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Spectroscopic analyses on interaction of melamine, cyanuric acid and uric acid with DNA
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 714–721
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Spectroscopic analyses on interaction of melamine, cyanuric acid and uric acid with DNA Jinhui Xie a, Dandan Chen a, Qiong Wu a, Jun Wang a,⇑, Heng Qiao b a b
College of Chemistry, Liaoning University, Shenyang 110036, PR China College of Environment, Liaoning University, Shenyang 110036, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Interaction of MEL, CYA and UA with
The interactions of DNA with melamine (MEL), cyanuric acid (CYA) and uric acid (UA) were studied by means of UV–vis, fluorescence, circular dichroism (CD) spectroscopy, viscosity and gel electrophoresis. The fluorescence quenching was used to study the interaction models of MEL, CYA and UA with DNA. The results show that MEL, CYA and UA are all able to bind to DNA and the binding strength order is DNA–UA > DNA–CYA > DNA–MEL.
DNA were studied by spectroscopy. Binding models of MEL, CYA and UA to DNA were confirmed by spectroscopy. Berberine was used to study the interactions of MEL, CYA and UA with DNA.
a r t i c l e
i n f o
Article history: Received 27 September 2014 Received in revised form 10 March 2015 Accepted 21 April 2015 Available online 27 April 2015 Keywords: Desoxyribonucleic acid (DNA) Melamine Interaction Spectroscopy Viscosity method
a b s t r a c t In this work, the interaction of DNA with melamine (MEL), cyanuric acid (CYA) and uric acid (UA) were studied, respectively, by means of UV–vis, fluorescence, circular dichroism (CD) spectroscopy, viscosity and gel electrophoresis methods. The fluorescence quenching was used to study the interaction models of MEL, CYA and UA with DNA, respectively, and the bimolecular quenching constant (Kq), apparent quenching constant (Ksv), effective binding constant (KA) and corresponding dissociation constant (KD) and binding site number (n) were calculated by adopting Stern–Volmer, Lineweaver–Burk and Double logarithm equations. The results show that MEL, CYA and UA are all able to markedly bind to DNA, and the binding strength order is DNA–UA > DNA–CYA > DNA–MEL. It is wished that these researches would facilitate the understanding of the formation of kidney stones and gout in the body after ingesting excess MEL. Ó 2015 Elsevier B.V. All rights reserved.
Introduction
⇑ Corresponding author. Tel.: +86 024 62207859; fax: +86 024 62202053. E-mail address: [email protected] (J. Wang). http://dx.doi.org/10.1016/j.saa.2015.04.060 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
Melamine (MEL) is a basic heterocyclic triazine organic compound extensively used in the manufacture of resins, plastics and glues, but it is strictly banned in food products and additives [1]. However, because of its high nitrogen content, many
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unscrupulous traders always illegally add MEL into milk and pet food to enhance the protein content falsely [2], particularly, formula milk in infants [3–5]. A variety of effects from foods contaminated with MEL that increase the risks to human health have caused a high-powered attention [6]. Consequently, many research reports on the toxicity of MEL have been conducted widely and its effects highlight the safety of the global food supply [7]. Cyanuric acid (CYA) can also be found in pet food as a co-contaminant, and it is added intentionally or perhaps is a by-product of melamine (MEL) production [8]. Depending on the purification process, many chemical products related to MEL may contain a number of by-products which are structurally similar to MEL, including CYA [9–12]. And that, the uric acid (UA) as a structural analog of MEL is a human cell metabolism and food ribonucleic acid products of purine metabolism. Unfortunately, in human body the sustained high blood UA concentrations can lead to gout easily [13]. In addition, some studies indicate that the MEL combined with CYA and UA can form stones so easily in the kidney, ureter or bladder [14]. Upon the formation of stones to treatment, in fact, the complete disappearance is a lengthy process, which would lead to chronic kidney inflammation and bladder carcinoma, even cause renal failure via physical blockage [14–16]. And yet, these small molecular compounds, MEL, CYA and UA, due to their special chemical properties, especially, their structural similarity with the bases compounds of DNA, will also affect the characteristics of human life for a long time. Therefore, it is necessary to study the interactions of MEL, CYA and UA with DNA by using any of the methods. As is well known, the deoxyribonucleic acid (DNA) is an important substance in the living organism, for example, the carrier of genetic information and the material base of gene expression. Thus, it plays a very important role in organism growth, development and reproduction [17]. In recent years, the studies of the binding of small molecular compounds, such as drugs [18–20], pesticides, fungicides, metals and compounds, etc. [21– 23] to DNA have been of great interest for many researchers. It is very useful to understand the binding mechanism of small molecular compounds with DNA for predicting the consequences of these interactions in the human body and design the structure of new and efficient drugs [24]. In addition, DNA is frequently the main molecular target for many natural and synthetic organic drugs [25]. Because of the special chemical properties, particularly, the structures displaying similarities with the base compounds of DNA, the MEL, CYA and UA can interact with DNA in different models and strengths. Through the interactions, they can change the structure of DNA, thereby affecting gene regulation and replication function resulting in the damage to human health. So, the DNA is the most suitable one to be selected as the target biological molecule. There are many methods to study the binding of small molecular compounds with DNA. According to the binding properties, the electronic absorption spectra, steady fluorescence spectra, circular dichroism (CD) spectra, cycling voltammetry and differential pulse voltammetry, viscosity measurement, agarose gel electrophoresis, H NMR measurement can be used [26–29]. So far, the interaction of MEL, CYA and UA with DNA has not been studied in detail. In this paper, the interactions of DNA with MEL, CYA and UA were studied by means of UV–vis, fluorescence circular dichroism (CD) spectroscopy, viscosity method and gel electrophoresis. According to the test results, the various parameters were calculated and the corresponding quenching mechanisms were also confirmed. The interaction model of MEL, CYA or UA with DNA and the conformation changes of DNA were also studied. It is hoped that this work could offer some valuable information in the field of researching toxicity and perniciousness of MEL.
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Experimental section Materials Melamine (MEL, analytical purity, Sinopharm Chemical Reagent Co., Ltd, China), Cyanuric acid (CYA, analytical purity, Sinopharm Chemical Reagent Co., Ltd, China) and Uric acid (UA, analytical purity, Sinopharm Chemical Reagent Co., Ltd, China) were purchased and used directly without further purification. Desoxyribonucleic acid (DNA, analytical purity, Sinopharm Chemical Reagent Co., Ltd, China) was used to study the interaction with MEL, CYA or UA. Berberine hydrochloride (BR, analytical purity, Shenyang Chemical Reagent Plant, China) was selected as the fluorescence probe. Ethidium bromide (EB, analytical purity, Sinopharm Chemical Reagent Co., Ltd, China) was selected as stained in the gel electrophoresis experiment. Tris (hydroxyl-methyl) aminomethane (Tris), NaCl and HCl (analytical purity, Shenyang Chemical Reagent Plant, China) were used to prepare the Tris– HCl–NaCl (pH = 7.4 and [Tris–HCl] = [NaCl] = 50 mmol L1) buffer solution and to adjust the solution acidity and maintain the ionic strength. And the trizmabase, acetic acid and ethylenediaminetetraacetic acid (EDTA) (analytical purity, Shenyang Chemical Reagent Plant, China) were used to prepare the TAE buffer solution. The other chemical reagents were all analytical reagent grade, and double distilled water was used for preparing solution. DNA stock solutions (5.00 104 mol L1) were prepared by dissolving DNA with Tris–HCl–NaCl (pH = 7.40 and [Tris– HCl] = [NaCl] = 50.00 mmol L1) buffer solution. The solutions were stored in refrigerator at 0–4 °C. The MEL, CYA and UA solutions (5.00 104 mol L1) were also prepared by dissolving MEL, CYA or UA with Tris–HCl–NaCl (pH = 7.40 and [Tris– HCl] = [NaCl] = 50.00 mmol L1) buffer solution, respectively. A stock solution of BR was prepared by adding 0.0102 g BR into flask of 500 mL and diluted to mark with Tris–HCl–NaCl (pH = 7.40 and [Tris–HCl] = [NaCl] = 50.00 mmol L1) buffer solution and mixed thoroughly. The concentration of stock solution of BR is 5.00 105 mol L1 and the solutions were stored in cool and dark place. The molecular structures of melamine (MEL), cyanuric acid (CYA) and uric acid (UA) and Berberine hydrochloride (BR) are presented in Fig. 1. Apparatus and instruments The fluorescence measurements were performed on a fluorophotometer (Cary 300, Varian Company, USA) and the UV–vis absorption spectra were recorded with an UV–vis spectrophotometer (Cary 50, Varian Company, USA). The temperature was dominated at 25.0 ± 0.2 °C in the whole experiment. The solution pH value was measured with a pH meter (PHS-3C, Shanghai Leici
Fig. 1. Molecular structure of melamine, cyanuric acid, uric acid and berberine hydrochloride (BR).
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Instrument Company, LTD, China). circular dichroism (CD) measurements were performed by a J-810 spectro polarimeter (JASCO, Japan) using a 1.0 cm quartz cell. The viscosity measurement was carried out using Ubbelohde viscometer. Electrophoresis was carried out at 100 V in 1 TAE buffer solution in a gel electrophoresis unit (DL-UV312, Beijing Ding Guo Changsheng Biotechnology Co., Ltd.). Measurement of the probe proportion The BR spectra in the presence of DNA were measured by UV–vis spectrophotometer and fluorophotometer. All measurements were carried out in Tris–HCl–NaCl (pH = 7.40 and [Tris– HCl] = [NaCl] = 50.00 mmol L1) buffer solution. The excitation and emission wavelength were fixed at 365 nm and 531 nm, respectively. The scan rate was 600 nmmin1 and the scan range was 300–500 nm, the slits width of the excitation and emission were all 5.0 nm. The UV–vis spectra and the fluorescence spectra of BR in DNA solutions were shown in Fig. 2. BR competition binding assay and measurement of binding parameters To six 25.00 mL volumetric flasks, six 5.00 mL BR stock solutions and six 5.00 mL DNA stock solutions with the 1:10 ratio of [BR] and [DNA] were added and mixed adequately. And then a series of different volumes of MEL, CYA and UA solutions were also added into the above flasks, respectively. They were all diluted to the mark with Tris–HCl–NaCl (pH = 7.4 and [Tris– HCl] = [NaCl] = 50 mmol L1) buffer solution. The final concentration of MEL, CYA or UA solutions was varied from 0.00 mmol L1 to 10.00 mmol L1 at 2.00 mmol L1 increment. The UV–vis absorption spectra of a fixed concentration of BR and DNA with various concentrations of MEL, CYA or UA solutions were measured, respectively. The volumetric flasks were placed in the room temperature for 30 min for equilibrium. The 600 nmmin1 scan rate and the 300–500 nm scan range were fixed and the absorption
spectra were obtained at room temperature. The UV–vis spectra of all sample solutions were shown in Fig. 3. Then the above solutions were transferred into the quartz cell and the fluorescence measurements were performed. The fluorescence spectra of all the sample solutions were shown in Fig. 4. The curves of fluorescence quenching spectra were obtained at 25.00 ± 0.02 °C. The maximal intensities of the intrinsic fluorescence of DNA–BR solutions were recorded at 531 nm for the calculation of quenching parameters. The corresponding results were shown in Fig. 5 and Table 1, respectively. Determination of circular dichroism (CD) spectroscopy Circular dichroism (CD) spectra were recorded on Applied Photophysics Chirascan Circular Dichroism Spectrometer with Stop Flow. The CD spectra were obtained by taking the average of three scan experiments from 300 to 210 nm. While measuring the absorption spectra, an equal amount of DNA was added to both the used compound (MEL, CYA or UA) solution and the reference solution to eliminate the absorbance of the DNA itself and the CD contribution by the DNA and Tris–HCl–NaCl was subtracted through base line correction. Four 25.00 mL brown volumetric flasks, four 5.00 mL DNA stock solutions and four 3.00 mL MEL, CYA or UA stock solutions were added into the above flasks, respectively. All observed CD spectra were corrected for buffer signal and results were expressed as ellipticity in millidegree. MEL, CYA and UA stock solution was diluted to the mark with Tris–HCl–NaCl (pH = 7.4 and [Tris–HCl] = [NaCl] = 50 mmol L1) buffer solution, respectively. The final concentration of MEL, CYA and UA solutions were all 1.00 105 mol L1. The CD spectra of all sample solutions were determined and the corresponding results were offered in Fig. 6. Viscosity measurement The viscosity measurement of 1.00 105 mol L1 DNA solution was performed at 20.00 ± 0.01 °C in the absence and presence
Fig. 2. UV–vis (a-1) and fluorescence (b-1) spectra and the corresponding absorbances at 348 nm (a-2) and intensities at 531 nm (b-2) of DNA + BR solution with DNA concentration (from 0.00 105 mol L1 to 30.00 105 mol L1 at 2.00 105 mol L1 intervals). ([BR] = 1.00 104 mol L1, [Tris–HCl] = [NaCl] = 50.00 mmol L1, pH = 7.40, Tsolu = 25.00 ± 0.20 °C and Vtotal = 25.00 mL, kex (DNA–BR) = 348 nm and kem (DNA–BR) = 531 nm.).
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Fig. 3. Fluorescence spectra of (a) DNA–BR + MEL, (b) DNA–BR + CYA and (c) DNA–BR + UA with MEL, CYA and UA concentrations (from 0.00 105 mol L1 to 10.00 105 mol L1 at 2.00 105 mol L1 intervals). ([DNA] = 1.00 105 mol L1, [BR] = 1.00 104 mol L1, [Tris–HCl] = [NaCl] = 50.00 mmol L1, pH = 7.40, Tsolu = 25.00 ± 0.02 °C and Vtotal = 25.00 mL. kex (DNA–BR) = 365 nm and kem (DNA–BR) = 531 nm.).
Fig. 4. UV–vis spectra of (a) DNA–BR + MEL, (b) DNA–BR + CYA and (c) DNA–BR + UA solutions with MEL, CYA and UA concentrations (from 0.00 105 mol L1 to 10.00 105 mol L1 at 2.00 105 mol L1 intervals). ([DNA] = 1.00 104 mol L1, [BR] = 1.00 105 mol L1, [Tris–HCl] = [NaCl] = 50.00 mmol L1, pH = 7.40, Tsolu = 25.00 ± 0.20 °C and Vtotal = 25.00 mL.).
Fig. 5. Stern–Volmer plot (a), Lineweaver–Burk plot (b) and Double logarithm plot (c) of DNA–BR + MEL, DNA–BR + CYA and DNA–BR + UA solutions with MEL, CYA and UA concentrations (from 0.00 105 mol L1 to 10.00 105 mol L1 at 2.00 105 mol L1 intervals). ([DNA] = 1.00 105 mol L1, [BR] = 1.00 104 mol L1, [Tris– HCl] = [NaCl] = 50 mmol L1, pH = 7.40, Tsolu = 25.00 ± 0.02 °C and Vtotal = 25.00 mL.).
of three compounds of MEL, CYA and UA. Digital stopwatch with least count of 0.1 s was engaged for flow times measurement with accuracy of ±0.1 s. The relative specific viscosity (g/g0)1/3, where g is the viscosity of DNA in presence of MEL, CYA or UA and g0 is the viscosity of DNA alone, were plotted against binding ratio r (r = [MEL, CYA or UA]/[DNA]), according to the theory of Cohen and Eisenberg. Viscosity values were calculated from the observed flow time of DNA-containing solutions (t) corrected for that of the buffer alone (t0), g = (t t0)/t0. The results were presented in Fig. 7.
constant voltage (100 V) for about 1.00 h in 1 TAE buffer (Tris– acetic acid–EDTA (TAE) buffer) using 1.0% agarose gel. At the end of electrophoresis, i.e. the end of DNA migration, the electric current was turned off. Then, the gel was stained by immersing it in water containing ethidium bromide (0.5 lg/ml) for 30–45 min at room temperature and visualized under ultravoilet light, followed by photographed with a Sony NEX-3NL/P Digital Camera. The results were given in Fig. S1 and the binding abilities were observed from the intensity of bands.
Gel electrophoresis assays
Results and discussions
The binding of MEL, CYA or UA with CT-DNA was monitored using agarose gel electrophoresis. The reaction mixture of CT-DNA and different concentrations of MEL, CYA or UA (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 103) in buffer solution was incubated for 1.5.0 h at 25 °C. The electrophoresis was performed at a
UV–vis and fluorescence spectra of BR and DNA It has generally been known that the fluorescence intensity of DNA itself is very weak, so it is hard to monitor the interactions of these small molecule compounds with DNA by fluorescence
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Table 1 Quenching constants (KSV and Kq), binding constants (KLB), dissociation constant (KD), stability constants (KA) and binding site numbers (n) calculated according to Stern–Volmer plots, Lineweaver–Burk plots and Double logarithm plots of DNA–BR + MEL, DNA–BR + CYA and DNA–BR + UA solutions with MEL, CYA and UA concentrations (from 0.00 105 mol L1 to 10 105 mol L1 at 2.00 105 mol L1 intervals). ([DNA] = 1.00 105 mol L1, [BR] = 1.00 104 mol L1, [Tris–HCl] = [NaCl] = 50 mmol L1, pH = 7.40, Tsolu = 37.00 ± 0.02 °C and Vtotal = 25.00 mL.). System
Stern–Volmer plot
R2
KSV (L/mol)
Kq (L/mol s)
DNA + MEL DNA + CYA DNA + UA
F0/F = 0.0203[MEL] + 1 F0/F = 0.0352[CYA] + 1 F0/F = 0.0445[UA] + 1
0.9996 0.9954 0.9932
0.203 104 0.352 104 0.445 104
0.203 1012 0.352 1012 0.445 1012
System
Lineweaver–Burk plot
R2
f
KLB (L/mol)
KD (mol/L)
DNA + MEL DNA + CYA DNA + UA System
1/[(F0 F)/F0] = 39.907/[MEL] + 2.3443 1/[(F0 F)/F0] = 21.906/[CYA] + 1.9007 1/[(F0 F)/F0] = 15.945/[UA] + 1.9349 Double logarithm plot
0.9969 0.9975 0.9920 R2
0.42657 0.52612 0.51682 KA (L/mol)
0.58744 104 0.86766 104 1.21348 104 n
1.70230 104 1.15253 104 8.24076 104 DG0 (kJ/mol)
DNA + MEL DNA + CYA DNA + UA
log[(F0 F)/F] = 0.8945log[MEL] + 2.8711 log[(F0 F)/F] = 0.8674log[CYA] + 2.9997 log[(F0 F)/F] = 0.8326log[UA] + 2.95651
Fig. 6. Circular dichroism (CD) spectra of DNA in the absence (a) and presence of MEL (b), CYA (c) or UA (d). ([DNA] = [MEL] = [CYA] = [UA] = 1.00 105 mol L1, [Tris–HCl] = [NaCl] = 50 mmol L1, pH = 7.40, Tsolu = 25.00 ± 0.02 °C and Vtotal = 25.00 mL.).
emission method [30]. In recent years, there are many reports on the interaction mechanisms between small molecule compounds and DNA using fluorescent probes such as acridine orange (AO), ethidium bromide (EB), oxidize yellow homodimers, nile blue, neutral red and so on [31–33]. By virtue of the derivative fluorescence, the changes of conformation and composition of DNA can be effectively investigated. However, they are also toxic and harmful to the environment and human health, especially, for some probes whose combination model with DNA is irreversible due to the strong affinity. Berberine hydrochloride (BR) is a natural isoquinoline plant alkaloid endowed with diverse pharmacological and biological activities [34]. BR is known as a DNA binder and its binding affinities have a wide range of features by employing several analytical techniques including absorption, fluorescence, NMR and electrospray ionization mass (ESI-MS) spectrometries [35]. For several years, it has been demonstrated that the BR can bind to DNA by an intercalative model, thus it was employed as a spectral probe to investigate the binding model of small molecule compounds with DNA [36,37]. So, in this work the BR was selected as a fluorescent probe to examine the change of structure and composition of DNA caused by MEL, CYA or UA.
0.9970 0.9985 0.9952
0.74319 104 0.99931 104 0.90469 104
0.8945 0.8674 0.8326
21.724 22.446 22.203
It has been known that the fluorescence intensity of BR in aqueous solution can increase with successive addition of DNA due to the formation of more and more DNA–BR complex through intercalative model of binding [38]. If the added MEL, CYA or UA can interact with DNA, they may quench the fluorescence intensity of DNA–BR as they compete with BR for binding sites in DNA. In order to verify this inference, it is essential to determine the optimal ratio of BR to DNA. The absorbance and fluorescence intensities of a series of assay solution containing constant concentration of BR and various concentrations of DNA were measured. As shown in Fig. 2(a-1 and a-2), in the absorption spectra of DNA–BR solution the absorbance of BR displays a hypochromic effect with the addition of increasing amounts of DNA and the absorption wavelength also presents a slight red-shift. These results revealed that there was a strong interaction between BR and DNA and the interaction model should be intercalation binding as previously reported elsewhere. From Fig. 2(b-1), it can be seen that the maximum fluorescence intensity of DNA–BR increases gradually with the addition of DNA. In this studied system, apparently, the best ratio is between 10:1 and 15:1 via the analysis of Fig. 2(b-2). Nevertheless, due to their special chemical structures, the MEL, CYA and UA are not as strongly as the interaction of BR with DNA. According to the report by Kakali Bhadra, the ratio of DNA–BR was about 8.6:1 [35]. So, in this work, the 10:1 is approximately adopted as the best proper ratio of DNA and BR in DNA–BR system.
Fluorescence and UV–vis spectra of DNA–BR solution with MEL, CYA or UA concentrations Fig. 3 shows the fluorescence intensity changes of DNA– BR + MEL, DNA–BR + CYA and DNA–BR + UA solutions along with the increase of the concentration of MEL, CYA and UA, respectively. In these three systems, the ratio of DNA and BR is 10:1. A maximum emission peak appears at kem = 531 nm when exited at kex = 365 nm, which should mainly belong to the characteristic peak of DNA–BR complexes. The quenching in fluorescence intensity of DNA–BR solution is noticed with the increasing amounts of MEL, CYA or UA after adding MEL, CYA or UA to DNA–BR mixed solution. These results implied that, through competing reaction, the MEL, CYA or UA also interact with DNA. The addition of MEL, CYA or UA molecules causes the amount of DNA–BR complex’s decrease, which displays the gradual decline of intensity of DNA– BR solution. However, the degrees of fluorescence quenching of DNA–BR caused by MEL, CYA or UA are different. The order of the degrees of the fluorescence quenching was DNA– BR + UA > DNA–BR + CYA > DNA–BR + MEL.
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determine the binding degree between the small molecular compound and DNA. The interaction of MEL, CYA or UA with DNA molecules in aqueous solution could be studied by measuring the change of the fluorescence of DNA–BR (excited by 365 nm wavelength) along with the increase of MEL, CYA and UA concentrations, respectively. As a hypothetical dynamic quenching process, the data of fluorescence intensities could be analyzed by using Stern–Volmer Eq. (1) [40]:
F 0 =F ¼ 1 þ K SV ½Q ¼ 1 þ K q s0 ½Q
Fig. 7. Effect of MEL, CYA and UA concentrations (from 0.00 105 mol L1 to 5.00 105 mol L1 at 1.00 105 mol L1 intervals) on relative viscosity of DNA solution. ([DNA] = 1.00 104 mol L1, [Tris–HCl] = [NaCl] = 50.00 mmol L1, pH = 7.40 and Tsolu = 20.00 ± 0.01 °C.).
Based on the structural feature and chemical composition of MEL, CYA and UA, the interaction strength to DNA could primarily be judged. UA (uric acid) is a product of purine metabolism in vivo, whose structure is similar to that of the base compounds of the DNA. Therefore, it is forecasted that the UA (uric acid) is more effective to interact with DNA. For DNA–CYA system, due to the presence of hydroxyl groups (–OH), the CYA (Cyanuric acid) could easily form the hydrogen bonds with DNA. However, the CYA (cyanuric acid) is different from UA in molecular structure and that the interaction strength with DNA is not as strong as UA. For DNA–MEL system, because of the presence of the amino groups (–NH2), the MEL also could form the hydrogen bonds in the interaction of MEL and DNA. And yet, the electronegativity of nitrogen (N) of MEL is less than that of the oxygen (O) of CYA. To sum up, the fluorescence quenching order is maintained as DNA–UA > DNA– CYA > DNA–MEL. Fig. 4 shows that the addition of DNA to the BR solution induces a red-shift obviously, forming a new peak compared with the controlled BR solutions [35]. The absorbance of BR solution at 345 nm and 425 nm gradually increased and showed blue-shift along with the increasing concentration of MEL, CYA or UA. It can be conjectureed that the addition of MEL, CYA or UA molecules causes the amount of DNA–BR complex to decrease and release the BR molecules from DNA–BR complex, which displays the increase of the absorbance of BR. These results implied that the MEL, CYA or UA could also interact with DNA through competing reaction. It could be inferred that the BR, which intercalated into the double helix conformation of DNA, might be squeezed out by MEL, CYA or UA, respectively, in these three systems. This phenomenon may demonstrate that new DNA complexes (DNA–MEL, DNA–CYA and DNA–UA) were formed during the competitive reaction, but their hyperchromic effects were weak even with the increase of the concentrations of MEL, CYA or UA. Obviously, the combination models of MEL, CYA and UA with DNA were different from that of BR with DNA. Binding characteristics of MEL, CYA or UA with DNA in the presence of BR It is known that the enhanced fluorescence of BR upon the addition of DNA could be quenched by the addition of a second small molecular compound at least partly [39]. The extent of fluorescence quenching of BR bound to DNA was utilized to
ð1Þ
where F0 and F are the fluorescence intensities in the absence and presence of MEL, CYA or UA at 531 nm, respectively. Kq is the quenching rate constant of bimolecular, s0 is the average life time of the fluorophore without MEL, CYA or UA, KSV is the Stern– Volmer dynamic quenching constant and [Q] is the concentration of MEL, CYA or UA [41]. Fig. 5(a) displays the Stern–Volmer plots of DNA–BR solutions in the presence of MEL, CYA or UA with various concentrations. It can be found that three plots exhibit a comparatively good linear relationship. Thus, as shown in Table 1 from Stern–Volmer Eq. (1), for these three systems their Kq could be obtained. And that, the values of the Kq are all greater than the maximum scatter collision quenching constant (2.0 1010 L mol1 s1) of various quenchers with biopolymers. Hence, according to the literatures [25,42,43], all these evidences suggest that the probable quenching mechanism of MEL, CYA or UA with DNA is mainly a static quenching. Moreover, the apparent quenching constant (Kq) arrange in the order of DNA–BR + UA > DNA–BR + CYA > DNA–BR + MEL. It indicated that the UA is more effective than CYA and MEL to quench the fluorescence of DNA–BR molecules in aqueous solution. For reconfirming the static quenching mechanisms, the data of fluorescence quenching of DNA–BR solutions along with the increase of MEL, CYA or UA concentrations are also analyzed according to the Lineweaver–Burk (modified Stern–Volmer or double reciprocal) Eq. (2).
1=½ðF 0 FÞ=F 0 ¼ 1=ðfK LB ½QÞ þ 1=f
ð2Þ
The F0 and F are also the fluorescence intensities of DNA–BR solutions at 531 nm in the absence and presence of MEL, CYA or UA with various concentrations, respectively. The f is the fraction of accessible fluorescence and KLB is the static fluorescence quenching association constant. Using the plots obtained from Fig. 5(b), the KLB could be obtained for DNA–BR + MEL, DAN-BR + CYA and DNA–BR + UA systems. And that, as can be seen from Table 1, for these three systems, their Lineweaver–Burk plots all have a better linear relationship than the corresponding Stern– Volmer plots. Thus, it could be confirmed again that the fluorescence quenching mechanism of DNA molecules by MEL, CYA or UA is mainly a static quenching procedure indeed. It also indicates that a complex between DNA–BR and MEL, CYA or UA has formed, which caused a decrease of fluorescence intensity of DNA–BR solution. From Fig. 5(c), the equilibrium constants (KA) and the binding site numbers (n) could be calculated by using the equation: (F0 F)/F = KA [Q]n. By using logarithmic treatment the following Double logarithm Eq. (3) can be obtained [44].
log½ðF 0 FÞ=F ¼ log K A þ nlog½Q
ð3Þ
Apparently, the KA and n could be measured by the intercept and slope by plotting log [(F0 F)/F] against log [Q]. According to Fig. 5(c), KA and n for DNA–BR + MEL, DNA–BR + CYA and DNA– BR + UA were also obtained, respectively. It can be found that there is a strong binding force between MEL, CYA or UA with DNA–BR, which results in a stable binding site being formed. The correlation coefficients are greater than 0.99, indicating that the interaction of MEL, CYA or UA with DNA–BR agrees well with the site-binding
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model. It could be seen that the plots also exhibited a good linear relationship and these n values are all equal to about 1. It indicates that the binding model of MEL, CYA or UA to DNA–BR was approximately 1:1. The KA for DNA–BR + UA system is slightly bigger than that for DNA–BR + MEL and DNA–BR + CYA systems, which indicates that the binding of DNA with UA was the tighter one. Utilizing KA, the free energy change (DG0) can be calculated from the relationship (4):
DG0 ¼ RTln K A
ð4Þ
The calculated results are all given in Table 1. Thus, R = 8.314 kJ mol1 K1 and T = 293.15 K, the calculated DG0 are 21.724 kJ mol1 for DNA–BR + MEL system, 22.446 kJ mol1 for DNA–BR-CYA system and 22.203 kJ mol1 for DNA–BR + UA system, respectively. The large negative values indicate that the interaction of MEL, CYA or UA to DNA–BR was all spontaneous reaction processes. Circular dichroism spectra of DNA solutions in the presence of MEL, CYA or UA Circular dichroism (CD) spectrum is a spectroscopic technique widely used to study the binding model and interaction affinity of small molecular compounds with many biomolecules, particularly, DNA and protein [45]. The changes of the CD spectra in the spectral range of 200–300 nm wavelengths have played an extensive utilization to detect and follow DNA conformational changes, damage and/or cleavage upon interaction with small molecular compounds. In CD spectrum, CT-DNA shows the typical absorption of a right-handed B-form DNA. And the absorption consists of two bands, that is, a negative (first) one at 245 nm attributing to the right-handed helicity and a positive (second) one at 275 nm attributing to the base stacking. Generally, the classical intercalation of small molecular compounds leads to the changes of DNA CD spectra in intensities of these two bands due to enhancement of base stacking and stabilization of helicity, which enhances the intensities of both the CD bands. However, the simple groove binding and electrostatic interaction of small molecular compounds display low or no perturbations on the base stacking and helicity absorption bands [46,47]. Therefore, in this work, the effects on DNA conformation by interacting with MEL, CYA or UA were evaluated by CD spectroscopy. As shown in Fig. 6, in comparison with blank DNA sample, after addition of MEL, CYA or UA, the DNA solution still retains basic CD spectrum shape, but its first (negative) band and second (positive) band increase and decrease, respectively. For the first (negative) band, the order (from bottom to top) is DNA + UA > DNA + CYA > DNA + MEL > DNA, and for the second (positive) band, the order (from top to bottom) is DNA > DNA + MEL > DNA + UA > DNA + CYA. It indicates that the UA mainly affects the right-handed helicity, and the CYA has a major influence on the base stacking. Nevertheless, the MEL displays the influence on both right-handed helicity and base stacking at the same time, but its change degree is not strong compared with that of UA and CYA. According to the above analyses, it can be confirmed that the CD spectra changes clearly rule out the intercalative model of binding and non-covalent interaction between these three compounds and DNA. Most likely, MEL, CYA or UA interact with DNA via groove binding model. Viscosity changes of DNA solution in the presence of MEL, CYA or UA A useful technique to prove intercalation is viscosity measurement, which is sensitive to length change of DNA and regarded as the least ambiguous and the most critical test of binding model
in solution in the absence of crystallographic structural data or NMR spectra [48]. Generally, a classical intercalation binding of small molecular compounds causes an increase in the viscosity of DNA solution. This is because such DNA molecules demand a large enough space of adjacent base pairs to accommodate these small molecular compounds, lengthen the double helix. In contrast, a partial or non-classical intercalation binding causes the reduction of DNA viscosity. And the non-intercalation binding, such as electrostatic or grooved binding does not appreciably alter DNA viscosity [48,49]. To further clarify the nature of the interaction between MEL, CYA, or UA and DNA, viscosity measurements were carried out by varying the concentration of the added MEL, CYA, or UA to DNA solution. From Fig. 7 it is observed that, as the concentration of MEL, CYA or UA increase, the relative viscosity of DNA almost remains steady, which is in agreement with a non-intercalation model between MEL, CYA, or UA and DNA. Hence, the results demonstrate that the three kinds of small molecular compounds could all bind to DNA by the non-intercalation interaction, that is, groove interaction. It is consistent with the above results of absorption, fluorescence and CD spectra.
Gel electrophoresis of DNA in the presence of MEL, CYA or UA The bindings of MEL, CYA and UA with CT-DNA could also be studied by gel electrophoresis. We examined the effect of different concentrations of the MEL, CYA or UA on CT-DNA by monitoring the changes in intensity of the bands and electrophoretic mobility in an agarose gel which results were presented in Fig. S1. It was clear that the intensity of the recorded CT-DNA bands after binding to MEL, CYA or UA had almost nothing to be changed and the electrophoretic mobility had gradually slowed down with the increasing concentration of MEL, CYA or UA, as compared to the band of the free CT-DNA. It could easily be conjectured that the change in conformation of the DNA was due to the presence of the investigated MEL, CYA or UA, but their different binding abilities to DNA result in different electrophoretic mobility. Also, the binding sites of MEL, CYA or UA to DNA were different from that of EB. Therefore, the lengthes of lanes of CT-DNA are increased with the increasing concentration of MEL, CYA or UA. What is more the length sequence is DNA + UA > DNA + CYA > DNA + MEL. It is in a good agreement with binding constant values which were investigated previously.
Conclusion In this work, the interaction of DNA with MEL, CYA or UA was studied by UV–vis spectroscopy, fluorescence, circular dichroism (CD) spectroscopy, viscosity method and gel electrophoresis. The fluorescence spectral monitoring results, Stern–Volmer dynamic quenching constant (Ksv), quenching rate constant of biomolecule (Kq), static fluorescence quenching association constant (KLB) and equilibrium constants (KA), were obtained. These results suggested that the MEL, CYA and UA all could bind with DNA molecules. And that, the fluorescence quenching processes of DNA–BR caused by MEL, CYA or UA were all attributed to static quenching. The interaction strength of the MEL, CYA or UA and DNA molecules ranks in the order of DNA–UA > DNA–CYA > DNA–MEL. The viscosity methods and CD spectroscopy showed that the binding models of the interactions of DNA with MEL, CYA and UA all may belong to the groove binding. The bindings of MEL, CYA and UA with DNA were also studied by gel electrophoresis and the obtained sequence of binding strength is DNA–UA > DNA–CYA > DNA–MEL. These research results may be a valuable reference for understanding
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the formation mechanism of kidney stones and gout and have a certain help about the related disease treatment at the same time. Acknowledgments The authors greatly acknowledge the National Science Foundation of China (21371084), Innovation Team Project of Education Department of Liaoning Province (LT2012001), Public Research Fund Project of Science and Technology Department of Liaoning Province (2012004001), Shenyang Science and Technology Plan Project (F12-277-1-15 and F13-289-1-00) and Science Foundation of Liaoning Provincial Education Department (L2011007) for financial support. The authors also thank our colleagues and other students for their participating in this work.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Spectroscopic analyses on interaction of melamine, cyanuric acid and uric acid with DNA Jinhui Xie a, Dandan Chen a, Qiong Wu a, Jun Wang a,⇑, Heng Qiao b a b
College of Chemistry, Liaoning University, Shenyang 110036, PR China College of Environment, Liaoning University, Shenyang 110036, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Interaction of MEL, CYA and UA with
The interactions of DNA with melamine (MEL), cyanuric acid (CYA) and uric acid (UA) were studied by means of UV–vis, fluorescence, circular dichroism (CD) spectroscopy, viscosity and gel electrophoresis. The fluorescence quenching was used to study the interaction models of MEL, CYA and UA with DNA. The results show that MEL, CYA and UA are all able to bind to DNA and the binding strength order is DNA–UA > DNA–CYA > DNA–MEL.
DNA were studied by spectroscopy. Binding models of MEL, CYA and UA to DNA were confirmed by spectroscopy. Berberine was used to study the interactions of MEL, CYA and UA with DNA.
a r t i c l e
i n f o
Article history: Received 27 September 2014 Received in revised form 10 March 2015 Accepted 21 April 2015 Available online 27 April 2015 Keywords: Desoxyribonucleic acid (DNA) Melamine Interaction Spectroscopy Viscosity method
a b s t r a c t In this work, the interaction of DNA with melamine (MEL), cyanuric acid (CYA) and uric acid (UA) were studied, respectively, by means of UV–vis, fluorescence, circular dichroism (CD) spectroscopy, viscosity and gel electrophoresis methods. The fluorescence quenching was used to study the interaction models of MEL, CYA and UA with DNA, respectively, and the bimolecular quenching constant (Kq), apparent quenching constant (Ksv), effective binding constant (KA) and corresponding dissociation constant (KD) and binding site number (n) were calculated by adopting Stern–Volmer, Lineweaver–Burk and Double logarithm equations. The results show that MEL, CYA and UA are all able to markedly bind to DNA, and the binding strength order is DNA–UA > DNA–CYA > DNA–MEL. It is wished that these researches would facilitate the understanding of the formation of kidney stones and gout in the body after ingesting excess MEL. Ó 2015 Elsevier B.V. All rights reserved.
Introduction
⇑ Corresponding author. Tel.: +86 024 62207859; fax: +86 024 62202053. E-mail address: [email protected] (J. Wang). http://dx.doi.org/10.1016/j.saa.2015.04.060 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
Melamine (MEL) is a basic heterocyclic triazine organic compound extensively used in the manufacture of resins, plastics and glues, but it is strictly banned in food products and additives [1]. However, because of its high nitrogen content, many
J. Xie et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 714–721
unscrupulous traders always illegally add MEL into milk and pet food to enhance the protein content falsely [2], particularly, formula milk in infants [3–5]. A variety of effects from foods contaminated with MEL that increase the risks to human health have caused a high-powered attention [6]. Consequently, many research reports on the toxicity of MEL have been conducted widely and its effects highlight the safety of the global food supply [7]. Cyanuric acid (CYA) can also be found in pet food as a co-contaminant, and it is added intentionally or perhaps is a by-product of melamine (MEL) production [8]. Depending on the purification process, many chemical products related to MEL may contain a number of by-products which are structurally similar to MEL, including CYA [9–12]. And that, the uric acid (UA) as a structural analog of MEL is a human cell metabolism and food ribonucleic acid products of purine metabolism. Unfortunately, in human body the sustained high blood UA concentrations can lead to gout easily [13]. In addition, some studies indicate that the MEL combined with CYA and UA can form stones so easily in the kidney, ureter or bladder [14]. Upon the formation of stones to treatment, in fact, the complete disappearance is a lengthy process, which would lead to chronic kidney inflammation and bladder carcinoma, even cause renal failure via physical blockage [14–16]. And yet, these small molecular compounds, MEL, CYA and UA, due to their special chemical properties, especially, their structural similarity with the bases compounds of DNA, will also affect the characteristics of human life for a long time. Therefore, it is necessary to study the interactions of MEL, CYA and UA with DNA by using any of the methods. As is well known, the deoxyribonucleic acid (DNA) is an important substance in the living organism, for example, the carrier of genetic information and the material base of gene expression. Thus, it plays a very important role in organism growth, development and reproduction [17]. In recent years, the studies of the binding of small molecular compounds, such as drugs [18–20], pesticides, fungicides, metals and compounds, etc. [21– 23] to DNA have been of great interest for many researchers. It is very useful to understand the binding mechanism of small molecular compounds with DNA for predicting the consequences of these interactions in the human body and design the structure of new and efficient drugs [24]. In addition, DNA is frequently the main molecular target for many natural and synthetic organic drugs [25]. Because of the special chemical properties, particularly, the structures displaying similarities with the base compounds of DNA, the MEL, CYA and UA can interact with DNA in different models and strengths. Through the interactions, they can change the structure of DNA, thereby affecting gene regulation and replication function resulting in the damage to human health. So, the DNA is the most suitable one to be selected as the target biological molecule. There are many methods to study the binding of small molecular compounds with DNA. According to the binding properties, the electronic absorption spectra, steady fluorescence spectra, circular dichroism (CD) spectra, cycling voltammetry and differential pulse voltammetry, viscosity measurement, agarose gel electrophoresis, H NMR measurement can be used [26–29]. So far, the interaction of MEL, CYA and UA with DNA has not been studied in detail. In this paper, the interactions of DNA with MEL, CYA and UA were studied by means of UV–vis, fluorescence circular dichroism (CD) spectroscopy, viscosity method and gel electrophoresis. According to the test results, the various parameters were calculated and the corresponding quenching mechanisms were also confirmed. The interaction model of MEL, CYA or UA with DNA and the conformation changes of DNA were also studied. It is hoped that this work could offer some valuable information in the field of researching toxicity and perniciousness of MEL.
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Experimental section Materials Melamine (MEL, analytical purity, Sinopharm Chemical Reagent Co., Ltd, China), Cyanuric acid (CYA, analytical purity, Sinopharm Chemical Reagent Co., Ltd, China) and Uric acid (UA, analytical purity, Sinopharm Chemical Reagent Co., Ltd, China) were purchased and used directly without further purification. Desoxyribonucleic acid (DNA, analytical purity, Sinopharm Chemical Reagent Co., Ltd, China) was used to study the interaction with MEL, CYA or UA. Berberine hydrochloride (BR, analytical purity, Shenyang Chemical Reagent Plant, China) was selected as the fluorescence probe. Ethidium bromide (EB, analytical purity, Sinopharm Chemical Reagent Co., Ltd, China) was selected as stained in the gel electrophoresis experiment. Tris (hydroxyl-methyl) aminomethane (Tris), NaCl and HCl (analytical purity, Shenyang Chemical Reagent Plant, China) were used to prepare the Tris– HCl–NaCl (pH = 7.4 and [Tris–HCl] = [NaCl] = 50 mmol L1) buffer solution and to adjust the solution acidity and maintain the ionic strength. And the trizmabase, acetic acid and ethylenediaminetetraacetic acid (EDTA) (analytical purity, Shenyang Chemical Reagent Plant, China) were used to prepare the TAE buffer solution. The other chemical reagents were all analytical reagent grade, and double distilled water was used for preparing solution. DNA stock solutions (5.00 104 mol L1) were prepared by dissolving DNA with Tris–HCl–NaCl (pH = 7.40 and [Tris– HCl] = [NaCl] = 50.00 mmol L1) buffer solution. The solutions were stored in refrigerator at 0–4 °C. The MEL, CYA and UA solutions (5.00 104 mol L1) were also prepared by dissolving MEL, CYA or UA with Tris–HCl–NaCl (pH = 7.40 and [Tris– HCl] = [NaCl] = 50.00 mmol L1) buffer solution, respectively. A stock solution of BR was prepared by adding 0.0102 g BR into flask of 500 mL and diluted to mark with Tris–HCl–NaCl (pH = 7.40 and [Tris–HCl] = [NaCl] = 50.00 mmol L1) buffer solution and mixed thoroughly. The concentration of stock solution of BR is 5.00 105 mol L1 and the solutions were stored in cool and dark place. The molecular structures of melamine (MEL), cyanuric acid (CYA) and uric acid (UA) and Berberine hydrochloride (BR) are presented in Fig. 1. Apparatus and instruments The fluorescence measurements were performed on a fluorophotometer (Cary 300, Varian Company, USA) and the UV–vis absorption spectra were recorded with an UV–vis spectrophotometer (Cary 50, Varian Company, USA). The temperature was dominated at 25.0 ± 0.2 °C in the whole experiment. The solution pH value was measured with a pH meter (PHS-3C, Shanghai Leici
Fig. 1. Molecular structure of melamine, cyanuric acid, uric acid and berberine hydrochloride (BR).
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Instrument Company, LTD, China). circular dichroism (CD) measurements were performed by a J-810 spectro polarimeter (JASCO, Japan) using a 1.0 cm quartz cell. The viscosity measurement was carried out using Ubbelohde viscometer. Electrophoresis was carried out at 100 V in 1 TAE buffer solution in a gel electrophoresis unit (DL-UV312, Beijing Ding Guo Changsheng Biotechnology Co., Ltd.). Measurement of the probe proportion The BR spectra in the presence of DNA were measured by UV–vis spectrophotometer and fluorophotometer. All measurements were carried out in Tris–HCl–NaCl (pH = 7.40 and [Tris– HCl] = [NaCl] = 50.00 mmol L1) buffer solution. The excitation and emission wavelength were fixed at 365 nm and 531 nm, respectively. The scan rate was 600 nmmin1 and the scan range was 300–500 nm, the slits width of the excitation and emission were all 5.0 nm. The UV–vis spectra and the fluorescence spectra of BR in DNA solutions were shown in Fig. 2. BR competition binding assay and measurement of binding parameters To six 25.00 mL volumetric flasks, six 5.00 mL BR stock solutions and six 5.00 mL DNA stock solutions with the 1:10 ratio of [BR] and [DNA] were added and mixed adequately. And then a series of different volumes of MEL, CYA and UA solutions were also added into the above flasks, respectively. They were all diluted to the mark with Tris–HCl–NaCl (pH = 7.4 and [Tris– HCl] = [NaCl] = 50 mmol L1) buffer solution. The final concentration of MEL, CYA or UA solutions was varied from 0.00 mmol L1 to 10.00 mmol L1 at 2.00 mmol L1 increment. The UV–vis absorption spectra of a fixed concentration of BR and DNA with various concentrations of MEL, CYA or UA solutions were measured, respectively. The volumetric flasks were placed in the room temperature for 30 min for equilibrium. The 600 nmmin1 scan rate and the 300–500 nm scan range were fixed and the absorption
spectra were obtained at room temperature. The UV–vis spectra of all sample solutions were shown in Fig. 3. Then the above solutions were transferred into the quartz cell and the fluorescence measurements were performed. The fluorescence spectra of all the sample solutions were shown in Fig. 4. The curves of fluorescence quenching spectra were obtained at 25.00 ± 0.02 °C. The maximal intensities of the intrinsic fluorescence of DNA–BR solutions were recorded at 531 nm for the calculation of quenching parameters. The corresponding results were shown in Fig. 5 and Table 1, respectively. Determination of circular dichroism (CD) spectroscopy Circular dichroism (CD) spectra were recorded on Applied Photophysics Chirascan Circular Dichroism Spectrometer with Stop Flow. The CD spectra were obtained by taking the average of three scan experiments from 300 to 210 nm. While measuring the absorption spectra, an equal amount of DNA was added to both the used compound (MEL, CYA or UA) solution and the reference solution to eliminate the absorbance of the DNA itself and the CD contribution by the DNA and Tris–HCl–NaCl was subtracted through base line correction. Four 25.00 mL brown volumetric flasks, four 5.00 mL DNA stock solutions and four 3.00 mL MEL, CYA or UA stock solutions were added into the above flasks, respectively. All observed CD spectra were corrected for buffer signal and results were expressed as ellipticity in millidegree. MEL, CYA and UA stock solution was diluted to the mark with Tris–HCl–NaCl (pH = 7.4 and [Tris–HCl] = [NaCl] = 50 mmol L1) buffer solution, respectively. The final concentration of MEL, CYA and UA solutions were all 1.00 105 mol L1. The CD spectra of all sample solutions were determined and the corresponding results were offered in Fig. 6. Viscosity measurement The viscosity measurement of 1.00 105 mol L1 DNA solution was performed at 20.00 ± 0.01 °C in the absence and presence
Fig. 2. UV–vis (a-1) and fluorescence (b-1) spectra and the corresponding absorbances at 348 nm (a-2) and intensities at 531 nm (b-2) of DNA + BR solution with DNA concentration (from 0.00 105 mol L1 to 30.00 105 mol L1 at 2.00 105 mol L1 intervals). ([BR] = 1.00 104 mol L1, [Tris–HCl] = [NaCl] = 50.00 mmol L1, pH = 7.40, Tsolu = 25.00 ± 0.20 °C and Vtotal = 25.00 mL, kex (DNA–BR) = 348 nm and kem (DNA–BR) = 531 nm.).
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Fig. 3. Fluorescence spectra of (a) DNA–BR + MEL, (b) DNA–BR + CYA and (c) DNA–BR + UA with MEL, CYA and UA concentrations (from 0.00 105 mol L1 to 10.00 105 mol L1 at 2.00 105 mol L1 intervals). ([DNA] = 1.00 105 mol L1, [BR] = 1.00 104 mol L1, [Tris–HCl] = [NaCl] = 50.00 mmol L1, pH = 7.40, Tsolu = 25.00 ± 0.02 °C and Vtotal = 25.00 mL. kex (DNA–BR) = 365 nm and kem (DNA–BR) = 531 nm.).
Fig. 4. UV–vis spectra of (a) DNA–BR + MEL, (b) DNA–BR + CYA and (c) DNA–BR + UA solutions with MEL, CYA and UA concentrations (from 0.00 105 mol L1 to 10.00 105 mol L1 at 2.00 105 mol L1 intervals). ([DNA] = 1.00 104 mol L1, [BR] = 1.00 105 mol L1, [Tris–HCl] = [NaCl] = 50.00 mmol L1, pH = 7.40, Tsolu = 25.00 ± 0.20 °C and Vtotal = 25.00 mL.).
Fig. 5. Stern–Volmer plot (a), Lineweaver–Burk plot (b) and Double logarithm plot (c) of DNA–BR + MEL, DNA–BR + CYA and DNA–BR + UA solutions with MEL, CYA and UA concentrations (from 0.00 105 mol L1 to 10.00 105 mol L1 at 2.00 105 mol L1 intervals). ([DNA] = 1.00 105 mol L1, [BR] = 1.00 104 mol L1, [Tris– HCl] = [NaCl] = 50 mmol L1, pH = 7.40, Tsolu = 25.00 ± 0.02 °C and Vtotal = 25.00 mL.).
of three compounds of MEL, CYA and UA. Digital stopwatch with least count of 0.1 s was engaged for flow times measurement with accuracy of ±0.1 s. The relative specific viscosity (g/g0)1/3, where g is the viscosity of DNA in presence of MEL, CYA or UA and g0 is the viscosity of DNA alone, were plotted against binding ratio r (r = [MEL, CYA or UA]/[DNA]), according to the theory of Cohen and Eisenberg. Viscosity values were calculated from the observed flow time of DNA-containing solutions (t) corrected for that of the buffer alone (t0), g = (t t0)/t0. The results were presented in Fig. 7.
constant voltage (100 V) for about 1.00 h in 1 TAE buffer (Tris– acetic acid–EDTA (TAE) buffer) using 1.0% agarose gel. At the end of electrophoresis, i.e. the end of DNA migration, the electric current was turned off. Then, the gel was stained by immersing it in water containing ethidium bromide (0.5 lg/ml) for 30–45 min at room temperature and visualized under ultravoilet light, followed by photographed with a Sony NEX-3NL/P Digital Camera. The results were given in Fig. S1 and the binding abilities were observed from the intensity of bands.
Gel electrophoresis assays
Results and discussions
The binding of MEL, CYA or UA with CT-DNA was monitored using agarose gel electrophoresis. The reaction mixture of CT-DNA and different concentrations of MEL, CYA or UA (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 103) in buffer solution was incubated for 1.5.0 h at 25 °C. The electrophoresis was performed at a
UV–vis and fluorescence spectra of BR and DNA It has generally been known that the fluorescence intensity of DNA itself is very weak, so it is hard to monitor the interactions of these small molecule compounds with DNA by fluorescence
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Table 1 Quenching constants (KSV and Kq), binding constants (KLB), dissociation constant (KD), stability constants (KA) and binding site numbers (n) calculated according to Stern–Volmer plots, Lineweaver–Burk plots and Double logarithm plots of DNA–BR + MEL, DNA–BR + CYA and DNA–BR + UA solutions with MEL, CYA and UA concentrations (from 0.00 105 mol L1 to 10 105 mol L1 at 2.00 105 mol L1 intervals). ([DNA] = 1.00 105 mol L1, [BR] = 1.00 104 mol L1, [Tris–HCl] = [NaCl] = 50 mmol L1, pH = 7.40, Tsolu = 37.00 ± 0.02 °C and Vtotal = 25.00 mL.). System
Stern–Volmer plot
R2
KSV (L/mol)
Kq (L/mol s)
DNA + MEL DNA + CYA DNA + UA
F0/F = 0.0203[MEL] + 1 F0/F = 0.0352[CYA] + 1 F0/F = 0.0445[UA] + 1
0.9996 0.9954 0.9932
0.203 104 0.352 104 0.445 104
0.203 1012 0.352 1012 0.445 1012
System
Lineweaver–Burk plot
R2
f
KLB (L/mol)
KD (mol/L)
DNA + MEL DNA + CYA DNA + UA System
1/[(F0 F)/F0] = 39.907/[MEL] + 2.3443 1/[(F0 F)/F0] = 21.906/[CYA] + 1.9007 1/[(F0 F)/F0] = 15.945/[UA] + 1.9349 Double logarithm plot
0.9969 0.9975 0.9920 R2
0.42657 0.52612 0.51682 KA (L/mol)
0.58744 104 0.86766 104 1.21348 104 n
1.70230 104 1.15253 104 8.24076 104 DG0 (kJ/mol)
DNA + MEL DNA + CYA DNA + UA
log[(F0 F)/F] = 0.8945log[MEL] + 2.8711 log[(F0 F)/F] = 0.8674log[CYA] + 2.9997 log[(F0 F)/F] = 0.8326log[UA] + 2.95651
Fig. 6. Circular dichroism (CD) spectra of DNA in the absence (a) and presence of MEL (b), CYA (c) or UA (d). ([DNA] = [MEL] = [CYA] = [UA] = 1.00 105 mol L1, [Tris–HCl] = [NaCl] = 50 mmol L1, pH = 7.40, Tsolu = 25.00 ± 0.02 °C and Vtotal = 25.00 mL.).
emission method [30]. In recent years, there are many reports on the interaction mechanisms between small molecule compounds and DNA using fluorescent probes such as acridine orange (AO), ethidium bromide (EB), oxidize yellow homodimers, nile blue, neutral red and so on [31–33]. By virtue of the derivative fluorescence, the changes of conformation and composition of DNA can be effectively investigated. However, they are also toxic and harmful to the environment and human health, especially, for some probes whose combination model with DNA is irreversible due to the strong affinity. Berberine hydrochloride (BR) is a natural isoquinoline plant alkaloid endowed with diverse pharmacological and biological activities [34]. BR is known as a DNA binder and its binding affinities have a wide range of features by employing several analytical techniques including absorption, fluorescence, NMR and electrospray ionization mass (ESI-MS) spectrometries [35]. For several years, it has been demonstrated that the BR can bind to DNA by an intercalative model, thus it was employed as a spectral probe to investigate the binding model of small molecule compounds with DNA [36,37]. So, in this work the BR was selected as a fluorescent probe to examine the change of structure and composition of DNA caused by MEL, CYA or UA.
0.9970 0.9985 0.9952
0.74319 104 0.99931 104 0.90469 104
0.8945 0.8674 0.8326
21.724 22.446 22.203
It has been known that the fluorescence intensity of BR in aqueous solution can increase with successive addition of DNA due to the formation of more and more DNA–BR complex through intercalative model of binding [38]. If the added MEL, CYA or UA can interact with DNA, they may quench the fluorescence intensity of DNA–BR as they compete with BR for binding sites in DNA. In order to verify this inference, it is essential to determine the optimal ratio of BR to DNA. The absorbance and fluorescence intensities of a series of assay solution containing constant concentration of BR and various concentrations of DNA were measured. As shown in Fig. 2(a-1 and a-2), in the absorption spectra of DNA–BR solution the absorbance of BR displays a hypochromic effect with the addition of increasing amounts of DNA and the absorption wavelength also presents a slight red-shift. These results revealed that there was a strong interaction between BR and DNA and the interaction model should be intercalation binding as previously reported elsewhere. From Fig. 2(b-1), it can be seen that the maximum fluorescence intensity of DNA–BR increases gradually with the addition of DNA. In this studied system, apparently, the best ratio is between 10:1 and 15:1 via the analysis of Fig. 2(b-2). Nevertheless, due to their special chemical structures, the MEL, CYA and UA are not as strongly as the interaction of BR with DNA. According to the report by Kakali Bhadra, the ratio of DNA–BR was about 8.6:1 [35]. So, in this work, the 10:1 is approximately adopted as the best proper ratio of DNA and BR in DNA–BR system.
Fluorescence and UV–vis spectra of DNA–BR solution with MEL, CYA or UA concentrations Fig. 3 shows the fluorescence intensity changes of DNA– BR + MEL, DNA–BR + CYA and DNA–BR + UA solutions along with the increase of the concentration of MEL, CYA and UA, respectively. In these three systems, the ratio of DNA and BR is 10:1. A maximum emission peak appears at kem = 531 nm when exited at kex = 365 nm, which should mainly belong to the characteristic peak of DNA–BR complexes. The quenching in fluorescence intensity of DNA–BR solution is noticed with the increasing amounts of MEL, CYA or UA after adding MEL, CYA or UA to DNA–BR mixed solution. These results implied that, through competing reaction, the MEL, CYA or UA also interact with DNA. The addition of MEL, CYA or UA molecules causes the amount of DNA–BR complex’s decrease, which displays the gradual decline of intensity of DNA– BR solution. However, the degrees of fluorescence quenching of DNA–BR caused by MEL, CYA or UA are different. The order of the degrees of the fluorescence quenching was DNA– BR + UA > DNA–BR + CYA > DNA–BR + MEL.
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determine the binding degree between the small molecular compound and DNA. The interaction of MEL, CYA or UA with DNA molecules in aqueous solution could be studied by measuring the change of the fluorescence of DNA–BR (excited by 365 nm wavelength) along with the increase of MEL, CYA and UA concentrations, respectively. As a hypothetical dynamic quenching process, the data of fluorescence intensities could be analyzed by using Stern–Volmer Eq. (1) [40]:
F 0 =F ¼ 1 þ K SV ½Q ¼ 1 þ K q s0 ½Q
Fig. 7. Effect of MEL, CYA and UA concentrations (from 0.00 105 mol L1 to 5.00 105 mol L1 at 1.00 105 mol L1 intervals) on relative viscosity of DNA solution. ([DNA] = 1.00 104 mol L1, [Tris–HCl] = [NaCl] = 50.00 mmol L1, pH = 7.40 and Tsolu = 20.00 ± 0.01 °C.).
Based on the structural feature and chemical composition of MEL, CYA and UA, the interaction strength to DNA could primarily be judged. UA (uric acid) is a product of purine metabolism in vivo, whose structure is similar to that of the base compounds of the DNA. Therefore, it is forecasted that the UA (uric acid) is more effective to interact with DNA. For DNA–CYA system, due to the presence of hydroxyl groups (–OH), the CYA (Cyanuric acid) could easily form the hydrogen bonds with DNA. However, the CYA (cyanuric acid) is different from UA in molecular structure and that the interaction strength with DNA is not as strong as UA. For DNA–MEL system, because of the presence of the amino groups (–NH2), the MEL also could form the hydrogen bonds in the interaction of MEL and DNA. And yet, the electronegativity of nitrogen (N) of MEL is less than that of the oxygen (O) of CYA. To sum up, the fluorescence quenching order is maintained as DNA–UA > DNA– CYA > DNA–MEL. Fig. 4 shows that the addition of DNA to the BR solution induces a red-shift obviously, forming a new peak compared with the controlled BR solutions [35]. The absorbance of BR solution at 345 nm and 425 nm gradually increased and showed blue-shift along with the increasing concentration of MEL, CYA or UA. It can be conjectureed that the addition of MEL, CYA or UA molecules causes the amount of DNA–BR complex to decrease and release the BR molecules from DNA–BR complex, which displays the increase of the absorbance of BR. These results implied that the MEL, CYA or UA could also interact with DNA through competing reaction. It could be inferred that the BR, which intercalated into the double helix conformation of DNA, might be squeezed out by MEL, CYA or UA, respectively, in these three systems. This phenomenon may demonstrate that new DNA complexes (DNA–MEL, DNA–CYA and DNA–UA) were formed during the competitive reaction, but their hyperchromic effects were weak even with the increase of the concentrations of MEL, CYA or UA. Obviously, the combination models of MEL, CYA and UA with DNA were different from that of BR with DNA. Binding characteristics of MEL, CYA or UA with DNA in the presence of BR It is known that the enhanced fluorescence of BR upon the addition of DNA could be quenched by the addition of a second small molecular compound at least partly [39]. The extent of fluorescence quenching of BR bound to DNA was utilized to
ð1Þ
where F0 and F are the fluorescence intensities in the absence and presence of MEL, CYA or UA at 531 nm, respectively. Kq is the quenching rate constant of bimolecular, s0 is the average life time of the fluorophore without MEL, CYA or UA, KSV is the Stern– Volmer dynamic quenching constant and [Q] is the concentration of MEL, CYA or UA [41]. Fig. 5(a) displays the Stern–Volmer plots of DNA–BR solutions in the presence of MEL, CYA or UA with various concentrations. It can be found that three plots exhibit a comparatively good linear relationship. Thus, as shown in Table 1 from Stern–Volmer Eq. (1), for these three systems their Kq could be obtained. And that, the values of the Kq are all greater than the maximum scatter collision quenching constant (2.0 1010 L mol1 s1) of various quenchers with biopolymers. Hence, according to the literatures [25,42,43], all these evidences suggest that the probable quenching mechanism of MEL, CYA or UA with DNA is mainly a static quenching. Moreover, the apparent quenching constant (Kq) arrange in the order of DNA–BR + UA > DNA–BR + CYA > DNA–BR + MEL. It indicated that the UA is more effective than CYA and MEL to quench the fluorescence of DNA–BR molecules in aqueous solution. For reconfirming the static quenching mechanisms, the data of fluorescence quenching of DNA–BR solutions along with the increase of MEL, CYA or UA concentrations are also analyzed according to the Lineweaver–Burk (modified Stern–Volmer or double reciprocal) Eq. (2).
1=½ðF 0 FÞ=F 0 ¼ 1=ðfK LB ½QÞ þ 1=f
ð2Þ
The F0 and F are also the fluorescence intensities of DNA–BR solutions at 531 nm in the absence and presence of MEL, CYA or UA with various concentrations, respectively. The f is the fraction of accessible fluorescence and KLB is the static fluorescence quenching association constant. Using the plots obtained from Fig. 5(b), the KLB could be obtained for DNA–BR + MEL, DAN-BR + CYA and DNA–BR + UA systems. And that, as can be seen from Table 1, for these three systems, their Lineweaver–Burk plots all have a better linear relationship than the corresponding Stern– Volmer plots. Thus, it could be confirmed again that the fluorescence quenching mechanism of DNA molecules by MEL, CYA or UA is mainly a static quenching procedure indeed. It also indicates that a complex between DNA–BR and MEL, CYA or UA has formed, which caused a decrease of fluorescence intensity of DNA–BR solution. From Fig. 5(c), the equilibrium constants (KA) and the binding site numbers (n) could be calculated by using the equation: (F0 F)/F = KA [Q]n. By using logarithmic treatment the following Double logarithm Eq. (3) can be obtained [44].
log½ðF 0 FÞ=F ¼ log K A þ nlog½Q
ð3Þ
Apparently, the KA and n could be measured by the intercept and slope by plotting log [(F0 F)/F] against log [Q]. According to Fig. 5(c), KA and n for DNA–BR + MEL, DNA–BR + CYA and DNA– BR + UA were also obtained, respectively. It can be found that there is a strong binding force between MEL, CYA or UA with DNA–BR, which results in a stable binding site being formed. The correlation coefficients are greater than 0.99, indicating that the interaction of MEL, CYA or UA with DNA–BR agrees well with the site-binding
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model. It could be seen that the plots also exhibited a good linear relationship and these n values are all equal to about 1. It indicates that the binding model of MEL, CYA or UA to DNA–BR was approximately 1:1. The KA for DNA–BR + UA system is slightly bigger than that for DNA–BR + MEL and DNA–BR + CYA systems, which indicates that the binding of DNA with UA was the tighter one. Utilizing KA, the free energy change (DG0) can be calculated from the relationship (4):
DG0 ¼ RTln K A
ð4Þ
The calculated results are all given in Table 1. Thus, R = 8.314 kJ mol1 K1 and T = 293.15 K, the calculated DG0 are 21.724 kJ mol1 for DNA–BR + MEL system, 22.446 kJ mol1 for DNA–BR-CYA system and 22.203 kJ mol1 for DNA–BR + UA system, respectively. The large negative values indicate that the interaction of MEL, CYA or UA to DNA–BR was all spontaneous reaction processes. Circular dichroism spectra of DNA solutions in the presence of MEL, CYA or UA Circular dichroism (CD) spectrum is a spectroscopic technique widely used to study the binding model and interaction affinity of small molecular compounds with many biomolecules, particularly, DNA and protein [45]. The changes of the CD spectra in the spectral range of 200–300 nm wavelengths have played an extensive utilization to detect and follow DNA conformational changes, damage and/or cleavage upon interaction with small molecular compounds. In CD spectrum, CT-DNA shows the typical absorption of a right-handed B-form DNA. And the absorption consists of two bands, that is, a negative (first) one at 245 nm attributing to the right-handed helicity and a positive (second) one at 275 nm attributing to the base stacking. Generally, the classical intercalation of small molecular compounds leads to the changes of DNA CD spectra in intensities of these two bands due to enhancement of base stacking and stabilization of helicity, which enhances the intensities of both the CD bands. However, the simple groove binding and electrostatic interaction of small molecular compounds display low or no perturbations on the base stacking and helicity absorption bands [46,47]. Therefore, in this work, the effects on DNA conformation by interacting with MEL, CYA or UA were evaluated by CD spectroscopy. As shown in Fig. 6, in comparison with blank DNA sample, after addition of MEL, CYA or UA, the DNA solution still retains basic CD spectrum shape, but its first (negative) band and second (positive) band increase and decrease, respectively. For the first (negative) band, the order (from bottom to top) is DNA + UA > DNA + CYA > DNA + MEL > DNA, and for the second (positive) band, the order (from top to bottom) is DNA > DNA + MEL > DNA + UA > DNA + CYA. It indicates that the UA mainly affects the right-handed helicity, and the CYA has a major influence on the base stacking. Nevertheless, the MEL displays the influence on both right-handed helicity and base stacking at the same time, but its change degree is not strong compared with that of UA and CYA. According to the above analyses, it can be confirmed that the CD spectra changes clearly rule out the intercalative model of binding and non-covalent interaction between these three compounds and DNA. Most likely, MEL, CYA or UA interact with DNA via groove binding model. Viscosity changes of DNA solution in the presence of MEL, CYA or UA A useful technique to prove intercalation is viscosity measurement, which is sensitive to length change of DNA and regarded as the least ambiguous and the most critical test of binding model
in solution in the absence of crystallographic structural data or NMR spectra [48]. Generally, a classical intercalation binding of small molecular compounds causes an increase in the viscosity of DNA solution. This is because such DNA molecules demand a large enough space of adjacent base pairs to accommodate these small molecular compounds, lengthen the double helix. In contrast, a partial or non-classical intercalation binding causes the reduction of DNA viscosity. And the non-intercalation binding, such as electrostatic or grooved binding does not appreciably alter DNA viscosity [48,49]. To further clarify the nature of the interaction between MEL, CYA, or UA and DNA, viscosity measurements were carried out by varying the concentration of the added MEL, CYA, or UA to DNA solution. From Fig. 7 it is observed that, as the concentration of MEL, CYA or UA increase, the relative viscosity of DNA almost remains steady, which is in agreement with a non-intercalation model between MEL, CYA, or UA and DNA. Hence, the results demonstrate that the three kinds of small molecular compounds could all bind to DNA by the non-intercalation interaction, that is, groove interaction. It is consistent with the above results of absorption, fluorescence and CD spectra.
Gel electrophoresis of DNA in the presence of MEL, CYA or UA The bindings of MEL, CYA and UA with CT-DNA could also be studied by gel electrophoresis. We examined the effect of different concentrations of the MEL, CYA or UA on CT-DNA by monitoring the changes in intensity of the bands and electrophoretic mobility in an agarose gel which results were presented in Fig. S1. It was clear that the intensity of the recorded CT-DNA bands after binding to MEL, CYA or UA had almost nothing to be changed and the electrophoretic mobility had gradually slowed down with the increasing concentration of MEL, CYA or UA, as compared to the band of the free CT-DNA. It could easily be conjectured that the change in conformation of the DNA was due to the presence of the investigated MEL, CYA or UA, but their different binding abilities to DNA result in different electrophoretic mobility. Also, the binding sites of MEL, CYA or UA to DNA were different from that of EB. Therefore, the lengthes of lanes of CT-DNA are increased with the increasing concentration of MEL, CYA or UA. What is more the length sequence is DNA + UA > DNA + CYA > DNA + MEL. It is in a good agreement with binding constant values which were investigated previously.
Conclusion In this work, the interaction of DNA with MEL, CYA or UA was studied by UV–vis spectroscopy, fluorescence, circular dichroism (CD) spectroscopy, viscosity method and gel electrophoresis. The fluorescence spectral monitoring results, Stern–Volmer dynamic quenching constant (Ksv), quenching rate constant of biomolecule (Kq), static fluorescence quenching association constant (KLB) and equilibrium constants (KA), were obtained. These results suggested that the MEL, CYA and UA all could bind with DNA molecules. And that, the fluorescence quenching processes of DNA–BR caused by MEL, CYA or UA were all attributed to static quenching. The interaction strength of the MEL, CYA or UA and DNA molecules ranks in the order of DNA–UA > DNA–CYA > DNA–MEL. The viscosity methods and CD spectroscopy showed that the binding models of the interactions of DNA with MEL, CYA and UA all may belong to the groove binding. The bindings of MEL, CYA and UA with DNA were also studied by gel electrophoresis and the obtained sequence of binding strength is DNA–UA > DNA–CYA > DNA–MEL. These research results may be a valuable reference for understanding
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the formation mechanism of kidney stones and gout and have a certain help about the related disease treatment at the same time. Acknowledgments The authors greatly acknowledge the National Science Foundation of China (21371084), Innovation Team Project of Education Department of Liaoning Province (LT2012001), Public Research Fund Project of Science and Technology Department of Liaoning Province (2012004001), Shenyang Science and Technology Plan Project (F12-277-1-15 and F13-289-1-00) and Science Foundation of Liaoning Provincial Education Department (L2011007) for financial support. The authors also thank our colleagues and other students for their participating in this work.
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