Characterization of the binding mechanism and conformational changes of bovine serum albumin upon interaction with aluminum-maltol: a spectroscopic and molecular docking study

Dai Cheng abcd, Xuerui Wang ac, Jinlei Tang ac, Xinyu Zhang ac, Chunling Wang *ac and He Li *b
aState Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, 300457, Tianjin, China. E-mail: wangchunling@tust.edu.cn
bBeijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU), Beijing 100048, China. E-mail: lihe@btbu.edu.cn
cDemonstration Center of Food Quality and Safety Testing Technology, Tianjin University of Science and Technology, 300457, Tianjin, China
dTianjin Key Laboratory of Food Science and Health, School of Medicine, Nankai University, 300071, Tianjin, China

Received 17th April 2019 , Accepted 6th August 2019

First published on 7th August 2019


The widespread use of aluminum in the treatment of drinking water, food, agriculture and pharmaceuticals has greatly increased the risk of human exposure to excess aluminum, which is a serious health hazard to human beings. In our previous work, serum albumin was reported to have a specific affinity for aluminum. However, the mechanism of binding of aluminum to serum albumin was unclear. In this work, the interaction between bovine serum albumin (BSA) and aluminum-maltol (Al-Mal) was studied by molecular docking and spectroscopic analysis. The results show that the combination of Al-Mal and BSA is a spontaneous endothermic reaction. The binding force is mainly related to the hydrophobic force and hydrogen bonding; when the ratio of BSA to Al-Mal was 1[thin space (1/6-em)]:[thin space (1/6-em)]10, the random coils of BSA increased by 47.6%. In addition, the hydrophobicity of BSA was enhanced after combining with Al-Mal. This study can provide a theoretical evidence for the binding mechanism of food-borne aluminum and serum albumin.



Significance to metallomics

Aluminum (Al) accumulation in human body might harm the central nervous system, bone lesions, and hemopoietic system, and is a suspected causal factor of Alzheimer's disease. Therefore, investigating the mechanism of biological transportation of Al has become a significant issue to be solved. In this study, we observed that aluminum-maltol could induce conformation impairment and hydrophobic enhancement of bovine serum albumin. We expect that our experiment will provide useful information in clarifying the toxicity and biological transportation of Al in vivo and be helpful for further exploring the detoxification strategy of Al.

1. Introduction

Aluminum (Al) is widely found in nature and is the most abundant among the metallic elements in the earth's crust,1 but it is not an essential trace element required by the human body. With the rapid development of the Al industry, a large amount of Al enters the human body through various foodborne routes, such as drinking water, raw materials, migration of Al from containers, aluminized pesticide residue, pollution and aluminum-containing food additives. The aluminum intake by a body is mainly derived from the daily diet.2,3 Studies have shown that the accumulation of Al in the body can interfere with the nervous hematopoietic system, skeletal system, respiratory system and immune system,4 and affect the normal metabolism of the body, inhibit the role of enzymes in the body, and cause a variety of diseases.5 The brain is the main organ for Al accumulation.6 Al can enter into the central nervous system through the blood–brain barrier.7 However, further researches on the physiological toxicity mechanism of Al need to be conducted.8,9 The accumulation of Al in different organs of the human body is closely related to the transport of Al in the human body, and protein is closely related to the metabolism of Al in the human body. Fatemi et al.10 found that albumin was a strong chelating agent for Al, which can bind strongly to 34% Al in serum. In our previous work,11 serum albumin was identified to have specific binding affinity for Al by 8-HQ staining. It lays a foundation for revealing the important role of serum albumin in the transport mechanism of Al. However, the binding mechanism of Al to serum albumin and the effect on the structure of the protein after binding are unclear.

Serum albumin is a carrier protein, rich in the human body, accounting for about 60% of total plasma protein.12 It has important significance in the life process, with a variety of pharmacological and physiological functions, such as combining and transporting compounds and maintaining colloid osmotic pressure and acid–base balance. The affinity of a compound for serum albumin affects its absorption, distribution, metabolism and toxicity in the human body.13–15 Bovine serum albumin (BSA) is a classic model for studying the interaction between serum proteins and different compounds.16–19 BSA is structurally homologous to human serum albumin (HSA) and is a globular polypeptide consisting of 582 amino acids. Serum albumin possesses natural fluorescence properties because its molecular structure contains characteristic groups, such as tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe).20–23 Sudlow et al.24 defined two major binding sites for the interaction of serum albumin with various compounds and named them as site I and site II. The interaction between compounds and serum albumin always affects the normal physiological function of serum albumin as the result of overlapping binding sites or changes in protein conformation.25–27 Therefore, studying the interaction mechanism of Al with serum albumin is important for revealing the metabolism and transport of Al in the human body.

Herein, the molecular mechanism of the interaction between Al and serum albumin was studied by multispectral analytical techniques, including the quenching mechanism, binding constant, thermodynamic parameters, type of interaction and number of binding sites. At the same time, the conformational changes of BSA and the changes in the microenvironment of the amino acid residues were also examined. In addition, Al-Mal binding sites and binding patterns on BSA were investigated by molecular docking. These results help to understand the biotransport mechanism of aluminum and its effect on the damage of albumin function from the molecular level, and have certain reference significance for evaluating the safety of aluminum-containing foods.

2. Materials and methods

2.1 Materials

Bovine serum albumin (BSA), with purity greater than 98% was purchased from Biotopped (Beijing, China) and used without further purification. Aluminum chloride (AlCl3) and Maltol (≥99.0%) were purchased from Solarbio (Beijing, China).

The other reagents used in this study were of analytical reagent grades and the buffer solutions were prepared using double distilled water.

The stock solutions of BSA (3 × 10−5 M) and Al-Mal were prepared in 0.01 M phosphate buffer (containing 0.2 M NaCl) of pH 7.4 and was kept in the dark at 277 K.

2.2 Fluorescence spectroscopy

Fluorescence spectroscopy measurements were conducted by a RF-5301PC fluorescence spectrophotometer equipped with a 1.0 cm quartz cell. The BSA concentration was set as 5 × 10−6 mol L−1, and the Al-Mal solution was diluted to the range of 131.25–2100 × 10−6 mol L−1. 2000 μL of BSA solution was titrated by successive additions of 100 μL of the diluted Al-Mal solution, producing a mixture with a concentration of Al-Mal in the range of 6.25–100 × 10−6 mol L−1. After incubation at three different temperatures (298, 304 and 310 K),28 the intrinsic fluorescence of the protein solutions was measured upon excitation at 282 nm and the emission spectra was recorded in the wavelength range of 302 to 400 nm. The excitation and emission slit widths were both set at 3 nm.

The synchronous fluorescence spectroscopy was performed at room temperature using a RF-5301PC fluorescence spectrophotometer. During the scanning process, the excitation wavelength and the emission wavelength were kept at a fixed wavelength interval (Δλ). When Δλ = 15 nm, the spectrum in the wavelength range of 280–340 nm was recorded to show only the spectral characteristics of the tyrosine residue. A spectrum in the wavelength range of 300–370 nm was recorded at Δλ = 60 nm for characterizing the spectral characteristics of tryptophan residues.29

After adding ANS to BSA and Al-Mal systems, they were incubated for 10 min at ambient temperature. The exogenous fluorescence of the mixture solutions was measured upon excitation at 380 nm and the emission spectra were recorded in the wavelength range of 400 to 600 nm. The excitation and emission slit widths were both set at 3 nm.30

The three-dimensional fluorescence spectra were measured by a F-7100 spectrofluorophotometer (Hitachi, Japan). Three dimensional fluorescence spectra of the solution containing free BSA (5 μM) as well as that of BSA in the presence of Al-Mal (1[thin space (1/6-em)]:[thin space (1/6-em)]5 molar ratios of protein to ligand) were acquired by recording the emission spectra in the wavelength range of 200–600 nm at the excitation wavelength range from 200 to 350 nm with an increment of 5 nm.31

2.3 UV-vis absorption spectra

UV-visible spectroscopy was performed on an Eppendorf biospectrometer using a cuvette with 1 cm path length. The BSA concentration was kept constant (1 × 10−6 mol L−1), and the Al-Mal concentration was varied from 0 to 20 × 10−6 mol L−1. Absorbance values were recorded after each addition of Al-Mal solution and equilibration.32

2.4 CD measurements

The CD spectrum within a wavelength range of 200–260 nm was recorded on a Bio-Logic New MOS-450 CD spectrometer using a 1.0 mm optical diameter quartz cuvette at a scan speed of 60 nm min−1. The CD spectra were recorded after incubating BSA with Al-Mal for 10 minutes at different molar ratios ([Al-Mal]/[BSA] = 0[thin space (1/6-em)]:[thin space (1/6-em)]1, 5[thin space (1/6-em)]:[thin space (1/6-em)]1 and 10[thin space (1/6-em)]:[thin space (1/6-em)]1) at room temperature.33 The background baseline (pH 7.4 phosphate buffer solutions) from all measured CD spectra were subtracted.

2.5 Molecular modelling

Molecular docking is a common tool for analyzing the precise binding position of a ligand to a protein. The docking software AutoDock 4.2 and AutoDock Tools 1.5.634 were used to simulate the binding interactions of Al-Mal with BSA. The natural structure of BSA from the protein database (ID 4f5s)35 was downloaded and the atomic coordinates of the chain B and water molecules were deleted and stored in a new file to be used as input to the AutoDock Tools. The three-dimensional structure of Al-Mal was constructed and optimized using Chemdraw and Chem3D, and exported as a pdb file. Prior to molecular modelling calculation, polar hydrogen and the Gasteiger charges were added to the BSA structure and atom types were defined. The root of the ligand Al-Mal was determined and a rotatable bond was defined. In addition, a large grid box of 66 × 70 × 80 points was used to provide sufficient space for the movement and rotation of Al-Mal. The docking parameters and algorithms use the default settings. After running the docking procedure, PyMol was used to further analyze the conformation of the best scoring complex with the lowest binding free energy.

3. Results and discussion

3.1 Fluorescence quenching of BSA after the addition of aluminum-maltol

Fluorescence spectroscopy is a common and sensitive method for studying the conformation of protein molecules. It is widely used to investigate the mechanism of binding between small molecular compounds and proteins in solution. The intrinsic fluorescence of protein is generally derived from aromatic amino acid residues such as tryptophan, tyrosine and phenylalanine.36 The fluorescence emission spectra of BSA at an excitation wavelength of 282 nm, in the presence and absence of Al-Mal, are shown in Fig. 1a (298 K). When the emission wavelength was 343 nm, BSA showed a strong fluorescence emission peak. Furthermore, as the concentration of Al-Mal increased, the fluorescence intensity of BSA decreased in a dose-dependent manner, which suggests that Al-Mal interacts with the BSA and quenches the intrinsic fluorescence of BSA.37 The maximum emission wavelength of BSA is relatively unchanged, indicating that Al-Mal may induce hydrophobic groups located inside BSA to be exposed and interact with them.38
image file: c9mt00088g-f1.tif
Fig. 1 Fluorescence spectra of BSA in the presence of different concentrations of Al-Mal at 298 K (a), 304 K (b) and 310 K (c). [BSA] = 5 × 10−6 M. The molar ratios [Al-Mal]/[BSA] = 0, 1.25, 2.5, 5, 10, 20 from “1” to “6”; pH 7.4; λex = 282 nm. The inset shows the Stern–Volmer plots for the BSA–Al-Mal system under the corresponding experimental conditions.

Different kinds of molecular interactions can cause the quenching of the endogenous fluorescence of proteins.39 Generally, the fluorescence quenching process can be divided into two types, namely, dynamic quenching and static quenching. The most common method of distinguishing the quenching mechanism is to determine the fluorescence lifetime and temperature dependence. Dynamic quenching mainly depends on larger amounts of collision and diffusion; higher temperatures will result in faster molecular diffusion and more collision between molecules. Therefore, the dynamic quenching constant value of the fluorescent complex may increase regularly with increasing temperature. Conversely, the higher the temperature, the more unstable the complex and lower the static quenching constant value. In addition, studies have shown that the maximum scattering collision quenching constant (Kdif) of the protein is 2.0 × 1010 L mol−1 s−1.40 The fluorescence quenching data of Al-Mal–BSA system was analyzed by the Stern–Volmer equation to study the quenching mechanism:41

 
F0/F = 1 + Kqτ0[Q] = 1 + KSV[Q](1)
where F0 and F are the fluorescence intensities of the protein in the absence and presence of the quencher, respectively. Kq is the quenching rate constant of the biomolecule, τ0 is the average lifetime of the biomolecule without the quencher (about 10 ns for most biomolecules), KSV is the Stern–Volmer quenching constant, and [Q] is the concentration of the quencher.

Fig. 1 shows the fluorescence emission spectra of Al-Mal quenching of BSA and the corresponding Stern–Volmer plots at three different temperatures (298, 304 and 310 K). The Stern–Volmer curve is linear over the concentration range studied, indicating that the quenching mechanism in the Al-Mal–BSA system is a single type. The Kq and KSV values calculated at the three temperatures are summarized in Table 1. The results showed that the values of Kq are much greater than Kdif, and the value of KSV shows a decreasing trend with an increase in the temperature, indicating that the quenching mechanism of BSA by Al-Mal is caused by static quenching, and that the BSA–Al-Mal complex was formed during the reaction. Further, according to the study by Plotnikova et al.,42 the fluorescence maxima ratio for different metals and BSA decreases in the order Cu (F0/F = 1.52) > Al (1.32) > Pb (1.29) > Cd (1.14). Analysis of the maximum ratio of fluorescence spectra made it possible to conclude that more efficient BSA fluorescence quenching was observed in the case of Al-Mal compared to lead nitrate and cadmium nitrate.

Table 1 Stern–Volmer quenching constants (KSV and Kq) for the binding between aluminum-maltol and BSA
T (K) K SV (104, M−1) K q (1012, M−1 s−1) R
Al-Mal 298 1.2148 1.2148 0.9965
304 1.2105 1.2105 0.9988
310 1.2057 1.2057 0.9915


3.2 Binding constant and number of binding sites

When the quenching mechanism is static, the Scatchard equation is used to calculate the binding constant (KA) and the number of binding sites (N) of the reaction:43
 
image file: c9mt00088g-t1.tif(2)
where KA represents the binding constant, N is the number of binding sites per protein, and other parameters are the same as in eqn (1).

The calculated KA and N values are summarized in Table 2. The results showed that the binding constants of Al-Mal and BSA belonged to the moderate binding strength range;44 the KA value was positively correlated to the temperature, indicating that the binding reaction between Al-Mal and BSA was enhanced at higher temperatures, and the interaction between the two was an endothermic reaction. The number of binding sites of Al-Mal in BSA is hardly affected by temperature changes, indicating that BSA has only one strong binding site for Al-Mal in the experimental concentration range. Furthermore, the KA values obtained in the present study are comparable with that of the metal complexes reported earlier (M−1):Ru(II) arene complexes (104) by Ganeshpandian et al.45

Table 2 Association constants (by Lineweaver Burk equation) and thermodynamic parameters for the interaction between aluminum-maltol and BSA
T (K) K A (104, M−1) N ΔH (kJ mol−1) ΔS (J mol−1 K−1) ΔG (kJ mol−1)
298 0.4310 0.9056 39.0711 161.8206 −9.1514
304 1.3316 1.0198 −10.1224
310 1.7478 1.0527 −11.0933


3.3 Thermodynamic parameters and force types

The reaction between the compound and serum proteins belongs to weak interactions between the molecules. There are four representative types of non-covalent interactions, namely, hydrogen bonding, electrostatic interaction, hydrophobic effects and van der Waals interactions.46 Generally, the intermolecular interaction force can be determined by the relative magnitude of the thermodynamic parameters of the binding reaction. Under three different temperature conditions, the structure of BSA does not change significantly or degrade, and hence, enthalpy change (ΔH) can be regarded as a constant.47 Then, using the van't Hoff equation (eqn (3)), ΔH and the entropy change value (ΔS) of the reaction can be calculated48 and correspondingly the value of free energy change (ΔG) can be obtained from eqn (4):35
 
log[thin space (1/6-em)]KA = −ΔH/RT + ΔS/R(3)
 
ΔG = ΔHTΔS(4)
where KA is the association constant at the corresponding reaction temperatures (298, 304, and 310 K) and R is the general gas constant.

Ross and Subramanian49 elaborated on the relationship between the possible binding modes of ligand–protein interaction systems and the signs and sizes of thermodynamic parameters. That is, in the process of binding small molecules to proteins, the hydrophobic interaction plays a major role for ΔH > 0 and ΔS > 0; for ΔH < 0 and ΔS < 0, van der Waals forces and hydrogen bonds are mainly formed; whereas for ΔH ≈ 0 and ΔS > 0, the electrostatic force is more important.

The thermodynamic parameters for the interaction of Al-Mal with BSA are listed in Table 2. The value of ΔG is negative, indicating that the reaction between Al-Mal and BSA is spontaneous at the corresponding temperature.47 For Al-Mal, the values of ΔH and ΔS are both greater than 0, indicating that the bonding process is endothermic. More importantly, positive ΔH and ΔS values illustrate that the interaction between Al-Mal and BSA is hydrophobic.

3.4 Synchronous fluorescence spectroscopy

Synchronous fluorescence spectroscopy is a method for studying changes in the microenvironment of amino acid residues. It measures changes in polarity around the chromophore and changes in the position of the maximum emission wavelength, which reflect the conformational changes of the protein. When the wavelength interval (Δλ) between the excitation and emission wave length is 15 nm, the resulting synchronous fluorescence spectrum shows only the characteristic fluorescence of the tyrosine residue; at Δλ = 60 nm, the characteristic fluorescence of the tryptophan residue can be obtained.50

The synchronous fluorescence spectra of Al-Mal and BSA systems are shown in Fig. 2. It is apparent from Fig. 2 that after the addition of Al-Mal, the maximum emission wavelengths of tyrosine and tryptophan residues were red-shifted by 2 nm (from 301 to 303 nm) and 3 nm (from 342 to 345 nm), respectively. This indicates an increase in the polarity of the microenvironment surrounding the tyrosine and tryptophan residues.


image file: c9mt00088g-f2.tif
Fig. 2 Synchronous fluorescence spectra of BSA in the presence of different concentrations of Al-Mal at 25 °C. [BSA] = 5 × 10−6 M, The molar ratios [Al-Mal]/[BSA] = 0, 1.25, 2.5, 5, 10, 20 from “1” to “6”, pH 7.4, Δλ = 15 nm (a) and Δλ = 60 nm (b).

3.5 Three-dimensional fluorescence spectroscopy

Three-dimensional fluorescence spectroscopy is a high-resolution and highly selective analytical method that comprehensively and intuitively displays the microenvironment of amino acid residues in protein molecules and their conformational changes under different conditions. In recent years, three-dimensional fluorescence spectroscopy has been proved to be an effective scientific means for studying the interaction mechanism between small molecules and proteins.

Fig. 3 shows the three-dimensional fluorescence spectra of free BSA and the BSA–Al-Mal systems. Two common characteristic peaks can be seen in the three-dimensional spectrum, namely, the Rayleigh scattering peak (peak 1, λex = λem) and the second-order scattering peak (peak 2, 2λex = λem).29 Peak A (λex = 280 nm, λem = 340 nm) mainly represents the intrinsic fluorescence characteristics of the Trp and Tyr residues. Peak B (λex = 220 nm, λem = 340 nm) is mainly related to the intrinsic fluorescence of the polypeptide chain skeleton structure and protein secondary structure.


image file: c9mt00088g-f3.tif
Fig. 3 Three dimensional fluorescence spectra and contour map of interaction of BSA (a) in the absence and the presence of Al-Mal (b). [BSA] = 5 × 10−6 M. [Al-Mal] = 25 × 10−6 M.

It can be seen from Fig. 3 that after the addition of Al-Mal, the fluorescence emission intensity of peak A is reduced by 15.5%, indicating that the polarity of the microenvironment, in which the Trp and Tyr residues are located, changes. Similarly, the fluorescence emission intensity of peak B is also reduced, which implies that the combination of Al-Mal and BSA may induce a slight destabilization of the protein and a slight expansion of the polypeptide chain skeleton structure. This result in a change in the conformation of the protein, increasing the exposure of some previously buried hydrophobic regions. The above results indicate that Al-Mal interacts with BSA and results in changes in the conformation and microenvironment of the protein.

3.6 CD response of BSA to aluminum-maltol

CD spectroscopy is a fast, simple and accurate method for studying the secondary structure of proteins in dilute solutions. It is one of the important methods to study the interaction between proteins and small molecules.

The CD spectra of the Al-Mal–BSA system are shown in Fig. 4. The CD spectrum of free BSA displays two negative bands in the far ultraviolet region of 208 and 222 nm, indicating typical characteristics of the alpha-helical structure of the protein. As the concentration of Al-Mal increases, the ellipticity of BSA decreases slightly, indicating that the interaction between Al-Mal and BSA changes the secondary structure of BSA.


image file: c9mt00088g-f4.tif
Fig. 4 The CD spectra of BSA in the presence of increasing amounts of Al-Mal. [BSA] = 5 × 10−6 M, the molar ratios of Al-Mal to BSA were 0[thin space (1/6-em)]:[thin space (1/6-em)]1, 5[thin space (1/6-em)]:[thin space (1/6-em)]1 and 10[thin space (1/6-em)]:[thin space (1/6-em)]1.

The secondary structure components based on the original CD data was calculated on a computer using a certain fitting algorithm to quantify the conformational change, the results of which are shown in Table 3. Compared with the free BSA, the α-helix content decreased significantly from 77.0% to 63.0% when the molar ratio of Al-Mal to BSA was 10[thin space (1/6-em)]:[thin space (1/6-em)]1, and the content of b-sheet and random coil structures were increased from 2.0% to 6.0% and from 21.0% to 31.0%, respectively.

Table 3 Secondary structures of BSA affected by aluminum-maltol at 25 °C
System Assignment Content (%)
BSA α-Helix 0.77
β-Sheet 0.02
Random coils 0.21
Al-Mal[thin space (1/6-em)]:[thin space (1/6-em)]BSA = 5[thin space (1/6-em)]:[thin space (1/6-em)]1 α-Helix 0.68
β-Sheet 0.04
Random coils 0.27
Al-Mal[thin space (1/6-em)]:[thin space (1/6-em)]BSA = 10[thin space (1/6-em)]:[thin space (1/6-em)]1 α-Helix 0.63
β-Sheet 0.06
Random coils 0.31


The above results indicate that the binding of Al-Mal to BSA induces the rearrangement of the protein polypeptide chain, causing the original hydrogen bond network to be destroyed, which in turn leads to a certain degree of protein destabilization. Some of the amino acid residues in the hydrophobic region of BSA are slowly exposed due to the interaction of Al-Mal with BSA. In addition, the CD spectrum of BSA in the presence and absence of Al-Mal has a similar shape without any significant peak shift, indicating that the secondary structure of BSA is still dominated by α-helix even after the addition of Al-Mal. The above analysis indicates that the binding of Al-Mal can cause changes in the conformation and amino acid polarity of BSA. In addition, the loss of α-helix partially causes the Al-Mal to be better exposed to the hydrophobic regions, which further explains that the hydrophobic interaction plays an important role in the binding process of Al-Mal and BSA, which is consistent with the thermodynamic results.

3.7 UV-vis absorption studies

Ultraviolet-visible absorption spectroscopy is a commonly used analytical method for detecting the conformational changes in proteins and the formation of complexes. Fig. 5 shows the UV-vis absorption spectra of BSA in the presence and absence of Al-Mal. The characteristic peak of BSA at 280 nm is related to the transition of aromatic amino acid residues (Tyr and Trp) of BSA.
image file: c9mt00088g-f5.tif
Fig. 5 UV-vis absorption spectra of BSA in the absence and presence of Al-Mal. [BSA] = 1 × 10−6 M. The molar ratios [Al-Mal]/[BSA] = 0, 1.25, 2.5, 5, 10, 20 from “1” to “6”.

From Fig. 5, the absorbance intensity of BSA showed a hyperchromic effect with an increase in the Al-Mal concentrations, suggesting the complexation between BSA and Al-Mal,51 and is accompanied by a blue shift of about 3 nm. This trend is consistent with the trend measured by Tian et al.52 The above results indicate that the interaction between Al-Mal and BSA may affect the conformation and microenvironment of BSA. In addition, the enhancement of the absorption peak at 280 nm can be attributed to the exposure of tryptophan and tyrosine aromatic heterocyclic hydrophobic groups in BSA to some extent.53 The dynamic quenching is mainly caused by the collision and energy transfer from albumin to the molecules. According to this mechanism, the UV-vis spectrum of albumin would have no change upon the addition of Al-Mal. Inversely, according to static quenching mechanism, the formation of a complex between protein and the substance could increase or decrease the absorbance intensity in the UV-vis spectrum of protein. These results further confirm that fluorescence quenching is primarily a static quenching process.54 In the UV-visible spectrum, no new characteristic absorption peaks were observed, indicating that Al-Mal did not induce significant changes in the natural structure of BSA in the experimental concentration range.55 After the addition of Al-Mal, the UV spectrum of BSA had a small absorbance value in the wavelength range of 320–360 nm. Gel electrophoresis techniques are used to detect protein aggregation,56 but it was not observed from the electrophoresis results (Fig. S1, ESI), indicating that Al-Mal in the experimental concentration range caused no BSA aggregation. In order to further explore the reason as to why the UV-visible spectral absorbance of Al-Mal–BSA in the wavelength range of 340–360 nm does not fall to zero, we measured the individual ultraviolet-visible spectrum of Al-Mal solution with corresponding concentrations. It can be seen from Fig. S2 (ESI) that Al-Mal has a certain absorption value at 320–360 nm, and the measurement results are similar to the absorbance values of the Al-Mal–BSA mixture in the same wavelength range. Therefore, based on the results, it is presumed that the absorbance value of Al-Mal–BSA in this range may be related to the absorbance of the corresponding concentration of Al-Mal.

The UV spectrum obtained from the interaction of maltol with BSA is almost identical to that of Al-Mal.57 The only difference is that the increase in BSA absorbance after the addition of maltol is slightly slower than that of Al-Mal. The degree of binding of Al-Mal to BSA may be slightly stronger than that of maltol, indicating that Al(III) plays an important role in the combination of Al-Mal and BSA.

3.8 ANS fluorescence measurements

The hydrophobicity of the protein surface is a significant parameter of proteins and plays a decisive role in maintaining the stability and functional activity of proteins. ANS is a non-polar fluorescent probe. In aqueous solutions, when the ANS fluorescent probe is present alone, the fluorescence quantum yield is very low, but when it is bound to the hydrophobic region of the protein, the fluorescence intensity will be significantly increased, so that it is often used to characterize the surface hydrophobicity of proteins.

To investigate the effect of Al-Mal on the surface hydrophobicity of BSA, the ANS fluorescence spectra of the Al-Mal–BSA system was investigated and are shown in Fig. 6. These results showed that the hydrophobicity of BSA increased in the presence of Al-Mal, which was reflected by the changes in the ANS-binding fluorescence spectra and this occurred in a dose-dependent manner.58 This indicates that the formation of Al-Mal–BSA complex may lead to the exposure of some hydrophobic regions inside BSA, which leads to the structural change of BSA. This result is consistent with the results of the UV-visible absorption spectrum. The change in the ANS fluorescence intensity was less than the intrinsic fluorescence change, indicating that Al-Mal induced small changes in the hydrophobic surface of BSA.


image file: c9mt00088g-f6.tif
Fig. 6 Fluorescence quenching spectra of ANS bound BSA in the presence Al-Mal. λex = 380 nm, [BSA] = 5 × 10−6 M. The molar ratios [Al-Mal]/[BSA] = 0, 1.25, 2.5, 5, 10, 20 from “1” to “6”.

3.9 Molecular docking studies

After docking simulation using AutoDock, the binding conformation with the lowest binding energy was selected to obtain the two-dimensional interaction map between BSA and Al-Mal. The structure of the simulated BSA–Al-Mal composite is shown in Fig. 7. Molecular docking results indicate that Al-Mal is located in the subdomain of the site II. From the schematic diagram of the docking sites, it can be seen that the position of Al-Mal is closer to that of the Tyr-451 residue of BSA, which provides a good structural basis for quenching the fluorescence intensity of BSA after adding Al-Mal. The hydrogen bond interaction between Al-Mal and the residues of Ser-193 and Lys-431 indicates that the formation of hydrogen bond reduces the hydrophilicity of the complex and increases its hydrophobicity, thereby maintaining its stability. Therefore, molecular modelling results show that Al-Mal can bind to BSA, and the interaction between them is mainly via hydrophobic interactions and hydrogen bonds. The docking results are in good agreement with the previous experimental results.
image file: c9mt00088g-f7.tif
Fig. 7 The predicted conformation of BSA–Al-Mal complex with the lowest docking energy. BSA is shown in the animated form. The atoms of Al-Mal are color-coded as follows: C, green; H, white; O, red. The 2D detailed view shows the adjacent residues of the binding site of Al-Mal and BSA.

It can be observed that the changes in the ANS fluorescence data caused by Al-Mal is smaller than that of BSA endogenous fluorescence, indicating that Al-Mal has less influence on the hydrophobicity of BSA. Unlike most small molecules,59 the Al-Mal docking site is not located in the hydrophobic cavity near the Trp residue, which may affect the magnitude of the change in the ANS fluorescence spectrum. However, Al-Mal enhanced the ANS fluorescence intensity and showed a dose-dependent relationship. At the same time, an increase in the absorbance at 280 nm was observed in the UV-visible spectrum. Similarly, the formation of hydrogen bonds can be observed in the molecular docking results. These results indicate that Al-Mal enhances the hydrophobicity of BSA. Amino acid residues near the docking site of Al-Mal can also affect the hydrophobicity of BSA to some extent.

4. Conclusion

In conclusion, the fluorescence spectra and the UV-visible spectra reveal that Al-Mal and BSA form a ground state complex during the bonding process. The binding reaction is a spontaneous endothermic reaction. The binding force is mainly related to the hydrophobic force and hydrogen bonding. From three-dimensional fluorescence, circular dichroism and exogenous fluorescence spectroscopy studies, it was confirmed that the combination of Al-Mal and BSA increases the hydrophobicity of the protein and changes the secondary structure and amino acid polarity of BSA. By molecular docking, site II of BSA was identified as the most likely binding site for Al-Mal. These results will help to understand the molecular mechanism of the interaction between Al and serum albumin.

Conflicts of interest

No conflict of interest exists in the submission of this manuscript.

Acknowledgements

We thank all participants for their participation and kind assistance. This work was supported by funding from the National Natural Science Foundation of China (31801453), the Open Project Program of State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology (SKLFNS-KF-201829), the key program of the Foundation of Tianjin Educational Committee (2017ZD07), the fund of the Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU) and China Postdoctoral Science Foundation (2017M621059 and 2018T110194).

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c9mt00088g

This journal is © The Royal Society of Chemistry 2019