratios for HSA and patulin.
Molar ratio (patulin-HSA) |
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Random coil (%) |
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0 : 1 | 55.3 | 8.7 | 15.3 | 20.7 |
1 : 1 | 52.7 | 9.1 | 15.6 | 22.6 |
2 : 1 | 50.7 | 10.6 | 17.4 | 21.3 |
The CD spectra of the patulin-HSA system. Patulin concentrations were 0.0, 1.5, and 3.0
Table
To study the changes in HSA topography with the addition of patulin, the free HSA and HAS-patulin complexes were imaged by AFM in triplicate. Figure
(a) An AFM topography image of free HSA and (b) an AFM topography image of the HSA-patulin complex. Samples were adsorbed onto mica under tapping mode in a Tris-HCl buffer solution, and the scan size of the image is 1.5
As shown in Figure
Crystal structure analyses indicated that HSA contains the three following domains, which are structurally similar (I–III): I (residues 1–195), II (196–383), and III (384–585). The principal ligand binding sites on HSA are located in the hydrophobic cavities in subdomains IIA and IIIA, which correspond to sites I and II (according to the terminology of Sudlow et al.) [
(a) The interaction mode between patulin and HAS. (b) A projection of 8a. The HSA residues are represented by lines, and the patulin structure is represented by a ball-and-stick model. Hydrogen bonds between patulin and HSA are represented by dashed lines.
As shown in Figure
In this study, the interactions of patulin with HSA were investigated under simulated physiological conditions using different spectroscopic methods: AFM and molecular modeling. Steady-state and time-resolved fluorescence spectra suggest that a dynamic quenching mechanism occurred in patulin-HSA complexes. The binding parameters for the reaction were determined using the Stern-Volmer equation. The thermodynamic parameters obtained at different temperatures and the molecular modeling results indicate that hydrophobic interaction was the dominant binding force and also suggest the formation of hydrogen bonds between patulin and HSA. This finding is expected to elucidate the toxigenicity of patulin when it is combined with the biomolecular function effect, transmembrane transport, toxicological testing, and other experiments.
The provisional maximum tolerable daily intake
A no-observed-effect level
Human serum albumin
Circular dichroism
Atomic force microscopy
Tryptophan
Tyrosine
Phenylalanine.
The authors declare that they have no conflict of interests.
Li Yuqin conceived and designed the study, carried out atomic force microscopy and data analysis, interpreted the entire results, and drafted the paper. You Guirong carried out steady-state fluorescence spectra and data analysis. Yang Zhen carried out UV-Vis spectroscopy and helped to draft the paper. Liu Caihong carried out time-resolved fluorescence spectra and data analysis. Jia Baoxiu carried out circular dichroism spectra and data analysis. Chen Jiao carried out molecular modeling and data analysis. Guo Yurong participated in the design of the study and interpreted the results. All authors read and approved the final paper.
This work was supported by the China Agriculture Research System (Cars-28).