Role of hydrogen-bonding and photoinduced electron transfer (PET) on the interaction of resorcinol based acridinedione dyes with Bovine Serum Albumin (BSA) in water

Journal of Luminescence 164 (2015) 146–153

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Role of hydrogen-bonding and photoinduced electron transfer (PET) on the interaction of resorcinol based acridinedione dyes with Bovine Serum Albumin (BSA) in water Rajendran Kumaran a,n, Mahalingam Vanjinathan a, Perumal Ramamurthy b a Department of Chemistry, Dwaraka Doss Goverdhan Doss, Vaishnav College (Autonomous), 833, Gokul Bagh, E.V.R. Periyar Road, Arumbakkam, Chennai 600106, Tamil Nadu, India b National Centre for Ultrafast Processes, University of Madras, Taramani Campus Chennai 600113, Tamil Nadu, India

art ic l e i nf o

a b s t r a c t

Article history: Received 1 September 2014 Received in revised form 30 January 2015 Accepted 10 March 2015 Available online 4 April 2015

Resorcinol based acridinedione (ADDR) dyes are a class of laser dyes and have structural similarity with purine derivatives, nicotinamide adenine dinucleotide (NADH) analogs. These dyes are classified into photoinduced electron transfer (PET) and non-photoinduced electron transfer dyes, and the photophysical properties of family of these dyes exhibiting PET behavior are entirely different from that of nonPET dyes. The PET process in ADDR dyes is governed by the solvent polarity such that an ADDR dye exhibits PET process through space in an aprotic solvent like acetonitrile and does not exhibit the same in protic solvents like water and methanol. A comparison on the fluorescence emission, lifetime and nature of interaction of various ADDR dyes with a large globular protein like Bovine Serum Albumin (BSA) was carried out in aqueous solution. The interaction of PET based ADDR dyes with BSA in water is found to be largely hydrophobic, but hydrogen-bonding interaction of BSA with dye molecule influences the fluorescence emission of the dye and shifts the emission towards red region. Fluorescence spectral studies reveal that the excited state properties of PET based ADDR dyes are largely influenced by the addition of BSA. The microenvironment around the dye results in significant change in the fluorescence lifetime and emission. Fluorescence enhancement with a red shift in the emission results after the addition of BSA to ADDR dyes containing free amino hydrogen in the 10th position of basic acridinedione dye. The amino hydrogen (N–H) in the 10th position of ADDR dye is replaced by methyl group (N–CH3), a significant decrease in the fluorescence intensity with no apparent shift in the emission maximum was observed after the addition of BSA. The nature of interaction between ADDR dyes with BSA is hydrogen-bonding and the dye remains unbound even at the highest concentration of BSA. Circular Dichroism (CD) studies show that the addition of dye to BSA results in a decrease in the alpha helical content of the protein. The shape and the pattern of CD bands after the addition of ADDR dye to BSA remain largely unaltered. & 2015 Elsevier B.V. All rights reserved.

Keywords: Photoinduced electron transfer (PET) Hydrogen-bonding Fluorescence enhancement Resorcinol based acridinedione dyes Bovine Serum Albumin (BSA)

1. Introduction The concept of photophysics and photochemistry of fluorescent dyes in a heterogeneous medium containing large peptides, protein or macromolecules in aqueous solution is of importance in the field of chemistry and biology [1–3]. Fluorescence spectral techniques and methods serve as a vital link in establishing and elucidating the nature of interaction of dye with protein. The most probable location and orientation of the dye molecule in aqueous solution containing a protein [2,4–11] is ascertained even at a very low concentration. Most of the biophysical and biochemical studies of non-homogeneous n

Corresponding author. Tel.: þ 91 44 2475 6655; fax: þ91 44 2475 4349. E-mail address: [email protected] (R. Kumaran).

http://dx.doi.org/10.1016/j.jlumin.2015.03.010 0022-2313/& 2015 Elsevier B.V. All rights reserved.

systems are largely focused on the changes in the microscopic level and are widely applicable to elucidate the structure of a large protein molecule. Fluorescence constitutes the most widely used experimental approach in determining the nature of dye–protein interaction. A large globular protein consists of several hydrophilic, hydrophobic and hydrogen-bonding moieties. The presence of hydrogenbonding and hydrophobic interactions largely influences the ground and excited state properties of the dye molecule. The dyes are often aromatic heterocyclic compounds accompanied with poor solubility in water and have high affinity to hydrophobic binding sites. The fluorescent dyes can be the analogs of natural ligands and drugs [7,8] or serve as the binding site markers in competition experiments with various ligands [9,10]. Their role in the studies of ligand binding properties of serum albumins is very

R. Kumaran et al. / Journal of Luminescence 164 (2015) 146–153

important in the field of protein chemistry. Fluorescent dyes are employed as an important tool for albumin determination in the presence of other proteins and also in clinically important media, such as blood plasma and urine [12]. Even though the studies of dyes binding to serum albumin have been well established over several years [13], BSA is widely employed as the test object for various probes [3,14–20]. The solubility of BSA in water provides the researchers to explore the phoptophysical properties of water soluble fluorophores with BSA. BSA is one of the most available and extensively studied of all proteins. It is the major constituent of blood plasma of many species, accounting for about 60% of its total protein content and providing about 80% of the blood osmotic pressure [21,22]. It is a carrier in the blood of many low-polar metabolites and drugs [23,24]. BSA is a large globular protein, (66.4 k Da) and consists of 583 amino acids. BSA has a relatively smaller proportion of tryptophan amino acid with 17 intra-chain disulfide bonds of a single polypeptide chain folded into tertiary globular conformation forming three domains [25,26]. The importance of fluorescence technique on the probe– protein interaction has been elucidated from the studies on the interaction of various Intramolecular Charge Transfer (ICT) and PET probes [27–39]. Fluorescence spectroscopy is used as a fascinating tool in determining location of the probe molecule in the heterogeneous medium of the protein molecule [5,11,40–42]. Acridinedione is a bifunctional molecule and acts as an electron donor or acceptor, and undergoes various interesting reactions in the excited state. The nature of the substituents in the 9th and 10th positions of the basic acridinedione dyes (Scheme 1) results in a large variation in their photophysical and photochemical properties. Acridinedione dyes belong to the family of laser dyes [43,44] and these dyes have biological importance because of their structural similarity with coenzyme nicotinamide adenine dinucleotide (NADH) [43,45,46] wherein it plays a vital role in the electron source in the reduction of oxygen in the respiratory chain [47]. Acridinedione dyes [4,46,48–54] are classified into PET (Scheme 1) and non-PET based dyes (Supporting information Scheme S1) based on the electron donating moieties present in the para position of the phenyl ring at the 9th position (Scheme 2). The electron transfer occurs through space between the donor moiety present at the para position of the phenyl ring at C9 carbon (N(CH3)2 (ADDR1 and ADDR3) or OCH3 (ADDR2)) to the acceptor moiety (C¼O) of the acridinedione ring in ADDR dyes [4,48–52,55]. The suppression of PET process resulting in a fluorescence enhancement has been well established in ADDR2 dyes with solutes containing hydrogen-bonded self assemblies like urea derivatives [48], amides [50] and guanidine hydrochloride [55]. Further, the PET process through space is observed in all solvents for ADDR2 dye and this has been authenticated from the interaction of urea derivatives with ADDR2 dye in water and methanol [48]. Interaction of ADDR dyes with hydrophobic nanocavities [56,57] and polymers [58] also results in a fluorescence enhancement and a large variation in the fluorescence lifetime properties. Further, the addition

147

Scheme 2. Basic structure of resorcinol based acridinedione (ADDR) dyes.

of metal ions and anions to ADDR dyes exhibits excellent signaling properties as ON–OFF sensors [59,60]. PET process in organic molecules and drugs containing a donor and acceptor moieties has relevance to many aspects in the field of chemistry and biology. The electron transfer (ET) process plays a crucial role in biological systems such as in the respiratory chain and photosynthetic processes [61]. A marked variation in the fluorescence spectral properties of dyes is visualized on binding with ligands and substrates, which occurs through an ET phenomenon. In general, the ET process is based on the nature of the functional groups in the host and guest molecules and is influenced by the solvent polarity, viscosity, hydrogen-bonding, hydrophobic and electrostatic interactions. A fluorescence quenching or enhancement of a dye reveals the nature of interaction and the preferred site of binding with the protein molecule. The type of interaction provides valuable information about the excited state properties of the fluorophore and the binding constant values [5,11,40–42,62]. ADDR1 dye exhibits dual emission characteristics and the presence of two different emission states is locally excited (LE) and PET promoted CT state [51,52]. In the present study, a fluorescence enhancement is observed after the addition of BSA to ADDR1 dye. In ADDR1 dye both PET and Intramolecular Charge Transfer (ICT) mechanism operate in aprotic solvents like acetonitrile. The PET process does not operate in water and this is attributed to the protonation of the amino nitrogen [51]. The N–H hydrogen (10th position) forms hydrogen-bonding with several metal ions resulting in shift in the emission maxima and this has been reported [51–54]. In the present investigation, we probe how the photophysical studies of a PET dye are influenced by a large globular protein. The mechanism of fluorescence enhancement and shift in the emission maxima in PET dyes ADDR1 and ADDR after the addition of BSA are investigated. The photophysical studies of ADDR1 dye with BSA are compared with that of ADDR2 dye with BSA, wherein the PET process in ADDR2 dye occurs through space irrespective of the nature of the solvent and the solute [4,48,50,55,63]. Further, the excited state characteristics of ADDR3 dye (structural similarity with ADDR1) with BSA in water are also investigated.

2. Materials and methods

Scheme 1. Structure of ADDR dyes having different substituents in the 9th and 10th positions.

BSA fraction V powder pH 6.0–7.0 was purchased from SRL chemicals India Ltd. and was refrigerated at around 4 °C. Resorcinol based acridinedione dyes (ADDR1–3) were prepared by following the procedure reported [45,64,65]. The concentration of the dyes used for fluorescence spectral measurements was 3.0570.05  10  5 M such that the absorbance of ADDR dyes in the spectral range 370–380 nm was less than 0.2 BSA stock solutions which were freshly prepared in triple distilled water. The concentration of BSA for CD spectral was around 1.0  10  6 M (the concentration of BSA was fixed such that the MRE value (θ) at 20872 nm was around  80).

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3. Experimental methods: photophysical studies

4. Results and discussion 4.1. Absorption spectral studies The absorption spectrum of ADDR1 dye in water shows maxima around 255 75 and 3757 5 nm in water (Fig. 1). The λmax at 375 75 nm is assigned to the ICT from the nitrogen to carbonyl group of acridinedione fluorophore and this state is influenced by the polarity of the medium [52]. Presence of electron donating substituents in the 9th position (donor) is not exhibiting any change in the absorption maximum. Substitution at the 10th position by N-methyl moiety of ADDR1 dye exhibits a shift towards the red region and this is attributed to its ICT nature [52]. The absorption spectrum of ADDR1 dye after the addition of BSA is shown in Fig. 1 (inset). It was observed that the addition of BSA does not influence the ICT absorption maximum (37575 nm) of ADDR1 dye and the increase in the absorbance in the spectral range of 250–300 nm is attributed to the strong absorbance of BSA. The increase in the absorbance less than 300 nm does not govern the excited state properties of ADDR dyes, since the wavelength of excitation is above 375 nm only. A similar pattern was also observed in the interaction of ADDR2 dye with BSA in water [4]. It has been well established that there is no significant change in the absorbance at the ICT absorption maximum of several PET and non-PET based ADDR dyes on the addition of BSA, such that the ground state characteristics of ADDR1 dye is not influenced by addition of BSA [4]. Contrary to the absorption spectrum, the emission spectrum recorded by exciting the dye at its absorption maximum is dependent on the electron donating property of the functional groups present at the 9th position of ADDR dyes and the variation in the fluorescence lifetime and emission depends on the nature of the solute and solvents used [50–60].

0.4

Absorbance

Absorption spectra were recorded using an Agilent 8453 diode array spectrometer and emission spectra were recorded in MPF44B fluorescence spectrophotometer interfaced with PC through RISHCOM-100 multimeter. The emission spectrum was recorded by carrying out a corrected spectrum following the literature reported [66,67] and the instrument voltage was kept constant during the course of recording the fluorescence intensity. Time resolved fluorescence decays were obtained by the time correlated-single-photon-counting (TCSPC) method [4]. A diode pumped Millenia V CW laser (Spectra Physics) was used to pump the Ti–sapphire rod in a Tsunami pico second-mode locked laser system (Spectra Physics). The 750 nm (85 MHz) beam from the Ti–sapphire laser was passed through a pulse picker (Spectra Physics, GWU 23PS) to generate 4 MHz pulses. The second harmonic output was generated by a flexible harmonic generator (Spectra Physics, GWU 23PS). A vertically polarized 377 nm laser was used to excite the sample. The fluorescence emission of ADDR dyes was monitored at magic angle (54.7°). This was counted by a MCP-PMT apparatus (Hamamatsu R3809U) after being passed through the monochromator and was proceeded through a constant fraction discriminator (CFD), a time-to-amplitude converter (TAC) and a multichannel analyzer (MCA). The instrument response function for this system is around 52 ps. The fluorescence decay obtained was further analyzed by using IBH (UK) software (DAS-6). CD spectral measurements were carried out in JASCO J-715 spectropolarimeter equipped with a thermostated cell holder, using a quartz cell of 0.1 cm path length. The equipment was calibrated using ammonium-d10-camphor sulfonic acid as prescribed.

0.5

0.3

0.2

0.1

0 210

260

310

360

410

460

Wavelength(nm) Fig. 1. Absorption spectra of ADDR1 dye (3.05  10  5 M) in water. Inset: absorption spectra of ADDR1 dye in the absence and presence of BSA in water. (1) ADDR1 dye (3.05  10  5 M), (2) ADDR1 dye þBSA 0.3  10-4 M, (3) ADDR1 dyeþ BSA 0.6  10  4 M, and (4) ADDR1 dyeþBSA 1.2  10  4 M.

4.2. Emission spectral studies In all PET based ADDR dyes, the first excited state (Local Excited (LE) state) remains largely localized on the acridinedione itself and exhibits emission maxima around 437 nm in water [4,48–51]. For ADDR1 dye in acetonitrile, the PET process from donor to the acceptor produces a low lying anomalous CT state around 570 nm in addition to the LE state emission (o430 nm) [51,52]. On contrary, the PET nature of ADDR1 dye in water is completely lost due to the protonation of the dimethylamino group. (The studies were not carried out in acetonitrile and methanol due to insolubility of BSA in these solvents.) In protic solvents like water and methanol, protonation of the dimethylamino group increases the oxidation potential of donor group thereby resulting in a large variation in the fluorescence lifetime compared to that in aprotic solvents [51,52]. The pH of ADDR dyes is highly sensitive to the variation in the pH of the solution. We carried out all the spectral studies in triple distilled water and the pH was maintained at 6.85 70.5. The pH of the dye remains unaltered even in the presence of very high concentration of BSA (dye:BSA is 1:10) and the pH of BSA in the absence of ADDR dyes was found to be around 6.80 70.6. It is well known that the addition of BSA to PET based acridinedione dye (ADDR2) leads to a fluorescence enhancement with no significant shift in the emission maxima. The increase in the fluorescence intensity of ADDR2 dye is attributed to the suppression of the PET process and this results in a large variation in the fluorescence lifetime and anisotropy value [4]. The PET nature of the dye is not lost even in the presence of very high concentration of BSA, and this is authenticated from time-resolved fluorescence spectral studies of ADDR2 dye with BSA. It was reported that the fluorescence enhancement of ADDR2 dye on the addition of BSA or urea derivatives or amides is independent of pH or viscosity of the medium, and the suppression of PET process occurs through space between the donor and acceptor moiety [4]. 4.2.1. ADDR1–BSA vs ADDR2–BSA emission spectral characteristics It was observed that the initial addition of BSA quenches the native LE state emission of ADDR1 dye (436 nm) and also 50% quenching of the fluorescence intensity (Fig. 2). On the subsequent

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149

2 8

Emission Intensity

1.6

1.2

0.8

1

0.4

2

0 400

450

500

550

Wavelength (nm) Fig. 2. Emission spectra of ADDR1 dye in the absence and presence of BSA in water λex 378 nm. ADDR1 dye concentration fixed at 3.05  10  5 M. (1) ADDR1 dye alone, (2) ADDR1 dyeþ BSA 0.3  10  4 M, (3) ADDR1 dyeþBSA 0.6  10  4 M, (4) ADDR1 dye þBSA 1.2  10  4 M, (5) ADDR1 dyeþ BSA 1.5  10  4 M, (6) ADDR1 dyeþBSA 1.8  10  4 M, (7) ADDR1 dyeþ BSA 2.1  10  4 M, and (8) ADDR1 dyeþBSA 3.0  10  4 M.

475

ADDR2 Dye ADDR1 Dye

Emission Maximum, nm

470 465 460 455 450 445 440 435

0.0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3.0

-4

BSA x 10 M Fig. 3. Shift in the emission maximum of ADDR dyes on the addition of BSA. ■ – ADDR1 dye. ▲ – ADDR2 dye.

addition of BSA to ADDR1 dye, fluorescence enhancement accompanied with a significant shift in the emission maxima results (439–470 nm). The extent of red shift in the emission maxima is around 3072 nm for ADDR1 dye (Fig. 3), whereas it is less than 5 nm in the case of ADDR2 dye (Supporting information Fig. S1). The large red shift in the emission maxima of ADDR1 dye over ADDR2 dye clearly illustrates that the excited state characteristics of ADDR1 dye is governed by the increasing concentration of protein. It is well known that PET promoted CT state emission results in ADDR1 dye in acetonitrile and the addition of methanol in acetonitrile results in the stabilization of the CT state [52]. This is due to the increase in the polarity of the medium. The addition of BSA results only one emission maximum and this is correlated to the new LE state emission fluorescence of ADDR1 dye and this does not correspond to the native LE state of ADDR1 dye in the absence of BSA. (This is ascertained based on the increase in the fluorescence

Fig. 4. Extent of fluorescence enhancement of ADDR dyes on the addition of BSA. ● – ADDR1 dye. ■ – ADDR2 dye.

lifetime and relative amplitude of the dye on each addition of BSA). No new CT state emission results from ADDR1 dye (as observed in acetonitrile) making the dye a suitable probe for study in aqueous medium. Interestingly, the red shift observed in ADDR1 dye after the addition of BSA (32 nm) is more predominant compared to that of ADDR2 dye (5 nm) as shown in Fig. 3. However, the pattern observed on the extent of fluorescence enhancement of both ADDR1 and ADDR2 dyes after the addition of BSA is almost similar (Fig. 4). The fluorescence enhancement observed in ADDR2 dye after the addition of BSA is attributed only due to the suppression of PET process through space [4], whereas the same observed in ADDR1 dye does not occur completely through PET process. The excited state properties of ADDR1 dye are found to be entirely different on the introduction of a protein molecule such that the microenvironment around the dye is altered due to hydrogenbonding and hydrophobic moieties present in the BSA. If suppression of PET process alone had been responsible for the fluorescence enhancement, a same pattern would have been resulted for ADDR3 dye after the addition of BSA. On the contrary, a decrease in the fluorescence intensity of ADDR3 dye was observed on the initial addition of BSA, which is similar to that of ADDR1 dye, but a further increase in BSA concentration results in no drastic change in the fluorescence intensity or shift in the emission maximum. Moreover, the emission intensity of ADDR3 dye after the addition of BSA almost remains the same at higher concentration of BSA (Supporting information Fig. S2) and this clearly shows that the LE state emission of ADDR3 dye is not largely influenced by BSA. In an ADDR dye containing p-C6H4N(CH3)2 and N–CH3 moieties in the 9th and 10th positions respectively, the excited state characteristics almost remain unaltered on the introduction of a large protein molecule. The presence of several hydrophobic moieties alone does not bring the change in the fluorescence intensity and lifetime. Although ADDR1 and ADDR3 dyes differ only in the substitution at the 10th position, a marked variation in the emission intensity and red shift in the emission maximum of ADDR1 dye is observed after the addition of BSA. On the contrary, significant shift in the emission maximum towards the red region was not observed after the addition of BSA to ADDR3 dye. This clearly shows that the excited state properties of ADDR dyes having free N–H in the 10th position, which is favorable for hydrogen-bonding are governed by the concentration of BSA. From steady-state fluorescence measurements, it is evident

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Scheme 3. Schematic representation of hydrogen-bonding between ADDR1 dye with Bovine Serum Albumin (BSA). The introduction of BSA results in hydrogen-bonding at the N–H hydrogen thereby resulting in a red shift in the emission maxima.

12000

9000

(F-FO )/(F-Fx)

that the presence of N(CH3)2 or OCH3 moiety in the para position of the phenyl ring in the 9th position of the acridinedione ring results in a fluorescence enhancement accompanied with a red shift. This is attributed to the hydrogen-bonding interaction of the amino nitrogen or the carbonyl oxygen of amino acid moieties of BSA with the N–H hydrogen of ADDR1 dye (10th position) as shown in Scheme 3. A similar result was also observed in the ADDR1 dye [52] with BSA. The interaction of ADDR3 dye with BSA was also studied, but no significant change in the fluorescence intensity or red shift was observed. When the N–H hydrogen of ADDR1 dye is replaced by N-methyl group the possibility of hydrogen-bonding interaction is ruled out because of the absence of hydrogen-bonding donor moieties (N–H hydrogen). From quantum mechanical (QM) studies it was shown that the N–H hydrogen of ADDR dyes is not hydrogenbonded with oxygen atom of water or methanol [58]. It has been established that the addition of BSA [4] and non-fluorescent solutes like urea derivatives, guanidine hydrochloride and amides to nonPET based ADDR dyes results in no characteristic change in the fluorescence intensity or lifetime [48,50,58], whether it has N–H or N–CH3 moieties in the 10th position. In order to establish a quantitative relationship between dye and BSA, we carried out binding studies of probe–protein interaction. The binding constant of protein interaction with drugs and fluorescent dyes is generally in the order of above 105 [M  1], which signifies a higher binding constant. A very high binding constant is correlated to very strong interaction between the dye and BSA. Using modified Benesi–Hildebrand equation [68], a plot of (F–F0)/ (Fx  F0) versus 1/ [BSA] of ADDR1 dye–BSA provides the nature of most probable interaction. F0 represents the fluorescence intensity of the ADDR1 dye in the absence of BSA. Fx represents the fluorescence intensity of the dye at an intermediate concentration of BSA and F represents the fluorescence intensity at maximum concentration of BSA (3.0  10  4 M). The graph is found to be an exponential curve as shown in Fig. 5. This reveals that there exists no clear 1:1 binding of dye with protein molecule and the variation in the binding pattern is governed by the concentration of the protein. (Fig. 5 (regions 1 and 2)). Our previous report reveals that ADDR2 dye [4] is presumably confined to the hydrophobic site of BSA and this is evident by a very high binding constant value (6.370.2  103 M  1). A high binding constant value of ADDR2–BSA signifies that the dye prefers to reside in the hydrophobic domain of BSA rather than in the hydrophilic phase [4] and this is further established from fluorescence lifetime studies with specific site binding fluorophores like phenyl butazone and fluflenamic acid [4] with ADDR–BSA system. On the contrary, the binding constant value of ADDR1 dye–BSA could not be ascertained due to large variation in binding nature at low and high concentrations of BSA,

2

6000

1 3000

0

5

10

15

20 -1

25

30

35

-4

[BSA] x 10 M Fig. 5. Plot of (F–F0/Fx–F0) of ADDR1 dye vs [BSA]  1.

whereas the binding constant value of ADDR2 dye with BSA is in agreement with that of many fluorescent dyes [34,40–41,69–72]. 4.3. Circular Dichroism (CD) spectral studies Circular Dichroism (CD) spectral measurements of BSA [73] with ADDR1 and ADDR2 dyes were carried out in water in the absence of buffer. The CD results are expressed in terms of mean residue ellipticity (MRE) in deg cm2 d mol  1 based on the equation [74]. MRE ¼Observed CD (m deg)/10Cpnl where Cp is the molar concentration of BSA (1.5  10  6 M), n denotes the total number of amino acid residues present in BSA (583) and l represents the path length (0.1 cm) of the cuvette. The MRE value at 208 nm is calculated from the equation

α−Helix (%) = [( − MRE (208 nm) − 4000)/(33, 000 − 4000)] × 100 where MRE (208 nm) 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 the pure α-helix of BSA at 208 nm. The CD spectrum for BSA [75] in aqueous solution is characteristic of macromolecules with high α-helical content, monitored by the two well-defined ellipticities values at 208 and 220 nm respectively. In the present study, α-helicity of native BSA as well as BSA with ADDR1 and ADDR2 is calculated using the above equation and the values given in Table 1. The CD spectrum of BSA with ADDR dyes is shown in Fig. 6 and the α-helicial content of unbound BSA at 208 nm is found to be 60.73%. A drastic

R. Kumaran et al. / Journal of Luminescence 164 (2015) 146–153

151

Table 1 Circular Dichroism (CD) spectra of Bovine Serum Albumin (BSA) with acridinedione dyes (ADDR) in water. [Dye]  10  5 M

Mean Residual Ellipticity (θ) at 220 nm

Mean Residual Ellipticity (θ) at 208 nm

Alpha helicity (%) (220 nm)

Alpha helicity (%) (208 nm)

1.0 1.0

– ADDR1 3.05 70.05 ADDR2 3.05 70.05

 76.3176  44.1079

 79.3665  47.0389

58.93 39.88

60.73 41.61

 41.0611

 44.3264

38.07

40.01

1.0

2

Log Counts

Bovine Serum Albumin [BSA]  10  6 M

6 1

1 4

8

12 Time, ns

16

Fig. 7. Fluorescence decay of ADDR 1 dye in the absence and water. λex 377 nm. (1) Laser profile, (2) ADDR1 dye, (3) 0.3  10  4 M, (4) ADDR1 dyeþ BSA 0.6  10  4 M, (5) 1.2  10  4 M, (6) ADDR1 dyeþ BSA 1.8  10  4 M, and (7) 3.0  10  4 M.

Fig. 6. Circular Dichroism (CD) spectra of ADDR dyes with BSA.

decrease in α-helicity is observed on the addition of ADDR1 and ADDR2 dyes to BSA. This decrease in α-helix content indicates that the binding of the hydrogen-bonding moieties of the ADDR dyes complexes with BSA thereby induces the conformational changes in BSA. There are reports which signify that addition of denaturants also results in a conformational change of the secondary structure of BSA which is attributed to the presence of hydrogenbonding functional groups, [76–79] which are also observed in the present study. Results from CD spectral studies of BSA with ADDR dyes exhibit a decrease in the helical structure from 60% to 40% at 208 nm. This is in good agreement with the conformational studies of protein folding and unfolding processes. However, the CD spectra of BSA in both the absence and presence of ADDR dye are similar in shape, which shows that the structure of BSA after the addition of ADDR dye is predominantly α-helix and no significant conformational changes result. The native structure of BSA is not perturbed by the introduction of a dye molecule. From CD spectral studies it is clear that the BSA–ADDR dye complex can exist in the aqueous solution through hydrogen-bonding network, the presence of hydrophobic moieties in ADDR dyes preferentially promotes unfolding of BSA rather than disrupting the secondary structure of BSA, and the native structure of BSA is not completely perturbed by the introduction of a dye molecule. 4.4. Time resolved fluorescence spectral studies The fluorescence lifetime of ADDR1 and ADDR3 dyes are 8.5270.12 and 8.7370.15 ns respectively in water. From the fluorescence lifetime studies, it is evident that the PET nature of ADDR1

20 presence ADDR1 ADDR1 ADDR1

24 of BSA in dye þBSA dye þBSA dye þBSA

and ADDR3 dyes is lost in protic solvents, whereas the fluorescence lifetime of ADDR2 dye is around 500710 and 400710 ps in water and methanol respectively [4]. Fluorescence lifetime of 500710 ps illustrates that the PET nature of ADDR2 dye still exists in protic solvents like water and this lifetime component is stable still even at any concentration of BSA added to it. It is known that the addition of BSA results in the introduction of several hydrophobic moieties in aqueous solution and influences the fluorescence lifetime of ADDR dyes [4]. The fluorescence decay profile of ADDR1 dye in the absence and presence of BSA is shown in Fig. 7 and that of ADDR2 dye with BSA is provided in Supporting information (Fig. S4). Addition of BSA to ADDR2 dye results in a triexponential lifetime and these lifetimes are attributed to the free dye component in aqueous phase (unbound dye), dye situated in the hydrophilic and hydrophobic domain. The relatively long lifetime of ADDR1 and ADDR3 dyes is due to the absence of PET phenomenon and this is similar to that of non-PET based acridinedione dyes' behavior in water. The lifetime of non-PET based ADDR dyes is of the order of 8.570.2 ns and the addition of BSA results in no significant change in the fluorescence lifetime [4]. On the addition of BSA to ADDR1 dye the fluorescence lifetime decay exhibits a bi-exponential decay pattern and the fluorescence lifetime of free dye and dye bound to BSA is given in Table 2. A gradual decrease in the fluorescence lifetime and the relative amplitude of the major lifetime component (free ADDR1 dye in water) is observed after the addition of BSA, and a gradual increase in the relative amplitude of the minor lifetime component (dye bound to BSA) results. The marked change in the fluorescence lifetime of ADDR1 dye supports the emission spectral pattern of ADDR1 dye with BSA. The LE state emission is highly susceptible to increasing concentration of BSA and we observed that the fluorescence lifetime of ADDR1 dye is not constant when compared to that of ADDR2 dye. The minor component lifetime of ADDR1 dye (200 ps) of 11% amplitude observed after the addition of BSA suggests that there exists a relatively lower proportion of dye bound to BSA in the aqueous phase. Further, the relative amplitude of ADDR1 dye bound to BSA (12%) is found to be less than that of ADDR 2 dye bound to BSA (64%) in water (Table 3). We have carried out specific site binding studies of ADDR2 dye [4] with BSA in the presence of phenyl butazone and flufenamic acid in water. We have proposed a

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Table 2 Fluorescence lifetime decay analysis of ADDR1 dye–BSA in water. Bovine Serum Albumin [BSA]  10  4 M

Fluorescence lifetime (τ1) in nanoseconds (ns)

Fluorescence Relative lifetime (τ2) in amplitude A1 (%) nanoseconds (ns)

0 0.6 1.2 1.8 3.0

8.52 70.12 8.17 70.12 7.88 70.11 7.63 70.12 7.51 70.11

0.2617 0.08 0.1687 0.04 0.1857 0.05 0.1967 0.08

100 97 88 85 89

Relative amplitude A2 (%)

Chi square (χ2)

3 12 15 11

1.049 1.098 1.149 1.145 1.028

bulk viscosity factor had been involved in the fluorescence enhancement, addition of BSA to PET or non-PET dyes should have also resulted in a fluorescence enhancement; in the present study this was not observed. Only ADDR1 and ADDR2 dyes exhibit a fluorescence enhancement which clearly reveals that the excited state properties of these dyes on the addition of BSA is not influenced by viscosity of the medium. Here we also report that the LE state of ADDR1 dye induces a new emissive state in aqueous medium which is influenced by the addition of BSA. The fluorescence enhancement is not attributed to the suppression of PET process as observed in the case of ADDR2 dye wherein, the hydrogen-bonding interaction at the N–H hydrogen results a large variation in the LE state emission which in turn promotes a new CT state towards the red region.

Table 3 Fluorescence lifetime decay analysis of ADDR2 dye–BSA in water. BSA [M]  10  4

τ1 (ns)

τ2 (ns)

τ3 (ns)

B1

B2

B3

χ2

6. Conclusion

0 0.06 0.12 0.24 0.3 0.6 1.2 1.8 2.7 3.0

0.517 0.01 0.517 0.02 0.517 0.03 0.517 0.01 0.517 0.01 0.517 0.01 0.517 0.01 0.517 0.01 0.517 0.01 0.517 0.01

– 0.85 7 0.01 1.41 70.04 1.3 70.1 1.7 70.2 2.0 7 0.2 2.17 0.1 2.4 7 0.1 2.5 7 0.1 2.7 7 0.1

– 6.32 7 0.02 6.187 0.09 6.29 7 0.09 6.38 7 0.09 6.80 7 0.08 6.95 7 0.05 7.137 0.05 7.217 0.05 7.40 7 0.05

100 89 89 80 78 68 51 43 38 36

– 5 2 5 5 7 11 14 17 19

– 6 9 15 17 25 38 43 45 45

1.05 0.97 1.13 1.14 1.00 1.10 1.20 1.04 1.06 1.12

The bichromophoric system ADDR1 dye exhibits single fluorescence in protic solvents and only fluorescence observed is the LE state emission. The observation based on photophysical studies reveals that the increase in the fluorescence intensity after the addition of BSA is due to the formation of a new state which is neither governed by PET nor by ICT state. The addition of BSA results in a large variation in the polarity of the medium surrounding the dye molecule, due to the hydrogen-bonding interaction and the presence of hydrophobic moieties present in BSA. ADDR1 and ADDR2 dyes significantly decreases the alpha helical structure of BSA and do not disrupt the secondary structure.

mechanism, wherein ADDR2 dye preferably resides in the hydrophobic domain of BSA, rather than in the hydrophilic domain. This was well supported by time-resolved fluorescence lifetime studies of ADDR2 dye and another PET based dye (dimedone based acridinedione dye (ADD1)) with BSA in water [4], wherein both these dyes are structurally similar, the relative amplitude of dye located in the hydrophobic domain varies. ADDR2 dye is predominantly located in the hydrophobic domain of BSA rather than in the aqueous phase and the nature of interaction is largely hydrophobic, whereas ADDR1 dye remains unbound with the protein molecule and the nature of interaction is through hydrogenbonding. In general hydrogen-bonding interaction influences the conformational changes in protein, and in the present study ADDR1 dye promotes more unfolding of the native structure of BSA than ADDR2 dye.

5. Mechanism of fluorescence enhancement The mechanism of fluorescence enhancement of a dye–BSA interaction in aqueous solution is governed by two different mechanisms [4,5,11,40–42,62]. One mechanism signifies that the fluorescence enhancement is attributed to the changes in the polarity around the probe molecule. The gradual addition of protein results in a decrease or increase in the polarity of the medium around the vicinity of the dye and causes an increase in the fluorescence intensity accompanied with a blue shifted or red shifted emission. The shift in the emission maxima is attributed to the change in the microenvironment around the fluorophore resulting in the stabilization or destabilization of the CT state. Interaction of BSA and human serum albumin (HSA) with fluorescent probes results in a change in the polarity around the fluorophore and this has been well documented in the literature [26,27]. The second mechanism signifies that an intermolecular energy transfer from the protein molecule to the dye results in a fluorescence enhancement [80–82]. Apart from these mechanisms, the fluorescence enhancement is also correlated to the binding of the dye to the protein molecule resulting in the formation of a stable complex in the excited state [71]. If the

Acknowledgments Financial support by DST-IRHPA (DST-FU is DST-Femto Upconversion Maintenance Support) and UGC-INNOVATIVE Program (UGC Assistance for Masters Degree in Photonics and Biophotonics-Teaching and Research in Interdisciplinary and Emerging Areas-NCUFP/Co 2134 dated 25.10.2004) is acknowledged. R.K. and M.V. thank the Management, D.G. Vaish- nav College (Autonomous), Chennai, for their support.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jlumin.2015.03. 010.

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