Insights into the Binding of 2-Aminobenzothiazole with Human Serum Albumin (HSA): Spectroscopic Investigation and Molecular Modeling Studies

Abstract

As one of the important thiazole derivatives, 2-aminobenzothiazole (2-ABT) has been widely used as a structural unit in the synthesis of antioxidants, anti-inflammatories, herbicides, antibiotics, and thermoplastic polymers. In this study, the interaction of 2-ABT with human serum albumin (HSA) was investigated in vitro under simulated physiological conditions, using multi-spectroscopic techniques and a molecular modeling study. The binding constant and binding sites were determined through fluorescence quenching spectra. The site-competitive replacement experiments revealed that the precise binding site of 2-ABT on HSA was site II (subdomain IIIA). Moreover, molecular docking results illustrated the electrostatic interaction between Glu 450 and 2-ABT, in accordance with the conclusions from the calculated thermodynamic parameters and the effect of ionic strength. The effect of 2-ABT on the conformational changes of HSA were evaluated by ultraviolet–visible (UV-Vis) absorption, three-dimensional (3D) fluorescence, synchronous fluorescence, and circular dichroism (CD) spectroscopy. This work facilitates comprehensive understanding of the binding of 2-ABT with HSA, contributing to evaluate the molecular transportation mechanism and biotoxicity of 2-aminobenzothiazole derivatives in vivo.

Keywords

Human serum albumin 2-Aminobenzothiazole Multispectroscopic technology Molecular modeling

INTRODUCTION

As a well-known model protein and the most abundant protein in the plasma (about 7.0 × 10⁻⁴ mol L⁻¹),¹ serum albumin serves as a carrier for many endogenous and exogenous compounds, including fatty acids, amino acids, vitamins, drugs, and contaminants.^2,3 Composed of a single polypeptide chain consisting of 585 amino acid residues,⁴ human serum albumin (HSA) is characterized by high α-helical content and many disulfide bonds.⁵

The compounds containing a thiazole ring have shown useful biological properties. For instance, one of these important thiazole derivatives, 2-aminobenzothiazole (2-ABT, Scheme 1) has been widely used as a structural unit in the synthesis of anti-oxidants, anti-inflammatories, herbicides, antibiotics, and thermoplastic polymers.⁶ The biotoxicity of 2-aminobenzothiazole derivatives, in other words, whether they can affect the structure and function of biomacromolecules, should be considered. In our previous research, we found that 2-ABT can interact with DNA in minor groove binding mode.⁷ There have been some other studies carried out in this field. Joanne Patman et al. synthesized a series of substituted 2-aminobenzothiazole compounds and evaluated them as nitric oxide synthase (NOS) inhibitors.⁸ Farukh Arjmand et al. indicated the strong binding between calf thymus DNA and the Cu (II) complex of a new ligand derived from the reaction of the Schiff base of 2-aminobenzothiazole and salicylaldehyde with KBH₄.⁹

Many studies on the interaction between organic materials and HSA at the molecular level have been reported, especially using optical spectroscopic methods.^10–14 In order to demonstrate the precise binding site of 2-ABT with HSA, molecular modeling studies were employed along with multi-spectroscopic technology. This combination of spectroscopic and molecular modeling study can be applied to investigate the interaction mechanism between small pollutants and biomacromolecules.^2,5,15

MATERIALS AND METHODS

Chemicals and Reagents. All chemicals were of analytical reagent grade and were used without further purification. All solutions were prepared using ultrapure water (18.25 MΩ cm).

HSA (Sigma) was dissolved to form a 5 × 10⁻⁵ mol L⁻¹ solution then preserved at 0–4 °C. This solution was further diluted as required.

We prepared a stock solution of 2-ABT (2.0 × 10⁻³ mol L⁻¹) by dissolving 0.03 g of 2-ABT purchased from TCI in 100 mL of water.

NaCl solution (0.5 mol L⁻¹) was prepared by dissolving 2.922 g of NaCl (Tianjin Damao Chemical Reagent Factory) into 100 mL of water.

Phosphate buffer (0.2 mol L⁻¹, mixture of NaH₂PO₄·2H₂O and Na₂HPO₄·12H₂O, pH 7.4) was used to control the acidity. NaH₂PO₄·2H₂O and Na₂ HPO₄·12H₂O were purchased from Tianjin Damao Chemical Reagent Factory.

Apparatus and Measurements. The fluorescence measurements were carried out as follows: to each 10 mL test tube, 1.0 mL of 0.2 mol L⁻¹ phosphate buffer (pH 7.4) and 1.0 mL of 5 × 10⁻⁵ mol L⁻¹ HSA were added, and then different amounts of 2-ABT were added. A series of samples were made at the same time and measured in order. All fluorescence spectra were recorded on an F-4600 fluorescence spectrophotometer (Hitachi Japan) with 1.0 cm path length fluorescence cuvette using 5/5 nm slit widths. The fluorescence emission spectra were measured at λ_ex = 278 nm (scan speed 1200 nm/min). The synchronous fluorescence spectra were collected at λ_ex = 250 nm, Δλ = 15 and 60 nm. The three-dimensional (3D) fluorescence spectra were excited at 200 nm and measured with the emission wavelength from 200 to 500 nm (scan speed 30 000 nm/min). The site-competitive replacement experiments were conducted as the addition of 2-ABT at different concentrations to the HSA-site marker system while the effect of ionic strength was investigated with the addition of varying amounts of NaCl solution.

Scheme 1.

Structure of 2-aminobenzothiazole (2-ABT).

Ultraviolet spectra were measured on a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan) equipped with a conventional quartz cell of 1.0 cm path length. The wavelength range of HSA was from 300 to 200 nm.

Circular dichroism (CD) measurements were made on a J-810 Spectropolarimeter (Jasco, Tokyo, Japan) in a 1.0 cm cell from 190 to 260 nm at room temperature and subtracted from the spectrum of buffer alone. Bandwidth was 1 nm and scanning speed was 200 nm/min. The content of the α-helix was calculated and compared at different molar ratios of 2-ABT/HSA.

All pH measurements were made with a pHs-3C acidity meter (Pen shun, Shanghai, China).

Molecular Modeling Study. The molecular modeling experiment was performed with AutoDock 4.0. The structure of 2-ABT was generated by Materials Studio 4.4. With the aid of AutoDock, the ligand root of 2-ABT was detected and rotatable bonds were defined.

The crystal structure of HSA was available in the Protein Data Bank (PDB ID: 1BJ5). Water was removed from the PDB file. Essential hydrogen atoms and Gasteiger charges were added with the aid of AutoDock tools (ADT). After enclosing in the grid defined by AutoGrid, which had 0.375 Å spacing, the grid map was calculated using the AutoGrid program. Maximum number of iterations was 300 and maximum number of successes/failures in a row was 4. Docking simulations were performed according to a local search. The output from AutoDock was rendered with RasMOL.

RESULTS AND DISCUSSION

Interaction Characteristics of 2-ABT with HSA. Fluorescence Quenching Spectra. Fluorescence measurement was employed to evaluate the interaction between 2-ABT and HSA. The emission spectra of HSA in the presence of different concentrations of 2-ABT are shown in Fig. 1. The intrinsic fluorescence of HSA was at 334 nm when the excitation wavelength was set at 278 nm, while 2-ABT displayed no fluorescence under the same conditions. The fluorescence intensity of HSA decreased gradually with 2-ABT increasing but almost no peak position shift had been observed, indicating that 2-ABT quenched the intrinsic fluorescence of HSA with little change to the local dielectric environments.

Fig. 1.

Fluorescence spectra of HSA with different concentrations of 2-ABT. Conditions: HSA: 5×10⁻⁶ mol L⁻¹; 2-ABT / (10⁻⁵ mol L⁻¹): ( a ) 0; ( b ) 1.6; ( c ) 3.2; ( d ) 4.8; ( e ) 6.4; ( f ) 8.0; ( g ) 9.6; ( h ) 11.2.

Quenching Mode Determination . Fluorescence quenching can be divided into dynamic quenching and static quenching. To determine the quenching mode of 2-ABT with HSA, the fluorescence maximums were determined using the dynamic quenching Stern–Volmer equation:^16,17

F_{0} / F = 1 + K_{q} τ_{0} C = 1 + K_{sv} C

(1)

where τ₀ is the average life expectancy of the fluorescent molecule, and F and F₀ are the fluorescence intensity of HSA with and without 2-ABT, respectively. K_q and K_sv are the quenching rate constant and Stern–Volmer quenching constant, respectively.

In this paper, K_q and K_sv at two different temperatures are listed in Table I. The fluorescence quenching of HSA by 2-ABT showed a linear relationship with its concentration. Generally, the fluorescent lifetime of biological macromolecules is approximately 10⁻⁸ s.¹⁸ At 297 K, the quenching constant K_q was then calculated to be 1.06×10¹² L mol⁻¹ s⁻¹ (K_sv was 1.06×10⁴ L mol⁻¹). This K_q is much bigger than the biggest diffusion control collision constant between the small molecular compound and biological macromolecule (2×10¹⁰ L mol⁻¹ s⁻¹).¹⁹ Therefore, the quenching type of HSA by 2-ABT should be static quenching from the formation of the ABT-HSA complex, rather than dynamic quenching.

TABLE I.

Stern–Volmer quenching constants for the interaction of 2-ABT with HSA at 297 K and 313 K.

pH	T (K)	K_sv (×10⁵ L mol⁻¹)	K_q (×10¹² L mol⁻¹ s⁻¹)	R ^a	S.D.^b
7.4	297	0.106	1.06	0.9954	0.0431
	313	0.092	0.92	0.9910	0.0530

R is the correlation coefficient.

S.D. is the standard deviation for the K_sv values.

Moreover, fluorescence spectra at two different temperatures were measured because static quenching is different from dynamic quenching by the temperature effects. As shown in Fig. 2, the quenching rate constant decreased as the temperature increased, confirming the static quenching mode.²⁰ The main reason was that increasing temperature adversely affected the stability of the ABT-HSA complex.²¹ On the contrary, K_q would increase because higher temperature leads to larger diffusion coefficients, which is characteristic of dynamic quenching.

Fig. 2.

Stern–Volmer plot of the fluorescence quenching of HSA by 2-ABT at 297 K and 313 K.

Binding Parameters and Binding Forces . Since the quenching mode has been determined to be static quenching, the binding constant (K_A) and the number of binding sites (n) can be calculated using Eq. 2.²²

lg [(F_{0} - F) / F] = lg K_{A} + n lg C

(2)

K_A and n, calculated from the double-logarithm curve (Fig. 3), are shown in Table II. The number of binding sites (n) was close to 1, indicating that 2-ABT interacted with HSA in the molar ratio of 1 : 1.

TABLE II.

Binding parameters and relative thermodynamic parameters of the ABT-HSA system.

T (K)	K_A (×10⁴ Lmol⁻¹)	n	R ^a	ΔH° (kJmol⁻¹)	ΔS° (Jmol⁻¹K⁻¹)	ΔG° (kJmol⁻¹)
297	3.62	1.138	0.9976	−17.69	27.71	−25.92
313	2.51	1.105	0.9857		27.70	−26.36

R is the correlation coefficient for the K_A values.

Fig. 3.

Plot of lg [(F₀ – F)/F)] versus lg [Q] for adding various amount of 2-ABT to HSA.

Non-covalent interactions between small molecules and biomacromolecules involve four modes of binding forces: hydrogen bonds, hydrophobic force, electrostatic force, and van der Waals' interactions. The thermodynamic parameters, such as the enthalpy (ΔH°) and entropy (ΔS°), are important in confirming the binding mode. If ΔH° > 0, ΔS° > 0, hydrophobic interaction plays a major role in the reaction; if ΔH° ≈ 0, ΔS°> 0, the main force is the electrostatic effect; if ΔH° < 0, ΔS° < 0, the main forces are van der Waals and hydrogen bond interactions.²³

Since the change of temperature has no significant effect, ΔH° can be considered a constant and can be approximated from Eq. 3. The free-energy change (ΔG°) and the entropy change (ΔS°) of the binding reaction follow Eqs. 4 and 5, respectively:

ln (\frac{K_{2}}{K_{1}}) = (\frac{1}{T_{1}} - \frac{1}{T_{2}}) (\frac{{Δ H}^{0}}{R})

(3)

{Δ G}^{0} = - R T ln K^{0}

(4)

{Δ G}^{0} = {Δ H}^{0} - T {Δ S}^{0}

(5)

where K₁ and K₂ are the binding constants (analogous to K_A in Eq. 2) at T₁ and T₂, and R is the universal gas constant.

The values of the thermodynamic parameters were ΔH° = −17.69 kJ mol⁻¹, ΔG° =–25.92 kJ mol⁻¹, and ΔS° = 27.71 J mol⁻¹ K⁻¹ at 297 K (shown in Table II). The negative sign for ΔG° and ΔH° means that the binding process was spontaneous and enthalpy-driven, respectively. Furthermore, the negative ΔH° and positive ΔS° indicate that electrostatic force played the major role during their interaction.

TABLE III.

Effects of site probe on the binding constant of 2-ABT to HSA.

K (without site probe; 10⁴ L mol⁻¹)	K (with PB; 10⁴ L mol⁻¹)	K (with FA; 10⁴ L mol⁻¹)	K (with Dig; 10⁴ L mol⁻¹)
3.62	2.72	0.78	4.07

Effect of Ionic Strength . In order to confirm the electrostatic force between 2-ABT and HSA directly, the influence of ionic strength on the fluorescence intensity of the ABT-HSA system was performed. Figure 4 demonstrates that the fluorescence intensity increased with the addition of NaCl.

Fig. 4.

Effect of ionic strength on fluorescence quenching. Conditions: 2-ABT: 5.0 × 10⁻⁶ mol L⁻¹; HSA: 5 × 10⁻⁶ mol L⁻¹.

When pH = 7.4, 2-ABT takes on a positive charge through protonation. There is competition between it and Na⁺ for the residues, which are negatively charged. As a consequence, the electrostatic force of the ABT-HSA system was gradually weakened by Na⁺. More and more free 2-ABT exists in the solution, leading to the increasing fluorescence intensity.

Specific Binding Sites Identification . HSA is a heart-shaped protein composed of three homologous domains called I–III, with each domain including two subdomains (A and B) to form a cylinder (see Fig. S1 in the supplemental material, available online).^2,3,24 The principal regions of binding sites for albumin are located in subdomains IIA and IIIA.²⁴ However, the data obtained above cannot accurately identify the correct binding site. Herein, site-competitive replacement experiments are necessary,^25–27 in which phenylbutazone (PB) for site I (subdomain IIA), flufenamic acid (FA) for site II (subdomain IIIA), and digitoxin (Dig) for site III are involved.²⁸

The changes in K_A after adding the site markers are shown in Table III. There is little difference in the absence and presence of phenylbutazone or digitoxin, suggesting 2-ABT was not displayed by these two drugs. Nevertheless, flufenamic acid gave a significant displacement of 2-ABT, indicating the binding site of 2-ABT on HSA was site II (subdomain IIIA).

Conformational Investigations. UV-Vis absorption, 3D fluorescence, synchronous fluorescence, and circular dichroism spectroscopy were employed in order to evaluate the effect of 2-ABT on the conformational changes of HSA.

Ultraviolet–Visible Absorption Spectroscopy . UV-Vis absorption spectroscopy has been widely used in the investigation of the structural changes of protein in the formation process of protein–ligand complexes. In this study, we measured the UV-Vis absorption spectra of HSA in different amounts of 2-ABT using 2-ABT in corresponding concentrations as the reference solution (Fig. 5). The strong absorption peak of HSA at about 208 nm reflects the framework conformation of the protein,^23,29 while the weak peak at about 280 nm represents the aromatic amino acids (Trp, Tyr, and Phe). As the concentration of 2-ABT increased, the intensity of the peak at 208 nm decreased gradually with a red shift. These results clearly indicated that the binding of 2-ABT to HSA induced loosening and unfolding of the protein skeleton.³⁰ Additionally, the static quenching was ascertained because the dynamic quenching can change the excited state of a fluorophore but has no effect on the absorption spectra while the formation of a non-fluorescence ground-state complex may change the absorption spectra of a fluorophore.^31,32

Fig. 5.

UV absorption spectra of HSA in the presence of different concentrations of 2-ABT (2-ABT in corresponding concentration was used as the reference solution). Conditions: HSA: 1×10⁻⁶ mol L⁻¹; 2-ABT / (10⁻⁵ mol L⁻¹): ( a ) 0; ( b ) 1.6; ( c ) 3.2; ( d ) 4.8; ( e ) 6.4; ( f ) 8.0.

Three-Dimensional Fluorescence Spectroscopy . Three-dimensional fluorescence spectroscopy is used to further investigate the conformation change of HSA after binding with 2-ABT. The 3D spectra of HSA and the ABT-HSA system are shown in Figs. 6A and 6B, respectively.

Fig. 6.

Three-dimensional fluorescence spectra of (A) HSA and (B) ABT-HSA system. Conditions: [HSA] = [ABT] = 2.5×10⁻⁶ mol ·L⁻¹; λ_ex = 200–320 nm, λ_em = 200–500 nm.

The peaks at the left and right were the first-order Rayleigh scattering peak (λ_em = λ_ex) and the second-order Rayleigh scattering peak (λ_em = 2λ_ex), respectively. Both of these two Rayleigh scattering peaks were enhanced after interaction because of the formation of the ABT-HSA complex, which led to the increase in the macromolecule diameter.

In the middle of the spectra, there were two intrinsic fluorescence peaks between two Rayleigh scattering peaks. The intensity of peak 1 (λ_ex = 230 nm and λ_em = 330 nm), which represented the fluorescent behavior of the polypeptide backbone structures, was related to the secondary structure of protein,²⁷ whereas peak 2 (λ_ex = 278 nm and λ_em = 330 nm) demonstrated the spectral behavior of the chromophore (mainly tryptophan and tyrosine residues).

After binding with 2-ABT, peak 1 decreases only a little (about 0.36%), suggesting that the peptide structure of HSA had been changed slightly. However, the quenching of peak 2 was much more obvious (4.34%) because the microenvironment of the chromophore may have been changed.

Synchronous Fluorescence Spectroscopy . Synchronous fluorescence spectroscopy is a common method to evaluate the molecular environment in the vicinity of a chromophore such as tryptophan (Trp) and tyrosine (Tyr), which involves simultaneous scanning of the excitation and emission monochromators while maintaining a constant wavelength interval between them. The shift in the emission maximum (λ_em) reflects the changes of polarity around the chromophore molecule.³³ Considering that tyrosine residues and tryptophan residues have similar excitation spectra and their conventional fluorescence spectra overlap strongly, the synchronous fluorescence spectra are characteristic of tyrosine and tryptophan when the wavelength intervals (Δλ) are stabilized at 15 and 60 nm, respectively.^34,35 The results at these two different wavelength intervals are presented in Figs. 7A and 7B.

Fig. 7.

Synchronous fluorescence spectra of HSA: (A) Δλ = 15 nm; (B) Δλ = 60 nm; (C) the quenching of HSA synchronous fluorescence by 2-ABT. Conditions: HSA: 5×10⁻⁶ mol L⁻¹; 2-ABT / (10⁻⁵ mol L⁻¹): ( a ) 0; ( b ) 1.6; ( c ) 3.2; ( d ) 4.8; ( e ) 6.4; ( f ) 8.0; ( g ) 9.6.

As the concentration of 2-ABT increased gradually, the synchronous fluorescence intensity decreased and both of the peaks of tyrosine and tryptophan residues shifted towards a longer wavelength significantly, which indicated that the hydrophobicity of these residues decreased and the residues buried in nonpolar hydrophobic cavities were moved to a more hydrophilic environment.³⁶ However, the slope was higher when Δλ was 60 nm (Fig. 7C), suggesting that 2-ABT was vicinal to tryptophan residues compared to tyrosine residues.

Circular Dichroism Spectroscopy . Circular dichroism (CD) is a sensitive technique to monitor the conformational changes in biomacromolecules, especially protein, upon interaction with pollutants or drugs. The CD spectra of HSA in the presence of different 2-ABT concentrations are shown in Fig. 8. It was inferred that the binding of 2-ABT to HSA induced obvious conformational changes in the secondary structure of HSA through the phenomenon that CD signal weakened, which was in accordance with the 3D fluorescence spectroscopy.

Fig. 8.

CD spectra of HSA in the presence of 2-ABT. Conditions: HSA: 1× 10⁻⁷ mol L⁻¹; 2-ABT / (10⁻⁷ mol L⁻¹): ( a ) 0; ( b ) 8; ( c ) 16.

The CD spectra of HSA exhibited two negative bands at about 208 and 218 nm in the ultraviolet region, which was characteristic for α-helix of proteins.²³ The amount of α-helix in the secondary structure of HSA after interaction with different concentrations of 2-ABT were calculated using Eqs. 6 and 7:

M R E = \frac{O b s e r v e d C D (m deg)}{C_{P} n l \times 10}

(6)

where C_P is the molar concentration of the protein, n is the number of amino acid residues (585), and l is the path length of the cell (1 cm).

α - H e l i x (%) = \frac{- M R E_{208} - 400}{33000 - 4000} \times 100

(7)

where MRE₂₀₈ is the observed MRE value at 208 nm, 4000 is the MRE of the β-form and random coil conformation cross at 208 nm, and 33 000 is the MRE value of a pure α-helix at 208 nm.

At different molar ratios of HSA to 2-ABT (1 : 8 and 1 : 16), the α-helicity decreased from 56.02% in free HSA to 47.46% and 44.03%, respectively. These results suggested that 2-ABT combined with the amino acid residues of the main polypeptide chain of HSA, which might affect its physiological function.

As mentioned above, the binding of 2-ABT to HSA can induce obvious conformational changes in the secondary structure of HSA, changing the microenvironment of residues.

Molecular Modeling Study. Molecular modeling studies have been widely used to ascertain the binding site on the basis of spectroscopy methods.^37,38 Since the binding site has been determined in the section above to be site II (subdomain IIIA), the simulation box should be set to this region, including Leu 387, Cys 392, Phe 395, Phe 403, Leu 407, Arg 410, Tyr 411, Leu 430, Cys 438, Arg 445, Ala 449, Glu 450, Leu 453, Arg 485, and so on. The docking results are shown in Fig. 9A while the precise binding site is illustrated in the online supplemental material in Fig. S2.

Fig. 9.

The binding mode between 2-ABT and HSA: (A) HSA is displayed in cartoons while the residues within 5 Å around 2-ABT are in surface mode. (B)

The positively charged 2-ABT mainly interacted with the negatively charged residues through electrostatic force, coincident with the conclusion above. Five residues within 5 Å around 2-ABT were demonstrated in Fig. 9B. The acidic amino acid Glu 450 displayed electrostatic attraction to 2-ABT, with the result that 2-ABT turned towards it. This phenomenon was aggravated by Arg 485, which was positively charged.

CONCLUSIONS

In this article, we investigated the interaction between 2-ABT and HSA at the molecular level. The electrostatic interaction binding mode was indicated by the calculated thermodynamic parameters and the effect of ionic strength. The precise binding site was illustrated using molecular modeling on the basis of site-competitive replacement experiments. Furthermore, HSA conformation change verified the formation of the ABT-HSA complex, which may affect the normal function of the transport protein during the blood transportation process. These results contribute to evaluate the biotoxicity or side effects of 2-aminobenzothiazole derivatives, such as anti-inflammatories, herbicides, and antibiotics.

Footnotes

ACKNOWLEDGMENTS

The work is supported by NSFC (20875055). Ministry of Education of China (708058), and Key Science-Technology Project in Shandong Province (2008GG10006012) are also acknowledged.

Notes

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