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Investigation of the interaction of DNA and neutral red by fluorescence spectroscopic analysis
Chinese Chemical Letters 18 (2007) 569–572 www.elsevier.com/locate/cclet
Investigation of the interaction of DNA and neutral red by fluorescence spectroscopic analysis Yong Nian Ni a,b,*, Xue Zhi Zhong a a
b
Department of Chemistry, Nanchang University, Nanchang 330047, China The Key Laboratory of Food Science of MOE, Nanchang University, Nanchang 330047, China Received 4 January 2007
Abstract The binding characteristics of neutral red (NR) with DNA were investigated by fluorescence spectrometry. Chemometrics approach as singular value decomposition (SVD) was used to evaluate the number of spectral species in the drug–DNA binding process, and then the intrinsic binding constant of 1.6 104 in base pairs and the binding site number of 0.97 were obtained from the Scatchard plot. # 2007 Yong Nian Ni. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Neutral red; DNA; Binding constant; Fluorescence; Chemometrics; Singular value decomposition
The interclaction of NR with DNA involves a two-step procedure. If the molar ratio of NR to DNA is higher than 1.33, the binding process is characterized by a binding constant of about 106 with the binding number close to 1. However, both the constant and the binding number will decrease with lower molar ratios of the interacting components [1]. In this work, there is a large excess of DNA in the interacting system, binding constant of 1.6 104 (pH 7.4) was obtained based on the enhancement of the drug fluorescence. 1. Experimental The concentrations of ct-DNA (Beitai Biochemical Co., Chinese Academy of Sciences) per nucleotide phosphate were calculated according to the absorbance at 260 nm by using eDNA = 6600 L/mol cm [2]. A 1.00 104 mol/L stock solution of neutral red (The Third Reagent Factory, Shanghai) was prepared by directly dissolving its crystals in water, and diluting to the required volume. Phosphate buffer solutions (PBS, pH 7.4) with different ionic strength used in all experiments were prepared by Na2HPO412H2O, NaH2PO412H2O and NaCl. All reagents were of analyticalreagent grade. Fluorescence intensities were measured by a Perkin-Elmer LS-55 luminescence spectrometer with the following setting: 540 nm as excitation wavelength, 618 nm as emission wavelength, 10 nm as excitation slit and 15 nm as emission slit. * Corresponding author at: Department of Chemistry, Nanchang University, Nanchang 330047, China. E-mail address: [email protected] (Y.N. Ni). 1001-8417/$ – see front matter # 2007 Yong Nian Ni. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2007.03.027
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Y.N. Ni, X.Z. Zhong / Chinese Chemical Letters 18 (2007) 569–572
2. Results and discussion The interaction procedure of NR with ct-DNA in pH 7.4 PBS was characterized by the fluorescence spectra (see Fig. 1). It is shown that the fluorescence emission spectral is significantly enhanced by increasing the DNA concentration. Chemometrics approach, such as singular value decomposition (SVD) [3], is an effective tool to resolve the measured data: M = USVt. The matrix U contains the so-called basis spectra, S is a diagonal matrix containing the singular values and V is a matrix containing amplitude vectors. Here it was used to calculate the number of significant spectral species from the fluorescence spectral data. Fig. 2 shows the weighted basis spectra (US) corresponding to the first three singular values. There are two prominent fluorescence emission spectra, while the third one is very small. This means that there are two main species, NR and NR–DNA, in the binding system (note that no fluorescence can be observed for DNA).
Fig. 1. Fluorescence emission spectra of (1) free NR (3 105 mol/L) and (2–24) bound NR–DNA (DNA concentration was increased from 7.496 104 to 8.995 103 mol/L with a step-increase of 3.748 104 mol/L).
Fig. 2. The product US spectra obtained by SVD method from fluorescence data as shown in Fig. 1.
Y.N. Ni, X.Z. Zhong / Chinese Chemical Letters 18 (2007) 569–572
571
Fig. 3. The residual between the experimental data matrix of fluorescence emission spectra and calculated matrices obtain for different numbers of singular values: (A) one, (B) two and (C) three assumed to be significant.
To verify the above conclusion, further chemometrics analysis, the residual matrix, D, was calculated after extraction of each component [4]: D = M USVt. From Fig. 3 it can be seen that the residual data of B and C are the same, this means there are two significant fluorescence components in the binding procedure. So this conclusion can be confirmed that in fact there are only two significant spectral species and binding of neutral red to DNA is truly a two-state process, i.e. the free and bound forms of NR. A simple binding model G + B = C may be fit to fluorescence emission data, where G is the ligand, B is the DNA binding site, and C is the complex. When drug fluorescence emission is enhanced, the concentration of the bound probe can be calculated by the equation [5]: Cb = Ct(F F 0)/(P 1)F 0, where Cb and Ct are the concentration of the bound probe and the total concentration of the probe added, respectively. F is the observed fluorescence emission under a particular set of DNA and drug concentrations, F 0 is the emission of the same drug concentration in the absence of DNA, and P is the ratio of the fluorescence of the completely bound drug to the free drug, P = F b/F 0 [6]. Using the fluorescence enhancement, the association constants K for the complex of neutral red with DNA was calculated. The binding parameters have been calculate using the Scatchard’s procedure [7]: r ¼ nK rK CF where K is the intrinsic binding constant and n is the binding site number in base pairs. CF = Ct Cb. Fig. 4 shows the Scatchard plot of r/CF versus r, where r = Cb/[DNA]total. Intrinsic binding constant of 1.6 104 and binding site number of 0.97 were obtained from line 1 in Fig. 4, which was in the case of a large excess of DNA. It indicated that this system has two types of binding mode in the molar ratio range studied.
Fig. 4. Scatchard plot for NR binding to DNA. CNR = 3 105 mol/L and pH 7.4.
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Y.N. Ni, X.Z. Zhong / Chinese Chemical Letters 18 (2007) 569–572
Acknowledgments The financial support from the National Natural Science Foundation of China (No. 20562009), the Natural Science Foundation of Jiangxi Province (No. 0620041), and the foundation of the State Key Laboratories of the Chemo/ Biosensing and Chemometrics of Hunan University (No. 2005-22), and the program for Changjiang Scholars and Innovative Research Team in Universities (No. IRT0540). References [1] [2] [3] [4] [5] [6] [7]
C.Z. Huang, Y.F. Li, P. Feng, Talanta 55 (2001) 321. Y.N. Ni, D.Q. Lin, S. Kokot, Talanta 65 (2005) 1295. Y.N. Ni, The Application of Chemometrics in Analytical Chemistry, Science Press, Beijing, 2004, p. 162. X.G. Qu, J.B. Chaires, Methods Enzymol. 321 (2000) 353. J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933. A. Blake, A.R. Peackocke, Biopolymers 6 (1968) 1225. Y. Li, X.J. Yao, J. Jin, X.G. Chen, Z.D. Hu, Biochim. Biophys. Acta 2007 (1774) 51.
Investigation of the interaction of DNA and neutral red by fluorescence spectroscopic analysis Yong Nian Ni a,b,*, Xue Zhi Zhong a a
b
Department of Chemistry, Nanchang University, Nanchang 330047, China The Key Laboratory of Food Science of MOE, Nanchang University, Nanchang 330047, China Received 4 January 2007
Abstract The binding characteristics of neutral red (NR) with DNA were investigated by fluorescence spectrometry. Chemometrics approach as singular value decomposition (SVD) was used to evaluate the number of spectral species in the drug–DNA binding process, and then the intrinsic binding constant of 1.6 104 in base pairs and the binding site number of 0.97 were obtained from the Scatchard plot. # 2007 Yong Nian Ni. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Neutral red; DNA; Binding constant; Fluorescence; Chemometrics; Singular value decomposition
The interclaction of NR with DNA involves a two-step procedure. If the molar ratio of NR to DNA is higher than 1.33, the binding process is characterized by a binding constant of about 106 with the binding number close to 1. However, both the constant and the binding number will decrease with lower molar ratios of the interacting components [1]. In this work, there is a large excess of DNA in the interacting system, binding constant of 1.6 104 (pH 7.4) was obtained based on the enhancement of the drug fluorescence. 1. Experimental The concentrations of ct-DNA (Beitai Biochemical Co., Chinese Academy of Sciences) per nucleotide phosphate were calculated according to the absorbance at 260 nm by using eDNA = 6600 L/mol cm [2]. A 1.00 104 mol/L stock solution of neutral red (The Third Reagent Factory, Shanghai) was prepared by directly dissolving its crystals in water, and diluting to the required volume. Phosphate buffer solutions (PBS, pH 7.4) with different ionic strength used in all experiments were prepared by Na2HPO412H2O, NaH2PO412H2O and NaCl. All reagents were of analyticalreagent grade. Fluorescence intensities were measured by a Perkin-Elmer LS-55 luminescence spectrometer with the following setting: 540 nm as excitation wavelength, 618 nm as emission wavelength, 10 nm as excitation slit and 15 nm as emission slit. * Corresponding author at: Department of Chemistry, Nanchang University, Nanchang 330047, China. E-mail address: [email protected] (Y.N. Ni). 1001-8417/$ – see front matter # 2007 Yong Nian Ni. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2007.03.027
570
Y.N. Ni, X.Z. Zhong / Chinese Chemical Letters 18 (2007) 569–572
2. Results and discussion The interaction procedure of NR with ct-DNA in pH 7.4 PBS was characterized by the fluorescence spectra (see Fig. 1). It is shown that the fluorescence emission spectral is significantly enhanced by increasing the DNA concentration. Chemometrics approach, such as singular value decomposition (SVD) [3], is an effective tool to resolve the measured data: M = USVt. The matrix U contains the so-called basis spectra, S is a diagonal matrix containing the singular values and V is a matrix containing amplitude vectors. Here it was used to calculate the number of significant spectral species from the fluorescence spectral data. Fig. 2 shows the weighted basis spectra (US) corresponding to the first three singular values. There are two prominent fluorescence emission spectra, while the third one is very small. This means that there are two main species, NR and NR–DNA, in the binding system (note that no fluorescence can be observed for DNA).
Fig. 1. Fluorescence emission spectra of (1) free NR (3 105 mol/L) and (2–24) bound NR–DNA (DNA concentration was increased from 7.496 104 to 8.995 103 mol/L with a step-increase of 3.748 104 mol/L).
Fig. 2. The product US spectra obtained by SVD method from fluorescence data as shown in Fig. 1.
Y.N. Ni, X.Z. Zhong / Chinese Chemical Letters 18 (2007) 569–572
571
Fig. 3. The residual between the experimental data matrix of fluorescence emission spectra and calculated matrices obtain for different numbers of singular values: (A) one, (B) two and (C) three assumed to be significant.
To verify the above conclusion, further chemometrics analysis, the residual matrix, D, was calculated after extraction of each component [4]: D = M USVt. From Fig. 3 it can be seen that the residual data of B and C are the same, this means there are two significant fluorescence components in the binding procedure. So this conclusion can be confirmed that in fact there are only two significant spectral species and binding of neutral red to DNA is truly a two-state process, i.e. the free and bound forms of NR. A simple binding model G + B = C may be fit to fluorescence emission data, where G is the ligand, B is the DNA binding site, and C is the complex. When drug fluorescence emission is enhanced, the concentration of the bound probe can be calculated by the equation [5]: Cb = Ct(F F 0)/(P 1)F 0, where Cb and Ct are the concentration of the bound probe and the total concentration of the probe added, respectively. F is the observed fluorescence emission under a particular set of DNA and drug concentrations, F 0 is the emission of the same drug concentration in the absence of DNA, and P is the ratio of the fluorescence of the completely bound drug to the free drug, P = F b/F 0 [6]. Using the fluorescence enhancement, the association constants K for the complex of neutral red with DNA was calculated. The binding parameters have been calculate using the Scatchard’s procedure [7]: r ¼ nK rK CF where K is the intrinsic binding constant and n is the binding site number in base pairs. CF = Ct Cb. Fig. 4 shows the Scatchard plot of r/CF versus r, where r = Cb/[DNA]total. Intrinsic binding constant of 1.6 104 and binding site number of 0.97 were obtained from line 1 in Fig. 4, which was in the case of a large excess of DNA. It indicated that this system has two types of binding mode in the molar ratio range studied.
Fig. 4. Scatchard plot for NR binding to DNA. CNR = 3 105 mol/L and pH 7.4.
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Y.N. Ni, X.Z. Zhong / Chinese Chemical Letters 18 (2007) 569–572
Acknowledgments The financial support from the National Natural Science Foundation of China (No. 20562009), the Natural Science Foundation of Jiangxi Province (No. 0620041), and the foundation of the State Key Laboratories of the Chemo/ Biosensing and Chemometrics of Hunan University (No. 2005-22), and the program for Changjiang Scholars and Innovative Research Team in Universities (No. IRT0540). References [1] [2] [3] [4] [5] [6] [7]
C.Z. Huang, Y.F. Li, P. Feng, Talanta 55 (2001) 321. Y.N. Ni, D.Q. Lin, S. Kokot, Talanta 65 (2005) 1295. Y.N. Ni, The Application of Chemometrics in Analytical Chemistry, Science Press, Beijing, 2004, p. 162. X.G. Qu, J.B. Chaires, Methods Enzymol. 321 (2000) 353. J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933. A. Blake, A.R. Peackocke, Biopolymers 6 (1968) 1225. Y. Li, X.J. Yao, J. Jin, X.G. Chen, Z.D. Hu, Biochim. Biophys. Acta 2007 (1774) 51.