REACTION OF ACRIDIKE ORANGE AND PROFLAVINE WITH POLYADENYLIC ACID
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The Interaction of Acridine Orange and Proflavine with Polyadenylic Acid’
by Gordon G. Hammes and Colin D. Hubbard Department of Chemistry, Cornell University, Ithaca, N e w York 14860
(Received N a r c h 7, 1966)
A kinetic study of the reactions between acridine orange and polyadenylic acid and between proflavine and polyadenylic acid has been made with the temperature-jump method a t pH 7.5 where the polynucleotide is single stranded. I n both systems a discrete relaxation time, which is independent of both reactant concentrations, can be discerned from the observed relaxation spectrum. This single relaxation process which is characteristic of an intramolecular process subsequent to polymer-dye complex formation, is probably a consequence of “stacking” of dye molecules along the polymer chain. Base-dye interactions appear to retard t’he rate of the “stacking” process markedly and conformational changes may be rate determining in the “stacking” interaction.
Introduction The relaxation effect which occurs in temperaturejump experiments with poly-a-L-glutamic acid (PGA) and acridine orange, 2,8-diniethyldianiinoacridine (AO), solutions at pH 4.7 and at pH 7.5 can be quantitatively described by two relaxation times, 71 and ~ 2 .A ~study of the dependence of 71 and 7 2 on polyglutaiiiic acid and acridine orange concentrations yielded data which could not be quantitatively reconciled with any of several possible mechanisms of interaction between the dye and the polymer. However, the processes occurring are intraniolecular and can be identified either with the aggregation or “stacking” of the dye upon the polymer chain or to solvent and counterion substitution by the dye subsequent to a very rapid initial interaction. Unfortunately, the data did not permit a sharp distinction between these possibilities to be made. The forward and revzrse rate constants for the A 0 nionomer-dimer react on were also measured, and if “stacking” is directly involved in the rate-controlling steps of the AO-PGA interaction, this phenomenon occurs considerably more slowly than in free solution. In the present study, the interaction of acridine orange and its diamine analog proflavine (2,8-dianiinoacridine, PR) with the synthetic polynucleotide polyadenylic acid (poly A) has been investigated by the temperature-jump method and by absorption spectroscopy at pH 7.5 where the poly A is single stranded and both dyes are protonated. For both dyes the observed relaxation effect is a complex spectrum of
relaxation processes which virtually encompasses the whole time range of the temperature-jump apparatus to 1.0 sec). A disused (approximately 2 X crete relaxation effect in the millisecond range can be distinguished for both dyes. The relaxation time for this effect can be estimated, and because it is independent of the concentrations of poly A and the dye, this part of the relaxation spectrum can be identified with an intramolecular process. Correlation of equilibrium spectral measurements with the kinetic results suggests that the observed discrete relaxation effect is characteristic of the “stacking” of the dye molecules into dimers along the poly A chain. or of conformational changes necessary to facilitate “stacking.” A knowledge of the time constants characteristic of dye “stacking” may be useful in understanding the “stacking” of nucleic acids. Some preliminary data obtained with the temperature-jump method for the acridine orangecalf thymus DXA system are also reported.
Experimental Section Acridine orange from the Kational Aniline Division was recrystallized twice from methanol. Proflavine sulfate from Mann Research Laboratories was recrystallized from water; the crystals were washed with di(1) This work was supported by a grant from the National Institutes of Health (G3113292). (2) G. G. Hainmes and C. D. Hubbard, J . Phys. Chem., 70, 1615 (1966).
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ethyl ether and dried under vacuum over phosphorus pentoxide. Polyadenylic acid was supplied from Miles Chemical Co. as the potassium salt with agiven weightaverage molecular weight of 100,000 to 2,000,000 and a number-average of 40,000 to 70,000. Calf thymus DNA was purchased from Worthington Biochemical Corp. Chloroquine diphosphate (chloroquine, CQ, is 7-chloro4-(4-diethylamino-l-niethylbutylamino) quinoline) was generously donated by Dr. R. 0. Clinton of the Sterling Winthrop Research Institute, Rensselaer, N. Y. All other materials were standard reagent grade chemicals. Doubly distilled water \vas used for the preparation of all solutions. Solutions of poly A were made by gently stirring a suspension of the material in a NaC1-Tris-HC1 medium of pH 7.5 at 4"; the concentrations were estimated spectrophotometrically by measuring the absorption at 257 mp.3 DNA solutions were made by a carefully controlled, very slow stirring of a suspension of DNA into a similar medium of pH 7.0 at 4'. Each DNA solution made was subjected to a hyperchromicity t e ~ t . ~ *This 5 showed that the sample was at least 95% native. The concentration of DNA was estimated simultaneously. Solutions of the two acridine dyes and chloroquine were made by weighing out suitable amounts for stock solutions and diluting aliquots of these as required. The absorption spectra of all solutions were measured with a Beckman Model DU spectrophotometer fitted with a thermostated cell housing. Polymer dye solutions were made by combining appropriate amounts of the stock solutions of each component and making up to a standard volume with addition of suitable quantities of NaCl and/or Tris-HC1 buffer to the desired ionic strength. The ionic strength of the solutions was 0.1 M (usually 0.01 M in Tris-HC1 buffer and 0.09 M in NaCl), although some solutions were used in which the ionic strength was considerably lower. The pH of the solutions was 7.5 for the poly A-AO, poly A-PR, and poly A-CQ systems, 7.0 for the DNA-A0 system, 5.9 for the DNA-A0 system, and 5.9 for the DNA-CQ system as measured with a Radiometer pH meter. The temperature-jump apparatus has previously been described in detaiL'j-8 Temperature-jump experiments on the poly A-A0 and poly A-PR systems were performed over a range of poly A concentration Ill (the molarity is from about 4 X to 3 X expressed as the monomer concentration) and at A 0 and PR concentrations of 5.0 X and 2.5 X M. Kinetic runs were performed at 25.0" and the ionic strength indicated in Table I. The wavelengths used for observation of the chemical relaxation were 486 mp for the poly A-A0 system and either 420, 440, or 470 The Journal of Physical Chemistry
GORDON G. HAMMESAND COLIND. HUBBARD
mp for the poly A-PR system. Kinetic runs on all systems were preceded by the testing of the component parts of each solution in the temperature-jump cell to ensure that the observed relaxation effects are due entirely to the polymer-dye interaction. The precaution of excluding light from solutions containing acridine dyes was carried out in the manner described previously.2 The construction and performance of the A ow apparatus used is described e l s e ~ h e r e . ~
Results The spectral changes occurring in solutions of poly A and A 0 are qualitatively similar to those observed for the PGA-A0 system and are consistent with spectra reported for basic poly A-A0 so1utions:'O at large values of the ratio [poly A]/[AO], the predominant absorption peak is near 500 mp and is accompanied by a less intense band with a maximum at 464 mp; at
'960 4;O
4;O
440 4kO
4O ;
460
440
5bO
5;O
5;O
! '0
A (mp)
Figure 1. Spectra of poly A-A0 solutions a t pH 7.5, hl: 25.0°, ionic strength = 0.1 hl, [AO] = 2.5 X , [poly A] = 0 M ; - -, [poly AI = M; 4.24 x 10-3 31; -. -, [poly A] = 2.12 X - - - -, [poly A] = 3.39 X fil.
(3) G. Felsenfeld and A. Rich, Biochim. Biophys. Acta, 26, 457 (1957). (4) E. Chargaff, "The Nucleic Acids," Vol. I, E. Chargaff and J. N. Davidson, Ed., Academic Press Inc., New York, N. Y., 1955, p 307. ( 5 ) R. D. Hotchkiss, Methods Enzymol., 3, 708 (1957). (6) G. G. Hammes and 3. I. Steinfeld, J . Am. Chem. SOC.,84, 4639 (1962). (7) G. G. Hammes and P. Fasella, ibid., 84, 4644 (1962). (8) R. E. Cathou and G. G. Hammes, ibid., 86, 3240 (1964). (9) J. E. Erman and G. G. Hammes, Rev. Sci. Instr., 37,746 (1966). (10) D. F. Bradley and M.K. Wolf, Proe. Natl. Acad. Sci. U . S., 45, 944 (1959).
REACTION OF ACRIDINE ORANGEAND PROFLAVINE WITH POLYADENYLIC ACID
289 1
Table I: Relaxation Data for Poly A-Acridine Dye Systems at 25"
Poly A-A0
Poly A-PR
4.24 x 2.12 x 1.06 x 4.24 x 1.69 x 3.39 x 4.08 x 2.04 x 1.02 x 4.08 x 1.63 x 3.27 x 4.05 x 2.03 x 1.02 x 4.05 x 1.62 x 3.24 x
10-5
10-3 10-3 10-4 10-4 10-5 10-3 10-3 10-3 10-4 10-4 10-5
2.5 x 2.5 x 2.5 x 2.5 x 2.5 x 2.5 x 2.5 x 2.5 x 2.5 x 2.5 x 2.5 x 2.5 x 5.0 x 5.0 x 5.0 x 5.0 x 5.0 x 5.0 x
10-6 10-6 10-5 10-6 10-5 10-5 10-5 10-5
10-5 10-5 10-6 10-6 10-5 10-5 10-5 10-5 10-6 10-6
lo-%-$
x,
8ec -1
mr
1, Ma
170 85 42.4 17 6.76 1.36 163 81.5 40.8 16.3 6.52 1.31 81 40.5 20.3 8.1 3.24 0.65
0.168 0.169 0.172 0.178 0.228 0.107 0.173 0.164 0.166 0.215 0.192 0.194 0.148 0.159 0.175 0.237 0.264 0.211
486 486 486 486 486 486 486 486 486 486 486 486 494 494 494 494 494 494
0.1 0.1 0.1 0.1 0.1 0.1 0.05 0.026 0.026 0.026 0.026 0.026 0.05 0.026 0.026 0.026 0.026 0.026
No relaxation 0.96 1.20 1.60 1.17 1.01 0.90 1.29 0.94 1.43
440
0.1
470 440 440 440 440 440 470 470 470
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
1.82 x 10-3
2 . 5 x 10-5
72.8
7 28 x 3.64 x 1 82 x 9.1 x 4.55 x 2.28 x
2.5 x 2.5 x 2.5 x 2.5 x 2.5 x 2.5 x 5.0 x 5.0 x 5.0 x
29.1 14.6 7.28 3.64 1.82 0.91 18.2 10.9 3.64
9 I
x
5.46 x 1.82 x a
10-3 10-3 10-3 10-4 10-4
[Poly AI/ [dye1
10-4 10-4
10-5 10-5 10-5 10-4 10-4 10-4
10-5 10-5 10-5 10-6 10-5 10-5 10-5 10-5 10-5
I = ionic strength.
smaller values of the ratio [poly A]/[AO] the shorter wavelength peak becomes slightly more intense while the peak at 500 inp diminishes. This type of behavior is accentuated when the ionic strength of similar solutions is lowered. A typical set of spectra is illustrated in Figure 1. Solutions of proflavine of concentration less than 5 X 10-5 ill exhibit a broad absorption band in the visible region with a maximum at 444 mp. The peak is shifted to longer wavelengths in the presence of high concentrations of poly A, but remains at the same position or shifts to a slightly shorter wavelength when the poly A concentration is lowered. This behavior is similar to that observed with AO, but the changes in wavelength are much smaller, and with all values of the ratio [poly A]/ [PR] investigated the band was not separated into two peaks. At intermediate values of [poly A]/[PR] the band is broadened and has a less well-defined maximum which lies at wavelengths between the long and short wavelength limits of approximately 455 and 440 mp, respectively. I n the spectra
of the poly A-A0 system the peaks are separated by about 40 mp. Typical poly A-PR spectra are shown in Figure 2. A much less detailed study was made of the spectra of the DNA-AO, DNA-CQ, and poly A-CQ systems. However, the observed characteristics of the first two systems were essentially identical with those previously reported. 1 1 , l 2 The results obtained with the poly A-A0 system will be considered first. A typical relaxation effect is initially curved, but subsequently the amplitude of the effect decreases virtually linearly with time. If a straight line is drawn through the latter sloping part of the trace and extrapolated to the zero-time ordinate, then the distance between this extrapolated line and the actual trace, at any time t, is the amplitude of the first relaxation effect since the straight line is the limit(11) A. K. Peacocke and J. N. H. Skerrett, Trans. Faraday SOC.,52, 261 (1956). (12) S. N. Cohen and K. L. Yielding, J. Bid. Chem., 240, 3123 (1965).
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GORDONG. HAMMES AND COLIND. HUBBARD
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1.0
09
4 ~ ' ' ( m's e c ) ' ' ' ' ' ~ 2
0.1
..\\I 410
420
430 440
450
460
470
480
490
Imp1
Figure 2. Spectra of poly A-PR solutions at pII 7.5, M: 25.0", ionic strength = 0.1 M, [PI11 = 2.5 X -, [poly A] = 0 M ; - -, [poly A] = 1.82 X lo-* M; , [poly A] = 7.28 X IO-' M; - - - -, [poly A] = 1.82 X l W 4 Ai'; - -, [poly A] = 2.28 X 1 O F M.
.
ing value of this relaxation process at infinite time. A plot of the logarithm of the amplitude against 1 is linear. Figure 3 shows a typical oscilloscope trace and the semilogarithmic plot extracted from it by the above procedure. The relaxation time obtained in this manner is to a good approximation independent of the concentrations of poly A and A 0 (in the limited conceutration range of the latter employed) and appears to be independent of the ionic strength of the solution. However, when the value of [poly A]/[AO] is reduced to less than about 6, a faster relaxation process can be distinguished. The niethod given above was applied to evaluation of the relaxation times of both processes, the faster of which is of the order of 0.7 msec, while the magnitude of the slower relaxation is of the same order of magnitude as its counterpart at high poly A concentrations. The precision with which the relaxation times can be measured is very poor when both processes are observed; this is due to the fact that the amplitude of the faster process is very small and that both processes have relaxation times of the same order of magnitude. I n view of the small concentration range in which the faster process can be distinguished, and the polydisperse nature of the poly A, a detailed interpretation will not be attempted. The faster relaxation process is probably always part of the over-all relaxation spectrum but only becomes discernible at very low concentrations of poly A. The complexity of the relaxation spectrum prevents a more exact evaluation of the relaxation parameters. The J o u d of Phys*d Chemistry
4
6
8
t
IO
12
Figure 3. A typical relaxation effect and plot of logarithm of amplitude of light intensity change, SA, us. time. [Poly A] = 4.24 X IO-' M , [AO] = 2.5 X M, pH 7.5, T = 6.05 msec. The time scale on the photograph is one large division of the horizontal axis = 2 msec. Details of the method for obtaining the plot are described in the text.
Measurement of the relaxation time characterizing the slowest part of the over-all relaxation effect was attempted by use of both the Guggenheim13and the Swinborne methods," but the difficulty of distinguishing between chemical relaxation effects and absorption changes of the solution due to convection within the temperaturejump cell after 200 msec has elapsed after the temperature jump makcs any estimate extremely dubious. Attempts to characterize quantitatively the slowest part of the relaxation spectrum by a concentration-jump method and by direct mixing of polymer and dye in a flow apparatus were not successful because of the very small amplitude of the relaxation effect in the former experiments and because of the lack of reproducibility in measurement of the slow reactions in the latter method. However, the results indicate a relaxation process with a relaxation time of approximately 0.5 to 2.0sec is occurring. The fart that only an order of magnitude can be placed upon the relaxation time of the slowest part of the relaxation effect prevents evaluation of its dependence on the reactant concentrations and the molecularity of the process involved. Therefore, attention is confined to the faster relaxation process. Qualitatively, the observed relaxation effect for the poly A-PR system is similar to that for the poly A-A0 system in that it is a spectrum which extends over the time range measurable by the temperaturejump technique, and has a step in the fint part of the spectruni which yields a linear semilogarithmic plot obtained in the manner already described. Nevertheless, significant differences from the poly A-A0 system (13) E. A. Guggenheim. Phil. dfw.,2, 538 (1926) (14) E.S. Swinborne. J . Chem. Soe.. 2371 (1900).
REACTION OF ACRIDINE ORANGE AND PROFLAVINE WITH POLYADENYLIC ACID
exist: the relaxation effect is not observed a t values of [poly A]/[PR] greater than about 30 in an electrolyte medium of ionic strength 0.1 and increases in amplitude as the concentration of poly A is reduced. When the concentration of poly A is decreased further so that [poly A ] is approximately equal to [PR],the amplitude again diminishes. The relaxation spectrum arising from the interaction of DNA with AO, observed at 486 mp, is similar to that described for the PGA-A0 system,z but is not quantitatively described by two relaxation times as was found for the latter system. However, rough estimates of the maximum and minimum relaxation times of the spectrum can be made by use of the initial and final slopes of the usual plots of the logarithm of the signal amplitude us. time. The minimum relaxation time is in the range of 70-400 psec and estimates for the maximum relaxation time are from 0.5 to 7.0 msec. The variations in both times are random within the concentration range of DKA employed (0.108-6.35 X 10-4 J I ; the molarity refers to the concentration of phosphorus). I n contrast to the poly A-A0 system, a very slow relaxation process was not observed. Large absorbance differences between a solution of chloroquine and a solution of chloroquine in the presence of poly A or D S A occur between 325 and 345 mp) but no relaxation effects were observed.
Discussion TWOfeatures of the poly A-A0 spectra are in contrast to the PGA-A0 spectra; a blue shift of the shorter wavelength band does not occur when the value of the ratio [poly A]/[hO] is reduced to unity, and the band which at large values of [PGA]/[AO] remains at the same wavelength as that of the monomer band in free solution shows a small red shift in the poly A-A0 system. Such differences have previously been observed in analogous systerns,lO and are not prejudicial to the general interpretation of the spectra, namely, that the band in the poly A-A0 spectra at approximately 500 nip is characteristic of an unstaclied dye-poly A coniplex while the peak at 464 nik arises from a dimer d y e poly A complex. The solutions used in the teniperature-jump experiments on the poly A-A0 system most probably contain a mixture of monomer dye complex and dimer dye coniplex. Proflavine does not aggregate in free solution a t conX,but is thought to centrations less than 5 x interact with adjacent molecules of proflavine when these are bound to DKA. The existence of two types of binding sites in DNA for proflavine has been demonstrated by equilibrium studies;" these have been identified as a binding which is electrostatic, that is, the
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attraction between the negatively charged phosphate residues and the cationic dye molecules, and an interaction between aromatic rings of the dye and the bases of the DSA. JIutual interaction between bound proflavine molecules has also been proposed. An analogous interpretation to that for the poly A-A0 spectra has been used for the poly A-PII spectral measurements. The shift of the proflavine peak to longer wavelengths at large values of the ratio [poly A]/ [PR] means that proflavine is bound in an unstaclied manner to the polymer, and the short wavelength peak represents a complex containing proflavine dimer stacks or possibly higher aggregates along the polymer chain. The lack of resolution into two bands indicates that when there is a mixture of species in solution, the absorption peaks of each complex species superpose resulting in a single broad band. An acceptance of this explanation of the changes in the absorption spectra allows a qualitative understanding of the relaxation data. The data obtained restrict us to a qualitative discussion of the mechanism of interaction between bound acridine dyes. The independence of the relaxation time of the poly A-A0 and poly A-PR systems upon either the concentration of poly A or the concentration of dye, except at very low poly A concentrations, almost certainly implicates an intramolecular process which takes place subsequently to initial binding on the polynucleotide chain. The relaxation effect of the poly A-PR system is observed only when the relative concentrations of the reactants are those which yield dimer dye-poly A complexes, as judged by the absorption spectrum. In the case of AO-poly A complexes, dimeric stacks are present over the entire concentration probed. The presence in the equilibrium mixture of a species having aggregated dye groups along the polymer chain is a requirement for the observed relaxation effect to occur. This is coriqisterit with the fact that no relaxation effect is observed i n the DSACQ and poly A-CQ systems where the cation is thought not to interact with itself in solution or when bound to D;\TA.'* However, the kinetic studies themselves do not distinguish dimer stacks from any other type of "interacting dye" structure, so these results cannot be considered as proof of the interpretation given to the spectra. The fact that a relaxation tinic characteristic of interniolecular complex formation cannot be nieasured enables a lower limit to be placed upon the bimolecular rate constant for binding. This lower limit is about 2 X lo8 J1-l set.-', which means that complex formation occurs at a rate controlled essentially by diffusion together of the reactants. However, the dependence upon reactant concentration of the fastest reVolume 70, S u m b e r 5
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GORDON G. HAMMES AND COLIND. HUBBARD
Table 11: Characteristic Time Constants of “Stacking” Interactions a t 25” Conformation of macromolecule
System
...
AO-AO“
“Stacking” relaxation time, sec
PGA-AO“ PGA-AO” poiy A-AO~
Coil Helix Single strand
4.7 7.5 4.7 7.5
