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Study on the interactions between CuL2 and Morin with DNA
Journal of Inorganic Biochemistry 91 (2002) 470–474 www.elsevier.com / locate / jinorgbio
Study on the interactions between CuL 2 and Morin with DNA a a, a a a b Yumin Song , Jingwan Kang *, Zhihua Wang , Xiaoquan Lu , Jinzhang Gao , Liufang Wang a
b
Department of Chemistry, Northwest Normal University, Lanzhou 730070, China State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730070, China
Received 27 August 2001; received in revised form 25 February 2002; accepted 28 February 2002
Abstract Absorption, fluorescence spectral and viscometric studies have been carried out on the interaction of Morin (29,3,49,5,7-pentahydroxyflavone, Scheme 1) and its Cu complex, CuL 2 ?2H 2 O [L5Morin (29-OH group deprotonated), Scheme 2] with calf thymus DNA. CuL 2 shows different spectral characteristics from that of Morin in the presence of DNA. Increasing fluorescence is seen for CuL 2 with DNA addition whereas decreased fluorescence is observed for Morin. Quenching fluorescence is observed for the DNA–EB system when CuL 2 is added whereas slightly quenched fluorescence is seen for the DNA–EB system with Morin addition. The relative viscosity of DNA and the DNA–EB system increases with the addition of CuL 2. Hypochromism and a smaller shift are observed in the UV–visible spectra of CuL 2 in the presence of DNA and the denatured temperature of DNA is decreased in the presence of CuL 2 . The above results suggested that Morin and CuL 2 can both bind to DNA, but the binding mode is different. The complex binds to DNA mainly by intercalation, while Morin binds in a nonintercalating mode. 2002 Elsevier Science Inc. All rights reserved. Keywords: Morin; DNA; CuL 2 ; Intercalation; Binding
1. Introduction A number of metal chelates have been used as probes of DNA structure in solution [1] as agents for mediation of strand scission of duplex DNA and as chemotherapeutic agents [2,3]. Barton and co-workers [4,5] have studied the interaction of enantiomers of Ru(phen) 331 with various DNA, the results lead them to the conclusion that there are two modes of interaction, intercalative and electrostatic binding. The d-isomer favors binding to the B-form DNA by intercalactive fashion, while the l-isomer is not favored. They developed a new type of probes of chiral complex for B-, Z-form DNA. They found that the intercalating ability appears to increase with the planarity of ligands. Kharatishvili et al. [6] also reported the effect on DNA binding in the presence of a planar intercalating ligand such as quinoline for both mononuclear and dinuclear Pt complexes. Mahadeven and Palaniandvavr [1] have studied copper(II) complexes of bis(pyrid-2-yl)-di / trithia ligands bound to calf thymus DNA and found that the coordination geometry and the ligand donor atom type
play a key role in deciding the mode and extent of binding of complexes to DNA. In our group, six new transition metal(II) complexes of Morin (29,3,49,5,7-pentahydroxyflavone) (Scheme 1) have been prepared and their antitumor activities were studied and were reported previously [7]. It was found that CuL 2 (Scheme 2) and ZnL 2 show higher antitumor activities than that of Morin. The phenomenon of Morin and its complexes exhibiting different antitumor activities lead us to consider the interaction of the two complexes and Morin with calf thymus DNA. Because nucleic acid is usually the target of some antitumor reagents in the organism, these reagents react with DNA thereby stopping the replication of DNA and inhibiting the growth of the tumor cell. Therefore these reagents have antitumor activities [8–10].
*Corresponding author. Fax: 186-931-797-1216. E-mail address: [email protected] (J. Kang). 0162-0134 / 02 / $ – see front matter 2002 Elsevier Science Inc. All rights reserved. PII: S0162-0134( 02 )00425-7
Scheme 1. The structure of Morin.
Y. Song et al. / Journal of Inorganic Biochemistry 91 (2002) 470 – 474
471
For viscosity measurements the viscometer (Ubbelohde Model, Instrument Factory of Fudan University, China) was thermostated at 25 8C in a constant temperature bath. A UV-3400 spectrophotometer (Hitachi, Japan), and a RF-540 spectrofluorophotometer (Hitachi) were used for all experiments.
3. Results and discussion
3.1. Relative binding
Scheme 2. The structure of the copper complex.
There is, therefore, considerable interest in the design of small molecules which react at specific sites along the DNA strand as reactive models for protein–nucleic acid interactions, in developing new probes of DNA structures, as an aid to drug design, and as tools in molecular biology [11]. We therefore wished to know that if the different antitumor activities of Morin and its complexes correlate well with the DNA binding mode and affinity. In this report we explore the interaction of the Morin compound and its Cu complex with calf thymus DNA by using a fluorometric method. The aim of this study is to find a relation between the better antitumor properties of the complex and the mode of DNA binding. The results of the study are that Morin binds in a nonintercalating mode while the complex binds by intercalation. Ethidium bromide (EB) is the most widely used fluorescence probe for DNA structure and has recently been employed in the examinations of the mode and process of metal complexes binding to DNA [12]. In this report we focus on the examination of the interaction of CuL 2 and Morin with DNA using several methods. The study in this report offers an opportunity to understand how the structure of molecules affect their binding mode and affinity of binding to DNA. Which will help us design new complexes which have biological and antitumor activities.
CuL 2 has fluorescence around 525 nm when excited at 445 nm while Morin fluorescences around 510 nm when excited at 432 nm. If DNA solution was added to the CuL 2 and the Morin solution, enhanced fluorescence and a shift to shorter wavelength for CuL 2 solution and quenched fluorescence for Morin are observed when excited under the given conditions. Fig. 1 shows the emission spectra of CuL 2 (Fig. 1A) and Morin (Fig. 1B) in the presence and absence of calf thymus DNA. The stronger enhancement for CuL 2 may be largely due to the increase of the molecular planarity of the complex and the decrease of the collisional frequency of the solvent molecules with the complex which caused by the planar aromatic group of the complex stacks between adjacent base pairs of DNA. The increase of the molecule’s planarity and the decrease of the collisional frequency solvent molecules with the complexes usually lead to emission enhancement. The binding of CuL 2 to DNA leading to a marked increase in emission intensity also agrees with those observed for other intercalators [10]. The emission quenching of Morin in the presence of DNA may be caused by the fact that, Morin
2. Experimental Analytical-reagent grade chemicals were used. The complex CuL 2 ?2H 2 O was synthesized by methods described previously [7]. Solutions of the complex and Morin were prepared by using EtOH and buffer. Calf thymus DNA was obtained from Humei Chemical (Beijing, China), DNA concentrations were determined spectrophotometrically with an extinction coefficient of 6600 M 21 cm 21 at 260 nm [10]. Experiments were carried out at pH 7.1 in buffer (53 10 23 M Tris, 5310 22 M NaCl). Solutions were prepared with distilled water. EB solution was prepared with buffer.
Fig. 1. Emission spectra: (A) CuL 2 system (excited at 445 nm, 3 ml solution): (a) 3.2 310 25 M CuL 2 ; (b) a10.02 ml DNA (1.5310 23 M); (c) a10.04 ml DNA; (d) a10.06 ml DNA; (e) a10.08 ml DNA; (f) a10.10 ml DNA; (g) a10.12 ml DNA; (h) a10.16 ml DNA; (i) a10.18 ml DNA; (j) a10.20 ml DNA; (B) Morin system (excited at 432 nm, 2.0310 24 M) in the absence (----) and presence (—) of increasing amounts of 1.5310 23 M DNA (40 ml per scan).
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Y. Song et al. / Journal of Inorganic Biochemistry 91 (2002) 470 – 474
DNA–CuL 2 system and DNA–Morin system, denaturing and viscosity.
3.2. Binding mode
Fig. 2. Emission spectra: (A) DNA–CuL 2 system (2.5310 24 M DNA, 8310 25 M CuL 2 ) in the absence (----) and presence (—) of increasing amounts of 2.0310 25 M EB (40 ml per scan), sample excited at 445 nm; (B) DNA–Morin system (1.2310 24 M DNA, 3.0310 25 M Morin) in the absence (----) and presence (—) of increasing amounts of 4.0310 24 M EB (10 ml per scan), excited at 432 nm.
being a small hydrophobic molecule, can be adsorbed by hydrophobic group on the surface of DNA. Since CuL 2 and Morin display different emission spectral characteristics in the presence of DNA, it is apparent that more than a single binding mode for the two compounds exists. This conclusion is further supported by studying the effect of EB on the emission spectra of the
In order to investigate the mode of CuL 2 and Morin binding to DNA, EB has been employed in examination of the reaction, as EB presumably binds initially to DNA by intercalation. The experiment of EB with CuL 2 binding to DNA was carried out in 3 ml solution of 2.5310 24 M DNA, 8310 25 M CuL 2 titrated with the 2.0310 25 M EB solution. Fig. 2 shows the emission spectra of the DNA– CuL 2 system (Fig. 2A) and the DNA–Morin?2H 2 O system (Fig. 2B) in the absence and presence of EB. When the concentration of EB was increased, the emission intensity of the DNA–CuL 2 system decreased and a small shift to longer wavelength were observed and a new emission spectrum appeared at 587 nm which is the characteristic spectra of the DNA–EB system [12]. This phenomenon meant that EB substituted for CuL 2 in the DNA–CuL 2 system which leads to a large decrease in the emission intensity of the DNA–CuL 2 system and an increase in emission intensity of the DNA–EB system. These changes with intercalation have been observed in several instances [13–15]. The emission intensity of the DNA–Morin system slightly changed as EB is added and a new emission spectra also appeared at 587 nm. Comparing these changes in the emission spectra of the DNA–CuL 2 system with the DNA–Morin system in the presence of
Fig. 3. Emission spectra (excited at 445 nm): (A) DNA–EB system (5.0310 25 M DNA) in the absence (----) and presence (—) of increasing amounts of 1.0310 24 M CuL 2 (20 ml per scan); (B) DNA–EB system (3.0310 26 M DNA) in the absence (----) and presence (—) of increasing amounts of 4.0310 24 M Morin (20 ml per scan).
Y. Song et al. / Journal of Inorganic Biochemistry 91 (2002) 470 – 474
EB, the difference is distinct. This phenomenon may also indicates that CuL 2 and Morin bind to DNA in different modes. Further support for the different modes of CuL 2 and Morin binding to DNA is given through the competitive binding experiment. Here EB has also been employed as a probe. The experiment was carried out in 3 ml solution of 3310 26 M EB, 5310 25 M DNA (at saturating binding levels [10]) titrated with 1310 24 M CuL 2 solution. Fig. 3 shows the emission spectra of the DNA–EB system in the presence of CuL 2 (Fig. 3A) and Morin (Fig. 3B). The emission intensity of the DNA–EB system decreased and the emission spectra of the DNA–CuL 2 system appeared at 525 nm as the concentration of CuL 2 increased. These changes observed here are often characteristic of intercalation [4]. This phenomenon meant that CuL 2 replaced EB from the DNA–EB system leading to the emission decrease of the DNA–EB system. The emission intensity was slightly quenched as Morin was added to the DNA–EB system, this did not mean that Morin replaced EB from the DNA–EB system. The binding of intercalative drugs to DNA has also been characterized classically through absorption titrations, following the hypochromism with binding of the CuL 2
473
complex to DNA. Fig. 4 displays a well-behaved titration of CuL 2 with calf thymus DNA. The spectra show clearly that addition of DNA yields hypochromism and a small red shift in the charge-transfer band of the complex. These spectral characteristics are attributable to a mode of interaction binding [16,17]. The binding constant Kb of CuL 2 to DNA can be determined with the following equation [18]: [DNA] /(eA 2 eF ) 5 [DNA] /(eB 2 eF ) 1 1 /Kb (eF 2 eB ) where eA , eF and eB correspond to A obsd / [CuL 2 ], the extinction coefficient for the free CuL 2 complex, and the extinction coefficient for the CuL 2 complex in the fully bound form, respectively. In plots of [DNA] /(eA 2 eF ) versus [DNA], Kb is given by the ratio of the slope to intercept. The obtained binding constant of CuL 2 binds to DNA (Kb , 2.2310 4 M 21 ) is lower than those for classical intercalators (ethidium-DNA, 7310 7 M 21 in 40 mM Tris– HCl buffer, pH 7.9 [19] and 1.4310 6 M 21 in 40 mM NaCl–25 mM Tris–HCl [20]). The Kb is also lower than the Kb (5.05310 4 M 21 ) of ZnL 2 to DNA [21]. This phenomenon clearly suggests that the CuL 2 complex may not fully intercalate into DNA. This agrees with that ZnL 2 has more antitumor activity than that of CuL 2 . The binding of intercalative drugs to DNA has also been characterized recently through the changes of viscosity of DNA. Table 1 summarizes the relative viscosity (hr ) of DNA and the DNA–EB system in the absence and presence of the ligand and its complex CuL 2 (avoiding from light after a 48 h reaction). The viscosity measurements were carried out by varying the concentration of the added ligand and CuL 2 . The values of relative viscosity (hr 5h /h0 ), where h is the viscosity of DNA and DNA–EB in the presence and absence of the ligand and CuL 2 , and h0 is the viscosity of the buffer. The intercalation of drugs with DNA usually causes the viscosity of DNA increase [20]. The viscosity experiment displays the hr of DNA and DNA–EB increased with the concentration of CuL 2 increase. But the Table 1 Relative viscosity (hr )
Fig. 4. UV–visible spectra of (2.0310 25 M) in the absence (----) and presence of (—) increasing amounts of DNA (1.0310 23 M, 0.1 ml per scan).
Concentration (10 25 M)
hr (DNA, 1310 23 M)
hr (DNA–EB, 1310 23 M, 2.0310 24 M)
CuL 2
0.0 2.5 5.0 7.5 10.0
1.309 1.341 1.363 1.384 1.392
1.430 1.439 1.467 1.481 1.513
L
0.0 5.0 10.0 15.0 20.0
1.309 1.316 1.334 1.342 1.330
1.431 1.462 1.483 1.444 1.498
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Y. Song et al. / Journal of Inorganic Biochemistry 91 (2002) 470 – 474
than that of CuL 2 in the absence of DNA. This phenomenon may be largely due to the intercalation of CuL 2 with DNA. The intercalation leads to the decrease of the collisional frequency of quenching molecules, so DNA plays a role of protection. Based on our investigation, although both CuL 2 and Morin can bind to DNA, the nature of the binding was found to be different for each of them. In the presence and absence of DNA, CuL 2 shows different spectral characteristics which agree with those observed for other intercalators. This phenomenon suggests that the CuL 2 binds to DNA mainly in the intercalating mode while Morin binds in a nonintercalating mode.
Fig. 5. The caver of thermally denatured (5.0310 25 M DNA, 3.0310 26 M EB, 3.0310 26 M CuL 2 ), (A) DNA–EB, (B) DNA–EB–CuL 2 .
extent of increase is lesser than that for the known intercalator EB [22]. In contrast to CuL 2 , the ligand does not exhibit a regular trend in the viscosity of DNA. The interaction of classical intercalators with DNA, such as ethidium, can increase the stability of helix of DNA, and cause the T m (denatured temperature) of DNA to increase [23,24]. Fig. 5 shows the behavior of thermally denatured of the DNA–EB system and the DNA–EB– CuL 2 system. The T m of DNA is 87 8C in the presence of EB, but in the presence of CuL 2 and EB, the T m is 83 8C. The interaction of CuL 2 with DNA may cause the T m to be decreased. This meant that CuL 2 complex effects the binding of EB to DNA and makes the stability of DNA decreased. Fig. 6 shows the effect of quenching agent K 4 [Fe(CN) 6 ] on fluorescence intensity of CuL 2 complex. Quenched fluorescence for CuL 2 is observed when K 4 [Fe(CN) 6 ] complex added. But in the presence of DNA, the effect of K 4 [Fe(CN) 6 ] on fluorescence intensity becomes smaller
Fig. 6. Effect of K 4 [Fe(CN 6 )] on fluorescence intensity of CuL 2 (4.03 10 25 M), (A) in the absence of DNA, (B) in the presence of DNA (1.0310 23 M).
Acknowledgements This work was supported by the Natural Science Foundation of GXGC NWNU.
References [1] S. Mahadevan, M. Palaniandavar, Inorg. Chim. Acta 254 (1997) 291. [2] S.J. Llippard, Acc. Chem. Res. 11 (1978) 211. [3] S.M. Hecht, Acc. Chem. Res. 19 (1986) 383. [4] C.V. Kumar, J.K. Barton, N.J. Turro, J. Am. Chem. Soc. 107 (1985) 5518. [5] J.K. Barton, A.T. Danishefsky, J.M. Golderg, J. Am. Chem. Soc. 106 (1984) 2172. [6] M. Kharatishvili, M. Mathieson, N. Farrell, Inorg. Chim. Acta 255 (1997) 1. [7] Zh. Qi, W.L. Fang, L. Xiang, J. Transition Met. Chim. 21 (1996) 23. [8] A.M. Pyle, T. Morii, J.K. Barton, J. Am. Chem. Soc. 112 (1990) 9432. [9] J.K. Barton, J.M. Goldberg, C.V. Kumar, N.J. Turro, J. Am. Chem. Soc. 108 (1986) 2081. [10] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 31 (1992) 9319. [11] A. Nishio, E.M. Uycki, Cancer Res. 43 (1983) 1951. [12] L.Sh. Du, Current Protocols for Molecular Biology, High Education Press, Beijing, 1993. [13] J.P. Rehman, J.K. Barton, Biochemistry 29 (1990) 1701. [14] Y. Pin, J. Lan, Y.B. Sheng, Chem. J. Chin. University 5 (1994) 1742. [15] J. Lan, Y. Pin, L.Q. Shang, Chem. J. Chin. University 17 (1996) 1345. [16] A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am. Chem. Soc. 111 (1989) 3051. [17] M.T. Carter, J. Allen, J. Am. Chem. Soc. 109 (1987) 7528. [18] A. Wolfe, G.H. Shimer, T. Meehan, Biochemistry 26 (1987) 6392. [19] M.J. Waring, J. Mol. Biol. 13 (1965) 269. [20] J.B. Leepecq, C. Paoletti, J. Mol. Biol. 27 (1967) 87. [21] Y.M. Song, J. Kang, J. Zhou, Zh.H. Wang, X. Lu, L. Wang, J. Gao, Spectrochim. Acta, Part A 56 (2000) 2491. [22] J.M. Veal, R.L. Rill, Biochemistry 30 (1991) 1132. [23] J.B. Chaires, Biochemistry 25 (1986) 8436. [24] C.V. Kumar, E.H. Asuncion, J. Am. Chem. Soc. 115 (1993) 8547.
Study on the interactions between CuL 2 and Morin with DNA a a, a a a b Yumin Song , Jingwan Kang *, Zhihua Wang , Xiaoquan Lu , Jinzhang Gao , Liufang Wang a
b
Department of Chemistry, Northwest Normal University, Lanzhou 730070, China State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730070, China
Received 27 August 2001; received in revised form 25 February 2002; accepted 28 February 2002
Abstract Absorption, fluorescence spectral and viscometric studies have been carried out on the interaction of Morin (29,3,49,5,7-pentahydroxyflavone, Scheme 1) and its Cu complex, CuL 2 ?2H 2 O [L5Morin (29-OH group deprotonated), Scheme 2] with calf thymus DNA. CuL 2 shows different spectral characteristics from that of Morin in the presence of DNA. Increasing fluorescence is seen for CuL 2 with DNA addition whereas decreased fluorescence is observed for Morin. Quenching fluorescence is observed for the DNA–EB system when CuL 2 is added whereas slightly quenched fluorescence is seen for the DNA–EB system with Morin addition. The relative viscosity of DNA and the DNA–EB system increases with the addition of CuL 2. Hypochromism and a smaller shift are observed in the UV–visible spectra of CuL 2 in the presence of DNA and the denatured temperature of DNA is decreased in the presence of CuL 2 . The above results suggested that Morin and CuL 2 can both bind to DNA, but the binding mode is different. The complex binds to DNA mainly by intercalation, while Morin binds in a nonintercalating mode. 2002 Elsevier Science Inc. All rights reserved. Keywords: Morin; DNA; CuL 2 ; Intercalation; Binding
1. Introduction A number of metal chelates have been used as probes of DNA structure in solution [1] as agents for mediation of strand scission of duplex DNA and as chemotherapeutic agents [2,3]. Barton and co-workers [4,5] have studied the interaction of enantiomers of Ru(phen) 331 with various DNA, the results lead them to the conclusion that there are two modes of interaction, intercalative and electrostatic binding. The d-isomer favors binding to the B-form DNA by intercalactive fashion, while the l-isomer is not favored. They developed a new type of probes of chiral complex for B-, Z-form DNA. They found that the intercalating ability appears to increase with the planarity of ligands. Kharatishvili et al. [6] also reported the effect on DNA binding in the presence of a planar intercalating ligand such as quinoline for both mononuclear and dinuclear Pt complexes. Mahadeven and Palaniandvavr [1] have studied copper(II) complexes of bis(pyrid-2-yl)-di / trithia ligands bound to calf thymus DNA and found that the coordination geometry and the ligand donor atom type
play a key role in deciding the mode and extent of binding of complexes to DNA. In our group, six new transition metal(II) complexes of Morin (29,3,49,5,7-pentahydroxyflavone) (Scheme 1) have been prepared and their antitumor activities were studied and were reported previously [7]. It was found that CuL 2 (Scheme 2) and ZnL 2 show higher antitumor activities than that of Morin. The phenomenon of Morin and its complexes exhibiting different antitumor activities lead us to consider the interaction of the two complexes and Morin with calf thymus DNA. Because nucleic acid is usually the target of some antitumor reagents in the organism, these reagents react with DNA thereby stopping the replication of DNA and inhibiting the growth of the tumor cell. Therefore these reagents have antitumor activities [8–10].
*Corresponding author. Fax: 186-931-797-1216. E-mail address: [email protected] (J. Kang). 0162-0134 / 02 / $ – see front matter 2002 Elsevier Science Inc. All rights reserved. PII: S0162-0134( 02 )00425-7
Scheme 1. The structure of Morin.
Y. Song et al. / Journal of Inorganic Biochemistry 91 (2002) 470 – 474
471
For viscosity measurements the viscometer (Ubbelohde Model, Instrument Factory of Fudan University, China) was thermostated at 25 8C in a constant temperature bath. A UV-3400 spectrophotometer (Hitachi, Japan), and a RF-540 spectrofluorophotometer (Hitachi) were used for all experiments.
3. Results and discussion
3.1. Relative binding
Scheme 2. The structure of the copper complex.
There is, therefore, considerable interest in the design of small molecules which react at specific sites along the DNA strand as reactive models for protein–nucleic acid interactions, in developing new probes of DNA structures, as an aid to drug design, and as tools in molecular biology [11]. We therefore wished to know that if the different antitumor activities of Morin and its complexes correlate well with the DNA binding mode and affinity. In this report we explore the interaction of the Morin compound and its Cu complex with calf thymus DNA by using a fluorometric method. The aim of this study is to find a relation between the better antitumor properties of the complex and the mode of DNA binding. The results of the study are that Morin binds in a nonintercalating mode while the complex binds by intercalation. Ethidium bromide (EB) is the most widely used fluorescence probe for DNA structure and has recently been employed in the examinations of the mode and process of metal complexes binding to DNA [12]. In this report we focus on the examination of the interaction of CuL 2 and Morin with DNA using several methods. The study in this report offers an opportunity to understand how the structure of molecules affect their binding mode and affinity of binding to DNA. Which will help us design new complexes which have biological and antitumor activities.
CuL 2 has fluorescence around 525 nm when excited at 445 nm while Morin fluorescences around 510 nm when excited at 432 nm. If DNA solution was added to the CuL 2 and the Morin solution, enhanced fluorescence and a shift to shorter wavelength for CuL 2 solution and quenched fluorescence for Morin are observed when excited under the given conditions. Fig. 1 shows the emission spectra of CuL 2 (Fig. 1A) and Morin (Fig. 1B) in the presence and absence of calf thymus DNA. The stronger enhancement for CuL 2 may be largely due to the increase of the molecular planarity of the complex and the decrease of the collisional frequency of the solvent molecules with the complex which caused by the planar aromatic group of the complex stacks between adjacent base pairs of DNA. The increase of the molecule’s planarity and the decrease of the collisional frequency solvent molecules with the complexes usually lead to emission enhancement. The binding of CuL 2 to DNA leading to a marked increase in emission intensity also agrees with those observed for other intercalators [10]. The emission quenching of Morin in the presence of DNA may be caused by the fact that, Morin
2. Experimental Analytical-reagent grade chemicals were used. The complex CuL 2 ?2H 2 O was synthesized by methods described previously [7]. Solutions of the complex and Morin were prepared by using EtOH and buffer. Calf thymus DNA was obtained from Humei Chemical (Beijing, China), DNA concentrations were determined spectrophotometrically with an extinction coefficient of 6600 M 21 cm 21 at 260 nm [10]. Experiments were carried out at pH 7.1 in buffer (53 10 23 M Tris, 5310 22 M NaCl). Solutions were prepared with distilled water. EB solution was prepared with buffer.
Fig. 1. Emission spectra: (A) CuL 2 system (excited at 445 nm, 3 ml solution): (a) 3.2 310 25 M CuL 2 ; (b) a10.02 ml DNA (1.5310 23 M); (c) a10.04 ml DNA; (d) a10.06 ml DNA; (e) a10.08 ml DNA; (f) a10.10 ml DNA; (g) a10.12 ml DNA; (h) a10.16 ml DNA; (i) a10.18 ml DNA; (j) a10.20 ml DNA; (B) Morin system (excited at 432 nm, 2.0310 24 M) in the absence (----) and presence (—) of increasing amounts of 1.5310 23 M DNA (40 ml per scan).
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Y. Song et al. / Journal of Inorganic Biochemistry 91 (2002) 470 – 474
DNA–CuL 2 system and DNA–Morin system, denaturing and viscosity.
3.2. Binding mode
Fig. 2. Emission spectra: (A) DNA–CuL 2 system (2.5310 24 M DNA, 8310 25 M CuL 2 ) in the absence (----) and presence (—) of increasing amounts of 2.0310 25 M EB (40 ml per scan), sample excited at 445 nm; (B) DNA–Morin system (1.2310 24 M DNA, 3.0310 25 M Morin) in the absence (----) and presence (—) of increasing amounts of 4.0310 24 M EB (10 ml per scan), excited at 432 nm.
being a small hydrophobic molecule, can be adsorbed by hydrophobic group on the surface of DNA. Since CuL 2 and Morin display different emission spectral characteristics in the presence of DNA, it is apparent that more than a single binding mode for the two compounds exists. This conclusion is further supported by studying the effect of EB on the emission spectra of the
In order to investigate the mode of CuL 2 and Morin binding to DNA, EB has been employed in examination of the reaction, as EB presumably binds initially to DNA by intercalation. The experiment of EB with CuL 2 binding to DNA was carried out in 3 ml solution of 2.5310 24 M DNA, 8310 25 M CuL 2 titrated with the 2.0310 25 M EB solution. Fig. 2 shows the emission spectra of the DNA– CuL 2 system (Fig. 2A) and the DNA–Morin?2H 2 O system (Fig. 2B) in the absence and presence of EB. When the concentration of EB was increased, the emission intensity of the DNA–CuL 2 system decreased and a small shift to longer wavelength were observed and a new emission spectrum appeared at 587 nm which is the characteristic spectra of the DNA–EB system [12]. This phenomenon meant that EB substituted for CuL 2 in the DNA–CuL 2 system which leads to a large decrease in the emission intensity of the DNA–CuL 2 system and an increase in emission intensity of the DNA–EB system. These changes with intercalation have been observed in several instances [13–15]. The emission intensity of the DNA–Morin system slightly changed as EB is added and a new emission spectra also appeared at 587 nm. Comparing these changes in the emission spectra of the DNA–CuL 2 system with the DNA–Morin system in the presence of
Fig. 3. Emission spectra (excited at 445 nm): (A) DNA–EB system (5.0310 25 M DNA) in the absence (----) and presence (—) of increasing amounts of 1.0310 24 M CuL 2 (20 ml per scan); (B) DNA–EB system (3.0310 26 M DNA) in the absence (----) and presence (—) of increasing amounts of 4.0310 24 M Morin (20 ml per scan).
Y. Song et al. / Journal of Inorganic Biochemistry 91 (2002) 470 – 474
EB, the difference is distinct. This phenomenon may also indicates that CuL 2 and Morin bind to DNA in different modes. Further support for the different modes of CuL 2 and Morin binding to DNA is given through the competitive binding experiment. Here EB has also been employed as a probe. The experiment was carried out in 3 ml solution of 3310 26 M EB, 5310 25 M DNA (at saturating binding levels [10]) titrated with 1310 24 M CuL 2 solution. Fig. 3 shows the emission spectra of the DNA–EB system in the presence of CuL 2 (Fig. 3A) and Morin (Fig. 3B). The emission intensity of the DNA–EB system decreased and the emission spectra of the DNA–CuL 2 system appeared at 525 nm as the concentration of CuL 2 increased. These changes observed here are often characteristic of intercalation [4]. This phenomenon meant that CuL 2 replaced EB from the DNA–EB system leading to the emission decrease of the DNA–EB system. The emission intensity was slightly quenched as Morin was added to the DNA–EB system, this did not mean that Morin replaced EB from the DNA–EB system. The binding of intercalative drugs to DNA has also been characterized classically through absorption titrations, following the hypochromism with binding of the CuL 2
473
complex to DNA. Fig. 4 displays a well-behaved titration of CuL 2 with calf thymus DNA. The spectra show clearly that addition of DNA yields hypochromism and a small red shift in the charge-transfer band of the complex. These spectral characteristics are attributable to a mode of interaction binding [16,17]. The binding constant Kb of CuL 2 to DNA can be determined with the following equation [18]: [DNA] /(eA 2 eF ) 5 [DNA] /(eB 2 eF ) 1 1 /Kb (eF 2 eB ) where eA , eF and eB correspond to A obsd / [CuL 2 ], the extinction coefficient for the free CuL 2 complex, and the extinction coefficient for the CuL 2 complex in the fully bound form, respectively. In plots of [DNA] /(eA 2 eF ) versus [DNA], Kb is given by the ratio of the slope to intercept. The obtained binding constant of CuL 2 binds to DNA (Kb , 2.2310 4 M 21 ) is lower than those for classical intercalators (ethidium-DNA, 7310 7 M 21 in 40 mM Tris– HCl buffer, pH 7.9 [19] and 1.4310 6 M 21 in 40 mM NaCl–25 mM Tris–HCl [20]). The Kb is also lower than the Kb (5.05310 4 M 21 ) of ZnL 2 to DNA [21]. This phenomenon clearly suggests that the CuL 2 complex may not fully intercalate into DNA. This agrees with that ZnL 2 has more antitumor activity than that of CuL 2 . The binding of intercalative drugs to DNA has also been characterized recently through the changes of viscosity of DNA. Table 1 summarizes the relative viscosity (hr ) of DNA and the DNA–EB system in the absence and presence of the ligand and its complex CuL 2 (avoiding from light after a 48 h reaction). The viscosity measurements were carried out by varying the concentration of the added ligand and CuL 2 . The values of relative viscosity (hr 5h /h0 ), where h is the viscosity of DNA and DNA–EB in the presence and absence of the ligand and CuL 2 , and h0 is the viscosity of the buffer. The intercalation of drugs with DNA usually causes the viscosity of DNA increase [20]. The viscosity experiment displays the hr of DNA and DNA–EB increased with the concentration of CuL 2 increase. But the Table 1 Relative viscosity (hr )
Fig. 4. UV–visible spectra of (2.0310 25 M) in the absence (----) and presence of (—) increasing amounts of DNA (1.0310 23 M, 0.1 ml per scan).
Concentration (10 25 M)
hr (DNA, 1310 23 M)
hr (DNA–EB, 1310 23 M, 2.0310 24 M)
CuL 2
0.0 2.5 5.0 7.5 10.0
1.309 1.341 1.363 1.384 1.392
1.430 1.439 1.467 1.481 1.513
L
0.0 5.0 10.0 15.0 20.0
1.309 1.316 1.334 1.342 1.330
1.431 1.462 1.483 1.444 1.498
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Y. Song et al. / Journal of Inorganic Biochemistry 91 (2002) 470 – 474
than that of CuL 2 in the absence of DNA. This phenomenon may be largely due to the intercalation of CuL 2 with DNA. The intercalation leads to the decrease of the collisional frequency of quenching molecules, so DNA plays a role of protection. Based on our investigation, although both CuL 2 and Morin can bind to DNA, the nature of the binding was found to be different for each of them. In the presence and absence of DNA, CuL 2 shows different spectral characteristics which agree with those observed for other intercalators. This phenomenon suggests that the CuL 2 binds to DNA mainly in the intercalating mode while Morin binds in a nonintercalating mode.
Fig. 5. The caver of thermally denatured (5.0310 25 M DNA, 3.0310 26 M EB, 3.0310 26 M CuL 2 ), (A) DNA–EB, (B) DNA–EB–CuL 2 .
extent of increase is lesser than that for the known intercalator EB [22]. In contrast to CuL 2 , the ligand does not exhibit a regular trend in the viscosity of DNA. The interaction of classical intercalators with DNA, such as ethidium, can increase the stability of helix of DNA, and cause the T m (denatured temperature) of DNA to increase [23,24]. Fig. 5 shows the behavior of thermally denatured of the DNA–EB system and the DNA–EB– CuL 2 system. The T m of DNA is 87 8C in the presence of EB, but in the presence of CuL 2 and EB, the T m is 83 8C. The interaction of CuL 2 with DNA may cause the T m to be decreased. This meant that CuL 2 complex effects the binding of EB to DNA and makes the stability of DNA decreased. Fig. 6 shows the effect of quenching agent K 4 [Fe(CN) 6 ] on fluorescence intensity of CuL 2 complex. Quenched fluorescence for CuL 2 is observed when K 4 [Fe(CN) 6 ] complex added. But in the presence of DNA, the effect of K 4 [Fe(CN) 6 ] on fluorescence intensity becomes smaller
Fig. 6. Effect of K 4 [Fe(CN 6 )] on fluorescence intensity of CuL 2 (4.03 10 25 M), (A) in the absence of DNA, (B) in the presence of DNA (1.0310 23 M).
Acknowledgements This work was supported by the Natural Science Foundation of GXGC NWNU.
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