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Time-resolved fluorescence studies of the interaction of the Eu3+ complexes of tetracycline analogues with DNA
ANALYTICA CHIMICA ACTA ELSEVIER
Analytica
Chimica
Acta 345 ( 1997) 2 13-2 I7
Time-resolved fluorescence studies of the interaction of the Eu3+ complexes of tetracycline analogues with DNA Xiao-jing Liu, Yuan-zong Li, Yun-xiang Ci* Department
Received 21 October
c$ Chemistry,
Peking
Uniwxtiry,
Beijing
100871,
China
1996; received in revised form 10 January 1997: accepted 22 January 1997
Abstract The interaction of the ELI” compIexes of tetracycline analogues with DNA has been examined by using LJV-visible absorption and fluorescence spectra. Among the tetracycline series, the europium fluorescence sensitized by tetracycline (TC) and oxytetracycline (OTC) were enhanced by DNA more than those of other antibiotics in the series. Therefore, EL?+-TCDNA and EL?+-OTC-DNA systems were investigated by time-resolved fluorescence. The Et?‘-OTC complex is a more sensitive fluorescence probe for selective determination of DNA than the EL?* -TC complex. Detection limits (signal/ noise=2) for DNA are 2.4 and 5.0 ng ml-’ for the OTC and TC systems, respectively. Keywor&: UV-visible spectrometry; fluorescence; Europium; Tetracycline; Oxytetracycline: DNA
1. Introduction The direct use of the fluorescence emission properties of nucleic acids to investigate their biological properties has been limited [ 1,2]. whereas the use of lanthanide(II1) cations as fluorescence probes for the structure and function of nucleic acids has increased markedly. Most of the research has been on Tb3+ and EL?+. Narayana et al. have studied Tb”+induced fluorescence of four-stranded G4-DNA that gave some useful information on DNA conformation [3]. The europium fluorescence sensitized by tetracycline (TC) in a micellar solution of Triton X- 100, via the formation of an organic chelate, was used for the sensitive detection of TC [4]. The similar intramolecular energy transfer from ligand (TC) to the lantha*Corresponding
author.
000%2670/97/$17.00 (‘, 1997 Elsevier Science B.V. All rights reserved P/f
SOOO3-2670(97)00084-6
nide ion was used to enhance the fluorescence of the ligand, which has been applied in liquid chromatographic analysis [5]. Recently, we have reported that the EL?+-TC complex is a sensitive fluorescence probe for determining double-stranded and singlestranded DNA without interference from RNA [6]. This finding aroused our interest for the systematic exploitation of the interaction of EL? complexes of TC analogues with DNA. It is expected that this study may shift out more sensitive fluorescence probes for determining DNA. In addition, it is well known that TC and its analogues have been widely used as antibiotics, and these antibiotics chelate with cations such as Mg2+ and Ca’+. This chelation plays a determining role in the bacteriostatic properties of the antibiotics [7,8]. Furthermore, it had been discovered that TC can combine with tumour cell [9], but it is not clear if TC interacts with the DNA of the tumour cell. Due to the
214
X.-j. Liu et al. /Analytica
Table 1 Tetracycline
derivatives
Chimica Acta 345 (1997) 213-217
studied
OH
OH
0
OH
0
Compound
RI
Tetracycline (TC) Oxytetracycline (OTC) 4-epi-tetracycline (4-epi-TC) Doxycycline (DOTC) Methacycline (MOTC) Chlortetracycline (CTC)
H H H H H Cl
R2 CH3 CH3 CH3
CK CH2
CH3
similarity of Ca2+ and Mg2+ with Eu3+ in binding with DNA, the signal from the interaction between the Eu3+-TC complex and DNA may provide some evidences for the interaction mechanism of these antibiotics in living systems. Here we report a study of the interaction of DNA with six Eu3+ complexes (Table 1).
2. Experimental 2.1. Materials Commercially prepared calf thymus (CT) DNA (Baitai Biochemicals, China) was suspended directly in 50 mM sodium chloride at a final concentration and used without further puriof 100 pg ml-’ fication. A stock solution of Eu3+ (1 .O mM) was prepared by dissolving europium oxide (Eu2O3, Beijing Xinxin Chemicals) in concentrated hydrochloric acid. The solution was evaporated to dryness, and the residue was dissolved in 0.1 M hydrochloric acid. Stock solutions of TC, OTC, 4-epi-TC, DOTC, MOTC and CTC were prepared by dissolving known amounts of their corresponding hydrochlorides (The Chinese Identification Institute for Drugs and Biological Products) in water. More dilute standard solutions were prepared by appropriate dilution with water. A 0.1 M Tris-HCl buffer (pH 8.5) was used.
-
R3
R4
R5
Rh
OH OH OH H OH OH
H OH H OH
NCH& NCHA H
H H
NCH& H
2.2. General experimental
NCH&
NCH& H N(CH&
H
procedures
Typically, samples containing appropriate concentrations of a TC, DNA and europium ion were made up to 10 ml in 50 mM Tris-HCl buffer (pH 8.5). The wavelengths used for fluorescence measurements were X,,,=395 nm and X,,,,=615 nm. Fluorescence readings are given as net fluorescence intensities (in arbitrary units of the instrument). Background fluorescence (from all the reagents except the one being evaluated) was subtracted from each value reported, except for those in excitation and emission spectra. Fluorescence spectra and intensities were measured on a Perkin-Elmer LS-SOB spectrofluorimeter with a pulsed xenon lamp and dual monochromators. The excitation and emission slits were maintained at 10 nm.
3. Results and discussion 3.1. Optimization
of the general procedure
First, the effect of pH on the fluorescence intensities of the Eu3+ complexes of the TC analogues with DNA was compared. The variations in fluorescence intensity as a function of pH in the six systems were similar. The result showed that the maximum emission of the Eu3+ complex occurs in the pH range 8.s9.7. In the subsequent study, pH 8.5 was used.
X.-j. Liu et al. /Analytica Table 2 Absorption
and fluorescence
Chimica Acta 34.5 (1997) 213-217
215
spectral data
Compound
Absorption x maX(nm)
Relative absorption intensity
X,,,, (nm)
x ,em, (nm)
TC OTC 4.epi-TC DOTC MOTC CTC Eu’ ’ -TC Eu3 ’ -OTC Euii4-epi-TC Eu’+-DOTC Eu7 ’ -MOTC EuZ ’ -CTC I%‘+ -TC-DNA Eu’ ’ -OTC-DNA Eu’+A-epi-TC-DNA Eu” -DOTC-DNA Eu’ ’ -MOTC-DNA Eu” -CTC-DNA
370 370 372 365 370 377 395 392 398 392 390 402 396 392 396 392 390 402
0.124 0.124 0.128 0.084 0.118 0.104 0.160 0.152 0.158 0.110 0.138 0.140 0.166 0.156 0.166 0.128 0.140 0.154
387 387 391 387 387 387 394 396 396 395 396 400 394 396 395 395 396 400
510 510 510 510 510 510 615 615 615 615 615 615 615 615 615 615 615 615
Secondly, the influence of Eu3+ concentration on the fluorescence intensity and the fluorescence intensity as a function of the concentration of TCs were further studied. The results showed maximum response at a mole ratio of 1 : 1 for Eu3+ and each TC analogue in the six systems. 3.2. Absorption
and fluorescence
Relative fluorescence intensity
1.oo 0.98 0.96 0.58 0.42 0.38 5.63 5.60 5.13 3.74 I .78 I .66 2x.2 29.3 19.9 13.3 3.97 3.59
(0
24-
spectra
Absorption spectral data of TC analogues, their complexes with Eu3+ as well as these complexes in the presence of DNA are summarized in Table 2. In the presence of Eu3+, the absorption bands of the TCs are red shifted 20-27 nm together with a slight increase in intensity, indicating complex formation between ELI”+ and the TCs. On addition of DNA, the absorption band remains unchanged except for a slight increase in intensity, which indicated that direct binding may not occur between DNA and the TCs. The slight increase in the absorption is related to ternary complex formation between Eu3+-TCs and DNA. In addition, from Table 2, it can be seen that maximum absorption wavelengths of all the TC analogues are nearly the same except for that of CTC, which has a slight red shift. This may be due to their structural difference. In CTC, there is a substituent (Cl) on
J 350
390
430
470
510
550
590
630
Wavelength (nm) Fig. 1, Excitation (left) and emission (right) spectra: tetracycline; (b)l.OPM Eu3++1.0 FM tetracycline; Et?‘+ 1.O pM tetracycline+1 pg ml. ’ CT DNA.
(a) 1.O pM (c)1.0 FM
aromatic ring D, which is the key moiety for the absorption of the whole molecule and is liable to be influenced by substitution. For the other TCs, the substituents are far from the aromatic ring, so they
216
X.-j. Liu ef al./Analytica
Chimica Acta 345 (1997) 213-217
have little effect on the peak wavelength. Among the six TC analogues, TC, 4-epi-TC and OTC as well as their corresponding complexes have relatively stronger peak intensities. The fluorescence excitation and emission spectra of TC analogues, their complexes with Eu3+ as well as these complexes in the presence of DNA were also studied. The fluorescence spectra of TC, Eu3+-TC and Eu3+-TC-DNA are shown in Fig. 1. The fluorescence data of the six TC analogues and their complexes are listed in Table 2. The results showed that upon addition of EL?+, the excitation wavelengths of the TC analogues are slightly red shifted, in agreement with the absorption spectra. The broad emission band of TC and its analogues is replaced with the much narrower and stronger emission band characteristic of the Eu” ion together with a decrease in fluorescence intensity of the free TC. The results also showed that the Eu’+TCs systems have nearly the same excitation wavelength with a maximum at 395 nm, and DNA does not change the excitation and emission wavelength of the binary complex. This is further evidence for the absence of direct interaction between TCs and DNA. Furthermore, they showed that DNA enhanced the fluorescence intensity of the Eu’+-TCs systems. The greater the fluorescence intensity of the binary complex, the stronger that of their corresponding ternary complexes with DNA. For example, the Eu3+--TC and the Eu3+-OTC complexes have relatively strong fluorescence intensities and their fluorescence intensities enhanced by DNA are greater than that of the other TC systems. In the Eu3+-TCs-DNA systems the excitation wavelengths of both binary and ternary complexes are very similar to those of the TCs themselves. Therefore, the fluorescence enhancement mechanism is different from that described earlier for Tb”+ or Eu’+, in which energy transfer from the nucleobases occured. In the present study, the energy transfer is
Table 3 The influence
of DNA on fluorescence
lifetime (T) of Ed+-TCs
evidently from TCs to Eu3+ both in the presence and in the absence of DNA. In order better to understand the fluorescence enhancement mechanism deeply, we studied the influence of DNA on the fluorescence lifetime. 3.3. Influence of DNA on fluorescence
lifetime
The fluorescence lifetime of tripositive lanthanide ions ranges from 1 ps to 2 ms, depending on the emitting ion and the medium. The decay curves corresponding to the Eu3+-TCs chelates and those in the presence of DNA were recorded. The values of fluorescence lifetime (T) are depicted in Table 3. It can be seen that, in the presence of DNA, the fluorescence lifetime of Eu3+ IS ’ generally increased. The extent of the increase is consistent with that of the fluorescence intensity increase. Among the six Eu3+-TCs, Eu’+OTC and Eu3+-OTC-DNA have the longest fluorescence lifetime in both the binary and the ternary systems. 3.4. The interaction
between TCS-Eu3+ and DNA
In the presence of 0.5 M Tris-HCl buffer (pH 8.5), a low concentration of HPOi- can prevent the interaction between the Eu”+-TC complex and DNA, indicating that the Eu3+-TC complex interacts with the phosphate group of DNA, and that Eu3+ acts as a bridge between the phosphate group of DNA and TCs. The binding of the DNA phosphate group to Eu3+ provides the chelated Eu”+ ion with a relatively hydrophobic environment which protects against nonradiative deactivation, which is responsible for the increase of fluorescence intensity and lifetime. According to these results we suggest that DNA does not change the process of energy transfer from TCs to Eu 3+, i.e. DNA does not change the process of fluorescence excitation, but it increases the fluorescence
systems
System
TC-Ed+
OTC-Ed+
4-epi-TC-Eu3-
DOTC-Ed+
MOTC-Ed+
CTC-Ed+
without DNA with DNA
39.2 97.7
38.9 139
27.0 75.1
38.9 67.0
27.0 36.1
15.9 21.0
The unit of 7 is ps.
X.-j. Liu et ul./Analytica
Table 4 Analytical
parameters
for the determination
of CT DNA
System
Linear range (ng ml ‘)
LOD” (ng ml
Eu’ ’ -TC-DNA
10.0-500 soo- IO00 5.0-500 500-l 500
5.0
Eu ’ +~OT(‘~DNA
“Limit of detection (signal/noise ratio=2). Vorrelation coefficient for seven measurements. ‘Relative standard deviation for seven measurements
’)
2.4
of 100 or 500 ng ml
lifetime and improves the fluorescence quantum yield the nonradiative deactivation, through decreasing resulting in the enhancement of the fluorescence intensity. 3.5. Determination ,fluorescence
217
Chimiccr Acta 345 (lYY7J 213-217
of DNA by using time-resolved
From the above discussion, it can be seen that of the six Et?+ complexes of TC analogues the fluorescence of the Eu3+-OTC and EL?--TC complexes are enhanced more by DNA. Therefore, these two systems were compared. The optimal time-resolved properties of the two systems were investigated. The delay time was selected as 60 us and optimal gate time was selected as 1 ms to give the maximal signal/noise ratio. Under the conditions established above, CT DNA was determined by time-resolved fluorescence in the two systems. The results are summarized in Table 4, which show that the ELI”+-OTC complex is a more sensitive fluorescence probe for determining DNA than the ELI”-TC complex. The detection limits for DNA are 2.4 and 5.0 ng ml--’ in the OTC and TC systems. respectively.
4. Conclusions No evident direct interaction between TCs and DNA was observed. The EL?+-TCs complexes may interact with the phosphate groups of DNA through
rh
RSD’
0.9938 0.9786 0.9962 0.9749
2.8 2.9 2.9 2.4
’ DNA.
Et?‘, bringing the TCs closer to DNA. The antibiotic function of TCs may follow the similar mechanism, i.e. the Ca2’ and Mg2+ in living systems may act as a bridge between TCs and DNA. The results showed that DNA did not influence the energy transfer from TCs to Eu’ ‘, but changes the Et?+ emission process leading to enhancement of the fluorescence intensity. Of the six Et?+-TCs complexes studied, the ELI”+OTC complex is the most sensitive fluorescence probe for determining DNA.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 29575 19 1).
References 111S. Udenfriend and P. Zaltzman. Anal. Biochem.. 3 (1962) 49.
PI H.C. Borresen, Acta Chem. Stand.. I7 ( 1963) 921. 1x1N. Narayana, B. Pumima and C. Dipankar. Biopolymers.
32 (1992) 1421. [41 J. George? and S. Ghazarian, Anal. Chim. Acta, 276 ( 1993) 401. ISI T.J. Wenzel and L.M. Collette, J. Chromatogr., 436 (1988) 299. 161 Y.X. Ci. Y.Z. Li and X.J. Liu. Anal. Chem., 67 (1995) 1785. 171 K.H. Ibsen and M.R. Urist. Proc. Sot. Exp. Bio. Med., I09 (1962) 797. L81 A. Cammarata and S.J. Yau, J. Med. Chem., I3 (1970) 93. [91 A.L. Dunn, C.D. Eskelson, J.F. Mcleay and R.E. Ogborn, Proc. Sot. Exp. Bio. Med., 104 (1960) I?_.
Analytica
Chimica
Acta 345 ( 1997) 2 13-2 I7
Time-resolved fluorescence studies of the interaction of the Eu3+ complexes of tetracycline analogues with DNA Xiao-jing Liu, Yuan-zong Li, Yun-xiang Ci* Department
Received 21 October
c$ Chemistry,
Peking
Uniwxtiry,
Beijing
100871,
China
1996; received in revised form 10 January 1997: accepted 22 January 1997
Abstract The interaction of the ELI” compIexes of tetracycline analogues with DNA has been examined by using LJV-visible absorption and fluorescence spectra. Among the tetracycline series, the europium fluorescence sensitized by tetracycline (TC) and oxytetracycline (OTC) were enhanced by DNA more than those of other antibiotics in the series. Therefore, EL?+-TCDNA and EL?+-OTC-DNA systems were investigated by time-resolved fluorescence. The Et?‘-OTC complex is a more sensitive fluorescence probe for selective determination of DNA than the EL?* -TC complex. Detection limits (signal/ noise=2) for DNA are 2.4 and 5.0 ng ml-’ for the OTC and TC systems, respectively. Keywor&: UV-visible spectrometry; fluorescence; Europium; Tetracycline; Oxytetracycline: DNA
1. Introduction The direct use of the fluorescence emission properties of nucleic acids to investigate their biological properties has been limited [ 1,2]. whereas the use of lanthanide(II1) cations as fluorescence probes for the structure and function of nucleic acids has increased markedly. Most of the research has been on Tb3+ and EL?+. Narayana et al. have studied Tb”+induced fluorescence of four-stranded G4-DNA that gave some useful information on DNA conformation [3]. The europium fluorescence sensitized by tetracycline (TC) in a micellar solution of Triton X- 100, via the formation of an organic chelate, was used for the sensitive detection of TC [4]. The similar intramolecular energy transfer from ligand (TC) to the lantha*Corresponding
author.
000%2670/97/$17.00 (‘, 1997 Elsevier Science B.V. All rights reserved P/f
SOOO3-2670(97)00084-6
nide ion was used to enhance the fluorescence of the ligand, which has been applied in liquid chromatographic analysis [5]. Recently, we have reported that the EL?+-TC complex is a sensitive fluorescence probe for determining double-stranded and singlestranded DNA without interference from RNA [6]. This finding aroused our interest for the systematic exploitation of the interaction of EL? complexes of TC analogues with DNA. It is expected that this study may shift out more sensitive fluorescence probes for determining DNA. In addition, it is well known that TC and its analogues have been widely used as antibiotics, and these antibiotics chelate with cations such as Mg2+ and Ca’+. This chelation plays a determining role in the bacteriostatic properties of the antibiotics [7,8]. Furthermore, it had been discovered that TC can combine with tumour cell [9], but it is not clear if TC interacts with the DNA of the tumour cell. Due to the
214
X.-j. Liu et al. /Analytica
Table 1 Tetracycline
derivatives
Chimica Acta 345 (1997) 213-217
studied
OH
OH
0
OH
0
Compound
RI
Tetracycline (TC) Oxytetracycline (OTC) 4-epi-tetracycline (4-epi-TC) Doxycycline (DOTC) Methacycline (MOTC) Chlortetracycline (CTC)
H H H H H Cl
R2 CH3 CH3 CH3
CK CH2
CH3
similarity of Ca2+ and Mg2+ with Eu3+ in binding with DNA, the signal from the interaction between the Eu3+-TC complex and DNA may provide some evidences for the interaction mechanism of these antibiotics in living systems. Here we report a study of the interaction of DNA with six Eu3+ complexes (Table 1).
2. Experimental 2.1. Materials Commercially prepared calf thymus (CT) DNA (Baitai Biochemicals, China) was suspended directly in 50 mM sodium chloride at a final concentration and used without further puriof 100 pg ml-’ fication. A stock solution of Eu3+ (1 .O mM) was prepared by dissolving europium oxide (Eu2O3, Beijing Xinxin Chemicals) in concentrated hydrochloric acid. The solution was evaporated to dryness, and the residue was dissolved in 0.1 M hydrochloric acid. Stock solutions of TC, OTC, 4-epi-TC, DOTC, MOTC and CTC were prepared by dissolving known amounts of their corresponding hydrochlorides (The Chinese Identification Institute for Drugs and Biological Products) in water. More dilute standard solutions were prepared by appropriate dilution with water. A 0.1 M Tris-HCl buffer (pH 8.5) was used.
-
R3
R4
R5
Rh
OH OH OH H OH OH
H OH H OH
NCH& NCHA H
H H
NCH& H
2.2. General experimental
NCH&
NCH& H N(CH&
H
procedures
Typically, samples containing appropriate concentrations of a TC, DNA and europium ion were made up to 10 ml in 50 mM Tris-HCl buffer (pH 8.5). The wavelengths used for fluorescence measurements were X,,,=395 nm and X,,,,=615 nm. Fluorescence readings are given as net fluorescence intensities (in arbitrary units of the instrument). Background fluorescence (from all the reagents except the one being evaluated) was subtracted from each value reported, except for those in excitation and emission spectra. Fluorescence spectra and intensities were measured on a Perkin-Elmer LS-SOB spectrofluorimeter with a pulsed xenon lamp and dual monochromators. The excitation and emission slits were maintained at 10 nm.
3. Results and discussion 3.1. Optimization
of the general procedure
First, the effect of pH on the fluorescence intensities of the Eu3+ complexes of the TC analogues with DNA was compared. The variations in fluorescence intensity as a function of pH in the six systems were similar. The result showed that the maximum emission of the Eu3+ complex occurs in the pH range 8.s9.7. In the subsequent study, pH 8.5 was used.
X.-j. Liu et al. /Analytica Table 2 Absorption
and fluorescence
Chimica Acta 34.5 (1997) 213-217
215
spectral data
Compound
Absorption x maX(nm)
Relative absorption intensity
X,,,, (nm)
x ,em, (nm)
TC OTC 4.epi-TC DOTC MOTC CTC Eu’ ’ -TC Eu3 ’ -OTC Euii4-epi-TC Eu’+-DOTC Eu7 ’ -MOTC EuZ ’ -CTC I%‘+ -TC-DNA Eu’ ’ -OTC-DNA Eu’+A-epi-TC-DNA Eu” -DOTC-DNA Eu’ ’ -MOTC-DNA Eu” -CTC-DNA
370 370 372 365 370 377 395 392 398 392 390 402 396 392 396 392 390 402
0.124 0.124 0.128 0.084 0.118 0.104 0.160 0.152 0.158 0.110 0.138 0.140 0.166 0.156 0.166 0.128 0.140 0.154
387 387 391 387 387 387 394 396 396 395 396 400 394 396 395 395 396 400
510 510 510 510 510 510 615 615 615 615 615 615 615 615 615 615 615 615
Secondly, the influence of Eu3+ concentration on the fluorescence intensity and the fluorescence intensity as a function of the concentration of TCs were further studied. The results showed maximum response at a mole ratio of 1 : 1 for Eu3+ and each TC analogue in the six systems. 3.2. Absorption
and fluorescence
Relative fluorescence intensity
1.oo 0.98 0.96 0.58 0.42 0.38 5.63 5.60 5.13 3.74 I .78 I .66 2x.2 29.3 19.9 13.3 3.97 3.59
(0
24-
spectra
Absorption spectral data of TC analogues, their complexes with Eu3+ as well as these complexes in the presence of DNA are summarized in Table 2. In the presence of Eu3+, the absorption bands of the TCs are red shifted 20-27 nm together with a slight increase in intensity, indicating complex formation between ELI”+ and the TCs. On addition of DNA, the absorption band remains unchanged except for a slight increase in intensity, which indicated that direct binding may not occur between DNA and the TCs. The slight increase in the absorption is related to ternary complex formation between Eu3+-TCs and DNA. In addition, from Table 2, it can be seen that maximum absorption wavelengths of all the TC analogues are nearly the same except for that of CTC, which has a slight red shift. This may be due to their structural difference. In CTC, there is a substituent (Cl) on
J 350
390
430
470
510
550
590
630
Wavelength (nm) Fig. 1, Excitation (left) and emission (right) spectra: tetracycline; (b)l.OPM Eu3++1.0 FM tetracycline; Et?‘+ 1.O pM tetracycline+1 pg ml. ’ CT DNA.
(a) 1.O pM (c)1.0 FM
aromatic ring D, which is the key moiety for the absorption of the whole molecule and is liable to be influenced by substitution. For the other TCs, the substituents are far from the aromatic ring, so they
216
X.-j. Liu ef al./Analytica
Chimica Acta 345 (1997) 213-217
have little effect on the peak wavelength. Among the six TC analogues, TC, 4-epi-TC and OTC as well as their corresponding complexes have relatively stronger peak intensities. The fluorescence excitation and emission spectra of TC analogues, their complexes with Eu3+ as well as these complexes in the presence of DNA were also studied. The fluorescence spectra of TC, Eu3+-TC and Eu3+-TC-DNA are shown in Fig. 1. The fluorescence data of the six TC analogues and their complexes are listed in Table 2. The results showed that upon addition of EL?+, the excitation wavelengths of the TC analogues are slightly red shifted, in agreement with the absorption spectra. The broad emission band of TC and its analogues is replaced with the much narrower and stronger emission band characteristic of the Eu” ion together with a decrease in fluorescence intensity of the free TC. The results also showed that the Eu’+TCs systems have nearly the same excitation wavelength with a maximum at 395 nm, and DNA does not change the excitation and emission wavelength of the binary complex. This is further evidence for the absence of direct interaction between TCs and DNA. Furthermore, they showed that DNA enhanced the fluorescence intensity of the Eu’+-TCs systems. The greater the fluorescence intensity of the binary complex, the stronger that of their corresponding ternary complexes with DNA. For example, the Eu3+--TC and the Eu3+-OTC complexes have relatively strong fluorescence intensities and their fluorescence intensities enhanced by DNA are greater than that of the other TC systems. In the Eu3+-TCs-DNA systems the excitation wavelengths of both binary and ternary complexes are very similar to those of the TCs themselves. Therefore, the fluorescence enhancement mechanism is different from that described earlier for Tb”+ or Eu’+, in which energy transfer from the nucleobases occured. In the present study, the energy transfer is
Table 3 The influence
of DNA on fluorescence
lifetime (T) of Ed+-TCs
evidently from TCs to Eu3+ both in the presence and in the absence of DNA. In order better to understand the fluorescence enhancement mechanism deeply, we studied the influence of DNA on the fluorescence lifetime. 3.3. Influence of DNA on fluorescence
lifetime
The fluorescence lifetime of tripositive lanthanide ions ranges from 1 ps to 2 ms, depending on the emitting ion and the medium. The decay curves corresponding to the Eu3+-TCs chelates and those in the presence of DNA were recorded. The values of fluorescence lifetime (T) are depicted in Table 3. It can be seen that, in the presence of DNA, the fluorescence lifetime of Eu3+ IS ’ generally increased. The extent of the increase is consistent with that of the fluorescence intensity increase. Among the six Eu3+-TCs, Eu’+OTC and Eu3+-OTC-DNA have the longest fluorescence lifetime in both the binary and the ternary systems. 3.4. The interaction
between TCS-Eu3+ and DNA
In the presence of 0.5 M Tris-HCl buffer (pH 8.5), a low concentration of HPOi- can prevent the interaction between the Eu”+-TC complex and DNA, indicating that the Eu3+-TC complex interacts with the phosphate group of DNA, and that Eu3+ acts as a bridge between the phosphate group of DNA and TCs. The binding of the DNA phosphate group to Eu3+ provides the chelated Eu”+ ion with a relatively hydrophobic environment which protects against nonradiative deactivation, which is responsible for the increase of fluorescence intensity and lifetime. According to these results we suggest that DNA does not change the process of energy transfer from TCs to Eu 3+, i.e. DNA does not change the process of fluorescence excitation, but it increases the fluorescence
systems
System
TC-Ed+
OTC-Ed+
4-epi-TC-Eu3-
DOTC-Ed+
MOTC-Ed+
CTC-Ed+
without DNA with DNA
39.2 97.7
38.9 139
27.0 75.1
38.9 67.0
27.0 36.1
15.9 21.0
The unit of 7 is ps.
X.-j. Liu et ul./Analytica
Table 4 Analytical
parameters
for the determination
of CT DNA
System
Linear range (ng ml ‘)
LOD” (ng ml
Eu’ ’ -TC-DNA
10.0-500 soo- IO00 5.0-500 500-l 500
5.0
Eu ’ +~OT(‘~DNA
“Limit of detection (signal/noise ratio=2). Vorrelation coefficient for seven measurements. ‘Relative standard deviation for seven measurements
’)
2.4
of 100 or 500 ng ml
lifetime and improves the fluorescence quantum yield the nonradiative deactivation, through decreasing resulting in the enhancement of the fluorescence intensity. 3.5. Determination ,fluorescence
217
Chimiccr Acta 345 (lYY7J 213-217
of DNA by using time-resolved
From the above discussion, it can be seen that of the six Et?+ complexes of TC analogues the fluorescence of the Eu3+-OTC and EL?--TC complexes are enhanced more by DNA. Therefore, these two systems were compared. The optimal time-resolved properties of the two systems were investigated. The delay time was selected as 60 us and optimal gate time was selected as 1 ms to give the maximal signal/noise ratio. Under the conditions established above, CT DNA was determined by time-resolved fluorescence in the two systems. The results are summarized in Table 4, which show that the ELI”+-OTC complex is a more sensitive fluorescence probe for determining DNA than the ELI”-TC complex. The detection limits for DNA are 2.4 and 5.0 ng ml--’ in the OTC and TC systems. respectively.
4. Conclusions No evident direct interaction between TCs and DNA was observed. The EL?+-TCs complexes may interact with the phosphate groups of DNA through
rh
RSD’
0.9938 0.9786 0.9962 0.9749
2.8 2.9 2.9 2.4
’ DNA.
Et?‘, bringing the TCs closer to DNA. The antibiotic function of TCs may follow the similar mechanism, i.e. the Ca2’ and Mg2+ in living systems may act as a bridge between TCs and DNA. The results showed that DNA did not influence the energy transfer from TCs to Eu’ ‘, but changes the Et?+ emission process leading to enhancement of the fluorescence intensity. Of the six Et?+-TCs complexes studied, the ELI”+OTC complex is the most sensitive fluorescence probe for determining DNA.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 29575 19 1).
References 111S. Udenfriend and P. Zaltzman. Anal. Biochem.. 3 (1962) 49.
PI H.C. Borresen, Acta Chem. Stand.. I7 ( 1963) 921. 1x1N. Narayana, B. Pumima and C. Dipankar. Biopolymers.
32 (1992) 1421. [41 J. George? and S. Ghazarian, Anal. Chim. Acta, 276 ( 1993) 401. ISI T.J. Wenzel and L.M. Collette, J. Chromatogr., 436 (1988) 299. 161 Y.X. Ci. Y.Z. Li and X.J. Liu. Anal. Chem., 67 (1995) 1785. 171 K.H. Ibsen and M.R. Urist. Proc. Sot. Exp. Bio. Med., I09 (1962) 797. L81 A. Cammarata and S.J. Yau, J. Med. Chem., I3 (1970) 93. [91 A.L. Dunn, C.D. Eskelson, J.F. Mcleay and R.E. Ogborn, Proc. Sot. Exp. Bio. Med., 104 (1960) I?_.