Interactions of serum albumins with antitumor agent benzo [a] phenazine—a spectroscopic study

Journal of Luminescence 131 (2011) 2195–2201

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Interactions of serum albumins with antitumor agent benzo [a] phenazine—a spectroscopic study Radhakrishnan Sivakumar, Selvaraj Naveenraj, Sambandam Anandan n Nanomaterials and Solar Energy Conversion Lab, Department of Chemistry, National Institute of Technology, Trichy 620015, India

a r t i c l e i n f o

abstract

Article history: Received 4 October 2010 Received in revised form 28 April 2011 Accepted 3 May 2011 Available online 18 May 2011

We present an investigation on the site specific interaction of antitumor agent benzo [a] phenazine (BAP) with serum albumins (HSA and BSA) and related photo-physical properties using absorption, emission and lifetime measurements. The absorption and emission measurements reveal that the binding of biomolecule benzo [a] phenazine took place near tryptophan moiety present in sub-domain IIA in serum albumins (HSA and BSA). In the selective excitation of benzo [a] phenazine at 365 nm, it was observed that the ground state of serum albumin quenches the excited benzo [a] phenazine through charge transfer exciplexation. The fluorescence decay analysis of serum albumins in the presence of benzo [a] phenazine shows decrease in lifetime, which confirms that photo-induced electron transfer takes place from serum albumins (HSA and BSA) to BAP. Also a suitable mechanism was proposed for the observed photo-induced electron transfer processes. Binding average distance (r) between the donor (serum albumins) and acceptor (benzo [a] phenazine) calculated using FRET theory confirmed their high probability of binding interaction. & 2011 Elsevier B.V. All rights reserved.

Keywords: Human serum albumin Bovine serum albumin Lifetime Benzo [a] phenazine Photo-induced electron transfer Exciplex formation

1. Introduction Phenazine based compounds are the most abundant natural products, having myriad of biological activities [1]. Phenazine (PZ) derivatives are used as drugs for the treatment of tuberculosis [2] and possess excellent activity against many other mycobacterial infections [3]. In this context, series of substituted angular benzo [a] phenazines were synthesized for their potential application as antitumor agents [4]. From benzo [a] phenazine, Silva et al. [5] synthesized a macrolactone, which shows potent activity against Mycobacterium tuberculosis. In addition, phenazine derivatives are extensively used as a photo-sensitizer [6] in photodynamic therapy (PDT) where the combination of light and photo-sensitizer generates highly reactive oxygen species or hydroxyl radicals near the tumor to selectively destroy the targeted tissue. Hence, there have been significant researches focused on understanding their excited state photo-physical and photo-chemical properties [7–15]. Particularly, most of the earlier work [9–12] focused on the triplet state of PZ derivatives because it undergoes efficient intersystem crossing. Besides the above biological and photochemical significance of phenazine derivatives, their interactions with biomolecules present in our body bring out different perspectives. Though the tumor localizing sensitizers are active only against targeted tissue, their transportation in blood stream often takes place via

n

Corresponding author. Tel.: þ91 431 2503639; fax: þ91 431 2500133. E-mail addresses: [email protected], [email protected] (S. Anandan).

0022-2313/$ - see front matter & 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2011.05.005

binding with serum albumins and other plasma proteins such as low and high density lipo-proteins. Therefore, the elucidation of interaction between drugs and serum albumins and their recognition to a particular part of serum albumin is a most essential step for understanding the molecular basis of biological activity and designing of novel drugs [16–20]. Most of these studies reveal the interaction of organic substances with serum albumins mainly recognized by the presence of multiple lipophilic binding sites on the surface of proteins. Hence here we made an attempt to investigate the interaction of bioactive molecule benzo [a] phenazine (BAP) with two serum albumins namely bovine serum albumin (BSA) and human serum albumin (HSA). The significance of such a study is based on the fact that, phenazine derivatives forms exciplexes with aromatic amines [21,22] and their stability mostly depends on the nature of the solvent [23]. Consequently, tryptophan is an aromatic amine present in serum albumins and it is expected that this may interact with benzo [a] phenazine. To address this particular point, spectroscopic measurements (UV–visible, fluorescence and lifetime) were performed and suitable mechanism was proposed.

2. Experimental section 2.1. Materials o-Phenylene diamine, 1,2-naphthoquinone, HSA and BSA were purchased from Sigma-Aldrich and were used without further purification. Serum albumin (BSA and HSA) solutions were

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0.30

prepared in pH 7.4 phosphate buffer solution (PBS) and stored at 0–4 1C. All other reagents used were of analytical reagent grade. Water from a Milli Q apparatus (Millipore, USA) was used throughout the experiments. All the experiments were performed at atmospheric conditions.

0.25

N

Measurements were carried out by taking 3 ml phosphate buffer solution of appropriate concentration of serum albumin and titrating manually by successive addition of increasing concentration (10 ml) of ethanol stock solution of BAP (180, 30 and 30 mM for absorbance, fluorescence and lifetime measurements, respectively). For reverse experiments, measurements were carried out by taking 3 ml phosphate buffer solution of appropriate concentration of BAP prepared from its ethanol stock solution and titrating manually by successive addition of increasing concentration (10 ml) of serum albumin (90 and 150 mM for absorbance and fluorescence, respectively). Since the stock solution of benzo [a] phenazine was prepared in ethanol, the quenching effect of ethanol was evaluated. The result showed that the effect of ethanol on the interaction of BAP with serum albumin could be negligible for the amount used in our experiment. Also appropriate blanks corresponding to the buffer were subtracted to correct the fluorescence background. UV–vis absorption spectra were recorded with a T90þ UV–vis spectrophotometer (PG Instruments, United Kingdom). Fluorescence measurements were performed on a RF-5301 PC spectrofluorophotometer (SCHIMADZU, Japan). The samples were degassed using pure argon gas for 15 min prior to each experiment. Fluorescence lifetime measurements were carried out in a picosecond time correlated single photon counting (TCSPC) spectrometer with a tunable Ti-sapphire laser (TSUNAMI, Spectra physics, USA) as the excitation source of 280 nm excitation wavelength. The fluorescence decay curves were analyzed using the software provided by IBH (DAS-6). Synthesized ligand reported in this manuscript was characterized using IR, NMR and GC–Mass spectrometers. FT-IR spectra were recorded using a JASCO-460 plus model spectrometer using KBr pellet techniques. 1H-NMR spectra were recorded with a Bruker 400 MHz NMR spectrometer at 298 K with TMS as internal standard. GC–Mass spectra were obtained using Clarus 560 GC/Mass spectrometer. 2.3. Synthesis of benzo [a] phenazine Benzo [a] phenazine was successfully synthesized by refluxing o-phenylene diamine and 1,2-naphthoquinone in ethanol for 6 h and purification was done by column chromatography using mobile phase (hexane:ethyl acetate; 9:1). The product was recrystallized from hexane as pale yellow needles (yield: 55%). Data: UV–vis (lmax): 3 peaks at 278, 388 nm (hump) and 405 nm (hump); IR (cm  1): 1637 (m, C¼N), 1358 (mw, C–N), 691–875 (mw, C–H bending) and 2926 (w, C–H stretching); 1H-NMR (in CDCl3) dppm: 7.72–7.89 (7H, m), 8.23 (1H, s), 8.30 (1H, s) and 9.32 (1H, s). Mass analysis (GC/MS): m/z¼230.

3. Results and discussion 3.1. Absorption measurements Bioactive molecule benzo [a] phenazine (BAP) shows one sharp peak at 278 nm (e ¼7.90  104 M  1 cm  1) and a broad peak around 400 nm with two humps at 388 nm (e ¼2.33  104 M  1 cm  1) and 405 nm (e ¼ 2.33  104 M cm  1) in the absorption spectra (Fig. 1), whereas serum albumins (HSA and BSA) shows absorption maxima at 280 nm due to tryptophan (Trp), tyrosine (Tyr) and phenylalanine

Absorbance

0.20

2.2. Methods

N 0.15

Benzo[a]phenazine

0.10 0.05 0.00

250

300

350

400

450

500

Wavelength (nm) Fig. 1. Absorption spectrum of the benzo [a] phenazine in phosphate buffer solution; [BAP]¼3 mM.

(Phe) residues. In order to find out the specific molecular interaction between benzo [a] phenazine (BAP) and serum albumins (HSA and BSA) in their ground state, absorption spectra of serum albumins were monitored in the absence and presence of benzo [a] phenazine by UV–visible spectrophotometer. Upon addition of BAP to serum albumin solution, the peak at 280 nm (Fig. 2a and b) was gradually increased with simultaneous enhancement in broad peak around 400 nm, which is a characteristic of BAP molecule. Since both BAP and SA have absorption maxima at 280 nm, it is unable to predict whether the increase in absorption might be due to interaction between the molecules or due to increase in concentration of BAP. As a consequence of this fact, further experiments were carried out by monitoring the absorbance of fixed concentration of BAP (3 mM) with increasing concentration of serum albumins (0–2.4 mM). It is interesting to note that the broad absorption spectrum around 400 nm (inset of Fig. 2a and b), which is a characteristic of BAP was found to be increased upon increasing the concentration of serum albumin, even though there may be an increase in the absorption maxima at 280 nm due to increase in concentration of SA. These suggest that there may be a ground state complex formation between SA and the biomolecule BAP through N-heterocycle interactions. Accordingly, the electronic spectra of N-heterocycle derivatives are very sensitive to the surrounding environments and hydrogen bonding interactions, which may completely alter the photo-physical properties by changing the relative energies of the n–p* and p–p* transitions [24]. 3.2. Fluorescence measurements The intrinsic fluorescence of serum albumins appears at 340 nm when excited at 280 nm, which is originating from tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) residues. Intrinsic fluorescence characteristics are very sensitive to its microenvironment. Actually, the intrinsic fluorescence of serum albumin were mainly contributed by the Trp residue alone due to the low quantum yield of Phe residue and the total quenching of Tyr residue (when it is self ionized or nearby an amino group, carboxylic acid, or Trp), which would be ultimately weakened if there is a slight change of the local surroundings of serum albumins, such as conformational transition, biomolecular binding and denaturation [25]. For this reason, the serum albumin samples were excited at 280 nm and the spectra were recorded in the range 300–550 nm in the absence and presence of BAP. Fig. 3a

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0.8

1.2

Increase in [ HSA ]

Absorbance

0.6

1.0

Absorbance

0.8

0.4

Increase in [ HSA ]

0.2

Increase in [ BAP ] 0.0 250

0.6

300

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0.4

Increase in [ BAP ]

0.2

0.0 250

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Wavelength (nm) 0.8

1.2 Increase in [ BSA ]

1.0

Absorbance

0.8

Increase in [ BAP ]

Absorbance

0.6

0.4

Increase in [ BSA ] 0.2

0.0 250

0.6

0.4

300

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400 450 500 550 Wavelength (nm)

600

650

700

Increase in [ BAP ]

0.2

0.0 250

300

350

400

450

500

550

600

650

700

Wavelength (nm) Fig. 2. Absorption spectrum of HSA (a) and BSA (b) in the absence and presence of BAP; [HSA] ¼[BSA] ¼5 mM; [BAP] ranges from 0, 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, 4.2, 4.8 and 5.4 mM. Inset shows the absorption spectrum of BAP in the absence and presence HSA (a) and BSA (b); [BAP] ¼ 3 mM; [HSA] and [BSA] ranges from 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1 and 2.4 mM. Titrations were carried out in phosphate buffer solution.

and b shows the fluorescence spectra of the serum albumins in the presence of various concentration of BAP. With gradual increase in BAP concentration, the fluorescence of serum albumins was found to be decreased accompanying the simultaneous enhancement in the fluorescence of BAP (lemi ¼469 nm). It could be seen from Fig. 3(a) and (b) that the intensity of the tryptophanyl fluorescence (lemi ¼336 nm for HSA and 340 nm for BSA) is specifically quenched with marginal blue shift in their emission maxima, unmasking the tyrosyl fluorescence (lemi ¼314 nm for both HSA and BSA) on increasing the concentration of BAP. However, even at higher concentration of BAP (2 mM), a shoulder is seen at 340 nm in the BSA–BAP system, but in the HSA–BAP system the tryptophan peak was almost diminished (see Fig. 4).

These suggest that quenching of fluorescence of HSA by BAP is more compared to that of BSA. Reason for such enhanced quenching processes for HSA compared to BSA may be ascertained from the crystal structure analyses, which revealed that HSA contains 585 amino acid residues with only one tryptophan (Trp), which is responsible for their fluorescence, located at position 214 along the chain whereas BSA contains 582 amino acid residues with two tryptophans located in positions 134 and 212 in which Trp-212 is in chemical microenvironment similar to Trp-214 in HSA and Trp-134 is located at the surface of the molecule [26]. The observed results in this system are almost similar to the interaction studies reported by Mathew and Balaram [27] for serum albumins–bilirubin system.

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25

200

Increase in [ BAP ]

HSA + BAP BSA + BAP

20

Intensity

Intensity

150

100

15

10

Increase in [ BAP ] 5

50

0

0

300

350

450 400 Wavelength (nm)

200

500

320

550

360 Wavelength (nm)

400

Fig. 4. Fluorescence spectra of HSA and BSA at higher concentration of BAP; lex ¼ 280 nm. [HSA]¼ [BSA] ¼0.2 mM; [BAP] ¼2 mM. Titrations were carried out in phosphate buffer solution.

Increase in [ BAP ]

2.5

150 HSA BSA

100 Increase in [ BAP ]

F0 / F

Intensity

2

1.5

50 1

0 300

350

400

450

500

550

Wavelength (nm)

0

Fig. 3. Fluorescence spectra showing quenching of intrinsic fluorescence of HSA (a) and BSA (b) with increase in BAP concentration; lexi ¼ 280 nm. [HSA] ¼[BSA] ¼0.2 mM; [BAP]¼ 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mM. Titrations were carried out in phosphate buffer solution.

Further, we investigate the extent of quenching by well known Stern–Volmer equation [28] as mentioned below F0 ¼ 1þ kq t0 ½Q  ¼ 1 þKSV ½Q  F

0.5

ð1Þ

where F0 and F are the fluorescence intensities in the absence and presence of quencher, kq is the fluorescence quenching rate constant of the biomolecules, KSV is the Stern–Volmer fluorescence quenching constant, t0 is the average lifetime of the biopolymer without quencher and [Q] is the concentration of the quencher. According to Stern–Volmer equation, F0/F versus [Q] (Stern–Volmer plot) was drawn for the quenching of serum albumins by BAP (Fig. 5). The resulted plots exhibit a good linear relationship (correlation coefficient (R)¼0.979 for HSA and 0.966 for BSA), which indicates the existence of possible binding that occurs only between N-heterocycle of BAP and tryptophan residue of SA [29]. From the slopes of the linear plots, the Stern– Volmer quenching constants (KSV) were calculated as 1.41  106 M  1 for HSA and 1.12  106 M  1 for BSA. The bimolecular quenching rate constants, kq of BSA and HSA are found to be 1.94  1014 and 2.80  1014 M  1 s  1, respectively. These kq values

2

4

6

8

Q x 10-7 M Fig. 5. Stern–Volmer plots for fluorescence quenching of HSA and BSA with increasing concentrations of BAP.

are greater than that of scatter procedure (1010 M  1 s  1), which show that the quenching mentioned above is not initiated by dynamic collision but from the ground state complex formation of static quenching. Accordingly, quenching constants exceeding the diffusion-controlled value were generally found for the interaction of serum albumins with other molecules [30–38]. But taking into account that, the absorption spectrum of BAP efficiently overlaps with emission spectrum of the serum albumins (Fig. 6); the increase in emission of BAP (Fig. 3a and b) may be due to the energy transfer from singlet excited state of tryptophan in serum albumins to benzo [a] phenazine. In order to determine the possibility of direct energy transfer between serum albumins and BAP, the fluorescence of BAP (lex ¼280 nm) was measured in the absence and presence of serum albumin (2 mM). The enhancement of fluorescence may be expected but the results are in controversy i.e., fluorescence of BAP was quenched by HSA (Fig. 7). Similar results were obtained in the case of BSA also (figure not shown). This decrease in fluorescence indicates that the electron transfer is more feasible compared to energy transfer between SA and BAP at the excited state. This reveals that BAP may act as an electron acceptor in the excited state. Thus, the

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5 0.00 300

0 350

400 Wavelength (nm)

0 400

450

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Wavelength (nm)

Fig. 6. Spectral overlap between (a) the absorption spectrum of BAP (acceptor) and (b) the fluorescence spectrum of HSA (donor) in phosphate buffer solution.

200

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BAP BAP + HSA Intensity

500 400 Intensity

450

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Increase in [ BSA ] 100

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200 0 400

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Wavelength (nm) Fig. 7. Fluorescence spectra of BAP in phosphate buffer solution in the absence and presence of HSA. [HSA] ¼2 mM (excitation wavelength lexi ¼ 280 nm).

initial interaction between serum albumins and benzo [a] phenazine involves the formation of an exciplex and subsequent excitation leads to charge transfer from serum albumin to BAP, which ties up with the nature and structure of the donor (serum albumins) and the acceptor (benzo [a] phenazine) molecules. It is well known that phenazine is an excellent photo-sensitizer with potential antitumor activity and their excitation in the longer wavelength results in the oxidation of targeted tissue. In this aspect, emission measurements for BAP were performed by excitation at 365 nm (which is not absorbed by the serum albumins) in the absence and presence of serum albumins. A progressive decrease in fluorescence intensity was observed (Fig. 8a and b) upon addition of serum albumins to BAP. Such decrease in fluorescence indicates that the ground state of serum albumin quenches the excited benzo [a] phenazine through charge transfer exciplexation. Here we could understand that the energy transfer from BAP to serum albumins was ruled out, since the absorption of serum albumins is at lower wavelength (280 nm) with respect to excitation wavelength (365 nm). So the electron transfer from serum albumins to BAP was the only way for this observed fluorescence quenching of BAP. On the contrary,

450

500 Wavelength (nm)

550

600

Fig. 8. Fluorescence quenching of BAP (2  10  6 M) with increasing concentration of HSA (a) and BSA (b). [HSA] and [BSA] ranges from 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 mM. Titrations were carried out in phosphate buffer solution.

Gentili et al. [39] observed that the fluorescence of flindersine was enhanced in the presence of BSA. Based on the observed results, we propose a mechanism as mentioned below for the interaction of serum albumins with BAP at different excitation wavelengths.

Upon excitation at 280 nm both serum albumin and BAP gets excited to form final products (SA þ þ BAP  ) by photo-induced electron transfer processes. Whereas excitation at 365 nm, BAP molecule only gets excited to form final products either by accepting electrons from tryptophan in serum albumin directly or by forming triplet state molecules by intersystem crossing processes. In general, the reason for such radical ion products formation can be ascribed to the formation of exciplex between tryptophan in serum albumin and BAP. Similar exciplex formation between a phenazine derivative, dibenzo [a,c] phenazine (DBPZ) and aromatic amines were reported by Dey et al. [40]. Thus, a

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longer wavelength excitation leads to an indirect evidence for the electron transfer mechanism.

3.3. Lifetime measurements For further insight into the fluorescence quenching processes, time resolved fluorescence lifetime measurements were carried out for BSA and HSA in the absence and presence of BAP at the excitation wavelength of 280 nm. The fluorescence decay curves of serum albumins were bi-exponential in the absence of benzo [a] phenazine, may be due to interaction of amino acids with their environment. Average lifetimes observed for HSA and BSA were 5.59 and 5.78 ns, respectively. Upon addition of BAP (0–0.3 mM) to these solutions, bi-exponential decay was observed along with the decrease in average fluorescence lifetime from 5.59 to 3.02 ns for HSA and 5.78 to 5.27 ns for BSA (refer Table 1; Fig. 9a and b for HSA and BSA, respectively). This reveals the fact that the serum albumin undergoes photo-induced electron transfer processes with BAP. The Stern–Volmer quenching plots for the time Table 1 Relative and average lifetime values of HSA and BSA in the absence and presence of benzo [a] phenazine. Benzo [a] phenazine (  10  7 M)

Lifetime values (ns) HSA

0 1 2 3

BSA

t1

t2

/tS

t1

t2

/tS

2.16 1.96 1.95 1.66

6.71 6.24 6.22 5.56

5.59 4.34 3.99 3.02

2.36 2.22 2.09 2.23

6.27 6.13 5.96 6.04

5.78 5.52 5.27 5.27

Counts

The fluorescence measurements proved that BAP formed a complex with HSA/BSA, and the distance between donor fluorophore and bound BAP molecules can be determined according to the Fosters non-radiative energy transfer theory. The efficiency of energy transfer is related to the distance between the donor and acceptor by R6 F ¼ 6 0 6 F0 R0 þ r

ð2Þ

where r is the distance between the donor and acceptor, and R0 is the critical distance when the transfer efficiency is 50%, which can be calculated by R60 ¼ 8:8  1025 ½k2 N 4 jJ

ð3Þ

where k2 is the spatial orientation factor related to the geometry of the donor and acceptor of dipoles, k2 ¼2/3 for random orientation as in fluid solution, N is the refractive index of the medium assumed to be 1.33, Q is the fluorescence quantum yield of the donor (0.15 for HSA and 0.118 for BSA) [41] and J is the overlap integral of the fluorescence emission spectrum of the donor serum albumins and the absorption spectrum of the acceptor BAP (Fig. 6), which could be calculated by R1 4 FðlÞeðlÞl dl J ¼ 0 R1 ð4Þ 0 FðlÞdl

1000 100 10

0

5

10

15

20

25

30

35

40

45

Time (ns) 10000 1000 Counts

3.4. Energy transfer between BAP and serum albumins

E ¼ 1

10000

1

resolved fluorescence quenching of serum albumins were also obtained by plotting t0/t versus [Q] and it was found linear (inset of Fig. 9a and b.), indicating that the quenching process is dynamic in nature. This also suggests that photo-induced electron transfer occurs between the excited serum albumin and BAP to form final products. The Stern–Volmer quenching constant and quenching rate constant found out from the lifetime measurements, of HSA (KSV ¼2.60  106 M  1, kq ¼4.65  1014 M  1 s  1) are greater than that of BSA (KSV ¼0.37  106 M  1, kq ¼0.64  1014 M  1 s  1), which follow the same trend as in the case of Stern–Volmer constants obtained from fluorescence quenching measurements. The Stern–Volmer constants obtained from lifetime measurements indicate that the quenching process was initiated through ground state formation. Therefore the quenching was initiated by static mechanism and then, at least in part, dynamic i.e., electron transfer.

where F(l) is the corrected fluorescence intensity of the donor in the wavelength range from l to (l þ Dl), with the total intensity normalized to unity and e(l) is the molar extinction coefficient of the acceptor at l. Using Eqs. (2)–(4), the following values were obtained: R0 ¼5.46 nm, E¼0.22 and r¼ 6.76 nm for HSA and R0 ¼5.77 nm, E¼0.25 and r ¼6.80 nm for BSA. The donor-toacceptor distance, r o7 nm and 0.5R0 or o1.5R0, indicated that the energy transfer from serum albumins to BAP may occur with high probability.

100

4. Conclusions 10 1

0

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20 25 Time (ns)

30

35

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Fig. 9. Fluorescence decay curve of HSA (a) and BSA (b) in the absence and presence of BAP. [BAP] ranges from 0, 0.1, 0.2 and 0.3 mM. Inset shows the Stern– Volmer plot for the time resolved fluorescence quenching of HSA and BSA by BAP, respectively. Titrations were carried out in phosphate buffer solution.

The interaction of benzo [a] phenazine and serum albumins were investigated using absorption, fluorescence and lifetime spectroscopic techniques. The mode of binding interaction was spontaneous and it was found that benzo [a] phenazine took place near tryptophan moiety present in sub-domain IIA in serum albumins (HSA and BSA). From the experimental results, the observed fluorescence quenching of serum albumin in the presence of BAP occurs through subsequent generation of radical ion pair through exciplex formation. This was further confirmed by

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the observed significant decrease in lifetime of the SA matrix, which reveals that the photo-induced electron transfer occurs between the excited serum albumin and BAP. Thus, we made an endeavor to describe the biomolecular interactions between serum albumins and antitumor agent (BAP), which opens new avenues for medical applications, biocompatible and novel therapeutics.

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Acknowledgments Author SA thanks DST, New Delhi, for the major research project (SR/S1/PC-49/2009) and DST, New Delhi (INT/AUS/P-1/07 dated 19th September 2007) and DEST, Australia, for the sanction of INDIA–AUSTRALIAN strategic research fund for their collaborative research. In addition, author SA thanks DST-FIST, New Delhi (SR/FT/CSI-190/2008 dated 16th March 2008) for the sanction of research fund towards development of new facilities. Also the authors thank Prof. P. Ramamurthy, Director, National Center for Ultra Fast Processes, University of Madras, Taramani Campus, Chennai, India, for analyzing lifetime measurements. The author (RS) thanks AICTE, New Delhi, for the NDF fellowship. References [1] J.B. Laursen, J. Neilsen, Chem. Rev. 104 (2004) 1663. [2] V.M. Reddy, G. Nadadhue, D. Daneluzzi, J.F. O’ Sullivan, P.R. Gangadharam, Antimicrob. Agents Chemother. 40 (1996) 633. [3] V.M. Reddy, J.F. O’ Sullivan, P.R. Gangadharam, J. Antimicrob. Chemother. 44 (1999) 615. [4] N. Vicker, L. Burgess, I.S. Chuckowree, R. Dodd, A.J. Folkes, D.J. Hardick, T.C. Hancox, W. Miller, J. Milton, S. Sohal, S. Wang, S.P. Wren, P.A. Charlton, W. Dangerfield, C. Liddle, P. Mistry, Alistair J. Stewart, W.A. Denny, J. Med. Chem. 45 (2002) 721. [5] R.S.F. Silva, M.C.F.R. Pinto, M.O.F. Goulart, J.D. Souza Filho, I.N. Jr., M.C.S. Lourenco, A.V. Pinto, Eur. J. Med. Chem. 44 (2009) 2334. [6] Beat B. Fischer, Anja Krieger-Liszkay, Rik I.L. Eggen, Environ. Sci. Technol. 38 (2004) 6307. [7] G.M. Badger, I.S. Walker, J. Chem. Soc. (1956) 122. [8] R. Hochstrasser, J. Chem. Phys. 36 (1962) 1808.

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