Introduction

A steel gray metal with corrosion-resistant properties, vanadium, is widely present in freshwater and seawater [1], and it is one of the most abundant heavy metals in the Earth’s crust, and the second most abundant transition element in the oceans [2]. Due to its widespread use across a multitude of industries such as smelting, foundry, textile, glass, and refinery [3], vanadium is regarded as the one of the most important elements for the twenty-first century [4].

Vanadium is mostly found in three oxidation states, + 3, + 4, and + 5 [5]. There are two main paths through which vanadium is absorbed in the organism: inhalation and ingestion [6]. The air in urban and industrialized areas is the most critical source of vanadium intake. Vanadium, in the form of V(IV) and V(V) oxides, VOx, is present in particulate form in the air, or absorbed to tiny dust particles and aerosols, and thus enters the lungs and the pulmonary system [7]. Studies have demonstrated that inhaled V2O5 causes pneumoconiosis [8]. There are two types of vanadium that are ingested orally: vanadate (H2VO4−, VV) present in drinking water and oxidovanadium (VO2+, VIV) [9]. Vanadium compounds possess a dual biological function: they are essential in trace amounts (0.05 μM) and toxic in excess (> 10 μM) [10]. Both deficiency and excessive levels of the compound can cause irreversible damage to various tissues and organs [11]. According to the International Agency for Research on Cancer (IARC), vanadium is listed in group 2B-possibly carcinogenic [12]. Generally, the toxicity of vanadium depends on many factors, such as the forms of vanadium complex (inorganic or organic), valence, dose, route of entry of this metal into the organism, and duration of exposure [13].

The two main serum proteins involved in vanadium species (VV and VIV) transport are human serum albumin and transferrin [14]. Serum albumin (SA) is the most abundant protein in plasma, and it has a wide range of physiological functions, including transportation, binding, and maintaining the osmotic pressure of the blood [15]. SA surface contains a number of binding sites, including hydrophobic pockets of varying sizes and shapes, as well as coordination domains that have sets of donor groups tailored to metals [16]. N-terminal binding site and multimetal binding site are two specific binding sites of metal ions in SA [17]. In most metal-protein interactions, BSA is commonly used as a model protein because its 76% structural homology with human serum albumin, stability, good physical properties, and low cost [18].

Many studies have found that vanadium compounds can change the structure of BSA [19,20,21,22,23]. However, the mechanism of various forms of vanadium in plasma is still unclear. The chemical form of a metal is key to understanding its toxicity and bioavailability [24]. Three forms of vanadium were selected as research objects in this study. The interaction mechanism between different forms of vanadium and BSA and the effect of two forms of vanadium on the structure of BSA were studied by multi-spectral method, which may provide reference for further understanding the toxic mechanism of different forms of vanadium to proteins.

Experimental

Materials

Oxidovanadium(IV) sulfate (VOSO4) was obtained from Xiya Reagent (China); sodium metavanadate (NaVO3) and oxidovanadium(IV) acetylacetonate (VO(acac)2) were obtained from Macklin (China); BSA was obtained from Sigma-Aldrich (USA). BSA stock solution was prepared in Tris–HCl buffer solution at pH 7.4. VOSO4/NaVO3 and VO(acac)2 stock solutions were prepared in water and ethanol, respectively.

Measurement of Fluorescence Spectra

Fluoromax-4NIR spectrofluorometer (Horiba, France) recorded the fluorescence spectra by setting at λex = 280 nm and λem = 290 ~ 500 nm. BSA solution (1.0 × 10−5 mol L−1) was successively titrated with VO(acac)2/NaVO3/VOSO4 both in the binary and the ternary system. In the binary system, the concentrations for VO(acac)2/NaVO3/VOSO4 were in range of 0 ~ 9.9 × 10−5 mol L−1. BSA and VOSO4/NaVO3 concentration were fixed at 1.0 × 10−5 and 5.0 × 10−5 mol L−1, respectively, and varied the concentration of VO(acac)2 from 0 to 9.9 × 10−5 mol L−1 in the ternary system.

The inner filter effects were eliminated by correction of fluorescence intensities using Eq. (1) [25].

$${F}_{\text{cor}}={F}_{\text{obs}}\times {e}^{\frac{{A}_{ex}+{A}_{em}}{2}}$$
(1)

The fluorescence intensities corrected and observed are represented by Fcor and Fobs represent. The absorbance of vanadium at excitation and emission wavelengths is represented by Aex and Aem, respectively.

Measurement of Three-Dimensional Fluorescence Spectra

The three-dimensional fluorescence spectra of BSA in the absence and presence of VO(acac)2/NaVO3/VOSO4 in the binary and the ternary system were recorded by setting λex = 200 ~ 500 nm with a 5-nm increment, and λem = 200 ~ 550 nm with a 2-nm increment.

Measurement of UV–vis Spectra

The absorption spectra were measured by a UV2800 spectrophotometer (Soptop, China) in the 200–300-nm range. BSA solution (1.0 × 10−6 mol·L−1) was successively titrated with VO(acac)2/NaVO3/VOSO4 both in the binary and the ternary system. In the binary system, the concentrations for VO(acac)2/NaVO3/VOSO4 was in range of 0 ~ 2.0 × 10−5 mol L−1. BSA and VOSO4/NaVO3 concentration were fixed at 1.0 × 10−6 and 1.0 × 10−5 mol L−1 and varied the concentration of VO(acac)2 from 0 to 2.0 × 10−5 mol L−1 in the ternary system. Sample spectra subtracted the spectra of buffer solutions without or with vanadium filters to correct background.

Measurement of Synchronous Fluorescence Spectra

The synchronous fluorescence spectra of BSA in the absence and presence of VO(acac)2/NaVO3/VOSO4 in the binary and the ternary system were recorded by setting the excitation and emission at a fixed wavelength interval of 15 nm and 60 nm, respectively.

Results and Discussion

Analysis of the Fluorescence Spectra

BSA fluorescence spectra at 350 nm (Fig. 1) decreased as the concentration of VO(acac)2/NaVO3/VOSO4 increased, indicating that VO(acac)2/NaVO3/VOSO4 quenched BSA fluorescence. VO(acac)2/NaVO3/VOSO4 quenched 68%, 37%, and 13% of BSA fluorescence at concentrations of 9.9 × 10−5 mol L−1, respectively. BSA fluorescence emission spectrum had a 4-nm redshift in the presence of VO(acac)2 and a 1-nm redshift in the presence of NaVO3, respectively, while VOSO4 did not significantly shift the BSA spectra, which implied that VO(acac)2 and NaVO3 affected the BSA conformation, especially VO(acac)2.

Fig. 1
figure 1

Fluorescence spectra of the BSA-vanadium system. CBSA = 1.0 × 10−5 mol L−1, Cvanadium = 0, 1.7, 3.3, 5.0, 6.6, 8.3, 9.9 × 10−5 mol L−1

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BSA fluorescence quenching is caused by dynamic and static quenching and described by the Stern–Volmer Eq. (2) [26].

$$\frac{{F}_{0}}{F}=1+{k}_{q}{\tau }_{0}\left[Q\right]=1+{K}_{sv}[Q]$$
(2)

where kq and Ksv represent the biomolecular quenching rate constant and Stern–Volmer quenching constant, respectively. F0 and F represent the fluorescence intensity of BSA in the absence and presence of VO(acac)2/NaVO3/VOSO4, respectively. τ0 represents the BSA lifetime. [Q] represents the concentration of VO(acac)2/NaVO3/VOSO4.

The main difference between dynamic quenching and static quenching is the relationship between quenching constant and temperature. As temperature increases, the complex becomes less stable during static quenching, leading to a reduction in Ksv, while higher temperature improves the collision efficiency during dynamic quenching, resulting in a rise in Ksv [27].

Table 1 showed that Ksv values in the VO(acac)2/NaVO3-BSA system reduced with rising temperature, whereas Ksv values in the VOSO4-BSA system increased with rising temperature, but all kq values were greater than 2.0 × 1010 L mol−1 s−1 (maximum diffusion collision quenching rate constant), indicating that the fluorescence quenching mechanism of VO(acac)2/NaVO3 to BSA was static whereas that of VOSO4 to BSA was a mixed process of dynamic and static [28]. The analysis of the three vanadium forms showed that the fluorescence quenching constants of VO(acac)2 to BSA were the largest, which suggested a stronger binding between VO(acac)2 and BSA.

Table 1 The interaction constants of VO(acac)2/NaVO3/VOSO4 and BSA
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Binding Constant

The binding affinity between VO(acac)2/NaVO3/VOSO4 and BSA can be measured by binding constant, which is calculated by Eq. (3) [29]:

$$log\left(\frac{{F}_{0}-F}{F}\right)=n\mathrm{log}[Q]+\mathrm{log}{K}_{b}$$
(3)

Kb and n represent the binding constant and number of binding sites, respectively. Table 1 showed the Kb and n values given by the intercept and slope of the log[(F0 − F)/F] vs log[Q] plot. The Kb values of different forms of vanadium and BSA differed greatly. VO(acac)2 and BSA had the largest Kb, about 105 L mol−1, indicating that VO(acac)2 formed a stable complex with BSA. A large Kb value may mean that VO(acac)2 was not metabolized very quickly by the body, and its accumulation in the blood may increase the risk of toxicity [30]. In reference [17], BSA assumed 1:1 complexes and a log K value of 3.0. Using electron paramagnetic resonance (EPR) spectroscopy, Sanna [22] found that VO2+ is complexed with serum transferrin and albumin. It was found that albumin could coordinate VO2+ ions and HSA could coordinate up to five to six equivalents of metal ions. A dinuclear complex (VO)2d HSA is formed in equimolar aqueous solutions or with an excess of protein.

Binding Forces

In the complex formed by ligands and BSA, there are four binding forces: hydrophobic interaction, electrostatic interaction, Van der Waals force, and hydrogen bond according to the summary of Ross et al. [31]. The thermodynamic parameters (ΔH, ΔS, and ΔG), which are obtained using the Van’t Hoff Eq. (4) and Eq. (5), are used to consider the binding forces.

$$\mathrm{ln}{K}_{b}=-\frac{\Delta H}{RT}+\frac{\Delta S}{R}$$
(4)
$$\Delta G=\Delta H-T\Delta S$$
(5)

From the calculated ΔH, ΔS, and ΔG (Table 1), it can be seen that the ΔH and ΔS of VO(acac)2/NaVO3-BSA system were both negative values, while in VOSO4-BSA system were positive values, suggesting that the binding forces of different forms of vanadium and BSA were different. VO(acac)2/NaVO3 bound to BSA by Van der Waals forces, while VOSO4 bound to BSA hydrophobic interaction.

Binding Distance

The binding distance between BSA and VO(acac)2/NaVO3/VOSO4 is given by Eq. (6) [32].

$$E=1-\frac{F}{{F}_{0}}=\frac{{R}_{0}^{6}}{({R}_{0}^{6}+{r}^{6})}$$
(6)

r and E represent the binding distance and energy transfer efficiency, respectively. R0 was estimated using Eq. (7) as the critical distance with 50% E.

$${R}_{0}^{6}=8.79\times {10}^{-25}{k}^{2}{N}^{-4}\Phi J$$
(7)

where k2, Φ, and N represent the spatial orientation factor of the dipole, the fluorescence quantum yield, and the average refractive index of the medium, respectively.

Equation (8) calculates J as an integral of the fluorescence emission spectrum of BSA and the absorption spectrum of VO(acac)2/NaVO3/VOSO4.

$$J=\frac{\sum F(\lambda )\varepsilon (\lambda ){\lambda }^{4}\Delta \lambda }{\sum F(\lambda )\Delta \lambda }$$
(8)

The adjusted fluorescence intensity of BSA in the wavelength range of λ-(λ + Δλ) and the molar absorption coefficients of VO(acac)2/NaVO3/VOSO4 are represented by F(λ) and ɛ(λ), respectively.

Nonradiative energy transfer occurs when the spectrum overlap between the energy donor and receiver is sufficient, and the distance between them is less than 7 nm according to Förster’s theory [33]. Energy transfer between BSA and VO(acac)2/VOSO4/NaVO3 was quite likely, according to the results in Table 2. The smallest r value in VO(acac)2-BSA system may indicated that VO(acac)2 bound to BSA more easily and may also explained why VO(acac)2 had greater fluorescence quenching on BSA [34].

Table 2 The values of J, E, R0, and r of the VO(acac)2/NaVO3/VOSO4 and BSA
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UV–vis Absorption Spectroscopy

The absorption spectra of BSA with different concentrations of VO(acac)2/VOSO4/NaVO3 (Fig. 2) were used to study BSA structure changes and the formation of vanadium-BSA complex.

Fig. 2
figure 2

UV–vis absorption spectra of BSA-vanadium system. CBSA = 1.0 × 10−6 mol L−1, Cvanadium = 0, 0.3, 0.6, 1.0, 1.3, 1.6, and 2.0 × 10−5 mol L−1. Insert: CBSA = 1.0 × 10−5 mol L−1, Cvanadium = 0, 0.3, 0.6, 1.0, 1.3, 1.6, and 2.0 × 10−4 mol L−1

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BSA absorption spectra at 214 nm decreased as the concentration of VO(acac)2/NaVO3/VOSO4 increased and with a slightly redshift. The change of the absorption peak corresponding to the peptide chain conformation of BSA [35] indicated that the secondary structure of BSA changed as VO(acac)2/NaVO3/VOSO4 binds to it. With the concentration of 2.0 × 10−5 mol L−1 VO(acac)2, the absorption peak of BSA shifted by 2 nm and the intensity decreased by 18%, which was more evident than the NaVO3-BSA system and VOSO4-BSA systems. In the presence of VO(acac)2, BSA absorption spectra at 278 nm showed a 3 nm blue shift and an obvious decrease, but NaVO3/VOSO4 had no significant effect on BSA absorption. According to the UV spectra, the change in this absorption peak corresponded to the microenvironment polarity of around amino acid residues, indicating that VO(acac)2 had a greater influence on the conformation of BSA than NaVO3/VOSO4.

Three-Dimensional Fluorescence Spectra

The three-dimensional fluorescence spectra of BSA with VO(acac)2/NaVO3/VOSO4 (Fig. 3) are used to study conformational changes of BSA. Two characteristic fluorescence peaks (peak 1 and peak 2) and a Rayleigh scattering peak (peak a) were observed. Peak 1 is associated with the fluorescence of amino acid residues, while peak 2 is associated with fluorescence spectral characteristics of the BSA polypeptide chain backbone [36].

Fig. 3
figure 3

The 3D fluorescence spectra of BSA-vanadium system. CBSA = 1.0 × 10−4 mol L−1, Cvanadium = 2.0 × 10−4 mol L−1

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As a result of binding to VO(acac)2/NaVO3/VOSO4, peak 1 and peak 2 intensities and positions of BSA changed, suggesting that the BSA polypeptide chain structure and polarity of tyrosine (Tyr) residues and tryptophan (Trp) residues changed. VO(acac)2 quenched the fluorescence intensity of peak 1 and peak 2 by 89% and 74%, respectively, and both peak 1 and peak 2 were redshifted by 10 nm. NaVO3/VOSO4 made peak 1 and peak 2 redshift 2 nm. The fluorescence quenching of BSA by NaVO3 was much greater than that by VOSO4. VO(acac)2/NaVO3/VOSO4 caused the changes in BSA backbone structure and amino acid residues microenvironment. VO(acac)2 had the greatest impact on the conformation of BSA, followed by NaVO3.

Synchronous Fluorescence Spectra

The synchronous fluorescence spectra of BSA with different concentrations of VO(acac)2/NaVO3/VOSO4 (Fig. 4) are used to study microenvironment changes of Tyr residues and Trp residues. Synchronous fluorescence spectra at 15- and 60-nm wavelength intervals are characteristic for Tyr residues and Trp residues, respectively [37]. VO(acac)2/NaVO3/VOSO4 quenched the fluorescence intensity of Tyr residues and Trp residues. Tyr spectra were accompanied by obvious redshift, which indicated that VO(acac)2/NaVO3/VOSO4 increased the microenvironment polarity around Tyr residues. VO(acac)2 made Tyr spectra redshift 4 nm and quenched fluorescence 82%, NaVO3 made Tyr spectra redshift 4 nm and quenched fluorescence 51%, and VOSO4 redshifted Tyr by 2 nm and quenched fluorescence by 37%. According to synchronous fluorescence spectra, NaVO3 induced the maximum shift in Trp residue, whereas VO(acac)2 induced the maximum fluorescence quenching of Trp residue. VO(acac)2 had the greatest effect on BSA conformation, followed by NaVO3. These results were in agreement with those of 3D spectra.

Fig. 4
figure 4

The synchronous fluorescence spectra of BSA-vanadium system. CBSA = 1.0 × 10−5 mol L−1, Cvanadium = 0, 2.0, 4.0, 6.0, 7.9, and 9.9 × 10−5 mol L−1

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Fourier transform infrared spectra

The secondary structure change of BSA induced by VO(acac)2/NaVO3/VOSO4 were further studied by FTIR spectra (Fig. 5). BSA exhibits a number of FTIR bands, out of which amide I and amide II related to the secondary structure of protein and polypeptide carbonyl hydrogen bonding pattern, respectively [38]. There was a shift in the amide I band (1600–1700 cm−1) and amide II (1500–1600 cm−1) under the presence of vanadium, which suggested vanadium binding with BSA disturbed its secondary structure, and verified the formation of BSA-vanadium complex.

Fig. 5
figure 5

The FTIR spectra of BSA-vanadium system. CBSA = 4.0 × 10−4 mol L−1, Cvanadium = 1.0 × 10−3 mol L−1

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Most investigations concentrate on the amide I band which is attribute to the stretching vibrations (C = O) absorption. Amide I band is an adjoint band of vibrational peaks of different secondary structures of BSA, such as α-helix, β-sheet, random coil, β-turn, and anti-parallel β-sheet, which makes the amide I band more sensitive to BSA secondary structure changes. Table 3 gives the quantitative results of each secondary structure content in the BSA-vanadium system. Binding of vanadium reduced the content of α-helix and β-turn in BSA, while increasing β-sheet, random coil, and anti-parallel β-sheet. During this study, all three forms of vanadium were found to alter the secondary structure of BSA. The α-helix are the main secondary structure of BSA, and from changes to the α-helix contents, VO(acac)2 had the largest effect on BSA secondary structure, followed by NaVO3 and VOSO4. Through FTIR, FT-Raman, and UV–Vis spectroscopy, Ferrer [20] investigated the ability of vanadate and vanadyl ions to produce conformational changes in BSA. As a result of incubating with oxovanadium (IV) and vanadate (V) species, the typical 1657 cm−1 band in the BSA spectrum was shifted toward lower frequencies. A curve-fitting procedure indicated a decrease in a-helix content and an increase in β-structures, turns, and random coils.

Table 3 The secondary structure content of the BSA-vanadium system
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The Effect of NaVO3/VOSO4 on the Interaction Between VO(acac)2 and BSA

According to the above experimental results, among the three forms of vanadium studied, VO(acac)2 had the strongest binding affinity with BSA and the most significant effect on the BSA conformation, which suggested that the interaction mechanisms between VO(acac)2 and BSA should be further studied. Since different forms of vanadium commonly coexist in vivo, we further investigated the binding process of VO(acac)2 and BSA in the presence of NaVO3/VOSO4. According to Table 1, the binding constants and thermodynamic constants of VO(acac)2 and BSA in the ternary system changed. The effect of NaVO3 appeared more obvious, not only increasing the binding affinity of VO(acac)2 and BSA, but also affecting their binding forces. These results suggested that NaVO3/VOSO4 participated in and influenced the binding of VO(acac)2 to BSA. The Kb values in the ternary system changed possibly due to competition and/or allosteric effect.

Binding Site

BSA provides characterized sites in different subdomains for various ligands to bind. Warfarin has been shown to bind to sub-domain IIIA (site I) of BSA, while ibuprofen bind to sub-domain IIA (site II) of BSA [39]. Therefore, warfarin and ibuprofen were used as site probes in competitive binding experiments to identify the binding site of VO(acac)2/NaVO3/VOSO4 on BSA. It was observed that the Kb values of vanadium-BSA system (Table 4) changed little in the presence of ibuprofen, whereas they changed significantly in the presence of warfarin, suggesting that ibuprofen and VO(acac)2/NaVO3/VOSO4 did not compete with each other for binding to BSA, while warfarin competed with VO(acac)2/NaVO3/VOSO4 for binding to the same site of BSA. Thus, it was speculated that VO(acac)2/NaVO3/VOSO4 bound to BSA in site I.

Table 4 The binding site parameters of vanadium-BSA system
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VOSO4 and VO(acac)2 bound to the same site on BSA, so there was probably binding competition between VO(acac)2 and VOSO4, which may explain why the binding constants of VO(acac)2 and BSA decreased in the presence of VOSO4. NaVO3 and VO(acac)2 are also bound to BSA competitively, but the Kb values of VO(acac)2 and BSA increased at 303 and 310 K in the presence of NaVO3, suggesting that another factor influenced the binding of VO(acac)2 and BSA besides competition. From fluorescence, three-dimensional fluorescence, and synchronous fluorescence, all three forms of vanadium changed the structure of BSA. However, NaVO3 had a greater effect than VOSO4, that is, NaVO3 altered the secondary structure of BSA and made it more strongly bound to VO(acac)2. This allosteric effect was more significant than that of competition [40, 41]. Using quantitative EPR measurements and circular dichroism (CD) data, Correia [19] concluded that in the (VIVOL2)n(HSA) species, the protein binds to vanadium through histidines.

Conclusion

The interaction mechanisms of binding affinity and binding forces between different forms of vanadium and BSA were different. The fluorescence quenching of BSA by VO(acac)2/NaVO3 was static quenching, while quenching by VOSO4 was dynamic and static mixed quenching. VO(acac)2/NaVO3 bound to BSA mainly through van der Waals forces, while VOSO4 bound to BSA mainly through hydrophobic interaction. The binding constants were quite different. VO(acac)2 had the strongest binding ability with BSA and had the most influence on BSA conformation. The binding constants of VO(acac)2 and BSA increased, and the main force changed to hydrophobic interaction in the presence of NaVO3. In general, it is believed that strong binding affinity means slower metabolism and more accumulation, which can cause harmful consequences [42]. It is therefore important to closely study the binding process of different forms of vanadium to transport proteins, especially the effect of vanadium on the structure and function of serum albumin when various forms of vanadium coexist.