Sept., 1955
INTERACTION OF DESOXYRIBONUCLEIC ACIDWITH ACRIFLAVINE
939
STUDIES ON THE INTERACTION OF DESOXYRIBONUCLEIC ACID WITH ACRIFLAVINE' BY HARRIET G. HEILWEIL A N D QUENTINVAN WINKLE Department of Chemistry, The Ohio State University, Columbus IO, Ohio Received February 86,1966
The interaction of a high molecular weight sample.of calf thymus desoxyribonucleic acid (DNA) with the fluorescent cationic dye acriflavine was studied in the concentration range where soluble complexes are formed and was found to be reversible. Determinations of the amount of binding were made over a wide range of equilibrium concentrations of unbound acriflavine, at several values of the experimental variables ionic strength, pH and temperature, by means of the partition analysis method. It was found that the binding sites for acriflavine on the DNA molecule are heterogeneous with respect to their intrinsic association constants and that the binding curves may be described in terms of a two-constant equation. The maximum number of sites available for complexing with acriflavine per DNA phosphate group is approximately 0.5 at ionic strength 0.002 a t pH 7. A method for the treatment of fluorescence uenching data was developed which takes into account the multiple e uilibria involved in macromolecular interactions. Txis method yields values for k l , the equilibrium constant for the first %NA-acriflavine association, in agreement with the independent partition analysis results. As is expected for an electrostatic interaction, kl decreases with increasing ionic strength and with decreasing pH, but the evidence indicates that secondary binding forces are important. The enthalpy changes are rather large and negative. The corresponding standard entropy changes are zero within experimental error, suggesting that the macromolecular configuration of DNA does not change significantly upon interaction with acriflavine.
Introduction It is expected that quantitative investigation of the interactions of desoxyribonucleic acid (DNA), under diverse experimental conditions, will provide information concerning the macromolecular configuration of DNA in solution and the mode of interaction of DNA in biological processes. Progress along similar lines has been made in the field of protein research,2 and the work on protein interactions has yielded methods and concepts many of which are applicable in the study of binding by DNA. Among the more recent DNA binding studies which have been reported are a number on the complexing with inorganic cations13-? with proteins,+12 and with small organic ions and molec u l e ~ . ' ~ - ' ~Several of these investigations are concerned with the interactions with DNA of certain biologically active quinoline and acridine der i v a t i v e ~ . Oster13 ~ ~ ~ ~ determined ~ that the quenching of the fluoresence of acriflavine, 3,6-diamino-l0methylacridinium chloride, by DNA is related to complex formation between the dye cation and the negatively charged phosphoric acid groups of the DNA. In the present paper fluorescence quenching data are used, in accordance with the mass law treatment of multiple equilibria,2 to derive con(1) Taken from a dissertation submitted b y H. G. Heilweil to the Graduate School of The Ohio State University in partial fulfillment of the requirements for the degree Doctor of Philosophy, 1954. (2) I. M.Klotr in H. Ncurath and K. Bailey, "The Proteins," Vol. I, P a r t B, Academic Press, Inc., New York, N. Y.,1953, p. 727. (3) J. Shack, R. J. Jenkins and J. M. Thompsett, J . B i d . Chem, 198,85 (1852), 203, 373 (1953). (4) A. Veis, THISJOURNAL,8'7, 189 (1953). (5) 5. Kats, J . A m . Chem. Soc., 1 4 , 2238 (1952). (6) C. Neuberg and I . S. Roberts, Arch. Biochem., 20, 185 (1949). (7) K. G. Stern and M. A. Steinberg, Biochim. Biophys. Acta. 11, 553 (1953). (8) E. P. Geiduschek and P. Doty, ibid., 9, 609 (1952). (9) R.F.Steiner, Arch. Biochem. Biophys.. 46, 291 (1953). (10) D.P. Riley and U. W. Amdt, Nature, 113, 294 (1953). ( 1 1 ) P. Alexander, Biochim. Biophys. Acta, 10, 595 (1953). (12) C. F. Crampton, R. Lipshitr and E. Chargaff, J . B i d . Chem., 206, 499 (1954). (13) G. Oater, Trans. Faraday Xoc., 47, 660 (1951). (14) L. F. Cavalieri and A. Angelos, J . A m . Chem. Xoc., 1 2 , 4686 (1950); L. F. Cavalieri. A. Angelos and M. E. Balia, dbid., I S , 4902 (1951). (15) N. B. Kurnick, ibid., 1 6 , 417 (1954). (16) J. L. Irvin and E. M. Irvin, J . B i d . Chem., 206, 39 (1954).
stants for the binding of acriflavine by a high molecular weight preparation of DNA. The results are corroborated by independent binding experiments carried out according to the partition analysis method described by Karush.17
Experimental Materials.-The sodium salt of DNA used in these studies was prepared from caIf thymus according to the procedure of Schwander and Signer's and the recommendations of Doty and B ~ n c e . 1 ~The nucleate solution was diluted 1: 1 with water before the first alcohol precipitation; the second alcohol precipitation was achieved by pouring one volume of aqueous nucleate into four of NaC1-saturated alcohol. The fibrous product was vacuum dried at 4 O , using no drying agent, and stored a t 4' in covered bottles in a desiccator care being taken to prevent contamination of the over PzO~, nucleate by P206 dust. The molecular weight was determined by I. J. Heilweil of this Laboratory from angular light scattering measurements in 0.2 M NaCl to be 1 X lo', based upon a measured refractive index increment of 0.160. In water, a t 259 mp, 238, and a t 231 mp, 89.4. In 0.2 M NaCI, at 259 mp, 204. The dry weight of nucleate samples was obtained by correcting for the moisture content after comparing, under identical humidity conditions, the sample weight with the weight of a reference sample. The dry weight of the reference sample had been determined by extrapolation to infinite time of a curve of reference sample weight, measured every few days over a month's time, versus reciprocal of vacuum drying time at 4 ' . For a particular sample, dry weight obtained in this way agreed with that determined by vacuum drying to constant weight a t 100". The DNA fibers were dissolved as needed in pre-cooled distilled water, using a magnetic stirrer a t moderate speeds for 8-12 hours at 4'. A sample of commercial acriflavine (National Aniline) was purified to remove proflavine hydrochloride by the method given by Albert.20 Analysis for C1 gave 13.52% compared with 13.65y0 calculated for ClIHI4NIC1. For all extinction measurements made on acriflavine solutions, there was mounted on the Beckman DU spectrophotometer an exit filter having zcro transmission for the green wave lengths of the fluorescence peak. At 452 mp, 16401 in water and at pH 5.5, ionic strength ( p ) = 0.1, 1720 at p H 7, p = 0.002, and 1810 at pH 7, p = 0.1 and 0.3, independent of the resence of dissolved n-hexanol. Deviations from Beer's f,w become significant at concentrations In n-hexanol saturated a t 25" higher than I X (17) F. Ilarush, J . A m . Chem. Xoc., I S , 1246 (1951). (18) H. Sohwander and R. Signer, Helu. Chim. Acta, 33, 1521 (1950). (19) P. Doty and B. H. Bunce, J . A m . Chem. Xoc., 7 4 , 5029 (1952). (20) A. Albert, "The Acridines," Edyaard Arnold and Co., London, 1951,p. 197.
940
HARRIET G. HEILWEIL AND QUENTINVAN WINKLE
with any of the buffers employed, 2340 a t 470 mM, and Beer's law is obeyed for concentrations at least as high as 5 X lO-'%. Since dilute acriflavine solutions were found to decrease slowly in concentration on standing, solutions were prepared fresh from the solid dye as needed, measured as soon as feasible, and kept in the dark as much as possible to minimize photooxidation. Potassium phosphate buffers, prepared with the aid of the data of Green,21were used throughout. Methods (a). Partition Analyses.--n-Hexanol was found to be a suitable organic solvent for the partition analysis study of the DNA-acriflavine ~ystem.1~Eastman Kodak Technical Grade n-hexanol was washed with 0.01N NaOH, then with distilled water, and, finally, saturated with the buffer to be used in a given experiment a t the temperature of the experiment. Equilibrations were carried out in pharmaceutical bottles with polyethylene snap-on caps. For each binding determination 10 ml. of washed n-hexanol was added to 10 ml. of aqueous solution containing DNA, acriflavine, and buffer a t the desired concentrations. For each set of experimental conditions a series of blanks containing no DNA was prepared in order to determine the partition coefficients of acriflavine between the two phases over the concentration range covered in the corresponding partition experiments. The bottles were mechanically shaken at about 60 cycles per minute, for four hours, either in a cold room a t 4 f 1' or in a constant temperature room a t 25 f 1'. The free dye concentration in the organic layer was determined either spectrophotometrically in 1 cm. cells for the more concentrated solutions, or by measurement of the fluorescence intensities of the dilute ones. In the case of the blanks, both the aqueous and organic layers were analyzed. Fluorescence measurements were made on an Aminco high sensitivity photometer designed by OsterZa(American Instrument Go., Silver Spring, Md.) a t 90' to the incident beam, with incident light of wave length 436 mp and a yellow-green exit filter. (b). Fluorescence Quenching Measurements.-Fluorescence quenching data were obtained by titrating a buffered acriflavine solution with a DNA solution a t the same concentrations of dye and buffer. A total of about 10 ml. of the DNA-acriflavine solution was added in suitable increments, from a buret graduated to 0.02 ml., to 15 ml. of the solution containing no DNA in the square cell provided with the Aminco photometer. The solution was stirred magnetically between additions. The procedure, including the measurement of fluorescence, was carried out in the constant teomperature room a t 25 i 1' or in the cold room at 4 i 1 , as required. It should be noted that the acriflavine concentrations used in the quenching experiments fell on the eseentially straight-line portion of the fluorescence-concentration curve.
Results The partition analysis results are presented in Fig. 1, in which r , the number of moles of acriflavine bound per mole of DNA nucleotide (assuming an average nucleotide weight of 33 1),is plotted against r/c, c being the equilibrium molar concentration of acriflavine. The reversibility of the interaction was tested by replacing known volumes of equilibrated organic layers with fresh n-hexanol (Fig. 1B). In addition, binding data taken a t a DNA concentration of 0.01% fall in with the results a t 0.00570 DNA (Fig. 1E). Although the method of equilibrium dialysis using cellophane membranes is not suited to quantitative study of acriflavine binding because of excessive membrane adsorption, the satisfactory agreement between results obtained by this technique and those from partition analysis (Fig. 1E)indicates that n-hexanol does not interfere in the DNA-acriflavine interaction. The curves in Fig. 1 were calculated from the equation (21) A. A. Green, J . Am. Chcm. Soc., 66, 2331 (1933). (22) G.Oster, Anal. Chsm., 26, 1165 (1953).
VOl. 59
Fig. 1.-Partition analysis results for DNA-acriflavine binding; [DNA10 = 0.005% except as noted. Points corremond to exDerimenta1 data. Curves calculated accorditig to the twb-constant method: curve A, p = O.OO!, pH 7.0 T =: 25". curve B, p = 0.10, p H 7.0, T = 25 ; curve 6, p = 0.36, pH 7.0, T = 25"; curve D, p = 0.10, pH 5.5, T = 25'; curve E: p = 0.10, pH 7.0, 2'4'. r_ = _nsKa __ C 1 KaC
+
+-1 nbKb + KbC
(1)
on the assumption that the n DNA binding sites can be divided into two groups, each containing ni sites having the same intrinsic binding constant, Ki.2a The intercepts lim ( r ) = n. r/c 0
and
lim ( r / c ) = naKe r+O
+ nb = n
(2)
+ nbKb = k~
(3)
were selected so as to give the best fit obtainable over the whole range of r . kl in equation 3 is the equilibrium constant for the formation of the complex containing one molecule of acriflavine per DNA molecule. Theoretical curves of r / c versus r constructed on the basis of adsorption isotherms derived by Sips24for two continuous distributions of binding energies could not be made to fit the data. When the logarithm of the function Q = r / ( n - r)c is plotted against r, as recommended by Scatchard,26 taking the value of n determined according to equation 2, the resultant straight lines yield, in accordance with (lim Q) = (kl/n),values for kl in C-W,
-0
(23) F. Karush. J . Am. Chem. SOC.,72, 2705 (1950). (24) R. Sips, J . Chem. Phus., 16, 490 (1048): 18, 1024 (19.90). (25) J. T. Edsall, G. Felsenfeld, D. S. Goodman and F. R. N. Curd, J . A m . Chem. Soc., 76, 3054 (1954).
Sept ., 1955
INTERACTION OF DESOXYRIBONUCLEIC ACIDWITH ACRIFLAVINE
'}
slow Symbol
C, x IO7
x IO+
0
I17
237
KII x
941
I
264
4
symbolc.,x
lo7
117 1140
c'
A
slow w5
P
K,,X
413 0088 108 0042 KII v )
10-5
453
I13
Co
2
:w[
Y
\
,^
bNA].
i
IO5,
Fig. 2.-Fluorescence quenching curves a t p H 7.0 and T = 25".
0 p
=
V
I
2
i
0.002,
satisfactory agreement with those obtained by the other extrapolation methods. The data at p = 0.002, however, were not readily treated in this manner, perhaps because of the large effect of experimental errors. Finally, values of kl were calculated from the partition analysis data according to a third method, that of Steiner,26which requires no knowledge of n. If it is assumed that the ratio, p , of the quantum yields of fluorescence of bound and free fluorescent molecules is the same for all bound molecules, then fluorescence quenching data may be expressed by the equation13
Fig. 3.-Fluo~esceiice quenching curveB a t p = 0.10, p H 7.0 and T = 25".
requires that the first complex, DNA-A, can exist in the absence of DNA-A2, DNA-A3, . . ., DNA-A, only in solutions very dilute with respect to dye. Accordingly, Oster's quenching method should be modified: A value of the quantity denoted as K11 is determined from each of several quenching curves obtained a t different dye concentrations co (equation 6 ) . Then, the value of Kll extrapolated to c g = 0 is kl. At co = 0, also, [DNA10 =. [DNA]. The quenching curves obtained in this investigation, contrary to the prediction of equation 4,go through a maximum, then slowly level off a t high [DNAIo,indicating that p is not constant for the (4) system. This is especially evident when the amount where F is the fluorescence intensity in the presence of binding is high, as, for example, in the run a t of quencher of a dye solution a t initial concentra- low ionic strength (Fig. 2 ) . Since K1l is insensitive tion col Fo the fluorescence intensity without to the value of p , it was decided to estimate for p a quencher, and cb is the equilibrium concentration of value which would hold fairly well in the range of bound dye. Oster13 made the further assumption low [DNAJo. This was accomplished by plotting that the DNA-acriflavine interaction is represented Fo/F against l/[DNA]o and extrapolating the risby ing portion of the curve to l/[DNA]o = 0 (equation 7). The initial curvature in quenching curves DNA A DNA-A, Kii = c ~ / [ D N A ] c (5) where binding is relatively extensive (Fig. 2 ) is atwhere A stands for acriflavine, and Ch = [DNA-A]. tributed to the fact that [DNAIo, rather than Kll was determined from the initial slope and limit- [DNA 1, is plotted. In these cases, the slope of the ing value of a quenching curve (Fo/F) - 1 versus linear portion, rather than the initial slope, was [DNAIo,the initial concentration of DNA, a t con- used in the calculation of Kll. Although the graph stant co, in accordance with the relationships of Kll versus co is slightly curved (Fig. 3, insert), the values of kl obtained by linear extrapolation to co = 0 when quenching data were available at only two dye concentrations are in satisfactory agreeand ment with those from partition analysis. In Table I are listed the results for kl obtained by the several methods discussed under the various conditions of p , pH, and temperature studied, toIt was necessary to assume that [DNA] G [DNA10 gether with the thermodynamic constants and the a t low co. Basic to all three of the treatments of the values for Ki,ni and n. Figure 4 shows the variapartition analysis data employed is the concept that tion of ICl and of log k, with p a t 25' and a t 4'. a number of independent binding sites are available Each open symbol represents a value for kl estion the DNA molecule and that these are simulta- mated from quenching data a t a single co and from a neously in equilibrium with acriflavine.2 This consideration of the slopes of Kl, versus CO, assumed independent of cg, as a function of p . (26) R. F. Steiner, Arch. Biochem. Biophys., 47, 56 (1953).
+
HARRIETG. HEILWEIL.4ND QUENTINV A N W I N K L E
942
CONSTANTS FOR i.b
PH T ("C.) kl X
kl X
Partition Anal.: Two-constant method Method of Scatchard Method of Steiner Quenching-slopemethod
x 10-5 - AFl", kcal./mole - AHlO, kcal./mole - Axlo, cal./OK./mole
klav
Ka X 10-6 x lo-' na
TABLEI INTERACTION OF DNA 0.002 0.10 7.0 7.0 25 25
THE
50.0
...
49.3 49.0 49 9.1 67 4.8 0.07 0.43 0.50
Kb
nb
n
The reversibility of the DNA-acriflavine interaction was checked by determining that the value of F for a given solution a t a given temperature is independent of the temperature a t which the components are mixed. Since F is time-independent in quenching titrations, equilibrium must be attained rapidly. Qualitative experiments to determine whether DNA interacts with several cationic compounds were carried out a t p = 0.1, pH 7, and 4'. While complexing with ethyl- and butylpyridinium bromides was not detected in equilibrium dialysis experiments covering a wide range of concentrations, considerable interaction was observed with the lauryl and cetyl derivatives. The doubly charged ethylene bis-quaternary pyridinium bromidez7 is bound to some extent, but, under the same conditions of concentration and p, benzidine di-hydrochloride is not.
3.63 3.48 3.35 2.90 3.3 7.5 8.1 1.9 3.9 1.3 0.09 0.20 0.285
WITH
VOl. 59
ACRIFLAVINE 0.30 0.10 5.5 7.0 25 25
0.10 7.0 4
1.20 0.99 1.03 0.92 1.1 6.8
1.93 1.84 1.78 1.96 1.9 7.2
9.55 9.43 9.39 9.22 9.4 7.6
2.8 1.8 0.03 0.18 0.208
2.5 1.8 0 06 0.20 0.262
12 3.4 0.08 0.21 0.285
presented here, the extrapolation to determine n is quite uncertain, and the effect of a large variation in n upon the theoretical binding curves is small, so that the values reported for n are estimates only. On the other hand, the uncertainties in extrapolating the partition analysis data to c = 0 by any of the three methods employed are a good deal smaller than the differences between the values of IC, for the different environmental conditions considered. If greater accuracy were required, however, partition analyses could be carried out a t lower dye concentrations by measuring the fluorescence of larger volumes of solutions than were used in these experiments. r
Discussion The distinct curvature in the plots of r/c versus r (Fig. 1) indicates that there are differences among the binding sites on the DNA macromolecule with respect to their affinities for acriflavine. It was not considered that electrostatic interactions among the binding sites play a large role, first, because the dye cations replace inorganic cations a t the sites,28 and, second, because an electrostatic correction was found inadequate to account for the curvature in DNA-rosaniline binding curves. l 4 Although the curves calculated on the basis of two intrinsic binding constants fit the partition analysis data fairly satisfactorily for any reasonable choice of intercepts, the values of K,, Kb, na and nb may not be interpreted too l i t e r a l l ~ l but ~ ! ~do ~ appear to show that a small number of sites with high intrinsic affinity exists and that a considerably larger number of sites have much lower affinities. That the twoconstant assumption is indeed an over-simplification is indicated by the disparity between the calculated curves and the partition analysis data in the region where the slope is changing rapidly. As is often the case in binding studies of the kind (27) Kindly given by Dr. J. L. Hartwell (J. L. Hartwell and M. A . Pogorelskin, J . Am. Chem. Soc., 72, 2040 (1950)). (28) G. Scatchard, Ann. N . 1'. Acad. Sci., 61, 660 (1949).
"Otl Symbol
E J 70 70 5.5 55
25'C 4 25 4
Blackened points i n d i c a t e results from both p a r t i t i o n analysis and fluorescence quenching Open p o i n t s obtained lrorn fluorescence quenching
M.
Fig. $.-Variation of k l with ionic strength.
The quenching-slope procedure, despite the approximations made in applying it to the DNA-acriflavine interaction, appears to provide a relatively
Sept., 1955
INTERACTION OF DESOXYRIBONUCLEIC ACIDWITH ACRIFLAVINE
rapid but reliable method for the determination of kl in interacting systems involving multiple equilibria where fluorescence quenching is appreciable and where a limiting slope a t infinitely low quencher concentration can be determined. In cases where the assumption holds that p is constant, equation 4 might be used to calculate c and, hence, an entire binding curve from quenching data. If it may be assumed that the quantum yield of fluorescence for the free dye remains constant in the presence of DNA, while the yield for the bound molecules depends upon the mechanism of the binding, then the variation of p in the present instance is further indication that the binding sites are heterogeneous with respect to their affinities for acriflavine. The values of kl obtained in this investigation are high relative to values reported in studies of other DNA interactions under comparable conditions of p , pH and temperature. Because DNA binding is very often dependent to a large extent upon the degree of polymerization of the preparation,l4vl6however, comparisons between the values of binding constants determined for different DNA samples must wait until more is known concerning this additional experimental variable. The decrease in kl accompanying the decrease in pH from 7.0 to 5.5 may be explained on the basis of increased competition of H + for the DNA binding sites, the ionization of acriflavine being independent of pH in this region. While the marked dependence of El upon ionic strength, especially at low ionic strengths, is expected for a basically electrostatic interaction, there is evidence, as 0sterl3 pointed out, that secondary forces are very important in the DNA-acriflavine complexing. First, fluorescence quenching, such as occurs on the interaction of DNA with a number of acridines, is believed to be associated with strong van der Waals'
943
bonding.29 The rather large negative values found for AH?, A H 2 and AHbo in the present study are in accord with this hypothesis. Furthermore, the qualitative findings reported here on the interaction of DNA with several organic cations, as well as the information on DNA complexing available in the literature, indicate that binding by DNA is favored for molecules which offer opportunities for secondary interactions. The standard entropy changes calculated for the DNA-acriflavine complexing are found, within experimental error, to be zero. It appears, then, that far-reaching structural changes probably do not occur upon binding, as they are not believed to occur on nucleic acidprotein interaction.1° For this reason, and, also, because equilibrium is reached rapidly in the DNAacriflavine system, it would seem that a proposed configuration for the DNA macromolecule should place the charged phosphate groups so that they would be readily accessible for electrostatic interaction. Several suggested structures for DNA, that of Crick and Watson30 included, satisfy this requirement. In addition, a structure similar to the one proposed by Crick and Watson allows that the planar hydrocarbon portion of a molecule like acriflavine slip between successive parallel planes occupied by purine and pyrimidine bases, the quaternary nitrogen oriented toward the DNA phosphate group, so as to permit strong van der Waals bonding as well as electrostatic interaction without requiring much change in the configuration of the DNA macromolecule. The authors gratefully acknowledge support of this work by the National Institutes of Health, Public Health Service,through a grant administered by the Ohio State University Research Foundation. (29) E. J. Bowen, Quart. Rev., 1, 1 (1947). (30) F. H. C. Crick and J. D. Watson, Proc. Roy. SOC.(London) 83.23, 80 (1954).
