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Spectrochimica Acta Part A 74 (2009) 835–838

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journa l homepage: www.e lsev ier .com/ locate /saa

hort communication

nantioselective binding of l,d-phenylalanine to ct DNA

ijin Zhanga,b, Jianhua Xuc, Yan Huangc, Shungeng Minb,∗

Shandong Analysis and Test Center, Shandong Academy of Sciences, Jinan 250014, ChinaDepartment of Applied Chemistry, College of Science, China Agricultural University, Beijing 100094, ChinaBeijing Synchrotron Radiation Laboratory, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China

r t i c l e i n f o

rticle history:eceived 28 December 2008

a b s t r a c t

The enantioselective binding of l,d-phenylalanine to calf thymus DNA was studied by absorption, circulardichroism, fluorescence quenching, viscosity, salt effect and emission experiments. The results obtained

eceived in revised form 8 July 2009ccepted 29 July 2009

eywords:t DNA,d-Phenylalanine

from absorption, circular dichroism, fluorescence quenching and viscosity experiments excluded theintercalative binding and salt effect experiments did not support electrostatic binding. So the binding ofl,d-phenylalanine to ct DNA should be groove binding. Furthermore, the emission spectra revealed thatthe binding is enantioselective.

© 2009 Elsevier B.V. All rights reserved.

roove bindingnantioselective

. Introduction

Protein has been used as one kind of chiral selectors for the chiraleparation of enantiomers [1]. Calf thymus DNA (ct DNA), one kindf chiral macromolecules, was also applied for the chiral separationf l,d-phenylalanine [2–4]. However, the mechanism of it has beennknown. Electrostatic binding, groove binding and intercalativeinding are three kinds of binding modes between small moleculesnd ct DNA [5]. The binding modes have been studied by means ofbsorption [6], fluorescence [7], circular dichroism [8], viscometry9], electrochemistry [10], Raman [11], NMR [12], melting studiesnd gel electrophoresis [13], and so on.

In the present study, a series of experiments have been per-ormed in order to explore the mechanism of l,d-phenylalanineinding to ct DNA. The results synthetically revealed that,d-phenylalanine bound to ct DNA with groove binding and l-henylalanine preferentially bound to ct DNA.

. Experiments and methods

.1. Materials

Ct DNA sample obtained from Sigma Chemical Co., Ltd. was dis-olved in 5.0 mmol/L Tris–0.5 mmol/L EDTA–HCl buffer (pH 7.4) andtored at 4 ◦C, whose concentration was determined by the molarbsorption coefficient value of 6600 dm3 (mol cm)−1 at 260 nm

∗ Corresponding author.E-mail address: minsgcau@sina.com (S. Min).

386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2009.07.019

[10]. l,d-Phenylalanine, l-phenylalanine andd-phenylalanine werepurchased from Shanghai Yuanju Biotechnologies Co., Ltd. andused directly. Other reagents were all analytical grade and useddirectly. Doubly distilled water was used for all the followingexperiments.

2.2. Absorption spectra

Absorption titrations were recorded on UV-1901 spectrometer(Beijing Purkinje General Instrument Co., Ltd., Beijing, China). Theabsorption titrations of phenylalanine were performed by keepingthe concentration of the l,d-phenylalanine constant while gradu-ally adding the solution of ct DNA. The experimental temperaturewas kept at room temperature (unless otherwise stated).

2.3. Circular dichroism spectra

Using a 1 cm path-length cuvette and subtracting the bufferbaseline, circular dichroism spectra were recorded in the range of290–200 nm on the synchrotron radiation vacuum ultraviolet cir-cular dichroism (Institute of High Energy Physics, Chinese Academyof Sciences, Beijing, China). The results were obtained by meanvalues of replicated measurements.

2.4. Fluorescence quenching experiments

In the absence and presence of ct DNA, [Fe(CN)6]4− wasgradually added to l,d-phenylalanine to observe the change offluorescence intensities of l,d-phenylalanine. All the fluorescenceintensities were plotted according to the following Stern–Volmer

8 ca Acta Part A 74 (2009) 835–838

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Fig. 1. Absorption spectra of l,d-phenylalanine (60.5 �M) in the presence of increas-ing amounts of ct DNA (1 → 6: 0, 30.3, 60.6, 90.9, 121.2, 151.5 �M).

To further clarify the binding of l,d-phenylalanine to ct DNA,viscosity experiments were also carried out. A classical interca-lation model results in the lengthening of DNA helix and theincrease of DNA viscosity. With the increase of the volume ratio

36 L. Zhang et al. / Spectrochimi

quation [14]:

F0

F= 1 + KSW[Q] (1)

here F0 and F are the corresponding fluorescence intensities of,d-phenylalanine in the absence and presence of quencher, respec-ively. KSW is the Stern–Volmer fluorescence quenching constantnd [Q] is the corresponding concentration of quencher.

.5. Viscosity experiments

Viscosity experiments were performed on an Ubbelodhe vis-ometer. ct DNA samples were prepared and immersed in aater-bath maintained at a constant temperature at 30.0 ± 1 ◦C.

he flow times were determined with a manually operated timernd mean values of replicated measurements were used to evaluatehe viscosity of the samples. Relative viscosity was calculated fromhe equation � = (t − to)/to, where t and to are the observed flowime of ct DNA in the presence and absence of l,d-phenylalanine.iscosity can be shown in the form of (�/�0)1/3 vs. the volume ratioetween l,d-phenylalanine and ct DNA, where � and �0 are the vis-osity of ct DNA in the presence and absence of l,d-phenylalanine8].

.6. Salt effect experiments

The Salt effect experiments were carried out by keeping theoncentration of l,d-phenylalanine and ct DNA constant whileradually adding the solution of NaCl.

.7. Emission spectra

Emission spectra were performed on a Hitachi F4500 flu-rescence spectrophotometer. Keeping the concentration ofhenylalanine constant (60.5 �M), the fluorescent titrations werexecuted by adding ct DNA solution gradually. l-phenylalaninend d-phenylalanie were excited at 218 nm and the correspondingmission was monitored at 290 nm. The emission and excita-ion slit widths used throughout the experiments were bothnm. All the corresponding fluorescence intensities were recordednd plotted according to the Stern–Volmer equation above andineweaver–Burk equation [15]:

F0 − F)−1 = F−10 + K−1

D F−1[Q]−1 (2)

here F0, F and [Q] are the same as those in Eq. (1), and KD is theinding constant between l,d-phenylalanine and ct DNA.

. Results and discussion

.1. Absorption spectra

The absorption spectra of l,d-phenylalanine in the presencef different concentrations of ct DNA are shown in Fig. 1. Thereppeared obvious hypochromism and weak red-shift, which coulduggest the intercalative binding of l,d-phenylalanine to ct DNA16]. However, the red-shift of no more than 1 nm was too smallo reach the conclusion [17]. It was necessary to consider otherxperiments to make a final conclusion.

.2. Circular dichroism spectra

Further clarification of the binding modes between the l,d-henylalanine and ct DNA was carried out by viscosity experiments.ig. 2 proved that the circular dichroism spectra of ct DNA in thebsence of l,d-phenylalanine were almost the same as that in theresence of l,d-phenylalanine.

Fig. 2. Circular dichroism spectra of ct DNA (2.27 mM) in the absence and presenceof l,d-phenylalanine (1.51 mM).

The major binding of l,d-phenylalanine to ct DNA could not bethe intercalative one which usually produces bigger changes in theintensity and position of the left and/or right band of the circulardichroism spectrum of native DNA [8].

3.3. Fluorescence quenching experiments

The binding of l,d-phenylalanine to ct DNA was further inves-tigated by fluorescence quenching experiments. If small moleculesintercalate into ct DNA base pairs, the double helix of ct DNAwould protect them from the fluorescence quenching of potassiumferrocyanide [18,19]. However, Fig. 3 showed that the fluores-cence quenching of potassium ferrocyanide to l,d-phenylalaninein the absence of ct DNA was nearly different from that in thepresence of ct DNA, which could not affirm the intercalation ofl,d-phenylalanine into ct DNA base pairs.

3.4. Viscosity experiments

Fig. 3. Fluorescence quenching of potassium ferrocyanide to l,d-phenylalanine(60.5 �M) in the absence and presence of ct DNA (60.6 �M).

L. Zhang et al. / Spectrochimica Acta Part A 74 (2009) 835–838 837

Fig. 4. Relative viscosity of ct DNA (3.03 mM) in the presence of increasing amountsof l,d-phenylalanine (6.05 mM).

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ig. 5. Emission spectra of phenylalanine (60.5 �M) with ct DNA (60.6 �M) in theresence of NaCl (1 → 7: 0, 0.0513, 0.1026, 0.1539, 0.2052, 0.2565, 0.3078 M).

f the l,d-phenylalanine and ct DNA, the relative viscosity of ctNA increased faintly and fixed at 1.1 (Fig. 4), which was smaller

han the classical viscosity of 1.4 [10]. Comprehensively consid-ring the results of absorption spectra, circular dichroism spectra,uorescence quenching experiments and viscosity experimental,he intercalative binding of l,d-phenylalanine to ct DNA could bexcluded and the electrostatic binding and groove binding wereossible.

.5. Salt effect experiments

NaCl was used to check the electrostatic interaction between ctNA and l,d-phenylalanine. As there exists the electrostatic attrac-

ion between Na+ and the anionic phosphate groups of ct DNA, NaClould protect small cationic molecules from binding to ct DNA [20].

ig. 6. Emission spectra of l-phenylalanine (60.5 �M) and d-phenylalanine (60.5 �M) in

Fig. 7. Plots of fluorescence quenching of ct DNA to l-phenylalanine and d-phenylalanine.

Fig. 5 showed that the addition of NaCl did not lead to the increas-ing fluorescence intensities of l,d-phenylalanine but its decrease,meaning that the binding of l,d-phenylalanine to ct DNA was notelectrostatic binding.

3.6. Emission spectra

The binding of l,d-phenylalanine to ct DNA was also studiedby fluorescence spectra. The emission spectra of l-phenylalanineand d-phenylalanine in the presence of different concentrationsof ct DNA are shown in Fig. 6(L) and (D), respectively. Thedecreasing fluorescence intensities of l-phenylalanine and d-phenylalanine illuminated the fluorescence quenching of ct DNAto l,d-phenylalanine and the strong interactions between the l,d-phenylalanine and ct DNA.

Fig. 7 shows that the shape of Stern–Volmer plots appearedto be linear, meaning that the fluorescence quenching of ct DNAto l-phenylalanine and d-phenylalanine could be predominantlydynamic or static. And the fluorescence quenching constants of KSWwere 11929 L/mol and 9983 L/mol, which proved that the fluores-cence quenching of ct DNA to l-phenylalanine andd-phenylalanineshould be static one [21], then the quenching constant wasconsidered as the binding constant of ST with DNA [15]. The double-reciprocal plots were made by (I0 − I)−1 vs. [Q]−1 so that the bindingconstants of 9618 L/mol and 8608 L/mol were obtained, whichdemonstrated that the binding of l-phenylalanine to ct DNA wasstronger than that of d-phenylalanine and l-phenylalanine prefer-entially bound to ct DNA. As these binding constants were smallerthan the reported values for some typical intercalators such asethidium bromide [15], the intercalative binding was impossible.

Furthermore, the enantioselective interaction between ct DNA andl,d-phenylalanine did not support the electrostatic binding. So thebinding of l-phenylalanine and d-phenylalanine to ct DNA weregroove binding.

the presence of ct DNA (1 → 8: 0, 30.3, 60.6, 90.9, 121.2, 151.5, 181.8, 212.2 �M).

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[19] W.Y. Li, J.G. Xu, X.Q. Guo, Q.Z. Zhu, Y.B. Zhao, Spectrochimica Acta Part A 53(1997) 781–787.

38 L. Zhang et al. / Spectrochimi

. Conclusion

The present study concentrated on the binding of l,d-henylalanine to ct DNA. The obvious hypochromism and weaked-shift in the absorption spectra were not helpful to under-tand the binding of l,d-phenylalanine to ct DNA. However, theircular dichroism spectra and viscosity changed little in the pres-nce of l,d-phenylalanine, and ct DNA was not found to protect,d-phenylalanine from the fluorescence quenching of potassiumerrocyanide, which excluded the intercalative binding of l,d-henylalanine to ct DNA. And NaCl had no competing effect onhe binding of l,d-phenylalanine to ct DNA so that the bindingas not electrostatic. Finally, the binding of l,d-phenylalanine to ctNA was testified to be groove binding. The emission spectra notnly confirmed this conclusion but also proved that the binding isnantioselective.

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