Micellar-mediated binding interaction between perylene and dl -phenylalanine: Insights from spectroscopic investigations

Journal of Molecular Liquids 168 (2012) 12–16

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Micellar-mediated binding interaction between perylene and dl-phenylalanine: Insights from spectroscopic investigations Sang Hak Lee a, Anil H. Gore b, Taslima Ferdous a, Seikh Mafiz Alam a, Govind B. Kolekar b,⁎ a b

Department of Chemistry, Kyungpook National University, Daegu, 702-701, Republic of Korea Fluorescence Spectroscopy Research Laboratory, Department of Chemistry, Shivaji University, Kolhapur-416004, Maharashtra, India

a r t i c l e

i n f o

Article history: Received 1 October 2011 Received in revised form 1 January 2012 Accepted 2 January 2012 Available online 14 January 2012 Keywords: DL-Phenylalanine Perylene Fluorescence Quenching

a b s t r a c t The interaction between perylene and DL-phenylalanine (DL-PA) was studied in micellar solution of sodium dodecyl sulfate (SDS) by spectroscopic methods including absorption, FTIR and steady state fluorescence spectroscopy. The fluorescence of perylene is quenched by DL-PA and quenching is in accordance with Stern–Volmer relation. The number of binding sites (n) and binding constant (K) was obtained by fluorescence method. The quenching rate constant (kq) was calculated from the fluorescence lifetime of perylene in SDS and was measured on time resolved fluorimeter (TRF) in the absence of DL-PA. The FTIR spectrum showed the conformational change of DL-PA in the presence of perylene. The analytical applications such as effect of foreign ion substance, effect of SDS were studied hence proposed quenching method is simple, selective and rapid. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The fluorescence and absorption of proteins and peptides are mostly due to the presence of three aromatic amino acids: tryptophan, tyrosine and phenylalanine [1,2]. This can be incorporated into peptide or protein chain which is a subject of extensive studies because of their use as internal probes in conformational analysis [3]. The phenylalanine is an essential amino acid to human health but they cannot be manufactured by the body, it must be obtained from food. DL-Phenylalanine (DL-PA) is a combination of L-phenylalanine, the natural form of phenylalanine found in proteins throughout the body, and D-phenylalanine, a mirror image of L-phenylalanine that can be synthesized in a laboratory. The body converts phenylalanine into tyrosine, another amino acid essential for making proteins, brain chemicals including dopamine and norepinephrine and thyroid hormones. Symptoms of phenylalanine deficiency include confusion, lack of energy, depression, decreased alertness, decreased memory, and diminished appetite. Phenylalanine is also one of the aromatic amino acids that exhibit ultraviolet radiation absorption properties with a large extinction coefficient. This characteristic is often used as an analytical tool to quantify the amount of protein in a sample. Phenylalanine plays a key role in the biosynthesis of other amino acids and some neurotransmitters. It is the most commonly found aromatic amino acid in proteins and enzymes with a molar ratio of 3.5% compared to the other amino acids, about double the amount of any other aromatic amino acid [4].

⁎ Corresponding author. Fax: + 91 0231 2692333. E-mail address: [email protected] (G.B. Kolekar). 0167-7322/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.molliq.2012.01.002

Recently it has been seen that the nitrile derivatized phenylalanine residue, p-cyano-phenylalanine and tryptophan constitute an efficient fluorescent resonance energy transfer (FRET) process. Determination of the distance between two fluorochromes is possible through FRET. The distances accessible by this technique range from 20 Å to 100 Å. These distances are comparable with the diameters of most biological molecules and this reason makes FRET technique enormously useful in a variety of biological applications [5,6]. The process was used to investigate the conformational distribution of a 14-residue peptide aqueous solution and also the membrane mediated helix folding kinetics of antimicrobial peptides [7]. It is one of the most commonly used methods for probing the distance between two sisters in biological systems. It is a very useful and powerful technique in probing the conformation, conformational change and its dynamics of biological molecules such as protein [8,9] and DNA [10]. Earlier methods used for its determination involved chromatographic separation [11] followed by spectrophotometry and fluorimetric analysis. The polynuclear aromatic hydrocarbons such as perylene, anthracene etc. are fluorescent and partially responsible for the blue fluorescence usually seen from gasoline [12]. The water insoluble perylene can be solubilized in non-fluorescent micellar solution [13]. According to the phenomenon of micellar solubilization, the solubility of organic substances increases with their incorporation into micelles of surfactant aqueous solutions. The micelles have attracted significant attention because of their ability to work as encapsulating systems by providing a high viscosity microenvironment and solubilization is very important industrially as well as biologically [14] and chemically it is very interesting. Solubilized polycyclic aromatic molecules have often been used as photosensitive probes in photochemistry

S.H. Lee et al. / Journal of Molecular Liquids 168 (2012) 12–16

2. Experimental 2.1. Materials All experiments were performed with analytical grade reagents and used directly without further purification. Doubly distilled and demineralized water was used throughout. Perylene from AccuStandard, Inc., DL-PA from Sigma and sodium dodecyl sulfate (SDS) from Fluka was obtained and used as received. 2.2. Apparatus The emission and excitation spectra were recorded at room temperature using a Hitachi F-4500 fluorescence spectrophotometer with a 450 W xenon lamp as an excitation source. Excitation and emission monochromator slit, increment, and integration time were set at 5 nm, 1 nm and 1 s respectively. All spectral data and derivative spectra were obtained by SPEX DM 3000F spectroscopy computer. The UV-1800 Shimadzu UV spectrophotometer was used to record the absorption spectrum. The FTIR spectra were recorded using a Jasco FT/IR-4100 Fourier Transform Infrared Spectrometer in the range of 1000 to 2000 cm − 1 at room temperature. Fluorescence lifetime was measured in time resolved fluorescence spectrometer (Edinburgh Analytical Instruments FL-900 CDT).

1.2

b 4000

1.0

3000

0.8

2500 0.6 2000

Intensity

Absorbance

3500

1500

0.4

1000 0.2 500 0.0

240

260

280

300

320

340

360

0 380

Wavelength, nm Fig. 1. Absorption (a) and emission (b) spectra of DL-PA in water. Condition: DL-PA, 1 × 10− 3 M.

The effect of increasing concentration of DL-PA on the absorption spectrum of perylene is shown in Fig. 2a. From this study it was found that absorption of perylene–DL-PA system increases regularly with the increase of concentration of DL-PA with a gradual shift from blue to red region (Fig. 2b). This may be due to the formation of ground state complex between DL-PA and perylene (Scheme 1).

a

1.5

a 1.0

e 0.5

0.0 230

240

250

260

270

280

Wavelength, nm

b 2.3. Preparation of solution

1.1 1.0 0.9

a

0.8 0.7

Absorbance

The perylene stock solution (2 × 10 − 6 M) was prepared by dissolving the calculated amount in SDS solution and kept overnight with stirring. The critical micelle concentration (CMC) of SDS is 8.1 × 10 − 3 M [10]. The concentration of SDS solution used in quenching experiments was 3 × 10 − 2 and 5 × 10 − 2 M. The stock solution of 1 × 10 − 3 M DL-PA was prepared by dissolving 8.25 mg of solid DL-PA in 50 ml doubly distilled water. The concentration of DL-PA was varied from 1 × 10 − 4 to 7 × 10 − 4 M in the quenching experiments. The solutions were deaerated before recording the fluorescence spectra.

4500

a

Absorbance

[15]. A number of studies have been devoted to the solubilization of aromatic compounds by aqueous micellar solutions of conventional surfactants made up of one hydrophilic head group and one hydrophobic chain. The solubilization of a series of aromatic compounds by micellar solutions of dodecylsulfonic acid [16,17], dodecylammonium perfluoroacetate [18], dodecylammonium trifluoroacetate [19], lithium 1-perfluoroundecanoate [20], and dodecyltrimethylammonium perfluorocarboxylate [21] was examined previously. However to the literature survey and best of our knowledge no one has reported the interactions of DL-PA with perylene in micellar solution. Therefore it is proposed to carry out the detailed and insightful interaction between DL-PA and perylene. In this manuscript the spectroscopic approach has been studied extensively, most of the efforts were focused on fluorescence measurement and characterization of the absorption, steady state fluorescence and FTIR spectroscopy. The binding constant and number of binding sites were calculated. The analytical applications such as effect of foreign ion substance, effect of SDS were studied by proposed quenching method. The method is simple, selective and rapid.

13

e

0.6 0.5 0.4 0.3 0.2 0.1 0.0

3. Results and discussion 3.1. Absorption characteristics of perylene–DL-PA system Fig. 1 shows absorption spectrum of DL-PA which lies in the range of 230–280 nm with sharp peak at 253 nm and emission spectrum showing sharp peak at 285 nm within the range of 260–380 nm.

-0.1 220

240

260

280

300

320

340

360

380

400

420

440

Wavelength, nm Fig. 2. (a) Absorption spectra for increasing amount of DL-PA in perylene (a→e). Conditions: DL-PA = 1.0, 2.0, 3.0, 4.0 and 5.0 × 10− 4 M; Perylene, 2 × 10− 6 M. (b) Absorption of perylene–DL-PA system increases regularly with the increase of concentration of DL-PA. Conditions: DL-PA = 1.0, 2.0, 3.0, 4.0 and 5.0 × 10− 4 M; Perylene, 2 × 10− 6 M.

14

S.H. Lee et al. / Journal of Molecular Liquids 168 (2012) 12–16

Scheme 1. Mechanism of fluorescence quenching.

Fluorescence Intensity, a.u.

10000

8000

a

6000

f

4000

2000

0

440

460

480

500

520

540

Wavelength, nm Fig. 3. Fluorescence spectra of perylene in the absence (a) and presence (b–f) of varying concentrations of DL-PA in SDS micellar solution (λex = 411 nm).Conditions: DLPA = 0.0, 1.0, 2.0, 3.0, 4.0 and 7.0 × 10− 4 M; Perylene, 2 × 10− 6 M.

3.2. Fluorescence quenching studies Fig. 3 shows the effect of increasing concentration of DL-PA on fluorescence emission of perylene in SDS. It was observed that, after the addition of DL-PA to the perylene solution in SDS results in the quenching of its fluorescence emission and no peak shift was noticed. It indicated that the complex formed between DL-PA and perylene is responsible for the kinetics of quenching of perylene. The kinetics of quenching is described by Stern–Volmer relation F 0 =F ¼ 1 þ K SV ½Q  ¼ 1 þ kq τ ½Q :

ð1Þ

3.3. Kinetics of quenching of perylene fluorescence The estimated values of kq given in Table 1 are of the order of 10 11 M − 1 S − 1. The values of quenching rate constant reported for donor–acceptor pairs in homogenous aqueous and nonaqueous medium are mostly of the order of 10 9 M − 1 S − 1 [24,25]. In comparison, the quenching rate constant observed for the present system in SDS micelle solutions is of a higher order. This observation is in support of efficient energy transfer from perylene to DL-PA in micelle environment. The efficiency of the energy transfer is calculated by using the relation [5] η ¼ 1−F d =F 0 :

ð2Þ

Where Fd is the fluorescent intensity of donor in the presence of acceptor, F0 is fluorescent intensity of donor without acceptor and the values estimated as a function of DL-PA concentration are given in Table 1. The values indicate that the efficiency of energy transfer increases with DL-PA concentration.

1.20 1.16 1.12

F 0/F

Where, F0 and F are the fluorescence intensities in the absence and presence of quencher, KSV is the Stern–Volmer constant, kq is the quenching rate constant and τ is the average lifetime of perylene molecule and [Q] is the concentration of quencher. Fig. 4 shows a plot of F0/F vs. concentration of DL-PA. According to Eq. (1), the graph is a straight line with intercept having a value of one on Y-axis and indicates validity of the Stern–Volmer relation [22]. The quenching rate constant, kq = 3.366 × 10 11 M − 1 S − 1 was calculated from slope of the graph using the lifetime of perylene in SDS without DL-PA. In general, molecular collisional quenching constant (kq) of various kinds of quencher to biopolymers is 2.0 × 10 10 M − 1 S − 1 [23]. But for perylene–DL-PA system higher quenching rate constant (3.366 × 10 11 M − 1 S − 1) was obtained. This shows that the quenching of perylene by DL-PA is not dynamic in nature. Therefore it depends on the formation of complex between perylene and DL-PA (Scheme 1).

1.08

Table 1 Quenching rate constant (kq) and efficiency of energy transfer (η).

1.04

Sr. no

1.00

Concentration of DL-PA M

0.96 0.92 0.0000

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0.0007

0.0008

Concentration (M) Fig. 4. Stern–Volmer plot of perylene in the absence and presence of varying concentrations of DL-PA in SDS micellar solution (λex = 411 nm). Conditions: DL-PA = 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 × 10− 4 M; Perylene, 2 × 10− 6 M.

1 2 3 4 5 6 7

1 × 10− 4 2 × 10− 4 3 × 10− 4 4 × 10− 4 5 × 10− 4 6 × 10− 4 7 × 10− 4

Concentration of SDS 3 × 10

−2

Concentration of SDS 5 × 10− 2 M

M

kq

η

3.366 × 1011

0.0085 0.0116 0.0298 0.0314 0.0638 0.0859 0.0969

kq

η

5.253 × 1011

0.0024 0.0130 0.0144 0.0184 0.0330 0.0352 0.0365

S.H. Lee et al. / Journal of Molecular Liquids 168 (2012) 12–16

3.4. Binding constant and number of binding sites

Table 2 Effect of interfering substances on fluorescence.

The following equation shows the relationship between the fluorescence intensity of quenching mechanisms: nQ þ M ¼ Q n ……M

ð3Þ

Where, M is the biomolecule with fluorophore Q is the quencher molecule and Qn……M is the quenched biomolecule. Then the resultant constant K is given by

K¼

15

Q n ……M : ½Q n :M

Interfering substance

Concentration (g L− 1)

Change in fluorescence intensity (%)

Na(I) K(I) Cu(II) Zn(II) Mn(VII) Thiourea EDTA SCN− Cr(VI) N03−

0.05 0.04 0.002 0.05 0.002 0.006 0.007 0.05 0.075 0.005

1.21 − 2.32 4.09 2.48 4.78 10.35 0.75 − 4.84 8.44 10.62

ð4Þ

If the overall amount of biomolecules (bound or unbound with the quencher) is Mo, then ½Mo ¼ ½Q n ……M þ ½M:

ð5Þ

Here [M] is the concentration of unbound biomolecules, then the relationship between fluorescence intensity and unbound biomolecule as [M]/[Mo] = F/F0, that is the binding process of perylene to DL-PA, which was analyzed by following Eq. (6) and employed to calculate the binding constant and number of binding sites [13]. log½F 0 −F=F  ¼ n log½Q  þ logK

ð6Þ

Where, K is the binding constant of DL-PA with perylene, which can be determined from the plot of log (F0 − F)/F vs. log [Q] as shown in Fig. 5. Thus we can measure binding constant K as K = 2.49 × 10 3 M − 1 and binding sites ‘n’ (1.39 ≈ 1.0) of DL-PA with perylene from the intercept and slope.

occupancy may occur at higher concentration, thereby increasing the intermolecular interactions [27]. In addition to this the increase in concentration of SDS results in increased probability of distribution of perylene molecules in micelle owing to micellar proximity effect [28]. Micelle with higher number of perylene molecules leads to efficient energy transfer process with DL-PA and the rate of quenching of perylene florescence by DL-PA is increased.

3.6. Effect of foreign substances and method of analysis The effect of foreign substances on the fluorescence determination of DL-PA with the proposed method was investigated. Initially, foreign substances to the DL-PA solution are taken in large excess and the fluorescence intensities are measured. When interference was found to be intensive, the tests were repeated with successive smaller amounts of foreign substances. According to the proposed method the metal ions shows small effect on the intensity of DL-PA. The tolerance limits for the ions are given in Table 2.

3.5. Effect of SDS 3.7. FTIR characteristics The quenching experiments were performed in SDS solution of two different concentration values of above CMC. The efficiency of energy transfer was calculated and is given in Table 1. It is seen that the quenching rate constant (kq) is higher in higher concentrations of SDS. The literature on micellar solution reveals that the spherical shape of micelle in dilute solution elongates more with concentration of surfactant and size of micelle increases [26]. Because of this, quenching rate constant (kq) is found to be higher in higher concentrations of SDS. At lower SDS concentrations, it is unlikely that more than one molecule will bind per micelle. Multiple

Fig. 6 shows the FTIR spectra of DL-PA in the absence and presence of perylene. From this figure we can verify whether the conformation of amino acid has changed or not. The change in transmittance, due to the addition of perylene in DL-PA, shows that the conformation of amino acid has been affected by the addition of perylene. The hydrogen oxygen linkage formed at the formation of the complex between DL-PA and perylene (Scheme 2) may be the reason for the change in the sensitization.

110

DL-PA ------- DL-PA - Perylene

-0.8 -1.0

100

%T

log [F0-F]/F

-1.2 -1.4

90

1415 1410

-1.6 80 -1.8 -2.0 70 -2.2 -3.0

1300 -3.2

-3.4

-3.6

-3.8

-4.0

1400

1500

1600

1700

Wavenumber, cm-1

log [Q] Fig. 5. Plot of log [F0 − F]/F vs. log [Q].

Fig. 6. The FTIR spectra of amino acid in the absence and presence of perylene. Condition: DL-PA, 1 × 10− 3 M; Perylene, 2 × 10− 6 M.

16

S.H. Lee et al. / Journal of Molecular Liquids 168 (2012) 12–16

Scheme 2. Mechanism for the interaction of DL-PA with perylene.

4. Conclusion The interaction between perylene and DL-PA in micellar environment is more efficient and not probable in water because of insolubility of perylene. The fluorescence of perylene in SDS solution was found to be quenched. The kinetic studies showed the validity of the Stern–Volmer relation. Quenching rate constant, binding constant and number of binding sites were calculated according to the relevant fluorescence quenching data. From FTIR spectra it can be shown that the conformation of DL-PA was changed by addition of perylene. This study helps in giving the great information about the effect of environment on amino acid structure. The quenching method is simple, selective and rapid. Acknowledgments We are grateful for the support extended by Indian National Science Academy (INSA), India and National Research Foundation (NRF), South Korea for their international collaboration scientist exchange program. References [1] J.B.A. Ross, W.R. Laws, K.W. Rousslang, H.R. Wyssbrod, in: J.R. Lakowicz (Ed.), Plenum Press, New York, 1992. [2] S.V. Konev, Fluorescence and Phosphorescence of Proteins and Nucleic Acids, Plenum Press, New York, 1967. [3] M.R. Eftink, in: C.H. Schulter (Ed.), Wiley, New York, 1991. [4] X.H. Pham, J.M. Kim, S.M. Chang, I. Kim, W.S. Kim, Journal of Molecular Catalysis B: Enzymatic 60 (2009) 87–92.

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