Research article Received: 26 January 2015,
Revised: 21 February 2015,
Accepted: 22 February 2015
Published online in Wiley Online Library: 31 March 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2904
Binding properties of palmatine to DNA: spectroscopic and molecular modeling investigations Ran Mi, Bao Tu, Xiao-Ting Bai, Jun Chen, Yu Ouyang and Yan-Jun Hu* ABSTRACT: Palmatine, an isoquinoline alkaloid, is an important medicinal herbal extract with diverse pharmacological and biological properties. In this work, spectroscopic and molecular modeling approaches were employed to reveal the interaction between palmatine and DNA isolated from herring sperm. The absorption spectra and iodide quenching results indicated that groove binding was the main binding mode of palmatine to DNA. Fluorescence studies indicated that the binding constant (K) of palmatine and DNA was ~ 104 L·mol1. The associated thermodynamic parameters, ΔG, ΔH, and ΔS, indicated that hydrogen bonds and van der Waals forces played major roles in the interaction. The effects of chemical denaturant, thermal denaturation and pH on the interaction were investigated and provided further support for the groove binding mode. In addition to experimental approaches, molecular modeling was conducted to verify binding pattern of palmatine–DNA. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: palmatine; DNA; groove binding; spectroscopic approach; molecular modeling
Introduction
1344
Alkaloids, natural products of plant origin, are gaining increasing attention for their potential therapeutic utility with high potency and low systemic toxicity. As the largest group of alkaloids, isoquinolines exhibit not only significant antimicrobial activity against a variety of organisms including bacteria, viruses, fungi, protozoans, helminths, and chlamydia, but also potential anticancer properties (1–3). Palmatine (Fig. 1) is one of the isoquinoline alkaloids distributed in many botanical families with significant anticancer activity. It has being extensively investigated due to its potential therapeutic actions, such as liverprotective and cardiovascular protective effects, clinical applications in the treatment of jaundice, dysentery, hypertension, inflammation, and liver-related diseases (4–6). In addition to its medicinal applications, palmatine is also used as a fluorescent probe of deoxyribonucleic acid (DNA) and RNA in biochemical researches (7,8). As a promising anticancer candidate with low toxicity, palmatine and its derivatives attract scientists to fully understand their anticancer effects and to elucidate the mechanisms using technologies of genomics, proteomics and other advanced approaches. DNA, apart from being a natural biological information carrier, has also been recognized as a key component in pharmaceutical and forensic industries and a crucial material in the development of advanced molecular devices (9–11). DNA is the target molecule of many drugs, especially antitumor drugs. The development of novel therapeutic intercalative agents for malignancy is one of the most important areas in modern medicinal chemistry (12). Consequently, understanding the binding affinity of small molecules with DNA is just as important as designing new and efficient drugs targeted to DNA. There are three important modes of DNA double-helix binding, viz. (i) the insertion of
Luminescence 2015; 30: 1344–1351
the molecules with the base pairs of nucleic acid (intercalation); (ii) the binding of molecules to the groove; and (iii) electrostatic interactions between the negatively charged phosphate groups of DNA and cationic portion of the molecules (13). However, the binding affinity and sequence specificity of small molecules with DNA can be influenced by various structural and electronic factors (14). Hence, understanding the reaction modes and kinetic mechanisms between the molecules and DNA is very important. This study is also helpful to function as a rational design system for the development of new efficient drugs targeted to DNA by serving as sensitive molecular reporters for monitoring nucleic acid structure, and so forth (15–17). In recent years, even though many researchers have discussed the interaction of palmatine with DNA, the binding mechanism of palmatine to DNA has not been fully investigated (18–21). Therefore, detailed information about the interaction of palmatine with DNA is essentially important as the experimental outcome for structure modification of this compound according to pharmaceutical need. In this study, the palmatine-DNA-binding mechanism was studied by UV-vis absorbance and fluorescence spectroscopy. In addition, theoretical study based on molecular docking provides further insight into the interaction between DNA and palmatine.
* Correspondence to: Y.-J. Hu, Hubei Collaborative Innovation Center for Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, Department of Chemistry, Hubei Normal University, Huangshi 435002, People’s Republic of China. Tel: +86 714 6515602. Fax: +86 714 6573832. E-mail: [email protected] Hubei Collaborative Innovation Center for Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, Department of Chemistry, Hubei Normal University, Huangshi 435002, People’s Republic of China
Copyright © 2015 John Wiley & Sons, Ltd.
Binding properties of palmatine to DNA
Results and discussion Fluorescence characteristics of the palmatine–DNA system
Our study provides a groove binding analysis of palmatineDNA interaction, which helps to design new efficient, safe probes for the fluorometric detection of DNA instead of traditional toxic and carcinogenic probes.
Experimental Materials Herring sperm DNA and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma-Aldrich (St. Louis, MO, USA); palmatine was obtained from Shanghai Jin Sui biological technology Co., Ltd (Shanghai, China); the Tris and Na2HPO4-NaH2PO4 (PB) buffers had a purity of no less than 99.5%, and NaCl, HCl, and so on were all of analytical purity. DNA solution was dissolved in PB buffer solution, and a stock solution of DNA was prepared by dissolving DNA in buffer and stored at 4°C for more than 12 h with gentle shaking to obtain homogeneity. The purity of DNA was verified by monitoring the ratio of absorbance at 260/280 nm (A260/A280). DNA concentrations were determined by using an extinction coefficient of 6600 mol1·cm1 (22) at 260 nm and expressed in terms of base molarity.
Equipments and spectral measurements The UV–visible light spectrum was recorded at room temperature on a UV-9000S spectrophotometer (Metash, China) equipped with 1.0 cm quartz cells. All fluorescence spectra were recorded on a F-4500 spectrofluorimeter (Hitachi, Japan) equipped with 1.0 cm quartz cells and a thermostat bath. All of the measurements were maintaining excitation and emission band passes at 10 nm. Appropriate blanks corresponding to the buffer were subtracted to correct the fluorescence background.
1.0
1.0
FL (a.u.)
Figure 1. Molecular structure of palmatine.
The fluorescence spectrum of palmatine in buffer solution shows a band at 530 nm upon excitation at 365 nm. Addition of DNA to the buffer solution of palmatine leads to the enhancement of fluorescence intensity immediately and gradually (Fig. 2). The experimental results indicated that palmatine can increase the fluorescence of the system after binding to DNA. This result can be explained by the fact that palmatine forms intermolecular fluorescent complexes with DNA (24). The inset in Fig. 2 shows that within the investigated concentrations, the increasing sensitivity of fluorescence of palmatine was proportional to the concentration of DNA. By stabilizing the concentration of DNA, and gradually increasing the concentration of palmatine, we found that the effect of palmatine on DNA fluorescence intensity (Fig. 3) was similar to the result in Fig. 2. However the fluorescence intensity increased with an increase in palmatine concentration. The inset in Fig. 3 shows that when the concentration of palmatine was higher than 12.0 × 105 mol·L1 the fluorescence intensity tended to be a constant. Obviously, when small molecule chromophore binds to the DNA bases or groove or backbone phosphates, its rotational motion should be restricted to the enhancement of a small molecule rigid flat structure and the fluorescence peak should be increased (25). Hence, the above results suggested that palmatine can bind with DNA. Because of the weak intrinsic fluorescence emission of DNA, acridine orange (AO) is used to mark DNA molecules. In recent years, it has been demonstrated that AO can bind to DNA as a fluorescence probe (22,26). The fluorescence of AO increases after binding with DNA by the formation of DNA–AO complexes. If palmatine binds to DNA, it would lead to a significant decrease in the fluorescence intensity by changing either the polar environment, the secondary and tertiary structure, or conformational stability of the DNA. It was observed in Fig. 4 that the emission spectra of the DNA-AO system decreased in the presence of palmatine. This result illustrates that palmatine can bind to DNA.
Molecular modeling
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0.8
T = 298K
0.8
0.6
0.4
0
5
4
8
-1
12
10 [DNA] (mol· L )
A 0.6
0.4
palmatine in buffer 400
450
500
550
600
650
700
Wavelength (nm) Figure 2. Fluorescence spectra of palmatine with various concentrations of DNA. Inset: Relative fluorescence intensity for palmatine as a function of the DNA for different 5 1 5 1 concentrations. c (palmatine) = 1.0 × 10 mol·L ; c (DNA)/(10 mol·L ), A–L: from 0.0 to 13.75 at increments of 1.25.
Copyright © 2015 John Wiley & Sons, Ltd.
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The molecular docking studies were performed by a Surflex Dock program in Sybyl-X 2.1 package (23), the crystal structure of B-DNA used for docking study was extracted from RCSB Protein Data Bank (PDB), PDB ID: 1VZK. Water was removed from the DNA PDB file. Polar hydrogen atoms and Gasteiger charges were added to prepare the DNA molecule for docking analysis. The protomol was generated using ligand mode with the threshold kept at 0.50 and the bloat 0. The structure of palmatine was drawn with the Sybyl-X 2.1 package, and energy optimized using the Tripos Force Field and charged using the Gasteiger-Huckel method. The more parameters were determined through numbers of attempts. For each docking simulation, the lowest binding energy conformer was searched out of 20 different conformers and was used for further analysis.
Fluorescence intensity (a.u.)
L
R. Mi et al. 1.0
2.0 T = 298K
0.8
1.5
0.6 0.4 0
3
6
9
5
A
0.6
-1
10 [palmatine] (mol·L )
0.4
0.2
A
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DNA-palmatine complex 1.0
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0
2
4
105 [palmatine] (mol·L-1)
0.5
DNA in buffer
400
450
0.0 500
550
600
650
700
Wavelength (nm)
1.0 (F0-F) / F
T = 298K
0.8
A
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0.0
0.6
0
3
6
5
9 -1
10 [palmatine] (mol· L )
L 0.4
0.2
0.0 480
540
570
600
630
Wavelength (nm) Figure 4. Fluorescence spectrum of DNA–AO with various concentrations of 4 palmatine. The inset corresponds to the Stern–Volmer plots. c (DNA) = 2.0 × 10 1 6 1 5 1 mol·L ; c (AO) = 1.8 × 10 mol·L ; c (palmatine)/(10 mol·L ), A–L: from 0.0 to 8.8 at increments of 0.8.
Absorption spectra
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To gain insight into the interaction between the small molecules with the DNA structure, analysis of visible absorption spectra was performed. In general, binding of small molecules to DNA produces hypochromic, hyperchromic and bathochromic shifts in the absorption band of the bound ligand. Hyperchromism and hypochromism are the spectral features of DNA concerning its double-helical structure. Hyperchromism means the breakage of the secondary structure of DNA while hypochromism means that the DNA-binding mode of the complex is due to electrostatic effect or intercalation, which can stabilize the DNA duplex. While the bathochromic shift is particularly dominant for intercalators (27,28). We recorded the absorption spectra of DNA in the absence and presence of palmatine, and the results are presented in Fig. 5. In this work, it was observed that the maximum absorption of free DNA was at 260 nm, and the absorption increased after the addition of different concentrations of palmatine. In Fig. 5 inset, the absorption value of DNA-palmatine complex was a little greater than the absorption value of free DNA and free palmatine and indicated
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250
300
350
400
450
Figure 5. UV absorption spectra of DNA with various concentrations of palmatine. Inset: Comparison of absorption at 260 nm between the DNA–palmatine complex and 5 1 the sum values of circular DNA and palmatine. c (DNA) = 1.0 × 10 mol·L ; c 5 1 (palmatine)/(10 mol·L ), A–K: from 0 to 5.0 at increments of 0.5.
that a weak hyperchromism existed between DNA and palmatine, thus groove binding possibly existed between DNA and palmatine. Moreover, a bathochromic shift implies the intercalation of ligand into the base stack of DNA, and no shift in this case could be due to groove binding rather than the intercalation of palmatine. Further convincing evidence to support the groove binding of palmatine to DNA was found from the fluorescence quenching studies. Iodide quenching experiment
DNA-AO-palmatine
510
DNA in buffer Wavelength (nm)
Figure 3. Fluorescence spectrum of DNA with various concentrations of palmatine. Inset: Relative fluorescence intensity for DNA as a function of the palmatine for differ4 1 5 1 ent concentrations. c (DNA) = 2.0 × 10 mol·L ; c (palmatine)/(10 mol·L ), A–K: from 1.25 to 13.75 at increments of 1.25.
Fluorescence intensity (a.u.)
DNA+palmatine
1.5
K
0.8
Absorbance
Fluorescence intensity (a.u.)
K
Absorbance
FL (a.u.)
1.0
To elucidate the mode of binding, fluorescence quenching of DNA-binding palmatine is studied in relation to the quenching of palmatine in buffered solution. Potassium iodide (KI) and K4[Fe(CN)6] are used as the quencher. A highly negatively charged quencher was expected to be repelled by the negatively charged phosphate backbone of DNA. Accordingly, intercalative bound small molecules should be protected from being quenched by anionic quencher, whereas the free buffered solution complexes and groove binding drugs should be quenched readily by anionic quenchers (16). Therefore, the negatively charged I and [Fe(CN)6]4 ions were selected to determine the binding mode of palmatine to DNA, as shown in Fig. 6. The fluorescence quenching data were analyzed to obtain the quenching constant by using the well known Stern–Volmer equation (25) F0 ¼ 1 þ K SV ½Q F
(1)
Where F0 and F denote the steady-state fluorescence intensities in the absence and in the presence of quencher (palmatine), respectively, KSV is the Stern–Volmer quenching constant, and [Q] is the concentration of the quencher. Hence, Eqn 1 was applied to determine KSV by linear regression of a plot of F0/F against [Q]. Fig. 6(A, B) shows the Stern–Volmer plots for the palmatine quenched by the negative ions (I and [Fe(CN)6]4 ) in the absence and presence of DNA, respectively. It was apparent that the negatively charged quenching effect was increased when palmatine bound to DNA. The results were possibly attributed to the palmatine molecule entering into the ‘space’ of groove but not
Copyright © 2015 John Wiley & Sons, Ltd.
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Binding properties of palmatine to DNA
palmatine-Ipalmatine-DNA-I-
0
(F -F) / F
0.4
0.2
0.0
(A) 0.5
0.0
1.0
1.5
2.0
4
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-1
10 [KI] (mol·L )
F0 1 1 1 þ ¼ ΔF f a K a ½Q f a
(2)
palmatine-DNA-Fe(CN)64-
0.6
0
Binding parameters. For a static quenching procedure, the data were analyzed according to the modified Stern–Volmer equation (31)
palmatine-Fe(CN)64-
0.8
(F -F)/ F
of weakly bound complexes and decreased static quenching (10). Consequently, quenching types can be determined on the basis of the calculation of KSV obtained at different temperatures. To confirm the quenching mechanism, the fluorescence quenching data at different temperatures (292, 298, 304 and 310K) were analyzed according to Stern–Volmer plots. The results are shown in Fig. S1. The calculation of KSV from Stern–Volmer plots is shown in Table 1. The results show that the Stern–Volmer plots constant KSV is inversely correlated with temperature, which indicates that the quenching mechanism of the palmatine–DNAbinding reaction is initiated by compound formation rather than by dynamic collision.
0.4
0.2
(B)
0.0 0
1
2
3
4
4
5
-1
4-
10 [Fe(CN) ] (mol·L ) 6
4
Figure 6. Fluorescence quenching of palmatine by I (A); and [Fe(CN)6] (B) in the 4 1 5 absence and presence of DNA. c (DNA) = 2.0 × 10 mol·L ; c (palmatine) = 4.0 × 10 1 mol·L .
buried deeply into the DNA, that being exposed to the buffer solution without protection from quenching by the anionic quencher. The values of quenching constants (KSV) of palmatine by I ion in the absence and presence of DNA were calculated to be 0.66 × 103 and 2.06 × 103 mol·L1, and by [Fe(CN)6]4 ion in the absence and presence of DNA were calculated to be 1.49 × 103 and 1.78 × 103 mol·L1, respectively, which provided direct evidence that palmatine with DNA was groove binding.
Effect of temperature A variety of molecular interactions can result in quenching, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching, and so on (29). Quenching mechanisms are usually classified as either dynamic quenching or static quenching. Dynamic and static quenching can be distinguished by their different dependence on temperature and viscosity (30). In this study, the effect of temperature on DNA–AO–palmatine fluorescence quenching was investigated.
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r=Df ¼ nK b rK b
(3)
Where r is the ratio of bound complex to total nucleotide concentration [DNA], Df is the molar concentration of free ligand, n is binding site multiplicity per class of binding sites, and Kb is the equilibrium binding constant. Fig. 7(A, B) shows the modified Stern–Volmer plots and Scatchard plots for the palmatine–DNA (AO) system at different temperatures, respectively. The corresponding results at different temperatures are shown in Table 2. The decreasing trend of Ka and Kb with increasing temperature was in accordance with KSV’s dependence on temperature, as mentioned above, which possibly resulted from the reduction in the stability of the palmatine–DNA complex.
Table 1. Stern–Volmer quenching constants of palmatine– DNA (AO) system at various temperatures pH
T (K)
10–3KSV/ (L·mol1)
Ra
S.D. b
7.2
292 298 304 310
11.77 8.66 7.96 5.26
0.9998 0.9994 0.9994 0.9996
0.0014 0.0018 0.0014 0.0008
R is the correlation coefficient. S.D. is standard deviation.
a
b
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Quenching mechanism. Quenching mechanisms include static and dynamic quenching. Higher temperatures result in faster diffusion and hence increased dynamic quenching, but the dissociation
In the present case, ΔF is the difference in fluorescence in the absence and presence of the quencher at concentration [Q], fa is the fraction of accessible fluorescence, and Ka is the effective quenching constant for the accessible fluorophores, which are analogous to associative binding constants for the quencher– acceptor system. The dependence of F0/ΔF on the reciprocal value of the quencher concentration [Q]1 is linear with the slope equaling the value of (faKa)1. The value f1 a is fixed on the ordinate. The con1 stant Ka is a quotient of the ordinate f1 a and the slope (faKa) . When small molecules bind to a set of equivalent sites on a macromolecule, the equilibrium binding constant and the number of binding sites can be also analyzed according to the Scatchard equation (32)
R. Mi et al. enthalpy change (ΔH) is calculated from the slope of the van ’t Hoff relationship. The free energy change (ΔG) is estimated from the following relationship ΔG ¼ ΔH TΔS
(5)
Table 2 summarizes the values of ΔH and ΔS obtained for the binding site from the slopes and ordinates at the origin of the fitted lines. The negative free energy (ΔG) means that the binding process is spontaneous. The negative enthalpy (ΔH) and entropy (ΔS) values of the interaction of palmatine and DNA indicate that the hydrogen bonds and van der Waals interactions played a major role in the reaction. Effect of different degeneration conditions
Figure 7. Modified Stern–Volmer plots (A) and Scatchard plots (B) of the palmatine– DNA (AO) system at different temperatures.
Thermodynamic parameters and binding modes. The interaction forces between drugs and biomolecules may include electrostatic interactions, multiple hydrogen bonds, van der Waals interactions, hydrophobic and steric contacts within the antibodybinding site, and so on (33). If the enthalpy change (ΔH) does not vary significantly in the temperature range studied, both the enthalpy change (ΔH) and entropy change (ΔS) can be evaluated from the van ’t Hoff equation ΔH ΔS ln K b ¼ þ RT R
(4)
where Kb is analogous to the associative binding constants at the corresponding temperature and R is the gas constant. The
In comparison with either acid or thermal unfolding, chemical agents such as GuHCl are more effective in disturbing the noncovalent interactions. Denaturation leads to changes in the secondary and tertiary structure and conformational stability of the biomacromolecules such as DNA, or proteins (22). The double helix of denaturated DNA would be weakened. The degenerationinduced perturbation of the drug–DNA binding interaction is attempted as a complementary corridor to explore the binding phenomenon (34). In this study, we introduced GuHCl to the palmatine–DNA (AO) system to investigate the effects on the binding; the ratios of the concentrations of denaturants and DNA were fixed as 3:1, 6:1 and 9:1, respectively. The binding parameters with increasing concentration of GuHCl were analyzed using the modified Stern–Volmer method and the Scatchard method respectively. The corresponding results are shown in Fig. S2(A, B) and Table 3. The results demonstrated that binding constants are obverse in pattern with respect to the increasing concentration of GuHCl during the fluorescence binding procedure. This inspection implies that GuHCl-induced denaturation of DNA leads to considerable weakening of the double helix and loosening of groove space, which makes palmatine more likely to combine with the groove. Hence, we can speculate that palmatine binds to the groove with DNA. Effect of thermal denaturation The thermal denaturation experiment provides further evidence for the groove binding of palmatine with DNA. DNA is a highly dynamic molecule in which the base pairs can demonstrate temporary breaking of a ‘closed’ pair and local separation of the two strands. Local openings may be activated by heating. At a certain
Table 2. Binding constants and relative thermodynamic parameters of palmatine–DNA interaction at pH 7.2 T (K)
Modified Stern–Volmer method 104Ka/ (L·mol1)
292 298 304 310
1.90 1.74 1.41 1.20
Scatchard method
Ra
104Kb/(L·mol1)
n
0.9996 0.9993 0.9991 0.9997
1.83 1.66 1.46 1.20
0.69 0.59 0.55 0.48
ΔH (kJ·mol1)
ΔG (kJ·mol1)
ΔS ( J·mol1·K1)
–31.30
–22.75 –22.47 –22.70 –22.09
–28.77
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R is the correlation coefficient.
a
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Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 1344–1351
Binding properties of palmatine to DNA Table 3. Binding parameters of palmatine–DNA interaction in the absence and presence of denaturants c (Denaturation)/c (DNA) Blank G1:3 G1:6 G1:9
Modified Stern–Volmer method 4
1
Scatchard method 4
10 Ka/ (L·mol )
R
1.40 1.81 1.77 1.88
0.9993 0.9995 0.9999 0.9999
a
10 Kb/ (L·mol1)
n
1.53 1.68 1.73 1.87
0.55 0.69 0.71 0.68
R is the correlation coefficient.
a
temperature, local openings of the double helix extend over the full molecule, resulting in a complete separation of the two strands (35). In the thermal denaturation study, we heated the native dsDNA solution in a water bath for 30 min at each temperature (60, 80 or 100°C) and then compared the binding parameters obtained from the two kinds of cooling methods (one cooling naturally, the other cooling in an ice–water bath). Fig. S3 (A, B) shows the modified Stern–Volmer plots and Scatchard plots for the palmatine–DNA (AO) system at different denatured temperatures, respectively. The corresponding results in Table 4 show that the binding parameters increased as the temperature increased, possibly resulting from the fact that higher temperature makes the double helix weaker and the space of the groove larger, both of which are more suited for small molecule (palmatine) binding DNA by groove binding. The binding constants of the DNA cooling naturally and the relevant one cooling in an ice–water bath showed nearly the same trend, which confirmed groove binding for DNA with palmatine. Effect of pH Solution pH plays a critically important role in a variety of chemical and biological processes. The requirement for pH of the humoral environment and intracellular environment is also very critical. The configuration of double-stranded DNA (dsDNA) structures and gene expression are closely related to environmental pH (36). Phosphate-buffered solution was used to study the effect of pH on the interaction between palmatine and DNA. The binding constants of the palmatine–DNA (AO) system are shown in Fig. S4.(A, B), the binding constants at different pH are presented in Table 5. It is obvious that the interaction of palmatine and DNA was greatly influenced by the pH values. The values of the binding constants decrease gradually with the decrease in pH
(
Revised: 21 February 2015,
Accepted: 22 February 2015
Published online in Wiley Online Library: 31 March 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2904
Binding properties of palmatine to DNA: spectroscopic and molecular modeling investigations Ran Mi, Bao Tu, Xiao-Ting Bai, Jun Chen, Yu Ouyang and Yan-Jun Hu* ABSTRACT: Palmatine, an isoquinoline alkaloid, is an important medicinal herbal extract with diverse pharmacological and biological properties. In this work, spectroscopic and molecular modeling approaches were employed to reveal the interaction between palmatine and DNA isolated from herring sperm. The absorption spectra and iodide quenching results indicated that groove binding was the main binding mode of palmatine to DNA. Fluorescence studies indicated that the binding constant (K) of palmatine and DNA was ~ 104 L·mol1. The associated thermodynamic parameters, ΔG, ΔH, and ΔS, indicated that hydrogen bonds and van der Waals forces played major roles in the interaction. The effects of chemical denaturant, thermal denaturation and pH on the interaction were investigated and provided further support for the groove binding mode. In addition to experimental approaches, molecular modeling was conducted to verify binding pattern of palmatine–DNA. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: palmatine; DNA; groove binding; spectroscopic approach; molecular modeling
Introduction
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Alkaloids, natural products of plant origin, are gaining increasing attention for their potential therapeutic utility with high potency and low systemic toxicity. As the largest group of alkaloids, isoquinolines exhibit not only significant antimicrobial activity against a variety of organisms including bacteria, viruses, fungi, protozoans, helminths, and chlamydia, but also potential anticancer properties (1–3). Palmatine (Fig. 1) is one of the isoquinoline alkaloids distributed in many botanical families with significant anticancer activity. It has being extensively investigated due to its potential therapeutic actions, such as liverprotective and cardiovascular protective effects, clinical applications in the treatment of jaundice, dysentery, hypertension, inflammation, and liver-related diseases (4–6). In addition to its medicinal applications, palmatine is also used as a fluorescent probe of deoxyribonucleic acid (DNA) and RNA in biochemical researches (7,8). As a promising anticancer candidate with low toxicity, palmatine and its derivatives attract scientists to fully understand their anticancer effects and to elucidate the mechanisms using technologies of genomics, proteomics and other advanced approaches. DNA, apart from being a natural biological information carrier, has also been recognized as a key component in pharmaceutical and forensic industries and a crucial material in the development of advanced molecular devices (9–11). DNA is the target molecule of many drugs, especially antitumor drugs. The development of novel therapeutic intercalative agents for malignancy is one of the most important areas in modern medicinal chemistry (12). Consequently, understanding the binding affinity of small molecules with DNA is just as important as designing new and efficient drugs targeted to DNA. There are three important modes of DNA double-helix binding, viz. (i) the insertion of
Luminescence 2015; 30: 1344–1351
the molecules with the base pairs of nucleic acid (intercalation); (ii) the binding of molecules to the groove; and (iii) electrostatic interactions between the negatively charged phosphate groups of DNA and cationic portion of the molecules (13). However, the binding affinity and sequence specificity of small molecules with DNA can be influenced by various structural and electronic factors (14). Hence, understanding the reaction modes and kinetic mechanisms between the molecules and DNA is very important. This study is also helpful to function as a rational design system for the development of new efficient drugs targeted to DNA by serving as sensitive molecular reporters for monitoring nucleic acid structure, and so forth (15–17). In recent years, even though many researchers have discussed the interaction of palmatine with DNA, the binding mechanism of palmatine to DNA has not been fully investigated (18–21). Therefore, detailed information about the interaction of palmatine with DNA is essentially important as the experimental outcome for structure modification of this compound according to pharmaceutical need. In this study, the palmatine-DNA-binding mechanism was studied by UV-vis absorbance and fluorescence spectroscopy. In addition, theoretical study based on molecular docking provides further insight into the interaction between DNA and palmatine.
* Correspondence to: Y.-J. Hu, Hubei Collaborative Innovation Center for Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, Department of Chemistry, Hubei Normal University, Huangshi 435002, People’s Republic of China. Tel: +86 714 6515602. Fax: +86 714 6573832. E-mail: [email protected] Hubei Collaborative Innovation Center for Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, Department of Chemistry, Hubei Normal University, Huangshi 435002, People’s Republic of China
Copyright © 2015 John Wiley & Sons, Ltd.
Binding properties of palmatine to DNA
Results and discussion Fluorescence characteristics of the palmatine–DNA system
Our study provides a groove binding analysis of palmatineDNA interaction, which helps to design new efficient, safe probes for the fluorometric detection of DNA instead of traditional toxic and carcinogenic probes.
Experimental Materials Herring sperm DNA and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma-Aldrich (St. Louis, MO, USA); palmatine was obtained from Shanghai Jin Sui biological technology Co., Ltd (Shanghai, China); the Tris and Na2HPO4-NaH2PO4 (PB) buffers had a purity of no less than 99.5%, and NaCl, HCl, and so on were all of analytical purity. DNA solution was dissolved in PB buffer solution, and a stock solution of DNA was prepared by dissolving DNA in buffer and stored at 4°C for more than 12 h with gentle shaking to obtain homogeneity. The purity of DNA was verified by monitoring the ratio of absorbance at 260/280 nm (A260/A280). DNA concentrations were determined by using an extinction coefficient of 6600 mol1·cm1 (22) at 260 nm and expressed in terms of base molarity.
Equipments and spectral measurements The UV–visible light spectrum was recorded at room temperature on a UV-9000S spectrophotometer (Metash, China) equipped with 1.0 cm quartz cells. All fluorescence spectra were recorded on a F-4500 spectrofluorimeter (Hitachi, Japan) equipped with 1.0 cm quartz cells and a thermostat bath. All of the measurements were maintaining excitation and emission band passes at 10 nm. Appropriate blanks corresponding to the buffer were subtracted to correct the fluorescence background.
1.0
1.0
FL (a.u.)
Figure 1. Molecular structure of palmatine.
The fluorescence spectrum of palmatine in buffer solution shows a band at 530 nm upon excitation at 365 nm. Addition of DNA to the buffer solution of palmatine leads to the enhancement of fluorescence intensity immediately and gradually (Fig. 2). The experimental results indicated that palmatine can increase the fluorescence of the system after binding to DNA. This result can be explained by the fact that palmatine forms intermolecular fluorescent complexes with DNA (24). The inset in Fig. 2 shows that within the investigated concentrations, the increasing sensitivity of fluorescence of palmatine was proportional to the concentration of DNA. By stabilizing the concentration of DNA, and gradually increasing the concentration of palmatine, we found that the effect of palmatine on DNA fluorescence intensity (Fig. 3) was similar to the result in Fig. 2. However the fluorescence intensity increased with an increase in palmatine concentration. The inset in Fig. 3 shows that when the concentration of palmatine was higher than 12.0 × 105 mol·L1 the fluorescence intensity tended to be a constant. Obviously, when small molecule chromophore binds to the DNA bases or groove or backbone phosphates, its rotational motion should be restricted to the enhancement of a small molecule rigid flat structure and the fluorescence peak should be increased (25). Hence, the above results suggested that palmatine can bind with DNA. Because of the weak intrinsic fluorescence emission of DNA, acridine orange (AO) is used to mark DNA molecules. In recent years, it has been demonstrated that AO can bind to DNA as a fluorescence probe (22,26). The fluorescence of AO increases after binding with DNA by the formation of DNA–AO complexes. If palmatine binds to DNA, it would lead to a significant decrease in the fluorescence intensity by changing either the polar environment, the secondary and tertiary structure, or conformational stability of the DNA. It was observed in Fig. 4 that the emission spectra of the DNA-AO system decreased in the presence of palmatine. This result illustrates that palmatine can bind to DNA.
Molecular modeling
Luminescence 2015; 30: 1344–1351
0.8
T = 298K
0.8
0.6
0.4
0
5
4
8
-1
12
10 [DNA] (mol· L )
A 0.6
0.4
palmatine in buffer 400
450
500
550
600
650
700
Wavelength (nm) Figure 2. Fluorescence spectra of palmatine with various concentrations of DNA. Inset: Relative fluorescence intensity for palmatine as a function of the DNA for different 5 1 5 1 concentrations. c (palmatine) = 1.0 × 10 mol·L ; c (DNA)/(10 mol·L ), A–L: from 0.0 to 13.75 at increments of 1.25.
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The molecular docking studies were performed by a Surflex Dock program in Sybyl-X 2.1 package (23), the crystal structure of B-DNA used for docking study was extracted from RCSB Protein Data Bank (PDB), PDB ID: 1VZK. Water was removed from the DNA PDB file. Polar hydrogen atoms and Gasteiger charges were added to prepare the DNA molecule for docking analysis. The protomol was generated using ligand mode with the threshold kept at 0.50 and the bloat 0. The structure of palmatine was drawn with the Sybyl-X 2.1 package, and energy optimized using the Tripos Force Field and charged using the Gasteiger-Huckel method. The more parameters were determined through numbers of attempts. For each docking simulation, the lowest binding energy conformer was searched out of 20 different conformers and was used for further analysis.
Fluorescence intensity (a.u.)
L
R. Mi et al. 1.0
2.0 T = 298K
0.8
1.5
0.6 0.4 0
3
6
9
5
A
0.6
-1
10 [palmatine] (mol·L )
0.4
0.2
A
1.0
DNA-palmatine complex 1.0
0.5
0
2
4
105 [palmatine] (mol·L-1)
0.5
DNA in buffer
400
450
0.0 500
550
600
650
700
Wavelength (nm)
1.0 (F0-F) / F
T = 298K
0.8
A
0.4
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0.0
0.6
0
3
6
5
9 -1
10 [palmatine] (mol· L )
L 0.4
0.2
0.0 480
540
570
600
630
Wavelength (nm) Figure 4. Fluorescence spectrum of DNA–AO with various concentrations of 4 palmatine. The inset corresponds to the Stern–Volmer plots. c (DNA) = 2.0 × 10 1 6 1 5 1 mol·L ; c (AO) = 1.8 × 10 mol·L ; c (palmatine)/(10 mol·L ), A–L: from 0.0 to 8.8 at increments of 0.8.
Absorption spectra
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To gain insight into the interaction between the small molecules with the DNA structure, analysis of visible absorption spectra was performed. In general, binding of small molecules to DNA produces hypochromic, hyperchromic and bathochromic shifts in the absorption band of the bound ligand. Hyperchromism and hypochromism are the spectral features of DNA concerning its double-helical structure. Hyperchromism means the breakage of the secondary structure of DNA while hypochromism means that the DNA-binding mode of the complex is due to electrostatic effect or intercalation, which can stabilize the DNA duplex. While the bathochromic shift is particularly dominant for intercalators (27,28). We recorded the absorption spectra of DNA in the absence and presence of palmatine, and the results are presented in Fig. 5. In this work, it was observed that the maximum absorption of free DNA was at 260 nm, and the absorption increased after the addition of different concentrations of palmatine. In Fig. 5 inset, the absorption value of DNA-palmatine complex was a little greater than the absorption value of free DNA and free palmatine and indicated
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250
300
350
400
450
Figure 5. UV absorption spectra of DNA with various concentrations of palmatine. Inset: Comparison of absorption at 260 nm between the DNA–palmatine complex and 5 1 the sum values of circular DNA and palmatine. c (DNA) = 1.0 × 10 mol·L ; c 5 1 (palmatine)/(10 mol·L ), A–K: from 0 to 5.0 at increments of 0.5.
that a weak hyperchromism existed between DNA and palmatine, thus groove binding possibly existed between DNA and palmatine. Moreover, a bathochromic shift implies the intercalation of ligand into the base stack of DNA, and no shift in this case could be due to groove binding rather than the intercalation of palmatine. Further convincing evidence to support the groove binding of palmatine to DNA was found from the fluorescence quenching studies. Iodide quenching experiment
DNA-AO-palmatine
510
DNA in buffer Wavelength (nm)
Figure 3. Fluorescence spectrum of DNA with various concentrations of palmatine. Inset: Relative fluorescence intensity for DNA as a function of the palmatine for differ4 1 5 1 ent concentrations. c (DNA) = 2.0 × 10 mol·L ; c (palmatine)/(10 mol·L ), A–K: from 1.25 to 13.75 at increments of 1.25.
Fluorescence intensity (a.u.)
DNA+palmatine
1.5
K
0.8
Absorbance
Fluorescence intensity (a.u.)
K
Absorbance
FL (a.u.)
1.0
To elucidate the mode of binding, fluorescence quenching of DNA-binding palmatine is studied in relation to the quenching of palmatine in buffered solution. Potassium iodide (KI) and K4[Fe(CN)6] are used as the quencher. A highly negatively charged quencher was expected to be repelled by the negatively charged phosphate backbone of DNA. Accordingly, intercalative bound small molecules should be protected from being quenched by anionic quencher, whereas the free buffered solution complexes and groove binding drugs should be quenched readily by anionic quenchers (16). Therefore, the negatively charged I and [Fe(CN)6]4 ions were selected to determine the binding mode of palmatine to DNA, as shown in Fig. 6. The fluorescence quenching data were analyzed to obtain the quenching constant by using the well known Stern–Volmer equation (25) F0 ¼ 1 þ K SV ½Q F
(1)
Where F0 and F denote the steady-state fluorescence intensities in the absence and in the presence of quencher (palmatine), respectively, KSV is the Stern–Volmer quenching constant, and [Q] is the concentration of the quencher. Hence, Eqn 1 was applied to determine KSV by linear regression of a plot of F0/F against [Q]. Fig. 6(A, B) shows the Stern–Volmer plots for the palmatine quenched by the negative ions (I and [Fe(CN)6]4 ) in the absence and presence of DNA, respectively. It was apparent that the negatively charged quenching effect was increased when palmatine bound to DNA. The results were possibly attributed to the palmatine molecule entering into the ‘space’ of groove but not
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 1344–1351
Binding properties of palmatine to DNA
palmatine-Ipalmatine-DNA-I-
0
(F -F) / F
0.4
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0.0
(A) 0.5
0.0
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F0 1 1 1 þ ¼ ΔF f a K a ½Q f a
(2)
palmatine-DNA-Fe(CN)64-
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Binding parameters. For a static quenching procedure, the data were analyzed according to the modified Stern–Volmer equation (31)
palmatine-Fe(CN)64-
0.8
(F -F)/ F
of weakly bound complexes and decreased static quenching (10). Consequently, quenching types can be determined on the basis of the calculation of KSV obtained at different temperatures. To confirm the quenching mechanism, the fluorescence quenching data at different temperatures (292, 298, 304 and 310K) were analyzed according to Stern–Volmer plots. The results are shown in Fig. S1. The calculation of KSV from Stern–Volmer plots is shown in Table 1. The results show that the Stern–Volmer plots constant KSV is inversely correlated with temperature, which indicates that the quenching mechanism of the palmatine–DNAbinding reaction is initiated by compound formation rather than by dynamic collision.
0.4
0.2
(B)
0.0 0
1
2
3
4
4
5
-1
4-
10 [Fe(CN) ] (mol·L ) 6
4
Figure 6. Fluorescence quenching of palmatine by I (A); and [Fe(CN)6] (B) in the 4 1 5 absence and presence of DNA. c (DNA) = 2.0 × 10 mol·L ; c (palmatine) = 4.0 × 10 1 mol·L .
buried deeply into the DNA, that being exposed to the buffer solution without protection from quenching by the anionic quencher. The values of quenching constants (KSV) of palmatine by I ion in the absence and presence of DNA were calculated to be 0.66 × 103 and 2.06 × 103 mol·L1, and by [Fe(CN)6]4 ion in the absence and presence of DNA were calculated to be 1.49 × 103 and 1.78 × 103 mol·L1, respectively, which provided direct evidence that palmatine with DNA was groove binding.
Effect of temperature A variety of molecular interactions can result in quenching, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching, and so on (29). Quenching mechanisms are usually classified as either dynamic quenching or static quenching. Dynamic and static quenching can be distinguished by their different dependence on temperature and viscosity (30). In this study, the effect of temperature on DNA–AO–palmatine fluorescence quenching was investigated.
Luminescence 2015; 30: 1344–1351
r=Df ¼ nK b rK b
(3)
Where r is the ratio of bound complex to total nucleotide concentration [DNA], Df is the molar concentration of free ligand, n is binding site multiplicity per class of binding sites, and Kb is the equilibrium binding constant. Fig. 7(A, B) shows the modified Stern–Volmer plots and Scatchard plots for the palmatine–DNA (AO) system at different temperatures, respectively. The corresponding results at different temperatures are shown in Table 2. The decreasing trend of Ka and Kb with increasing temperature was in accordance with KSV’s dependence on temperature, as mentioned above, which possibly resulted from the reduction in the stability of the palmatine–DNA complex.
Table 1. Stern–Volmer quenching constants of palmatine– DNA (AO) system at various temperatures pH
T (K)
10–3KSV/ (L·mol1)
Ra
S.D. b
7.2
292 298 304 310
11.77 8.66 7.96 5.26
0.9998 0.9994 0.9994 0.9996
0.0014 0.0018 0.0014 0.0008
R is the correlation coefficient. S.D. is standard deviation.
a
b
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Quenching mechanism. Quenching mechanisms include static and dynamic quenching. Higher temperatures result in faster diffusion and hence increased dynamic quenching, but the dissociation
In the present case, ΔF is the difference in fluorescence in the absence and presence of the quencher at concentration [Q], fa is the fraction of accessible fluorescence, and Ka is the effective quenching constant for the accessible fluorophores, which are analogous to associative binding constants for the quencher– acceptor system. The dependence of F0/ΔF on the reciprocal value of the quencher concentration [Q]1 is linear with the slope equaling the value of (faKa)1. The value f1 a is fixed on the ordinate. The con1 stant Ka is a quotient of the ordinate f1 a and the slope (faKa) . When small molecules bind to a set of equivalent sites on a macromolecule, the equilibrium binding constant and the number of binding sites can be also analyzed according to the Scatchard equation (32)
R. Mi et al. enthalpy change (ΔH) is calculated from the slope of the van ’t Hoff relationship. The free energy change (ΔG) is estimated from the following relationship ΔG ¼ ΔH TΔS
(5)
Table 2 summarizes the values of ΔH and ΔS obtained for the binding site from the slopes and ordinates at the origin of the fitted lines. The negative free energy (ΔG) means that the binding process is spontaneous. The negative enthalpy (ΔH) and entropy (ΔS) values of the interaction of palmatine and DNA indicate that the hydrogen bonds and van der Waals interactions played a major role in the reaction. Effect of different degeneration conditions
Figure 7. Modified Stern–Volmer plots (A) and Scatchard plots (B) of the palmatine– DNA (AO) system at different temperatures.
Thermodynamic parameters and binding modes. The interaction forces between drugs and biomolecules may include electrostatic interactions, multiple hydrogen bonds, van der Waals interactions, hydrophobic and steric contacts within the antibodybinding site, and so on (33). If the enthalpy change (ΔH) does not vary significantly in the temperature range studied, both the enthalpy change (ΔH) and entropy change (ΔS) can be evaluated from the van ’t Hoff equation ΔH ΔS ln K b ¼ þ RT R
(4)
where Kb is analogous to the associative binding constants at the corresponding temperature and R is the gas constant. The
In comparison with either acid or thermal unfolding, chemical agents such as GuHCl are more effective in disturbing the noncovalent interactions. Denaturation leads to changes in the secondary and tertiary structure and conformational stability of the biomacromolecules such as DNA, or proteins (22). The double helix of denaturated DNA would be weakened. The degenerationinduced perturbation of the drug–DNA binding interaction is attempted as a complementary corridor to explore the binding phenomenon (34). In this study, we introduced GuHCl to the palmatine–DNA (AO) system to investigate the effects on the binding; the ratios of the concentrations of denaturants and DNA were fixed as 3:1, 6:1 and 9:1, respectively. The binding parameters with increasing concentration of GuHCl were analyzed using the modified Stern–Volmer method and the Scatchard method respectively. The corresponding results are shown in Fig. S2(A, B) and Table 3. The results demonstrated that binding constants are obverse in pattern with respect to the increasing concentration of GuHCl during the fluorescence binding procedure. This inspection implies that GuHCl-induced denaturation of DNA leads to considerable weakening of the double helix and loosening of groove space, which makes palmatine more likely to combine with the groove. Hence, we can speculate that palmatine binds to the groove with DNA. Effect of thermal denaturation The thermal denaturation experiment provides further evidence for the groove binding of palmatine with DNA. DNA is a highly dynamic molecule in which the base pairs can demonstrate temporary breaking of a ‘closed’ pair and local separation of the two strands. Local openings may be activated by heating. At a certain
Table 2. Binding constants and relative thermodynamic parameters of palmatine–DNA interaction at pH 7.2 T (K)
Modified Stern–Volmer method 104Ka/ (L·mol1)
292 298 304 310
1.90 1.74 1.41 1.20
Scatchard method
Ra
104Kb/(L·mol1)
n
0.9996 0.9993 0.9991 0.9997
1.83 1.66 1.46 1.20
0.69 0.59 0.55 0.48
ΔH (kJ·mol1)
ΔG (kJ·mol1)
ΔS ( J·mol1·K1)
–31.30
–22.75 –22.47 –22.70 –22.09
–28.77
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R is the correlation coefficient.
a
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Luminescence 2015; 30: 1344–1351
Binding properties of palmatine to DNA Table 3. Binding parameters of palmatine–DNA interaction in the absence and presence of denaturants c (Denaturation)/c (DNA) Blank G1:3 G1:6 G1:9
Modified Stern–Volmer method 4
1
Scatchard method 4
10 Ka/ (L·mol )
R
1.40 1.81 1.77 1.88
0.9993 0.9995 0.9999 0.9999
a
10 Kb/ (L·mol1)
n
1.53 1.68 1.73 1.87
0.55 0.69 0.71 0.68
R is the correlation coefficient.
a
temperature, local openings of the double helix extend over the full molecule, resulting in a complete separation of the two strands (35). In the thermal denaturation study, we heated the native dsDNA solution in a water bath for 30 min at each temperature (60, 80 or 100°C) and then compared the binding parameters obtained from the two kinds of cooling methods (one cooling naturally, the other cooling in an ice–water bath). Fig. S3 (A, B) shows the modified Stern–Volmer plots and Scatchard plots for the palmatine–DNA (AO) system at different denatured temperatures, respectively. The corresponding results in Table 4 show that the binding parameters increased as the temperature increased, possibly resulting from the fact that higher temperature makes the double helix weaker and the space of the groove larger, both of which are more suited for small molecule (palmatine) binding DNA by groove binding. The binding constants of the DNA cooling naturally and the relevant one cooling in an ice–water bath showed nearly the same trend, which confirmed groove binding for DNA with palmatine. Effect of pH Solution pH plays a critically important role in a variety of chemical and biological processes. The requirement for pH of the humoral environment and intracellular environment is also very critical. The configuration of double-stranded DNA (dsDNA) structures and gene expression are closely related to environmental pH (36). Phosphate-buffered solution was used to study the effect of pH on the interaction between palmatine and DNA. The binding constants of the palmatine–DNA (AO) system are shown in Fig. S4.(A, B), the binding constants at different pH are presented in Table 5. It is obvious that the interaction of palmatine and DNA was greatly influenced by the pH values. The values of the binding constants decrease gradually with the decrease in pH
(
