Kenneth G. Strothkamp Drew University, Madison, NJ 07940 Ronald E. Strothkamp 135 Hofstra University, Hempstead, NY 11550
Ethidium bromide, a phenanthridine dye, binds to double-stranded DNA's through an intercalative mechanism, resulting in changes in the absorption and fluorescence properties of the dye (1,2).When intercalatively bound to a DNA, the ethidium fluoresces with an intensity from 20 to 100 times that of the free or nonintercalatively bound dye. It is therefore possible to quantitate this binding by monitoring the fluorescence of an ethidium-DNA solution. Ageneral discussion of fluorescence can be found in a variety of books on biochemical techniques (3,4). At any time the observed fluorescence intensity lobs of a given solution is equal to the sum of the fluorescenceintensities of the species in solution. If ethidium is the only fluorescent material under the experimental conditions, then lobs can be defined as where I b is the intensity of the fluorescence of the intercalatively bound dye, and Ifis the intensity of the remaining, or free, ethidium. Iband Ifare functions of the concentrations of the intercalatively bound cb and the remaining ethidium ct If the conditions of the experiment are optimal, then 1,.
= kbeb + kA%m - %)
(2)
A plot of experimentally determined r/&values versus r yields a value for n, the maximum number of binding sites per DNA base pair, from the x intercept. It also gives the intrinsic binding constant, K, from the slope of the line. The Scatchard equation applies only to systems with independent, identical binding sites. Proflavine, an acridine dye, can also bind to doublestranded DNA's. The binding of ethidium can be followed in the presence of proflavine to see ifthe binding of one dye affects the binding of the other and in what fashion. Abinding constant for proflavine can also be calculated from such data. Experimental Preparation of Solutions Caution:Ethidium bromide is both an irritant and a patent mutagen. Gloves should always be warn while using solutions of the dye. A face mask should also be used when weighing out the pure compound. All ethidium solutions should be saved for proper disposal.
A stock solution of ethidium in distilled water should be provided with a n approximate concentration of 3 x lo3 M. Students will determine the exact concentration of this solution by diluting a small portion 100-fold and measuring the absorbance a t 480 nm (& = 5,600 M-' cm-'1.
The constants kb and kf are determined experimentally c, is controlled, and cb is determined from monitoringl,b, as shown in eq 3.
Buffer
Once the concentration of the bound dye is known, the binding of the ethidium may be described in terms of the Scatchard equation (5):
Ethidium Solution A: Prepare 5 mL of a 3.0 x lo4 M solution from the ethidium stock solution.
Students need 100 mL of a 0.050 M Tris-CI, 1.0 M NaCl solution of pH 7.5. Prepare all of the following solutions in buffer.
Solution B: Prepare 10 mL of a 3.0 x from solution A. where r is the ratio of bound dye to DNAbase pair concentration; cf is the concentration of free dye; n is the maximum value of r; and K is the intrinsic binding constant of the ethidium.
M solution
DNA Solution C: 2 mL of an approximately 3 x lo3 M solution of nucleic acid from Salmon testes. This solution is
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prepared in advance by dissolving about 2 mg of DNA per mL of buffer. The exact molarity in base pairs is determined by measurement of the absorbance a t 260 n m of a 100-fold dilution of this solution (E = 1.3 x lo4 M-' em-' 1. Solution D: Prepare 10 mL of DNAsolution with a concentration of 3.0 x lo5 M from solntion C. Proflavine A stock solution should be provided, approximately 1.3 x 10-'M. Solution E: Prepare 2 mL of a working solution by diluting the available stock solution 50-fold. To determine the exact concentration of your working solution, dilute a portion by another 50-fold, and measure its absorbance at 445 nm (E = 3.6 x lo4 M-' ern-'). Fluorimetry Determination of Fluorimeter Settings Each fluorescencespectrometer should be set up according to the appropriate operating instructions for that instrument. Fill a fluorescence cuvette with 2.000 mL of buffer, 0.400 mL of solution C, and 50 pL of solution A. Mix well. Place the cuvette in the fluorimeter. Set the excitation wavelength to 525 nm, the excitation slit to 5 nm, and the emission slit to 3 nm. Slowly scan the emission wavelength from 560 to 640 nm to determine the emission maximum. Set the emission wavelength to the value that gave the maximum intensity. Set the fluorescence intensitv of this s a m ~ l to e 50% of the range of the instrument by appropriate adjustment of the gain or slit widths. These parameters are now set for the entire experiment. Determination of Fluorescence Parameter kr Place 2.500 mL of solution B in the cuvette, and record the fluorescenceintensity. Add a 20-pL aliquot of solution A, mix well, and record the intensity. Continue with 4 additional 20-pL aliquots of solution A, recording the fluorescence intensity after each addition. If the pmflavine study is to be done, add a 50-pL aliquot of solution E to this last solution in order to test for quenching. Be certain that your two dyes do not interact versus [ethidinml should with each other. A plot of lobs yield a straight line with slope kf. Determination of Fluorescence Parameter kb Place 2.400 mL of solution B in the cuvette, and add 50 pL of solution C. Mix well, and record the fluorescence intensity. Add successive aliquots of solution C, recording the
1 T i w f ~ g ~shows r e tne saturanon of a qkots of ernla ~m wltn F~g~re DhA for the delermlnat on of & Eacn I me me ntenslty ,eves OH, a fresh a lq~orof eth alum is adoea, lo lowed oy aoo Ilona DNA
78
Journal of Chemical Education
intensitv after each addition. until the intensitv no loneer increases, that is, until all the ethidium is bbund. ~ L i s should not take more than 3-4 aliquots of solution C. Add 20 pL of solution A, and record the intensity. Again add 50 pL aliquots of solution C until the intensity stops increasing. Repeat these 20-pL aliquots of solution A followed by 50-pL aliquots of solution C until the intensity goes off scale. You will want a t least 5 different ethidium concentrations as data points. If the intensity is increasing too rapidly, reduce your aliquots of solution A to 10 pL.Figure 1illustrates this portion of the experiment for the first two concentrations of ethidium. A lot of Ink. versus lethidiuml. where the ethidium concenGation isdetermined at the-first point of constant intensity, should yield a straight line with slope kb. Determination of the Binding Parameters for Ethidium to DNA Place 2.700 mL of DNA from solution D in the cuvette. and measure the fluorescence intensity. Any deviation from a zero reading should be com~ensatedfor bv rezeroing the instrument. Titrate this solution with Ahidium, using 20yL aliquots of solution A. Record the intensity after each addition. Take at least 10 readings. Determination of Ethidium Binding to DNA in the Presence ofAnother Drug Place 2.700 mL of solution D in the cuvette, and measure the fluorescence intensity. Add a 75-pL aliquot of solution E, mix well, and measure the intensity ~ g & n any , deviations from a zero reading should be compensated for by rezeroing. Titrate this solution with aliquots of solution A as in section C. Repeat the titration starting with a 150yL aliquot of solution E. Analysis The two graphs called for in part B yield straight limes with very good correlation coefficients. When fluorescence intensities are plotted in arbitrary units from 0 to 100, kb is about 7.7 x lo6 M-'. and ksis about 2.3 x lo5 M-'. These values demonstrate the large enhancement in fluorescence when ethidium binds to DNA. From part C, a plot of rlcrversus r will yield Kand n for ethidium as previously described. These data may be obtained by calculating ctOtd,cb, ct, and [DNA] for each of these points. (See'eqs 3 and 4.) For each data point, r = cd[DNAl. Remember that the DNA concentration slowly decreases as the titration is carried out. ~~~
Fgure 2 Scarchard pots for the o~ndmgof elh d l ~ mlo DNA n tne absence of profavne (m), a! 7 1 r lo* M profavne( 0 . .and a! 1 4 r M proftavne (A,
ent value of K (i.e., KobJ decreases as the concentration of inhibitor is increased while the x intercept remains constant. In competitive inhibition the fraction of binding sites occupied by each dye depends on the dye concentrations and binding constants. Since the x intercept corresponds to [ethidi&nl= m, all the sites are filled wiih ethidium regardless of the proflavine concentration. ~ d d i an competitive ~ inhibitor to a Scatchard analysis produces the relationship shown below.
I
0
0
0.2
0.4
0.6
1
0.8
c',
1.2
1.4
1.6
x lo5
where K' is the binding wnstant of the inhibitor, and c; is the concentration of free inhibitor. The derivation of eq 5 can be found in the article by LePecq and Paoletti (2). The reciprocal of eq 5 produces a straight-line relationship.
Figure 3. Replot of observed ethidium binding constants at different proflavine concentrations. The data from part D is treated as above. and rlcrversus for each of the two proflavine c o & n t r a r is again tions. 1\11 three sets of data are plutted on the ;ame grnph, and tvnicnl results are shown in Firmre 2. Student data for this experiment usually displays good linearity. However, points obtained a t low values of r often show some curvature. Such points are omitted at the graphing stage. A.
-
Additional Challenges
There are more elaborate schemes for treating the binding of dyes to DNA, and students wishing to go into more depth might try treating their data according to the analysis published by McGhee and von Hippel (6).However, this is not necessary because results using the Scatchard analysis are usually quite satisfactory. The data shown in Figure 2 in the absence of proflavine yield a binding constant of 3.3 x lo5 M-' and a n n value of 0.32. These results are well within the range reported for many DNA's using a variety of techniques (2,7,8). Competitive Inhibition
The additional results in Firmre 2 in the Dresence of nroflavine suggest that this dye& a competiiive inhibit& of ethidium bindine. This is in ameement with ~ublished data. In the of a compzitive inhibitor the appar-
The data from Figure 2 is plotted according to eq 6 in Figure 3. The concentration of free proflavine has been ap~roximatedbv the total ~roflavineconcentration at s e k a r t of each tkration. ~ r d m the slope and intercept of this graph, the binding constant of proflavine to DNA is estimated to be 1.2 x 105M-'. Conclusion This experiment introduces students to the widely used technique of fluorescence spectroscopy and generates data that can be analyzed by the classical binding scheme for small molecules to macromolecules. Acknowledgment We thank Mary E. Howe-Grant for suggesting this experiment to us and for providing information on procedures. Literature Cited 1.Wating, M. J. J. M d Bio! 1965.13.269-262. 2. LePecq, J-B.:Paoletti, C . J . Mol. E d . 1967.27.87-106. 3. Freifelder, D. Physical Biochemistry, 2nd ed.; W H. Freeman: NeuYork, 1932.
4. Boyer, R. F M d w n E*p4rim~ntalBiochemistry; Addison-Wesley: Reading. MA. 1986. 5. Scakhhard. G. Ann. N Y h d Sci. 1949.51.66&672. . 6. McGhee, J. D.: von Hippel, P. H. J . Mol. B i d . 1974,86.469489. 7. Hinton, D. M.;Bade,V. C . J B i d Chem. 1976.250, 1061-1070, 8. LePeeo. J.-B. In Methods of Bioch~mimlAnalvais: Glick. D.. Ed.: Interscience:New YO& 1971:Vo!. 20. pp 41-66.
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