2252
J . Phys. Chem. 1986, 90, 2252-2259
Quenching of the Fkorescence of DNA-Intercalated Ethidium Bromide by Some Transition-Metal Ions Stephen J. Atherton* Center f o r Fast Kinetic Research, Patterson No. 131, University of Texas at Austin, Austin, Texas 7871 2
and Paul C. Beaumont Research Division School of Natural Sciences, The North East Wales Institute, Deeside, Clwyd CH5 4BR, U.K. (Received: October 15, 1985)
We have studied the quenching of fluorescencefrom DNA-intercalated ethidium bromide excited states by the transition-metal ions Cu2+,Ni2+,and Co2+. Quenching by all three metal ions leads to biphasic fluorescence decay. We suggest that this results from an equilibrium between metal ions bound to the DNA phosphate groups and metal ions mobile around the DNA helix. A quantitative model is given and allows us to deduce that the metal ions may be arranged in order of increasing mobility as Cu2+ < Ni2+ < Coz+. The mobility of Co2+is a factor of ca. 20 less than in water. We conclude that the metal ions move from base to base at a rate between 4 X lo7 and 2 X lo8 s-’ and have a residence time on the phosphate group between ca. 5 and 20 ns. Quenching of fluorescence may occur from metal ions bound to any of the nearest 6 phosphate groups to the intercalated excited state, a distance of ca. 1 nm. Quenching is suggested to occur via electron transfer from excited ethidium bromide to the metal ion.
Introduction We have recently been interested in the quenching of fluorescence of DNA-intercalated ethidium bromide by transition-metal i0ns.l It is well established that some such ions are essential for the accurate and efficient replication of DNA and, indeed, play significant roles in many biological processes. Another motivation for pursuing such a line of study is the relative lack of excited-state quenching studies in DNA.2 In view of the wealth of data concerning excited-state quenching in proposed models of biological systems, e.g. micelles and ves~icles,~ we feel that study of an in vitro biochemical system using these techniques is overdue. Previous studies of excited-state quenching in DNA, which are pertinent to the present work, have included those of Prusik and Geacintov2cwho observed quenching of the triplet state of acridine orange intercalated within DNA by Ag’ and Mn2+ions. In this instance the triplet lifetime of acridine orange in the absence of quenchers was relatively long (tens of milliseconds) and on this time scale the metal ions are mobile along the DNA strand. Single-exponent decay of the triplet population was observed under all quenching conditions. Importantly, the rate of base to base “hopping” was determined as ?lo5 and 1 5 X lo2 s-I for Mn2+ and Ag’, respectively. At 77 K, double-exponential behavior was observed and attributed to the lack of metal ion mobility resulting in two populations of triplet states, one with metal ion within the quenching radius and one without. Unlike at room temperature these populations are noninterchangable on the time scale of triplet decay because of severely reduced metal ion mobility. This double-exponential behavior has also been observed at room temperature by Baguley and Le Bret,2f who studied fluorescence quenching of DNA-intercalated ethidium bromide as a function (1) Atherton, S. J.; Beaumont, P. C. Photobiochem. Phofobiophys. 1984, 8, 103. (2) (a) Green, B. Eur. J. Biochem. 1970, 14, 567. (b) Galley, W. C.; Purkey, R. M. Proc. Natl. Acad. Sci. U.S.A. 1972,69, 2198. (c) Prusik, T.; Geacintov, N. E. FEES Lett. 1976, 71, 236. (d) Le Bret, M.; Le Pecq,J.-B.; Barbet, J.; Roques, B. P. Nucleic Acids Res. 1977, 4 , 1361. (e) Prusik, T.; Kolubayev, T.; Morelli, M. J.; Brenner, H. C. Photochem. Photobiol. 1980, 31, 315. (f) Baguley, B. C.; Le Bret, M. Biochemisfry 1984, 23, 937. (3) (a) Infelta, P. P.; Grltzel, M.; Thomas, J. K. J . Phys. Chem. 1974, 78, 190. (b) Rodgers, M. A. J.; Wheeler, M. F. S. Chem. Phys. Lett. 1978,53, 165. (c) Turro, N. J.; Yekta, A. J. A m . Chem. Soc. 1978, 100, 5951. (d) Thomas, J . K. Chem. Reu. 1980,80, 283. ( e ) Rodgers, M. A. J.; Becker, J. C. J . Phys. Chem. 1980.84, 2762. (f) Grieser, F.; Tausch-Treml, R. J . Am. Chem. SOC.1980, 102, 7258. (9) Dederen, J. C.; Van der Auweraer, M.; De Schryver, F. C. J. Phys. Chem. 1981,85, 1198. (h) Atherton, S. J.; Baxendale, J. H.; Hoey, B. M. J. Chem. Soc., Faraday Trans I 1982, 78, 2167.
0022-3654/86/2090-2252$01.50/0
of added antitumor agents, e.g. amacrine. The double-exponential behavior was attributed to “a proportion of the ethidium bromide molecules being highly quenched, with the proportion varying for different amsacrine binding ratios”. Quenching was suggested to occur via reversible formation of electron-transfer complexes between the antitumor agent and ethidium bromide excited state. In the present work we describe the quenching of ethidium bromide excited-state fluorescence by the metal ions Ni2+,Cu2+, and Co2+in aqueous solutions of DNA. As stated previously,’ and as will be shown, double-exponential fluorescence decays are observed. We describe a quantitative model for this behavior which allows us to deduce quenching rate constants, the approximate range over which quenching can occur, and a measure of the mobility of the metal ions around the DNA. We have chosen the above metal ions as they are known to bind preferentially to the DNA phosphate group^,^ at least under the low metal ion to DNA phosphate ratios employed here. Ethidium bromide was chosen as a fluorescent probe as it is strongly intercalated within DNA (binding constant = 2.1 X lo6 M-l)s without regard to base pair composition.6 Also the fluorescence lifetime of ethidium bromide excited state is dramatically increased when intercalated in DNA (1.8 ns in pure water vs. 22.5 ns in DNA).’ In our experiments all ethidium bromide will be intercalated and if the metal ions perturb the DNA structure sufficiently to eject the ethidium bromide into the aqueous phase we would expect to see a component of fluorescence with a lifetime of 1.8 ns. The treatment of some of the data will be similar to that previously formulated for micelle quenching
experiment^.^^^^ Experimental Section Ethidium bromide and DNA (calf thymus, type 1) were from Sigma and used as received. Nickel(I1) chloride, cobalt(I1) chloride, copper(I1) sulfate, and sodium sulfate were all of the best available grade and used as received. Water was purified by a Millipore filtration system. DNA stock solutions were prepared by dissolution overnight in water containing the ap(4) Izatt, R. M.; Christensen, J. J.; Rytting, J. H. Chem. Rev. 1971, 71, 349. (5) Gaugain, B.; Barbet, J.; Capelle, N.; Roques, B. P.; Le Pecq, J.-B. Biochemisirv 1978. 17. 5078. (6) Le Pecq, J.-B.; Paoletti, C. J. Mol. Biol. 1967, 27, 8 7 . (7) Burns, V. W. F. Arch. Biochem. Biophys. 1969, 183, 420. (8) (a) Infelta, P. P. Chem. Phys. L e f f .1979, 61, 88. (b) Dederen, J. C.; Van der Auweraer, M.; De Schryver, F.C. Chem. Phys. Left. 1979,68,451.
0 1986 American Chemical Society
The Journal of Physical Chemistry, Vol. 90, No. 10, 1986 2253
Fluorescence of DNA-Intercalated Ethidium Bromide
1"'
I
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1.1 IO
10
2.2
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500
510
100
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310
280
210
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Figure 1. Fluorescence excitation spectra for solutions of ethidium bromide M) and Na2S04(5 X lo-) M) containing (a) 0.02% DNA; (b) 0.02% D N A + 1.6 X lo4 M Co2+; (c) 0.02% D N A + 1.6 X lo4 M NiZ+;(d) 0.02% D N A + 1.6 X M Cuz+; (e) 0. Scales a to d, 0 to 100; e 0 to 6.6. Emission monitored at 600 nm.
propriate salt (Na,SO,) concentration. The salt concentration was always greater than M to inhibit denaturation? Samples were freshly made up on the day of use and all remaining DNA stock was discarded at the end of the day. We encountered some irreproducibility among different batches of DNA from the same company, in that some batches failed to dissolve completely and a fine white suspension remained. These batches were not used in the experiments and we found all other batches gave the same results within experimental error. Steady-state absorption measurements were made on a Hewlett Packard 8540A UV-visible spectrometer. Steady-state fluorescence measurements were made on a Perkin-Elmer M P F 43A luminescence spectrometer. For the time-resolved fluorescence measurements we employed two laser systems. In one the 532-nm second harmonic, 200-ps pulse from a Quantel YG402 Nd:YAG laser was the excitation source. Fluorescence was observed perpendicular to the excitation beam via a monochromator (Spex Minimate), PM tube (Hamamatsu R306 using only 5 dynodes to produce a response time of 0.5 ns), digitizer (Tektronix R7912) assembly. In these experiments fluorescence was observed at 620 nm, through other observation wavelengths gave equivalent results. The digitizer was interfaced with a Digital Equipment PDPl 1/70 minicomputer for data analysis. For each sample ten separate decay traces were averaged to give the final result. The other laser system comprised a Spectra Physics Series 3000 Nd:YAG laser mode locked at 82 MHz. The 532-nm second harmonic of this laser pumped a Spectra Physics Model 375B dye laser containing rhodamine 6G. In this way a train of 82-MHz, ca. 6-ps pulses is produced tunable around the 590-nm dye maximum. The frequency of this train was then reduced with a Spectra Physics Model 344 cavity dumper and frequency doubled with the appropriate nonlinear crystal. In our experiments the dye laser was tuned to 610 nm, cavity dumped at 0.8 M H z and frequency doubled to 305 nm to provide the excitation source. Fluorescence was monitored perpendicular to the excitation source in a conventional single-photon-counting arrangement. A small portion of the beam was deflected onto a Hamamatsu R306 PM tube to provide the start pulse and the fluorescence was detected with a Hamamatsu R928 PM tube which provided the stop pulse. Signals were processed with a conventional array of time-resolved single-photon-counting equipment, Ortec fast amplifiers, discriminators, and time-to-height converter and a Tracor Northern TN-7200 multichannel analyzer. The multichannel analyzer was interfaced to the on-line PDPlI/70 for data analysis. The computer system and analysis software have been described previously.'o Monitoring fluorescence with either a horizontal or (9) Eichhorn, G. L. In Inorganic Biochemistry, Eichhorn, G . L., Ed.; Elsevier: Amsterdam, 1975; pp 1210-1243.
Figure 2. Effect of addition of Cu2+ on the fluorescence of DNA-intercalated ethidium bromide (EBr). (A) 0.02% DNA, M Na2S04, M EBr, semilog transform of fluorescence decay. Solid line represents best fit to a single exponential. Insert shows autocorrelation function. (B) as A with lo4 M Cu2+,semilong transform of fluorescence decay. Solid line represents best fit to a single exponential of the fast decaying portion of fluorescence, drawn to accentuate deviations at long time. Insert i shows the autocorrelation function for a double-exponential fit; insert ii, the autocorrelation function for a single-exponential fit. In both traces one horizontal division is 25 ns.
vertical polarization filter before the PM tube gave identical results under all conditions. There was no difference in results produced when either laser system was used which confirms that possible energy transfer from DNA to ethidium bromide under 305-nm excitation is unimportant in this work. We will be presenting data mainly obtained from the single-photon-counting system since this gave superior signal-to-noise characteristics.
Results Figure 1 shows the fluorescence excitation spectra of ethidium bromide in aqueous solution, DNA solution, and DNA solution containing the maximum amount of each metal used in the following study. Note that the addition of the metal ions produces little change in shape of the excitation spectrum over that obtained in metal-ion-free DNA solution. Certainly the figure gives no indication that the metal ions cause ejection of the ethidium bromide into the aqueous phase and provides evidence that these concentrations of metal ions do not disrupt the D N A structure. Similar conclusions were inferred from consideration of the absorption and emission spectra of the same solutions (not shown). Figure 2 shows the effect of addition of Cu2+ M) to a D N A solution (0.02% w/v) containing N a 2 S 0 4 M) and ethidium bromide M). Trace A is a semilog transform of the fluorescence in the absence of Cu2+. The autocorrelation function (inset) confirms monoexponential decay. In the absence (10) Foyt, D. C. Comput. Chem. 1981, 5 , 49.
Atherton and Beaumont I
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7
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Figure 3. Plots of the decay rate, k,, of the slower fluorescence component vs. total Cu2+concentration: (+) 0.01%DNA, 5 X M Na2S04, M EBr; (0)0.02% DNA, 5 X M Na2S04, M EBr; (A) 0.03% DNA, 5 X loT3M Na2S04, M EBr; (0)0.02% DNA, M Na2S04, M EBr; ( X ) 0.03% DNA, M Na2S04, M EBr. EBr stands for ethidium bromide.
of quenching metal ions, all conditions of DNA, salt, and ethidium bromide employed resulted in excellent first-order behavior with the same lifetime of 23 f 1 ns. Trace B shows the semilog transform of the fluorescence in the presence of Cu2+. Consideration of the autocorrelation functions for double- and singleexponential decay (inserts i and ii, respectively) confirms that we are observing double-exponential fluorescence decay. Addition of any of the metal ions Cu2+,Ni2+,or Co2+under all conditions of DNA, salt, or ethidium bromide always resulted in doubleexponential behavior. Further as the metal ion concentration increased the following effects on the fluorescence were observed. (i) The extent of the faster component increased at the expense of the slower component. Total signal height however remained constant. (ii) The decay rate of the fast component was nonlinear in metal ion concentration and in many instances remained roughly constant, independent of both the concentration and identity of the metal ion. (iii) The rate of the slower component increased in a manner depending upon which metal ion was employed. A selection of the observed data is shown in Table I, where kf denotes the rate of the faster exponential decay and k, denotes the rate of the slower expotential decay. The relative intensities of the two components have been expressed as the ratio of the total emission, I,,to the slower emission, I,. For each fluorescence signal both the exponential decays were extrapolated back to time zero, taken to be the middle of the exciting pulse. I, was then taken to be the sum of the two extrapolations. I , was found to be independent of metal ion concentration therefore there is no "static" quenching and we are observing all quenching phenomena in real time. Note that we have given two sets of data for each metal ion changing only one parameter between each set. We have plotted the rate of the slower portion of fluorescence as a function of total added metal ion for each metal, under varying
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Figure 4. Plots of the decay rate, k,, of the slower fluorescence component vs. total Ni2+concentration: (+) 0.01%DNA, 5 X M Na2S04, M EBr; (0)0.02% DNA, 5 X lo-) M Na2S04,2 X M EBr; ( X ) 0.03% DNA, 5 X 10-3 M Na2S04, EBr.
6
5
4
Figure 5. Plots of the decay rate, k,, of the slower fluorescence compoM nent vs. total Co2+ concentration: (+) 0.01% DNA, 2.5 X M EBr; (0)0.01% DNA, 5 X lo-' M Na2S04, M Na2S04, EBr; (D) 0.02 DNA, 5 X M Na2S04, M EBr; (X) 0.03% DNA, 5X M Na2S04, M EBr.
conditions of salt, DNA, and ethidium bromide concentrations. These plots are shown in Figures 3-5. Table I1 shows the bimolecular rate constants obtained from the slopes of these plots, assuming this is a diffusional quenching process. For both Cuz+ and Ni2+these constants were derived from the linear portion of
Fluorescence of DNA-Intercalated Ethidium Bromide TABLE I: Selection of Fluorescence Decay Parameters As Described in Text 0.01% DNA,
0 1
1.5 2 2.5 3 3.5 4 5 6 7 8 0.02% DNA, 0 2 3 4 6 8 10 12 14 16
M Na2S04, M EBr 4.48 1 1.22 4.49 1.18 1.26 4.49 1.24 1.76 4.74 1.27 1.56 4.86 1.40 1.59 4.94 1.38 1.63 5.06 1.47 2.22 5.42 1.42 2.00 5.58 1.62 2.45 6.03 1.73 2.55 6.36 1.77 2.41 6.60 1.82 M Na2S04, M EBr 4.57 1 0.89 4.20 1.28 0.87 4.22 1.35 1.39 4.48 1.28 1.48 4.73 1.38 1.68 5.12 1.53 2.13 5.64 1.74 1.96 5.99 1.94 2.34 6.75 1.95 2.43 7.14 2.14
[Ni2+]/10-5M kf/lOBs-' k,/107 S-' 4 / 1s 0.02% DNA. 5 X lo-' M N a S O , . M EBr 0 4141 1 2 4.48 4.75 1.12 3 1.81 4.65 1.17 4 1.83 4.76 1.26 5 2.80 5.12 1.34 6 2.35 5.33 1.43 7 2.47 5.64 1.46 8 2.69 6.10 1.51 9 3.50 6.47 1.60 10 3.76 6.58 1.67 0.02% DNA, M Na2S04, M EBr 0 4.42 1 2 1.94 4.48 1.14 4 2.56 4.74 1.25 6 3.00 5.20 1.33 8 2.55 5.46 1.45 10 3.30 5.90 1.51 12 3.07 6.18 1.58 14 2.55 6.29 1.66 [ C 0 ~ + ] / 1 0 -M ~ kf/108 s-I k,/107 s-I 4 / 1, 0.02% DNA, 5 X lo-' M Na2S04,5 X 10" M EBr 0 4.42 1 4 1.68 4.65 1.26 6 1.94 5.00 1.37 8 2.15 5.3 1 1.41 10 2.28 5.57 1.46 12 2.40 5.81 1.45 14 2.30 5.92 1.54 16 2.38 6.09 1.58 0.02% DNA, 5 X lo-) M Na2S04, 0 4.59 4 1.90 4.78 6 1.67 5.03 8 2.08 5.34 10 2.07 5.55 12 2.06 5.76 14 2.06 5.93 16 2.25 6.01
M EBr 1 1.27 1.38 1.43 1.47 1.51 1.53 1.55
the curves at higher metal ion concentration. Where no linear portion is observed the rate constants are omitted. It must be stressed that there are large errors in these rate constants, particularly for Cu2+ and Ni2+ where the curves are by no means linear and extracted rate constants are dependent on which points are included in the linearization. This is highly subjective on the part of the authors, through hopefully we have included enough
The Journal of Physical Chemistry, Vol. 90, No. 10, 1986 2255 TABLE 11: Bimolecular Rate Constants for Quenching of Slower Fluorescence Comwnent (See Text) [DNA]/(w/v%) [Na2S0,1/M [EBrl/M kh/lO" M-' s-I cu2+ 0.01 0.02 0.02 0.02 0.03 0.03
5 x lo-' 5 x 10-3 10-2 10-2 5 x 10-3 10-2
0.01 0.02 0.02 0.02 0.03
5 5 5 5 5
0.01 0.01 0.02 0.02 0.03
2.5 x 10-3 5 x 10-3 5 x 10-3 5 x 10-3 5 x 10-3
10-5 10-5 10-5 5 x 10" 10-5
10-5
8.9 5.1 2.6 2.8 4.6 2.2
Ni2+
x 10-3 x x x x
10-5 2 x 10-5 5 x 10-6
10-3 10-3 lo-' 10-3
10-5
3.8 3.1 3.1 3.1 2.6
10-5 10-5 5 x 10-6 10-5 10-5
2.6 1.8 1.1 1.1 0.99
10-5
co2+
raw data to enable any reanalysis on the part of the reader. We believe that the results obtained are sufficient to draw meaningful conclusions.
Discussion Addition of each of the three metal ions, to any of the systems considered, always leads to double-exponential decay of fluorescence. This behavior indicates that during the time scale of fluorescence there must be two noninterchangable populations of ethidium bromide excited states; any other scheme leads to monoexponential decay. In our case neither population decays with a lifetime longer than that observed in the absence of metal ions, and we believe that the changes in rate are due to quenching of the fluorescence by the metal ions. That perturbation of the fluorescence is due to a change in the structure of DNA caused by the metal ions is discounted, because even the faster component of fluorescence is considerably slower than that of water-solvated ethidium bromide (a. 5 ns vs. 1.8 ns). Thus structural disruption, if present, is insufficient to force ethidium bromide into the aqueous phase. Also we have given evidence that only those metal ions which significantly quench the fluorescence of ethidium bromide in aqueous solution affect the fluorescence of DNA-intercalated M ethidium bromide.' For example, the addition of up to Cd2+ions to a solution of ethidium bromide in DNA resulted in no change of fluorescence decay, even through DNA has a similar affinity for CdZ+as for the other ions used here." If perturbation of the fluorescence is due to structural disruption we would certainly expect to witness this in the presence of such high CdZ+ concentrations. Formation of a ground-state complex between the metal ions and ethidium bromide, whose fluorescence decay rate is faster than that of ethidium bromide, would also account for the faster fluorescence component. This is unlikely since the fluorescence excitation spectra of DNA-intercalated ethidium bromide are almost identical in the presence and absence of the three metal ions (Figure 1). Also we see no evidence for the formation of such a complex in the absence of DNA.' We observe two fluorescence decays, one arising from a population of ethidium bromide excited states strongly quenched by metal ions and one arising from a population less strongly perturbed. It is known that under the low ratios of metal ion to DNA phosphate employed here (maximum 0.26) the ions are bound to the D N A phosphate group^.^ Cu2+ has been shown to bind to the bases but only at metal-to-phosphate ratios above about 0.5.12 Thus we suggest that the fast component of fluorescence results (1 1) Beaumont, P. C.; Powers, E. L. In Proceedings of VIIth International Congress on Radiation Research, Broerse, J. J., Barendsen, G. W., Kal, H. B., Van der Kogel, A. J., Ed.; Martinus Nijhoff Amsterdam, 1983. (12) Venner, H.; Zimmer, Ch. Biopolymers 1966, 4 , 321.
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Atherton and Beaumont
from ethidium bromide intercalated at a site which has a metal ion bound to a nearby phosphate group, and the slower component results from ethidium bromide similarly intercalated but with no nearby metal ion. Quenching of this slower component is by metal ions that diffuse into the quenching sphere from other locations. As the metal ion concentration is increased the proportion of the first population will increase at the expense of the second and observation i above is readily explained. We now treat the data in a similar manner to that used in micelle quenching If the metal is bound to a phosphate group within the quenching radius of an ethidium bromide excited state the fluorescence is quenched and contributes to the fast decaying portion of fluorescence, I,. Residual low emission, Is, is due to excited ethidium bromide with no metal ion bound within the quenching radius. If E P represents a phosphate group within the quenching radius and P a phosphate group outside then we must consider the following equilibria: K
M+P,MP
(1)
K’
M
+E P e M E P
(2)
where M represents free metal ion, M P a metal ion bound to P, and MEP a metal ion bound to EP. It then follows that (3) The extent of the fast emission is proportional to the number of phosphate groups within the quenching radius of ethidium bromide occupied by metal ion, Le., If = C[MEP]. The constant, C, depends on the quantum yield of ethidium bromide fluorescence and the sensitivity of the detection apparatus. Similarly the extent of the slow emission is proportional to the number of those sites unoccupied by metal ion ( I , = C[EP]), and the total emission, It, is given by It = C([MEP] [EP]). Thus I, [MEP] [EP] _ -(4) I, PPI
+
+
Under the conditions of our experiments [MEP] is small with respect to [MP], in the worst possible case the ratio of phosphate to ethidium bromide is 60, and we may equate total metal ion concentration [MIT with [MP] if we assume that equilibria 1 and 2 are weighted to the right, i.e. the majority of metal ion is bound. Also since the ratio of phosphate to ethidium bromide is so high few phosphates will be within the quenching radius and [PI may be equated with total phosphate, [PIT. Then from eq 3 and 4
Plots of I,/I, vs. [MIT should be linear with slope S = K’/K[P],. From experiments performed at different DNA concentrations (different phosphate concentrations) the relation
may be used to obtain KIK’. Figures 6-8 show the data obtained for Cuz+. The reasonable linearity of these plots testifies to the accuracy of the interpretation. Note that as for Ni2+ and Co2+ (Figures 9 and 11) there is no effect of ethidium bromide concentration, as is required by the interpretation. The effect of DNA concentration shows that the quenching reaction is taking place between reactants bound to the DNA. Figure 8 shows the plots of 1/S vs. DNA concentration for Cu2+. Linear behavior is observed as predicted by the model and there appears to be a strong ionic strength dependence, Le., the lower the ionic strength the more effective is the quenching of fluorescence. If we calculate the slopes of the lines in Figure 8 values of 0.27 and 0.16 for KIK‘are obtained at salt concentrations of and 5 X M, respectively. In this calculation
Figure 6. Plots of I , / & vs. metal ion concentration, for Cu2+: (0)0.01% DNA, M Na2S04, 10” M EBr; (X) 0.02% DNA, M Na2S04, M EBr; (m) 0.02% DNA, M Na2S04, 5 X lod M EBr; ( 0 ) 0.03% DNA, lo-* M Na2S04, M EBR.
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Figure 7. Plots of IJ18 vs. metal ion concentration, for Cu2+: (0)0.01% DNA, 5 X lo-’ M Na2S04, M EBr; ( X ) 0.02% DNA, 5 X IO-’ M Na2S04, M EBr; (m) 0.03% DNA, 5 X M Na2S04, M EBr.
the average nucleotide molecular weight was taken as 342. As formulated the ratio of K’ to K represents a measure of “preference” of a Cu2+ ion for a phosphate group within the quenching radius over one outside. There is no obvious reason for there to be such a preference and if we equate K’with K the data tell us that there are ca. 4 and 6 phosphate groups within
Fluorescence of DNA-Intercalated Ethidium Bromide
The Journal of Physical Chemistry, Vol. 90, No. 10, 1986 2257
Figure 10. Plots of 1/S vs. DNA concentration for Ni2+,as described EBr. in the text. All solutions contained 5 X lo-' M Na2S04and
Figure 8. Plots of 1/ S vs. DNA concentration for Cu2+,as described in the text: (0) M Na2S04;(X) 5 X IO-' M Na2S04: All solutions M EBr. contained
q 1.0
/
Figure 9. Plots of l , / I s vs. metal ion concentration,for Ni2+: (e) 0.01% DNA, 5 X lo-) M Na2S04, M EBr; (m) 0.02% DNA, 5 X lo-' M Na2S04,2 X10-5 M EBr; (A) 0.02% DNA, 5 X IO-) M Na2S04,5 X IOd M EBr; (0)0.02% DNA, 5 X lo-) M Na2S04, M EBr; (+) 0.03% DNA, 5 X IO-) M Na2S04, EBr; (v)0.02% DNA, M Na2S04, M EBr.
the quenching radius of intercalated ethidium bromide for M salt respectively. In effect for lo-* M salt and 5 X quenching may only occur from metal ions positioned on any one of the four nearest phosphate groups to excited ethidium bromide; M salt the nearest six. In this way we may define for 5 X a distance over which quenching is occurring. We do not, however, receive any information on how salt concentration affects this distance. Figures 9 and 10 show the data gathered for quenching by Ni2+. Again reasonable linearity is obtained which would appear to justify the treatment. We see a large effect of salt and D N A concentrations but no effect of ethidium bromide concentration. M salt KIK' = 0.24, or quenching may occur from At 5 X metal ion bound to any of the four nearest phosphate groups. At the same salt concentration K/K'= 0.16 for Cu2+and this may reflect either quenching over a larger distance for Cu2+or a lower D N A binding constant for Ni2+ under these conditions. Figure 11 shows the data obtained for Co2+ quenching of ethidium bromide fluorescence. Clearly here the treatment breaks down and the assumptions used to formulate it are invalid.
Figure 11. Plots of I , / l s vs. metal ion concentration, for eo2+:(A)0.01% DNA, 2.5 X IO-) M Na2S04, M EBr; (X) 0.01% DNA, 5 X IO-) M Na2S04, M EBr; (0)0.02% DNA, 5 X M Na2S04, M EBr; (0)0.02% DNA, 5 X lo-) M Na2S04,5 X IO" M EBr; (+) 0.03% DNA, 5 X IO-' M Na2S04, M EBr.
However, again we see no effect of ethidium bromide concentration, and a strong effect of both salt and DNA concentrations. This allows us to deduce that quenching is again occurring via DNA bound reactants. The treatment as formulated demands only that the equilibria 1 and 2 are weighted to the right. The fact that it breaks down for Co2+suggests that the majority of this metal ion is not bound on the time scale of ethidium bromide fluorescence. At a given DNA concentration, as the metal ion concentration is increased the fraction bound to the phosphate groups will decrease. We would thus expect plots of eq 5 to exhibit downward curvature. This is certainly observed for Co2+and there are indications that this is also true for Cu2+ and Ni2+ (Figures 6 , 7, and 9). Further evidence that there is less metal ion bound to the phosphate groups in the case of Coz+is given by the behavior of the slower fluorescence decay (Figure 5 ) , on the assumption that this is due to diffusional quenching by unbound metal ion. Here the rate of decay is linear in total metal even at low metal con-
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The Journal of Physical Chemistry, Vol. 90, No. 10, 1986
centration, whereas for the other metals (Figures 3 and 4) low concentrations often make no significant difference. This is presumably due to stronger binding of these ions to the phosphate groups and hence their participation in the faster quenching process. In fact there is a trend in the linearity of these plots, which is that they are most linear for Co2+ and least linear for Cu2+. This coupled with the result that for the same salt conM, Cu2+is more effective than Ni2+ in centration, vis. 5 X producing the fast decay component ( K / K ’ = 0.16 for Cu2+vs. K / K ’ = 0.24 for Ni2+) leads us to suggest that the strength of binding of metal ions to DNA phosphate follows the order Cu2+ > Ni2+ > Co2+. This order is supported by previous work. In a review, Izatt et aL4 have tabulated values of the equilibrium constants for the binding of metal ions to DNA nucleotides. For each nucleotide the equilibrium constants decrease in the order Cu2+> Ni2+ > Co2+. These results reflect total binding to DNA and give no information concerning the nature of the binding process or the possibility of binding in different environments. If we suggest that the quenching of the slower component is due to unbound metal ion, the bimolecular rate constants in Table I1 present us with a problem, viz. where is the unbound metal ion? If it is in the aqueous phase then we are dealing with bimolecular rate constants orders of magnitude greater than diffusion controlled. These constants moreover were calculated on the basis that all metal ion is unbound. Although this will become more and more true as the metal ion concentration is increased, the extracted rate constants must be viewed as lower limits. Thus we cannot believe that the quenching of the slower fluorescence component in our experiments is because of reaction with metal ions which diffuse from the aqueous phase, and we feel we have no option but to assume that the majority of the metal ion is associated with DNA. We postulate the following. The majority of the metal ion is associated with DNA. Equilibria 1 and 2 are between metal ion bound to the phosphate groups and metal ion mobile around the DNA helix. This postulate is similar to the “site bound” and “territorially bound” ions in polyelectrolyte theory.I3 In these theories “site bound” ions would be defined as those bound to the DNA phosphate groups without the intervention of a hydration layer between ion and phosphate. Territorially bound ions are fully hydrated ions bound within a region centered on the axis of the DNA helix, but free to move freely within this region.’3b It is worth noting that polyelectrolyte theory suggests little “site binding” to DNA;’3e however, the extent of dehydration of Mg2+ when bound to DNA has been shown to be ~ignificant.’~ In terms of our model we suggest that bound metal ions are responsible for the fast decay process and the mobile population is responsible for the slower decay. The average time for which a metal ion is bound to a phosphate group must then be between 5 (taking the average value of 2 X loss-* for the rate of decay of the faster component, kf) and 23 ns (the rate of fluorescence decay in the absence of metal ions). Since the shorter the residence time the more mobile is the metal ion around the helix we place the metal ions in order of increasing mobility as Cu2+< Ni2+ < co2+. This order is also supported by the values of the rate constants in Table 11. For the three metal ions studied we have three incidences of identical conditions, 0.01%, 0.02%, and 0.3% DNA all with 5 X M N a 2 S 0 4 and 10-j M ethidium bromide. Considering the ratio of the rate constants in the order Cu2+: Ni2+:Co2+for each of these conditions we observe 4.9:2.1:1, 4.6:2.8:1, and 4.6:2.6:1 for 0.01%, 0.02%, and 0.03% DNA, respectively. We average these to 4.7:2.5:1. In the absence of DNA, however, the ratio for the three metals is 15.8:3.25:1.’ Thus in DNA solution the rate of eo2+quenching of the slower fluorescence component is increased a factor of 3.4 with respect to Cu2+ (13) (a) Manning, G. S . Biopolymers 1976, 15, 2385. (b) Manning, G. S . Biophys. Chem. 1977, 7, 95. (c) Manning, G. S. Biophys. Chem. 1977, 7, 141. (d) Manning, G. S . Q. Rev. Biophys. 1978, I Z , 179. (e) Manning, G. S. Arc. Chem. Res. 1979, 12, 443. (14) Skerjanc, J.; Strauss, U. P. J . A m . Chem. SOC.1968, 90, 308.
Atherton and Beaumont and a factor of 1.3 with respect to Ni2+. We attribute this to greater mobility, smaller phosphate residence time, of Co2+over Ni2+over Cu2+and would suggest a ratio of 3.4:1.3:1 for these parameters for Co2+, NiZ+,and Cu2+, respectively. An attempt at a value of mobility may be made if we correct the metal ion concentrations used in the plots of the slower rate constants for the “volume” of the DNA. If we take a helix diameter of 2 nm and a distance of 0.34 nm between base pairs,’j a 0.02% solution contains 0.19 cm3 of DNA per liter. Thus we must increase the metal ion concentrations in Figures 3 to 5 by factors of lo4, 5 X lo3, and 3.3 X lo3 for 0.01%, 0.02%, and 0.03% DNA, respectively. This certainly helps nullify the apparent DNA concentration dependence of these rate constants and in the case M Na2S04, gives a of say Co2+ for 0.02% DNA and 5 X bimolecular rate constant of 2.2 X lo7 M-I SKI.Contrastng this with the value of 4.3 X los M-’ s-’ obtained for this rate constant in water’ indicates a mobility of Co2+around the DNA helix, at this salt concentration, roughly a factor of 20 less than in water. Similar calculations can be made for each case. Salt Effects. Of the three parameters studied (kf, k,, and Zt/Z,) only kfappears independent of salt concentration. The bimolecular rate constants in Table I1 for the slow component of fluorescence are decreased with increasing salt. Polyelectrolyte theory predicts that the charge fraction of a given polyelectrolyte (e.g. DNA) remains virtually constant over a large range of ionic strength, at least up to 0.1 M.’3b Thus we would expect that as the salt concentration rises sodium ions would replace quencher ions, while maintaining the DNA charge fraction, and hence cause these rate constants to decrease. The lack of any salt dependence on the fast decay may be due to the operation of this quenching mode over a fixed distance. Although increasing salt will decrease the number of bound ions in accordance with the charge fraction rule, it may not affect the position of those which remain bound. These considerations also account for the salt effect on the plots of Zt/I, vs. [MI. As the salt concentration increases sodium ions replace quencher ions and the extent of the fast quenching process decreases. Thus for Cu2+ K / K ‘ = 0.21 and 0.16 at and 5 X M salt, respectively, Le., quenching via the fast mechanism is more effective M salt K/K’ is given as at lower salt. Also for Ni2+at 5 X 0.24 as oppoosed to 0.16 for Cu2+at equivalent salt, and we have given evidence that Ni2+is less strongly bound than Cu2+to DNA phosphate. Mechanism of Quenching. The possible mechanisms of quenching are Forster energy transfer, heavy atom effect, collisional energy transfer, and electron transfer. We rule out Forster energy transfer on the basis of insufficient overlap between the fluorescence spectrum of ethidium bromide and the absorption spectrum of the metal ions. We rule out heavy atom effects since the addition of Zn2+,Cd2+,and T1+ fail to effect the fluorescence.’ We also rule out collisional energy transfer due to the lack of any salt effect on the fast fluorescence decay rate. We thus favor quenching via electron transfer from ethidium bromide excited state to the metal ion.I6 These considerations are those used by Baguley and Le Bret2f to reach a similar conclusion for their system. Also Kemlo and SheperdI7 have concluded that rapid (> 1O8 M-I s-’ ) dynamic quenching of excited singlet states by metal ion is only possible either via electron transfer, or by energy transfer if there is appreciable overlap between the absorption spectrum of the metal ion and the emission spectrum of the excited state. If the quenching does occur via electron transfer then it is interesting to consider the fast decay component. We have sug(15) Freifelder, D. Molecular Biology; Science International: Boston, 1983; pp 102-103. (16) It is worth pointing out that we have used transient absorption and emission spectroscopy to show that methylviologen quenches the excited singlet state of ethidium bromide in DNA, and that we observe production of reduced viologen within the fluorescence decay time. Also we have evidence that Cu2+ quenching of the ethidium bromide excited state in the DNA system leads to the formation of the ethidium bromide cation. These results will presented in detail elsewhere. (17) Kemlo, J. A.; Sheperd, T. M. Chem. Phys. Lett. 1977, 47, 158.
J . Phys. Chem. 1986, 90, 2259-2264 gested that this quenching takes place by metal ions situated at any of the 6 nearest phosphate groups to ethidium bromide excited state. The 6 nearest phosphate groups to intercalated ethidium bromide are the 2 adjacent groups and the 4 groups one base pair away. The distance between the ethidium bromide and a metal ion at a phosphate group one base pair away is roughly 1 nm, if we take the radius of the helix as 1 nm and the base pair separation as 0.34 nm. Thus we have electron transfer across this distance with a rate of ca. 2 X lo8 s-I. Again this must be taken as an approximation. In fact there is a tendency for the rates to increase in the order Cu2+C Co2+ C Ni2+.
Conclusion For the systems we have studied, we believe we have demonstrated that fluorescence quenching techniques may yield useful information. We have given evidence that under our conditions the majority of metal ions are associated with DNA, and there is an equilibrium between metal bound at the phosphate groups and metal mobile around the helix. We have used our results to place the three metal ions studied in order of increasing mobility as Cu2+< Ni2+ C Co2+and have estimated that the mobility of Co2+,in DNA, under the stated conditions of salt concentration, is a factor of ca. 20 less than its mobility in water. We also conclude that rapid ca. 2 X lo8 s-I quenching can occur from metal ions bound to phosphate groups one base their away from intercalated ethidium bromide, a distance of roughly 1 nm. The equilibrium we are studying may be that between "site bound" and "territorially bound" metal ions as given by polyelectrolyte theory. If so our results suggest a significant degree of site binding for Ni2+ and particularly for Cu2+. Although this is in contradiction to earlier measurements we would point out
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that our technique allows for sensitive determination of fast equilibria between the two metal ion populations. If we convert our results into a "base to base hopping frequency" in the manner of Prusik and GeacintovZCthis must be somewhere between the lifetime of unquenched ethidium bromide excited state, in order to account for quenching of the slower fluorescence component, and the rate of decay of the fast component, to account for biphasic decay. These considerations give this frequency a value between 4 X lo7 and 2 X lo8 s-l for each metal studied, at least a factor of 400 greater than the lower limit given previously for Mn2+ by the above authors. In the quenching experiments of Baguley and Le Bret,2fdouble-exponential behavior was observed but the slower component of fluorescence decayed at the same rate as the fluorescence in the absence of quencher. With respect to our model we suggest that their quenchers are totally immobile on the time scale of ethidium fluorescence. Finally we are aware that we have tried throughout to simplify a complex system. For example, we have not considered structural effects on DNA due to salt concentration. However, we feel that our current model explains the data and will help us to predict the behavior of other metal ions and fluorescence probes in the DNA system. Acknowledgment. We thank Chris Lambert, Mike Rodgers, and Dr. E. L. Powers for helpful criticism. The Center for Fast Kinetic Research is supported jointly by the Biomedical Research Technology Program of the Division of Research Resources of N I H (RR 00886) and by the University of Texas at Austin. Partial support was provided from N I H Grant GM24235. Registry No. Cu, 7440-50-8; Ni, 7440-02-0; Co, 7440-48-4.
GENERAL PHYSICAL CHEMISTRY Effusion Studies of the Decomposition of Zinc Sulfate and Zinc Oxysulfate R. D. Brittain, K. H. Lau, D. R. Knittel, and D. L. Hildenbrand* SRI International, Menlo Park, California 94025 (Received: September 17, 1985)
The torsion-effusion method has been used to measure the SO3decomposition pressure over the crystalline solids ZnSO, and ZnO-2ZnS0, in the range of about 800 to 900 K. SO, is the only detectable vapor species under these conditions. Vapor composition was verified by mass spectrometry and by molecular weights obtained from simultaneous torsion and mass-loss effusion measurements. The vaporization process is kinetically limited in two respects, since SO2 and O2 are the dominant gaseous products at equilibrium and since the SO3effusion pressures show a pronounced dependence on orifice size. "Equilibrium" SO3pressures over both ZnS04 and ZnO.ZnSO4, derived by extrapolation of steady-state pressures to zero orifice area, are more than a factor of five lower than values calculated from established thermodynamic data, indicating the likelihood that the product solid phases are formed in a finely divided metastable condition. Addition of a few mole percent of Pt powder to Zn0.2Zn04 led to a dramatic pressure increase and to effusing gas compositions consistent with decomposition to SO, and 02,while for ZnS0, the effusing gas was converted to SO, and O2but the pressure increase was smaller and not entirely reproducible. The mechanistic implications of these results and correlations with other sulfate studies are discussed.
Introduction Previous of the decomposition pressure of MgSO4 at about 1000 K by the effusion method have shown that gross departures from chemical equilibrium are observed under these
dynamic conditions. In particular, SO3 is the sole gaseous decomposition product f o i moderate to -large effusion orifices, whereas so2 and 0 2 are the dominant equilibrium products in this system, with the equilibrium pressure ratio [ P ( S 0 2 ) / P ( S 0 3 ) ] 100 in the range of our measurements. It was also found that several metal and metal oxide additives are effective catalysts for the MgS04 decomposition, leading to substantia1 in observed pressure, and conversion of gaseous products to SO2 and . d
(1) Lau, K. H.; Cubicciotti, D.; Hildenbrand, D. L. J . Chem. Phys. 1977, 66, 4532.
0022-3654/86/2090-2259$01.50/0
0 1986 American Chemical Society
