Research article Received: 2 July 2014,

Revised: 24 September 2014,

Accepted: 6 October 2014

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bio.2812

Exploring the interaction of the photodynamic therapeutic agent thionine with bovine serum albumin: multispectroscopic and molecular docking studies† Perumal Manivel,a* Shanmugam Anandakumarb and Malaichamy Ilancheliana* ABSTRACT: This study explores the binding interaction of thionine (TH) with bovine serum albumin (BSA) under physiological conditions (pH 7.40) using absorption, emission, synchronous emission, circular dichroism (CD) and three-dimensional (3D) emission spectral studies. The results of emission titration experiments revealed that TH strongly quenches the intrinsic emission of BSA via a static quenching mechanism. The apparent binding constant (K) and number of binding sites (n) were calculated as 2.09 × 105 dm3/mol and n~1, respectively. The negative free energy change value for the BSA–TH system suggested that the binding interaction was spontaneous and energetically favourable. The results from absorption, synchronous emission, CD and 3D emission spectral studies demonstrated that TH induces changes in the microenvironment and secondary structure in BSA. Site marker competitive binding experiments revealed that the binding site of TH was located in subdomain IIA (Sudlow site I) of BSA. The molecular docking study further substantiates Sudlow site I as the preferable binding site of TH in BSA. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: bovine serum albumin; thionine; emission spectroscopy; circular dichroism; molecular docking

Introduction Serum albumins are the most abundant proteins in blood plasma which assist in the disposition and transportation of various exogenous and endogenous ligands to specific targets (1). The most important physiological functions of serum albumins are to maintain the osmotic pressure and pH of the blood. They also act as a plasma carrier through nonspecific binding with several hydrophobic steroid hormones across organ–circulatory interfaces such as the liver, intestine, kidney and brain (2). Interactions between serum albumins and ligands have attracted a great deal of interest for many years due to their application in a wide variety of biological, pharmaceutical, toxicological and cosmetic systems (3,4). Bovine serum albumin (BSA) has been studied extensively in kinetic and affinity drug tests as a replacement for human serum albumin (HSA) because of its stability, low cost, unusual ligand-binding properties and its structural homology with HSA, in particular (5). BSA has frequently been used as a model protein for investigating ligand-binding mechanisms and to understand the structural basis for designing new therapeutic agents (6). Therefore, detailed investigations of drug–protein interactions assume significance in understanding the structural features of the bioaffinity and pharmacokinetic behavior of a drug in a protein environment. BSA is a major component of blood plasma, accounting for ~60% of the total protein and corresponding to a concentration of 43 mg/mL. BSA is composed of a single-chain 583-amino-acid globular nonglycoprotein that is cross-linked with 17 cystine residues (eight disulfide bonds and one free thiol) (7). It has been

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categorized into three homologous domains: I (residues 1–195), II (196–383) and III (384–583), which are divided into nine loops (L1–L9) by 17 disulfide bonds. The loops in each domain consist of a sequence of large–small–large loops that form a triplet. There are two tryptophans (Trp) in BSA, with Trp213 embedded in the hydrophobic pocket of subdomain IB in a way that is similar to Trp214 in HSA and Trp134 located on the surface of subdomain IIA (8). Thionine (3,7-diamino-5-phenothiazinium) (TH) is a planar cationic phenothiazinium dye (Scheme 1). It has been widely studied for its intercalative interaction and photoinduced mutagenic actions on DNA (9–11). TH has been used as energy sensitizer, as a probe for investigation into various microenvironments, including micelles and polymeric matrices, in nanocomposite materials, high quantum efficiency photoelectrochemical cells and photochemical biosensors (12–16). It is also used to induce photodynamic inactivation of bladder cancer cells, Escherichia coli

* Correspondence to: Malaichamy Ilanchelian and Perumal Manivel, Department of Chemistry, Bharathiar University, Coimbatore – 641046, Tamil Nadu, India. E-mail: [email protected]; [email protected]

This paper is dedicated to Professor Ramasamy Ramaraj, School of Chemistry, Madurai Kamaraj University, Madruai-625021, India for his contribution to the fields of photochemistry and photoelectrochemistry.

a

Department of Chemistry, Bharathiar University, Coimbatore, India

b

Department of Bioinformatics, Bharathiar University, Coimbatore, India

Copyright © 2014 John Wiley & Sons, Ltd.

P. Manivel et al. Circular dichroism measurements

Scheme 1. Structure of thionine.

and Saccharomyces cerevisiae (17). In view of the enormous range of applications of TH, it is necessary to understand the binding mechanism of TH with biological systems to elucidate their potential applications and toxic effects on biological macromolecules. In this study, the binding interaction of biological photosensitizer TH with BSA was investigated using spectroscopic methods including absorption, emission, three-dimensional (3D) emission, synchronous emission and circular dichroism (CD) spectral studies. An attempt is also undertaken to unravel the effect of TH binding on the secondary structure of BSA to rationalize the applicability of the TH molecule as an effective phototherapeutic agent. The binding location of TH within BSA was further confirmed using a site marker experiment and molecular docking studies.

Experimental Materials BSA (fraction V), purchased from Himedia (India) was used without further purification. Warfarin, ibuprofen and digitoxin were obtained from TCI Chemicals (Japan) and used as received. TH dye was obtained from Himedia and was purified by column chromatography on neutral alumina using ethanol:benzene (7:3 v/v) containing 0.4% glacial acetic acid and then recrystallized from ethanol (18). The stock solution of BSA was prepared using phosphate buffer solution (PBS) at pH 7.40. The concentration of BSA was measured spectrophotometrically using previously reported procedures (19,20). All other reagents were of analytical grade and used as received. The water used in this experiment was doubly distilled over alkaline potassium permanganate using all-glass apparatus.

Absorption and emission spectral measurements Absorption spectral measurements were carried out using JASCO V-630 UV/vis spectrophotometer. Quartz cuvettes of path length 1 cm were used to record the absorption spectra. The emission spectral studies were performed with a JASCO FP6600 spectrofluorometer. All the titration experiments were carried out by adding an appropriate amount of TH to 1 mL of BSA solution in a 5-mL standard measuring flask in sequence and then made up to the mark with PBS. The BSA–TH solutions were mixed uniformly and the solution was allowed to equilibrate for 10 min before recording the spectra. BSA was excited at 295 nm and the emission was monitored at 348 nm. The emission and excitation slit widths used throughout the experiments were 5 and 2 nm, respectively. The synchronous emission spectra were recorded at Δλ =15 nm and Δλ =60 nm. The 3D emission spectra were recorded over the range of 200–500 nm at a scanning rate of 2000 nm/min. The emission and excitation slit widths for 3D emission spectra were 10 and 2 nm, respectively. All measurements were carried out at room temperature (25 °C). Stock solutions of BSA and TH were always freshly prepared before use.

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CD measurements were performed with a JASCO-180 spectropolarimeter using a 0.1 cm path length quartz cell. The CD spectra were recorded in the range of 200–300 nm with 0.1 nm step resolution and averaged over two scans at a speed of 50 nm/min. All observed spectra were baseline subtracted for buffer solution and the α-helical content was calculated on the basis of change of molar ellipticity value. Molecular docking studies Molecular docking experiments were performed using the docking software AUTODOCK 4.2 along with the AUTODOCK tools (ADT). The crystallographic coordinates of TH was obtained from the PubChem database. The native structures of BSA (PDB id: 4F5S) was retrieved from the Protein Data Bank. As required for Lamarckian Genetic Algorithm docking compilation, all water molecules were removed from the native structure of BSA with subsequent addition of hydrogen atoms followed by the calculation of Gasteiger charges. The grid size along the x-, y- and z-axes was set to 30, 30 and 30 Å respectively. The grid spacing was set as 1 Å and the grid center along the x-, y- and z-axes was set to 126, 44 and 78 Å, respectively, which encompasses the entire protein structure was used throughout the docking process. The AUTODOCK parameters used were, Genetic Algorithm (GA) population size =150, maximum number of energy evaluations =250,000 and a GA cross-over mode of two points. The lowest binding energy conformer was observed of 30 different conformers from total docking GA runs. MAESTRO 9.5 software was used for visualization of the docked conformations.

Results and discussion Emission spectral studies of BSA with TH The intrinsic emission property of BSA is very sensitive to ligand binding, changes in the surrounding microenvironment and ligand-induced conformational changes to the secondary structure of the protein. As a result, the intrinsic emission property of BSA has been widely used to assess the interaction of BSA with ligands or drug molecules. The emission spectra recorded for BSA with different concentrations of TH are shown in Fig. 1. The emission spectrum of BSA in the absence of TH shows an emission maximum at 348 nm, when excited at 295 nm. The choice of 295 nm as the excitation wavelength is to avoid the contribution of tyrosine (Tyr) residues (21). It can be seen from Fig. 1 that with the addition of increasing concentrations of TH, the emission intensity of BSA decreased gradually. It is well known that decreases in the emission intensity of BSA are generally associated with drug–protein interactions (22). As a result, the decrease in emission intensity of BSA upon the addition of increasing concentrations of TH could be attributed to the complexation of BSA with TH. However, the existence of an inner filter effect (IFE) may also affect the emission titration measurements. Therefore, the possible influence of IFE on emission quenching of BSA by TH was investigated further. Elimination of the IFE The IFE refers to the absorption (or partial dispersion) of light at the excitation or emission wavelength by the compounds

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PDT agent thionine with bovine serum albumin

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Figure 1. Emission spectra of BSA (3.40 × 10 mol/dm ) at various concentra3 6 6 6 tions of TH (in mol/dm ): (a) 0.00, (b) 0.34 × 10 , (c) 0.68 × 10 , (d) 1.02 × 10 , 6 6 6 6 6 (e) 1.36 × 10 , (f) 1.71 × 10 , (g) 2.05 × 10 , (h) 2.39 × 10 and (i) 3.07 × 10 , pH 7.40.

present in the solution. When a ligand is added to a protein solution, if the absorption of the ligand at the excitation wavelength is strong, less light reaches the center of the solution and the emission intensity of the protein is therefore reduced. Whereas, if the added ligand possesses considerable absorption at the emission wavelength of the fluorophore, it reduces the emitted light that reaches the detector, which in turn, decreases the emission intensity of the protein (23,24). The absorption spectra at increasing concentrations of TH are shown in Fig. S1, ESI. It is evident from the Fig. S1 that TH dye shows considerable absorption at the excitation (295 nm) and emission (348 nm) wavelengths of BSA. From Fig. S1, it can be emphasized that the addition of increasing concentrations of TH significantly influences the emission spectral studies of BSA–TH system via IFE. Thus, in this investigation, we carried out the emission titration experiments at low concentrations of TH (0.34 ×106 to 3.07 ×106 mol/dm3) to rule out the influence of IFE in emission spectral studies. It is pertinent to note that at low concentrations, the absorption value of TH is

Exploring the interaction of the photodynamic therapeutic agent thionine with bovine serum albumin: multispectroscopic and molecular docking studies.

This study explores the binding interaction of thionine (TH) with bovine serum albumin (BSA) under physiological conditions (pH 7.40) using absorption...
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