Comparative binding study of anti-tuberculosis drug pyrazinamide with serum albumins

Abstract

The interaction of anti-tuberculosis drug pyrazinamide (PYZ) with serum albumins (HSA and BSA) has been studied using spectroscopic and molecular docking approaches. The effects of PYZ on the protein conformation, topology and stability were determined using Circular Dichroism (CD), Dynamic Light Scattering (DLS) and Differential Scanning Calorimetry (DSC). The obtained binding constant (K_b) was ∼10⁴ M⁻¹ for both HSA and BSA, although a higher affinity of PYZ was found with BSA. A higher value for the Gibbs free energy reflects that PYZ interacts more favourably with BSA. A reduction in the hydrodynamic radii and increase in the secondary structural content of the protein confirm that the serum albumins stabilize on binding with PYZ. Furthermore, elevation of the transition melting point also supports the stabilizing action of PYZ. Furthermore, site specific markers and a molecular docking study confirmed the binding location of PYZ with HSA and BSA. The present study will be helpful for understanding the binding of PYZ and associated alterations in the stability and conformation of serum albumins.

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1. Introduction

Investigations of interactions of drugs with proteins have received considerable attention in the fields of chemistry, life sciences and clinical medicines for decades. Thus protein–drug interactions play a significant role in the regulation of biological functions. The nature and extent of these interactions influence the bio-safety, pharmacological response, delivery rate, therapeutic efficacy and design of new drugs.¹ Furthermore, the drug distribution, free concentration and metabolism are significantly affected by protein–drug interactions in the blood stream.² During a chemotherapeutic process, this phenomenon also has an influence on the drug stability and toxicity.³ Studying the interaction of anti-tuberculosis drugs with proteins is also essential for understanding their mode of action and the structural specificity of their binding reactions. These interactions also provide a structural guideline for rational drug design. This will help in the synthesis of novel and enhanced drug entities with greater clinical efficacy, a more selective activity, and lower toxicity level. The proteins which are commonly used as a carrier for drugs are serum albumins, lipoproteins, and glycoproteins. Serum albumins, the most abundant proteins found in systematic circulation in the plasma (50–60%), are synthesized in parenchymal cells of the liver and exported as a non-glycosylated proteins. Serum albumins act as a storehouse for the interacting bioactive material and also can be circulated through the body. The majority of drugs bind to albumin in a reversible manner and it functions as a carrier.^4–8 Binding of serum albumin to hydrophobic drugs also enhanced the solubility and modulated the delivery efficiency into the cell.⁹ It also has an influence on the pharmacokinetic and pharmacodynamic actions of the drugs. If the binding affinity is low the initial step of the pharmacokinetic process (drug absorption) is not feasible, while moderate binding indicates feasibility for absorption and distribution of the drugs. However, with a strong binding affinity, due to the stability of the complex (protein–drug), the drug distribution is limited to the required tissue, which in turn adversely effects the pharmacokinetics of the drugs. Particularly, serum albumins serve as a transport vehicle for several endogenous compounds including fatty acids, hemin, bilirubin, and tryptophan in the human body. They also maintain the osmolarity of the blood with respect to the tissue.^8,10–12 They also possess some enolase, esterase, and hydrolase activity. Additionally albumins play influential roles in drug deposition and preservation of the blood colloid osmotic pressure in the body.¹³

Human and bovine serum albumin are globular, non-glycosylated proteins that consist of 585 and 583 amino acid long single polypeptide chains. Both have multiple binding sites, which are distributed over the surface of the protein, that bind various ligands with high affinity.^13–16 They have three binding domains viz domain I, II and III; and they are further divided into A and B subdomains. There are two Sudlow sites; site I and site II, which correspond to subdomains II A and III A that bind drugs, ions and endogenous hormones. Site I has a hydrophobic wall and the opening is lined with positively charged residues, which enables them to bind different ligands. Site II is smaller and less flexible than site I.¹⁷ The binding interactions offered by site II comprise of hydrogen bonding, electrostatic interactions as well as hydrophobic interactions; whereas site I only offers hydrophobic interactions.

PYZ is an important drug that shortens tuberculosis therapy by disrupting the membrane energetics and inhibiting the membrane transport function in Mycobacterium tuberculosis.¹⁸ PYZ has attracted much attention due to its pharmacological properties in combination with rifampicin and isoniazid. It is a nicotinamide analog prodrug that is converted from pyrazinoic acid into a bactericidal form by the bacterial enzyme pyrazinamidase.¹⁹ Among therapeutic molecules, this drug is more considered for use because it is more active against a population of persistent, non-growing, tubercle bacteria than other tuberculosis drugs.^20,21 PYZ has been reported to act against female genital tuberculosis in conjugation with rifampin,²² as an antiuricosuric drug.²³ It seems to have non-specific targets and exerts anti-mycobacterial effects through various means like disruption of the cell membrane and acidification of the cytoplasm, which are required for Mycobacterium tuberculosis to persist and survive.²⁴ In spite of the vast pharmacological properties of PYZ, its mode of binding with serum albumins has not been elucidated. It is thus pertinent to study the interaction of PYZ with serum albumins to reveal how this drug could be modified to enhance its biological activities.

In the present study, the mode of binding of serum albumins with PYZ was studied using fluorescence spectroscopy and other biophysical techniques. Serum albumins exhibit marked changes in their fluorescence properties upon binding with ligands, as compared to their spectral characteristics when free in solution. Various thermodynamic parameters were also calculated. Binding to a drug causes alteration of the conformation and stability of the protein. A circular dichroism study was performed to gain insight into the structural changes that occurred due to binding with PYZ. Dynamic light scattering was also used to monitor the structural changes that occurred due to protein–drug interactions. We monitored the stability of the protein using differential scattering calorimetric (DSC) techniques. Furthermore, molecular docking was performed to reveal the involved amino acid residues and relative binding energies of the complex formed.

2. Materials and methods

Human serum albumin (A1887), bovine serum albumin (A7030) and PYZ (P7136) were purchased from Sigma Aldrich, India. All of the other reagents used were of analytical grade.

2.1 Sample preparation

HSA and BSA (100 μM) stock solutions were prepared using 20 mM phosphate buffer (pH 7.4). Protein concentrations were determined spectrophotometrically using the extinction coefficient at 280 nm, using a Perkin-Elmer Lambda double beam UV-visible spectrophotometer.^25,26 A PYZ solution (5 mM) was prepared using 20 mM phosphate buffer (pH 7.4) and diluted to various concentrations using the same buffer. All experiments were carried out in 20 mM phosphate buffer (pH 7.4) at 298 K.

2.2 pH determination

pH measurements were carried out with a Mettler Toledo pH meter (Seven Easy S20-K) using an Expert “Pro3 in 1” type electrode. The lowest alteration in the pH registered by the pH meter was 0.01 pH units.

2.3 Steady state fluorescence quenching measurements

Fluorescence measurements were performed using a Shimadzu fluorescence spectrophotometer, model RF-5301. The fluorescence spectra were obtained at 25 ± 0.1 °C with a 1 cm path length cell. Both the excitation and emission slits were set at 5 and 10 nm, respectively. The intrinsic fluorescence was measured by exciting the protein solution at 280 nm and 295 nm emission spectra were recorded in the range of 300–460 nm. Titration of PYZ (0–60 μM) into BSA and HSA (5 μM) solutions was carried out at 298 K.

The fluorescence quenching data were analysed according to the Stern–Volmer equation.²⁷

F₀/F = 1 + K_sv[Q] (1)

where the F₀ and F represent steady-state fluorescence intensities in the absence and presence of PYZ, respectively. K_sv is the Stern–Volmer quenching constant and [Q] is the concentration of the compound. K_sv was calculated using a linear regression plot of F₀/F versus [Q]. An inner filter correction was applied for the fluorescence intensities of the protein and ligand using the following equation:²⁸where F_corrected and F_observed are the corrected and observed fluorescence intensities, D is the dilution factor, and A_{280/295 nm} and A_{340 nm} are the sum of the absorbance of the protein and ligand at the excitation (280/295 nm) and emission (340 nm) wavelengths, respectively. Furthermore, to establish the mechanism of the quenching, whether it is static or dynamic, k_q was calculated using the following equation:

K_sv = k_qτ₀ (3)

where K_sv is the Stern–Volmer constant, k_q is the bimolecular quenching constant and τ₀ is the lifetime of the fluorophore in the absence of quencher. In static quenching, the quencher forms a stable complex with the molecule, however, in dynamic quenching, the quencher indirectly interacts with the molecule.

In order to determine the number of binding sites and other binding parameters. A modified Stern–Volmer equation was used.⁹

log(F₀/F − 1) = log K_b + n log[Q] (4)

where, the F₀ and F represent steady-state fluorescence intensities in the absence and presence of PYZ respectively, K_b is the apparent binding constant, n is the number of binding sites and Q is the concentration of the quencher i.e. PYZ. The fluorescence intensity at 340 nm was used to calculate the binding constant (K_b). Prior to the fluorescence spectra measurements, respective blank measurements were subtracted.

2.4 Time resolved fluorescence measurements

Time resolved fluorescence spectra were obtained using a time resolved single photon counting setup on a FL920 spectrometer (Edinburgh Instruments, UK). The excitation wavelength was set at 295 nm and 280 nm respectively for HSA and BSA. The instrument response function (IRF) was obtained using a LudoxTM suspension. The emission decay data at 340 nm and 350 nm respectively for HSA and BSA were analysed using FAST software, available freely online.

2.5 Synchronous fluorescence spectroscopy

Synchronous fluorescence spectroscopy was carried out by simultaneously scanning the excitation and emission mono-chromators. Conformational alteration around the tyrosine and tryptophan residues of HSA could be observed when the wavelength interval (Δλ) was 15 and 60 nm, respectively. The concentration of BSA and HSA was 5 μM and that of PYZ varied in the range of 0–60 μM. All parameters were kept the same as discussed in above section.

2.6 Circular dichroism measurements

Far-UV CD measurements (190–250 nm) were carried out using a JASCO-J815CD spectrometer equipped with a Peltier-type temperature controller at 298 K, using a quartz cell with a path length of 0.1 cm. Three scans were accumulated at a scan speed of 100 nm min⁻¹. 1 : 0, 1 : 5 and 1 : 12 molar ratios of BSA–PYZ and HSA–PYZ were taken. The percentage of alpha helical content was calculated using the Chen et al. method.²⁹

Near UV-CD measurements were carried out using a protein concentration of 15 μM, and 1 : 0, 1 : 5 and 1 : 12 molar ratios of BSA–PYZ and HSA–PYZ were used.

2.7 UV-visible spectroscopy measurements

UV-visible absorption measurements were carried out using a Perkin-Elmer Lambda 25 double beam UV-visible spectrophotometer attached to a Peltier temperature programmer 1 (PTP-1). A cuvette with a path length of 10 mm and a scanning speed of 1000 nm min⁻¹ were used. The concentration of protein was kept at 25 μM and 1 : 0, 1 : 05 and 1 : 12 molar ratios of HSA to PYZ were used.

2.8 Dynamic light scattering (DLS) measurements

DLS measurements were carried out at 830 nm using DynaPro-TC-04 dynamic light scattering equipment (Protein Solutions, Wyatt Technology, Santa Barbara, CA) equipped with a temperature-controlled micro-sampler. BSA and HSA (2 mg ml⁻¹) were incubated with PYZ for 12 h. The samples were spun at 10 000 rpm for 10 min and were filtered serially through 0.4 and 0.22 μm Whatman syringe filters directly into a 12 μL quartz cuvette. For each experiment, 20 measurements were carried out. The mean hydrodynamic radius (R_h) and polydispersity were analysed, using Dynamics 6.10.0.10 software at optimized resolution. Angle of detection was set at 90°. The R_h was estimated on the basis of an autocorrelation analysis of scattered light intensity data, and based on a translational diffusion coefficient, using the Stokes–Einstein relationship:where R_h is the hydrodynamic radius, k is the Boltzmann constant, T is temperature, η is the viscosity of water and D is the diffusion coefficient.

2.9 Differential scanning calorimetry

The differential scanning calorimetry measurements were carried out using a VP-DSC micro-calorimeter (Micro Cal, Nortampton, MA). Samples were prepared in phosphate buffer (pH 7.4). Prior to the experiment, the samples were degassed properly using a thermovac. The DSC measurements of HSA and BSA in the absence and presence of PYZ in a 1 : 12 ratio were performed from 25 °C to 90 °C at a scan rate of 1 °C min⁻¹. Furthermore, the data were analyzed using origin software provided with the instrument to obtain the unfolding transition (T_m) point.

2.10 Molecular docking and drug displacement experiments using site specific markers

A molecular docking study was performed using Autodock 4.2 and Autodock tools (ADT) using a Lamarckian genetic algorithm.³⁰ The crystal structures of BSA (PDB id: 4F5S) and HSA (PDB id: 1AO6), and a three dimensional structure of PYZ (CID: 1046) were obtained from the Brookhaven Protein Data Bank and PubChem, respectively. Chain A of the protein was selected, water molecules and ions were removed, and all the hydrogen atoms were added. Then partial Kollman charges were assigned to the protein. The protein was set to be rigid and there was no consideration of solvent molecules on docking. The grid size was set to be 120 Å, 120 Å and 120 Å along the X, Y and Z axes with a 0.534 Å grid spacing. The autodock parameters used were a GA population size of 150 and a maximum number of energy evolutions of 2 500 000. The 10 best solutions based on the docking score were retained for further analysis. Discovery studio 3.5 was used for visualization and for identification of the residues involved in binding. Different site specific markers, warfarin for site I and diazepam for site II, were used for performing displacement experiments.^31–33 Titration of PYZ was carried out, with the solution having the protein and site marker in a molar ratio of 1 : 1. The fluorescence emission spectra were recorded in a similar way as mentioned for the fluorescence measurements and the K_sv values for drug–protein–marker were evaluated using the Stern–Volmer equation.

3. Results and discussion

3.1 Steady state fluorescence quenching measurements

Fluorescence spectroscopy has been widely used to study protein–ligand interactions, which provides information on the mechanism of fluorescence quenching, binding constants and the number of binding sites.^34–38 The contribution from tryptophan fluorescence is very large compared to tyrosine and phenylalanine in the intrinsic fluorescence of the protein. BSA contains two tryptophan residues (Trp 134 and Trp 212), whereas HSA contains only one tryptophan residue (Trp 214).^39,40 The fluorescence intensities of the serum albumins in the absence and presence of PYZ, after excitation at 280 nm, are shown in Fig. 1. Furthermore, the fluorescence spectra after excitation at 295 nm are shown in ESI Fig. 1.† It can be clearly observed from the figure that the fluorescence intensity continuously decreases with increase of the concentration of PYZ, but a greater decrease in the fluorescence intensity for BSA shows a prominent interaction of PYZ with BSA, compared to HSA. The bimolecular quenching constant (k_q) values for PYZ binding with HSA and BSA were found to be 4.07 × 10¹² M⁻¹ s⁻¹ and 1.69 × 10¹² M⁻¹ s⁻¹ (at 280), and 3.64 × 10¹² M⁻¹ s⁻¹ and 8.60 × 10¹² M⁻¹ s⁻¹ (at 295), respectively, which are greater than the maximum collision quenching constant 2 × 10¹⁰ M⁻¹ s⁻¹. This inferred that HSA and BSA interact with PYZ through a static quenching mechanism. The binding constant and number of binding sites can be obtained by plotting a graph of log(F₀/F − 1) versus log[Q] as shown in Fig. 2 and the ESI Fig. 1.† All the binding parameters are summarised in Table 1. It is clear from the figures and Table 1 that PYZ binds with more affinity to BSA (∼10 times), compared with HSA. Although a contribution from dynamic quenching could not be denied at this stage, in order to make clear the process of the quenching, time resolved fluorescence measurements were carried out, which is discussed in the subsequent section. Furthermore, to check the specific binding pattern, selective excitation of tryptophan was done by exciting the protein at 295 nm and the fluorescence intensity profiles of BSA and HSA after excitation at 295 nm are shown in the ESI Fig. 1.†

Fig. 1 PYZ-induced fluorescence quenching of (A) HSA and (B) BSA. The concentration of protein was 5 μM and the concentration of PYZ was varied from 0–60 μM. The intrinsic fluorescence of the proteins was measured upon excitation at 280 nm in 20 mM sodium phosphate buffer, pH 7.4 at 298 K.

Fig. 2 (A) Stern–Volmer plot for the HSA/BSA–PYZ interactions. (B) Modified Stern–Volmer plot for the HSA/BSA–PYZ interactions at 298 K.

Table 1 Stern–Volmer quenching constants and binding parameters for the HSA/BSA–PYZ interactions at 298 K

Wavelength (nm)	Protein	Ksv (M^−1)	kq (M^−1 s^−1)	Kb (M^−1)	n	ΔG^0 (kcal mol^−1)	R^2
280 nm	HSA	2.29 × 10^4	4.07 × 10^12	1.77 × 10^4	1.02	−5.77	0.99
	BSA	1.06 × 10^4	1.69 × 10^12	4.89 × 10^4	1.07	−6.37	0.99
295 nm	HSA	2.05 × 10^3	3.64 × 10^12	1.09 × 10^3	0.93	−4.12	0.99
	BSA	5.32 × 10^3	8.60 × 10^12	9.12 × 10^3	1.05	−5.38	0.99

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3.2 Time resolved fluorescence measurements

In order to establish the mechanism of quenching and to exclude contributions from dynamic quenching processes, time resolved fluorescence measurements were carried out on serum albumin–PYZ complexes. The average fluorescence life-time (τ) for exponential iterative fitting was calculated from the decay times and relative amplitude using the following equation:

τ = α₁τ₁ + α₂τ₂ (6)

The obtained values for the relative fluorescence life-time for HSA and BSA in the absence and presence of PYZ are listed in the ESI Tables 1 and 2.† In the presence of PYZ, the average fluorescence life-time for HSA and BSA reduces from 5.62 ns to 5.33 ns and 6.18 to 5.82 ns, respectively. The insignificant reduction in the fluorescence life-time values implies that the mechanism of quenching is essentially static in manner. Thus, both the time resolved and steady state fluorescence measurements indicate a static mechanism of quenching mainly through ground state complex formation, which is further supported by the UV absorption study.

3.3 Synchronous fluorescence measurements

Synchronous fluorescence provides information on the micro-environmental changes around the tyrosine and tryptophan residues.⁴¹ When the difference (D-value) between the excitation and emission wavelengths is set at 15 and 60, this provides information on the tyrosine and tryptophan residues respectively. Synchronous fluorescence spectra of HSA and BSA are shown in Fig. 3 and 4 respectively. The synchronous fluorescence spectra of the HSA–PYZ interaction show that PYZ leads to a change in the microenvironment around both the fluorophores i.e., tyrosine and tryptophan, when the D-value is set at 15 and 60, respectively, as is evident from the reduction in the fluorescence intensity and the red shift (Fig. 3). When the D-value was set at 15, for the BSA–PYZ interaction, a smaller reduction in the fluorescence intensity without any red shift was observed (Fig. 4). This signifies no change in the microenvironment of the tyrosine residue. In contrast, when the D-value was set at 60, a greater decrease in the fluorescence intensity with a red shift suggested that interaction of PYZ with the protein results in a polar environment around the tryptophan residue of BSA. These results suggest that PYZ binds to HSA in such a manner as to push the tyrosine and tryptophan from a less polar environment to a more polar environment, whereas PYZ increases the polarity around only the tryptophan residue of BSA, which is also in complete accord with the previous reports.⁹

Fig. 3 Synchronous fluorescence spectra of HSA: (A) Δλ 15, (B) Δλ 60, using HSA (5 μM) in the presence of increasing concentrations of PYZ (0–60 μM).

Fig. 4 Synchronous fluorescence spectra of BSA: (A) Δλ 15, (B) Δλ 60, using BSA (5 μM) in the presence of increasing concentrations of PYZ (0–60 μM).

3.4 Circular dichroism measurements

Circular dichroism is a very important tool in structural biology and protein chemistry.^42–47 To decipher the structural and conformational changes induced in the serum albumins upon interaction with PYZ, we carried out far UV-CD measurements in the absence and presence of the drug. The CD spectra of the native serum albumin (HSA and BSA) possess two negative bands at 208 nm and 222 nm, which are characteristic features of the α-helical class of proteins.^9,48 Upon increasing the concentration of PYZ, the values of both minima start increasing, without any shift in their position, in the case of both of the serum albumins as shown in Fig. 5. These results are indicative of an increase in the helical content of the protein in the presence of increasing concentrations of PYZ. The change in the percentage helical content for the serum albumin is described in the ESI Table 5.† Near-UV CD spectroscopy is widely used to detect tertiary structure changes in proteins. Aromatic amino acids in the proteins have a signature signal of ellipticity in a specific wavelength range. Moreover, disulphide bonds have a characteristic wavelength range between 260–270 nm. When PYZ interacts with HSA and BSA, a major tertiary structure alteration occurred with BSA, which was confirmed from a shift of the minima and shoulders in the wavelength range of 260–270 nm (ESI Fig. 2†).

Fig. 5 Far-UV CD spectra of (A) HSA and (B) BSA in the absence and presence of PYZ.

3.5 UV-visible absorption spectroscopy

UV absorption spectroscopy is an important tool for exploring structural changes and complex formation.^49,50 UV-visible absorption spectra of the serum albumins in the absence and presence of PYZ are presented in the ESI Fig. 3.† It is clear from the figure that the absorption of the serum albumins (HSA and BSA) increases upon increasing the concentration of PYZ.⁴⁹ A greater absorbance increase was observed in the case of BSA, compared to HSA. Furthermore, it is clear that the maximum peak position shifted towards a shorter wavelength, which indicates complex formation between the drug and protein.

3.6 Dynamic light scattering measurements

Dynamic light scattering was employed to determine the hydrodynamic radii of HSA and BSA, alone and in the presence of PYZ. The hydrodynamic radii (R_h) of the native HSA and BSA and their complexes with PYZ are plotted in Fig. 6 and presented in the ESI Table 6.† A lower value of polydispersity (5.2–15.1) is indicative of homogeneity in the solution. The hydrodynamic radii of the native HSA and BSA were found to be 3.4 nm and 3.7 nm respectively, which are in agreement with previous reports.^51–53 The hydrodynamic radii (R_h) value of the HSA/BSA–PYZ complex was smaller than that of the native value. The reduction in the R_h value may be attributed to collapse of the protein as PYZ binds to the protein, which results in shrinkage of the molecular volume due to conformational alterations. Similar results were obtained in the case of limonene binding to BSA.⁹ The change in conformation induced by PYZ may be the cause of the reduction in the hydrodynamic radii of the proteins, as shown in Fig. 6 and summarized in the ESI Table 6.†

Fig. 6 Hydrodynamic radii patterns of HSA and BSA in the absence of PYZ (A & D respectively) and in the presence of PYZ: (B) HSA + PYZ (1 : 5); (C) HSA + PYZ (1 : 12); (E) BSA + PYZ (1 : 5); (F) BSA + PYZ (1 : 12).

3.7 Differential scanning calorimetry measurements

Differential scanning calorimetry was employed to determine the melting temperature^54,55 of HSA and BSA, alone and in the presence of PYZ. DSC provides information on the effect of ligand binding on the thermal stability of the protein, on the basis of ΔT_m (melting temperature). Representative differential scanning profiles of HSA and BSA, alone and in the presence of PYZ, are shown in Fig. 7. Serum albumins show a transition in melting curves due to their multi-domain structure. When albumins are exposed to thermal treatment, domain I is more likely to melt first. The melting temperature of the native HSA and BSA was found to be around 60.06 and 60.9 °C respectively. In the presence of PYZ, the melting temperatures of HSA and BSA were found to be enhanced by 3 and 4 °C, respectively.⁵⁶ These results advocate that the binding of PYZ stabilizes the protein structure, which is also in accordance with the CD data.

Fig. 7 (A) DSC thermograms of HSA and the HSA–PYZ complex (HSA : PYZ = 1 : 12), (B) DSC thermograms of BSA and the BSA–PYZ complex (BSA : PYZ = 1 : 12).

3.8 Molecular docking and drug displacement experiments using site specific markers

To gain insight into the binding of PYZ to HSA/BSA, molecular docking was performed. The best conformation, based on the binding energy, was selected for further analysis using the discovery studio. The molecular docking results are summarised in Fig. 8 and the ESI Table 7.† Fig. 8a and b represent the binding site of PYZ and amino acid residues involved in the binding to HSA, respectively. It is clear that PYZ interacts with Leu 219, Leu 238, Arg 257, Leu 260, Ser 287 and Ile 290 via hydrophobic interactions, and Ala 291 interacts through hydrogen bonding at site I (ESI Table 7† and Fig. 8b). Fig. 8c and d show the binding pocket of PYZ and the amino acid residues of BSA involved in the BSA–PYZ complex formation, respectively. It was deciphered from Fig. 8d and Table 3† that Tyr 401, Asn 405, Ala 406, Leu 429, Met 548, Lys 545 and Asp 549 interact with PYZ through hydrophobic interactions, and Lys 402 interacts via hydrogen bonding at site II. The majority of drugs bind at two primary sites known as Sudlow sites I and II on serum albumins. In order to confirm the binding site of PYZ with serum albumins, competitive binding experiments with site specific markers were carried out. Warfarin for site I and Diazepam for site II were used. To trace the binding site of PYZ with the serum albumins, K_sv values were calculated using the emitted fluorescence intensity data in the absence and presence of the markers, using the Stern–Volmer equation. There was significant change in the K_sv value in the presence of warfarin but no change was observed in the presence of diazepam in the case of HSA (ESI Table 3†). In contrast to this, opposite results were obtained in the case of BSA (ESI Table 4†). These results signify that PYZ binds to site I and site II on HSA and BSA, respectively.

Fig. 8 (A) Best conformation of PYZ docked into HSA. (B) Cartoon representation of the residues involved in binding of PYZ to HSA. (C) Best conformation of PYZ docked into BSA. (D) Cartoon representation of the residues involved in binding of PYZ to BSA.

4. Conclusion

All the results together infer that PYZ forms a complex with HSA and BSA. PYZ interacts through a static quenching manner; and the extent of the binding is greater with BSA than HSA. The value of n revealed that there is a single class of binding site on HSA and BSA for PYZ. Synchronous fluorescence study results confirmed that PYZ results in a change of the microenvironment around the aromatic fluorophores. Moreover, the increment in the alpha helical content, the compacting of the size and the increase in the transition melting point of HSA and BSA upon interaction with PYZ confirmed the stabilizing action of PYZ on serum albumins. The significance of this study lies in that the pharmaceutical industry needs a method of screening for compound/drug binding with HSA & BSA, and such a kind of study as that for the interaction of PYZ with HSA/BSA would be useful in the pharmaceutical industry, medicinal chemistry, life sciences and clinical medicine.

Acknowledgements

Facilities provided by I. B. U, Aligarh Muslim University are gratefully acknowledged. S. K. Chaturvedi and P. Alam are highly thankful to the Council of Scientific and Industrial Research, New Delhi, for financial assistance in the form of a senior research fellowship (SRF). M. K. Siddiqi is thankful to the Department of Biotechnology, Govt. of India for providing a fellowship in the form of JRF.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra10487h

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