INTERACTION OF PYRONINE-G WITH POLY (STYRENESULFONIC
ACID)
197
Interaction of Pyronine-G with Poly( styrenesulfonic acid)l by V. Vitagliano and L. Costantino Istituto Chimico, UniversitZl di Napoli, Naples, Italy
(ReceivedFebruary 84, 1969)
A spectrophotometric study of the interaction of poly(styrenesu1fonic acid) and pyronine-G has allowed the identification of three different species for the bound dye which have been recognized as monomer, dimer, and aggregate dye molecules. The experimental distribution among the three species is in agreement with the distribution computed by a statistical method including first- and second-neighbor interactions.
The interaction between cationic dyes and biological polymers has been the subject of a very extended literature in the recent years. The binding of acridine dyes to DNA and polyribonucleotides has been particularly studied using several techniques which include dialysis equilibrium,2 viscosity, 8 , 4 optical spectr0metry,~~5-~ induced optical rotation,1°-12 circular dichroism,13,14 f l ~ o r e s c e n c eX-ray , ~ ~ ~ ~on fiber^,^^^ and low angle X-ray ~ c a t t e r i n g . ~ The ~ ~ ~ ’great interest in the acridine dyes binding to polynucleotides is mainly due to the remarkable mutagenicity of acridines which can interfere in the replication of DNA by causing deletions or insertions of base pairs.’* As Bradley and Lifson recently pointed out,18 the accumulation of so much experimental knowledge has not yet allowed a unified picture of the binding process. On the other hand, the binding of dyes to synthetic polyelectrolytes has been generally neglected. Synthetic polyelectrolyte molecules are simpler systems and, to our opinion, the study of their interaction with dyes should give interesting information to help in approaching problems connected with biological polymers. The main feature of the binding of a lot of cationic dyes to biopolymers is the so-called “methachromasia;”20,21 this effect was recognized as due to different binding modes of the dye molecules which can exhibit different absorption spectra in the visible region. The absorption bands show that the dye molecules bind as monomer, or they interact with each other on the polyelectrolyte molecule to form dimers or aggregates similar to those existing in concentrated aqueous solut i o n ~ . ~ Bradley ~ - ~ ~ and Wolf5 proposed a simple model which accounts for this behavior in terms of dilution of the dye molecules along the polymer chain. The interaction of synthetic polyelectrolytes with methachromatic dyes is similar to that of biological polymer^.^^^^^ The tendency of dye molecules to stack each other is much higher,26-29and generally it is difficult to observe the spectrum of monomer bound dye. The Bradley and Wolf model does not apply satisfactorily in this case. The behavior of synthetic polyelectrolytes can be attributed to the higher flexibility of their chains5J6,28with respect to
those of biopolymers. I n this respect the poly(styrenesulfonic acid) has found more resemblance to DNA and to polyribonucleotides than to the synthetic electrolytes with a polyvinyl chain.26g2s,35The (1) This research has been carried out with the financial support of the Italian C. N. R. (2) A. R. Peacocke and J. N. H:Skerrett, Trans. Faraday Soc., 52, 261 (1956). (3) L. 9. Lerman, J . Mol. Biol., 3, 18 (1961). (4) D. 8. Drummond, N. J. Pritchard, V. F. W. Simpson-Gildemeister, and A. R. Peacocke, Biopolymers, 4, 971 (1966). ( 5 ) D. F. Bradley and M. K. Wolf, Proc. Nutl. Acud. Sci., 45, 944 (1959). (6) D. F. Bradley and G. Felsenfeld, Nature, 184,1920 (1959). (7) A. L. Stone and D. F. Bradley, J . Amer. Chem. Soc., 83, 3627 (1961). (8) D. S. Drummond, V. F. W. Simpson-Gildemeister, and A. R. Peacocke, Biopolymers, 3,135 (1965). (9) D. M. Neville, Jr., and D. R. Davies, J . Mol. Biol., 17, 57 (1966). (10) D. M. Neville, Jr., and D. F. Bradley, Biochim. Biophys. Acta, 50, 397 (1961). (11) A. Blakeand A. R. Peacocke, Nature, 206,1009 (1965). (12) A. Blake and A. R. Peacocke, Biopolymers, 4,1091 (1966). (13) S. F. Mason and A. J. LMcCaffery, Nature, 204,468 (1964). (14) B. J. Gardner and S. F. Mason, Biopolymers, 5,79 (1967). (15) L. S. Lerman, Proc. Natl. Acad. Sci., U.S., 49, 94 (1963). (16) V. Lussati, F. Mason, and L. S, Lerman, J. Mol. Biol., 3, 634 (1961). (17) We have not intended here to cover all the literature on the subject. (18) S. Brenner, L. Barnett, F. H. C. Crick, and A. Orgel, J. Mol. BioZ., 3,121 (1961). (19) D. F. Bradley and S. Lifson, “Molecular Associations in Biology,’’ B. Pullman, Ed., Academic Press, New York, N. Y.,1968, p 261. (20) L. Michaelis and 8. Granick, J . Amer. Chem. SOC.,67, 1212 (1945). (21) L. Michaelis, J . Phys. Chem., 54, l(1950). (22) E. Rabinowitch and L. F. Epstein, J . Amer. Chem. Soc., 63, 69 (1941). (23) V. Zanker, Z. Phys. Chem., (Leipsig), 199,225 (1952). (24) G. Barone, L. Costantino, and V. Vitagliano, Ric. Sci., 34 (IIA), 87 (1964). (25) M. E. Lamur and D. M. Neville, Jr., J. Phys. Chem., 69, 3872 (1965). (26) G. Barone, V. Crescenzi, F. Quadrifoglio, and V. Vitagliano, Ric. Sci., 36, 503 (1966). (27) V. Crescenzi, F. Quadrifoglio, and V. Vitagliano, J . Macromol. Sci., A l , 917 (1967). (28) G. Barone, R. Caramasza, and V. Vitagliano, Ric. Sci., 32 (IIA). 485 (1962). (29) M. X. Pal and M. Schubert, J . Phys. Chem., 65,872 (1961). Volume 74, Number 1 January 8, 1970
198
V. VITAGLIANO AND L. COSTANTINO
aim of this paper is to show the behavior of poly(styrenesulfonic acid) with a methachromatic dye very similar to the acridine orange.
r
l+
10-5 mol/l.) by mixing a concentrated PSSA solut'ion containing the dye and a dye solution in distilled water. A dilution cell was used which allowed volume changes from 3 to 50 ml.
Results
J
L
Pyronine-G hydrochloride
Experimental Section The pyronine-G (PG) hydrochloride, Fluka, was purified by dissolving the dye in methyl alcohol and precipitating with ether. The molar extinction coefficient of the monomer dye was found to be 46,500 a t X 550 mp ( E 45,900 at c = mol/l., E 45,600 at c = 1.5 X mol/l.) and did not change by further purification. This value is higher than the value reported by Koizumi and Mataga.ao Poly(styrenesu1fonic acid) (PSSA) was obtained by sulfonation of poly= 160,000), as already restyrene (Bios product, ported.31 Spectrophotometric measurements have been taken at room temperature with a Beckman DK-2 spectrophotometer. The measurements on PG a t varying concentration (see Figure 1) have been taken in aqueous HC1 solutions (0.001 mol/l.) to standardize the pH and the ionic strength; however, the dye spectra have been found not to vary in acid and neutral solutions. The stock solutions of the PG were found to be stable for weeks. The measurements with PSSA have been taken a t constant dye concentration (-1.5 X
r,
Free D y e in Aqueous HC1 Solutions. The behavior of PG in water is very similar to that of other methachromatic dyes well described in literature. 22-25 Increasing the dye concentration, the optical extinction coefficients curves change, clearly showing an equilibrium between two species (Figure 1, curves 2, 3, and 4) with two well-defined isosbestic points and maxima a t about 550 and 512 mp. I n agreement with the previously studied dyes an equilibrium between a monomeric (550-mp maximum) and a dimeric species (512-mp maximum) may be assumed. At higher concentrations a shift appears in the maximum of the dimer (Figure 1, curves 5 and 6) toward lower wavelengths, and the isosbestic points disappear. This is due to the aggregation of the dye molecules. By extrapolating the data of several runs the spectrum of the monomer (Figure 1, curve 1) and the limiting spectrum of the dimer (Figure 1, curve 7) were drawn. The equilibrium constant was estimated to be K = - -2a2c
1--a!
-
0.0012
The agreement between experimental and calculated values of the extinction coefficients is good up to a concentration of about 5 X mol/l. (see Figure 2). The value of the equilibrium constant is higher than
't
t Figure 1. Molar extinction coefficients of pyronine-G in aqueous HCl solutions (0.001 m): 1, extrapolated extinction coefficients of monomer dye; 2, dye concentration 1.486 X mol/l.; 3, dye concentration 4.406 X lo-* mol/l.; 4, dye concentration 9.18 X 10-4 mol/l.; 5, dye concentration 18.36 X lo-* mol/l.; 6, dye concentration 25.9 X mol/l.; 7, limiting extinction coefficients of dimer dye. The Journal of Physical Chemistry
I
I
10''
10"
I 10"
C
I 10''
I 10-2
I
10''
Figure 2. Comparison of experimental extinction coefficients of pyronine-G in aqueous HCl (0.001 m) solutions with the values computed through eq 1. (30) M. Koizumi and N. Mataga, J . Amer. Chem. SOC.,76, 614 (1964). (31) R.Hart and T. Timmerman, Compt. Rend. Congr. Intern. Chim. Ind., 8 l e Liege, (1958);Chim. Ind., 80,137 (1958); Chem. Abstr., 54, 8141f (1960).
INTERACTION OF PYRONINE-G WITH POLY (STYRENESULFONIC
199
ACID)
Table I: Optical Density Data of the Mixtures of Pyronine-G-Poly(styrenesulf0nic acid) a t Three mol/l.) Significant Wavelengths (Dye Concentration 1.45 X Nosa
P/Db
0 0.031 0.062 0.093 0.124 0.155 0.186 0.217 0.242 0.278 0.311 0,340 0,371 0.402 0.432 0.496 0.555 0.616 0.739 0.891
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17
18 19
i 20 21
i 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
38 39 40 j j j j j
(P/D)d
1,042 1.189 1.340 1.492 1.789 2.08 2.40 2.98 3.72 4.46 5.20 5.94 7.41 8.88
1.075 1.221 1.372 1,525 1.822 2.11 2.43 3.00 3.74 4.49 5.23 5.97 7.44 8.90
AexptPd
0.660 0.642 0,615 0.581 0.550 0.508 0.469 0.432 0.393 0.359 0.332 0,29& 0.265 0.244 0.220 0.190 0.169 0.155 0.137 0.127 0.127 0,132
... 0.142
AexptW
AexptP"'
0.115 0.121 0.124 0,137 0.152 0.170 0.188 0.211 0.227 0,255 0.272 0.286 0.310 0.330
A~orr~'~~
0.295 0,291 0.286 0.280 0,274 0.265 0.256 0.247 0.238 0,230 0,221 0,213 0.207 0.201 0.194 0.189 0.188 0.186 0.184
0.496 0,480 0.467 0.433 0.415 0.380 0.353 0.328 0.305 0.278 0.247 0.230 0.204 0.187 0.172 0.150 0.133 0,127 0.116 0,107
...
0.103 0.111 0.115 0.129 0.146 0.169 0.184 0.209 0.226 0.252 0.270 0.285 0.309 0.329 0.358 0.380 0.405 0.429 0.450 0.468 0,480 0.502 0.389 0.492 0.518 0.836 0.548
11.80 14.70 19.00 24.6 31.5 38.3 47.6 64.1 15.2 58.2 120 252 14,100
AoorP'
0.193 0.200
0,190 0.198 0.205 0,216 0.235
..
0.217 0,236 0.250 0.263 0.272 0.277 0.278 0.278 0.277 0,274 0,270 0,264 0,259 0.253 0,248 0,245 0,243 0.238 0.231 0.259
... 0.218 0.218
a Number corresponding to the graphs for Figure 3. +Ratio of polyelectrolyte to dye concentration (in the text the reverse DIP of this ratio has been used). c Corrected values of the ratios P / D including only the bound dye. d Experimental optical densities a t the wavelength of the maximum absorption of monomeric free dye (A -550 mp). 6 Experimental optical densities a t the wavelength of the maximum absorption of monomeric bound dye ( h -560 mp). Experimental optiCorrected optical densities (see text) a t h -560 mp. cal densities a t the wavelength of the maximum of dimeric bound dye ( h -517 mp). Corrected optical densities (see text) a t h -517 mp. ' Spectral graphs not registered in Figure 1. Data of a run not reported in Figure 3.
'
those of the similar dyes: acridine orange23f25*32 (K = 1.08 X and methylene blue24( K = 2.2 X The aggregation of dye in aqueous solution is a typical case of hydrophobic interaction^;^^ the presence of oxygen in the PG molecule and its possibility to build
up specific interactions with the water molecules (32) R. Caramazza, L. Costantino, and V. Vitagliano, Ric. Sci., 34, (IIA), 67 (1964). (33) G. NBmethy and H. A. Soheraga, J. Chem. Phys., 36, 3401 (1962).
Volume 74- Number 1 January 8, 1970
v. VITAGLUNO AND L. COSTANTINO
200
through hydrogen bonds should weaken these interactions and an increase of the K constant must be expected. Polyelectrolyte-Pyronine Intermtion. The methachromatic behavior of PG with polyelectrolytes has already been shown in the past." The interaction with poly(styrenesu1fonic acid) can be seen in Figure 3 where the absorption spectra of PG solution (1.45 X mol/l.) have been reported as a function of wavelength for various amounts of added PSSA; the concentration data are given in Table I.
" : e 6 1 .
l
/a'
0.5
0 0.Z
//
0.4
0.6
0.8
Figure 4. Dialysis equilibrium experiments for mixtures of PG and PSSA; concentration of free dye outside the dialysis bags &s a function of the DIP ratio inside the dialysis bags.
pears immediately under the DIP = 1 ratio and for DIP values of 0.05 to -0.04 the dye exists mainly as monomer. The PSSA behaves very similarly to the DNA; aud as in the case of DNA and acridine oranges we may talk of a strong binding for the monomeric dye and a weak binding for the aggregate dye36 (see Figure 4). We suggest that the high stability of the monomeric bound dye can be due to the benzene side rings in the PSSA molecules which might promote some kind of dye intercalation similar to that proposed for acridine orange and DNA.3 Spectra B of Figure 3 show very clearly the dilution of the dye along the PSSA molecule: decreasing the DIP ratio we see the contemporary appearance of the monomer band a t X 560 mp, and the shift of the maximum from -505 to -517 mp; no isosbestic points are present. I n agreement with the behavior of PG in aqueous solutions (Figure 1) these spectra may be attributed to the gradual disappearance of aggregate dye in favor of the dimer and monomer forms. For DIP ratios lower than 0.25 to -0.20 the probability of finding aggregates of more than two molecules becomes very small, and we see again an equilibrium between only two species: the monomer and the dimer with two quite well-defined isosbestic points a t X -527 mp and X -580 mp. The clear interpretation of the absorption spectra of Figure 3 B suggested trying to apply them a statistical treatment similar to that used by Bradley and W0lf.5 Recently, Lifsona7 published a very simple statistical mechanical method for computing the partition function of linear chain molecules easily applicable to the binding of dyes to linear chains.lg According to Lifson's treatment the partition function for such a I
Figure 3. Optical absorption spectra of PG 1.45 x 10-6 M in presence of different amounts of poly(styrenesu1fonic acid): A, excess of dye: . . DIP > 1 :. B.. excess of PSSA. D / P < 1. The PSSA concentration data. are given in Table I.
Increasing the polyelectrolyte concentration the graphs of Figure 3A show the gradual binding of the dye to the PSSA; the equilibrium between only two species (free and bound dye) is supported by the existence of isosbestic points. When the polyelectrolyte exceeds the dye amount (DIP < 1) the absorption spectra exhibit again the band of a monomer (Figure 3B) with a red shift of the maximum (X -560 mp) as found for other dye^.^.^^,^' It is possible to titrate the ionized groups of PSSA using PG with an accuracy higher than 1% as it can be done with acridine orange and DNA."." The appearance of the monomer dye has been attributed to the dilution of the dye over the polyelectrolyte molecule owing to the increased number of available binding sites. This effect has been very well discussedby Bradley and Wolf6 who studied the binding of acridine orange to a number of biological polymers including DNA. The Bradley and Wolf model, however, can be applied only to rigid polymers, and it was not found satisfactory for synthetic polyelectrolytes.as I n this respect the behavior of PSSA is peculiar; the absorption band of the monomer bound dye apT b Journal of Physiool Chem&u
~
(34) L. Costantino, A. M. Liquori, and v. Vitagliano. Biopolymers, 2, 1(1964). (35) L.Costantino, V. Creseenai. and V. Vitagliano, paper presented at the Tenth Meeting of the Italian Chemical Society. Padova. 1968. (36) Some experiments of dialysis equilibrium have shown that for D / P ratios lower than 0.04-0.02. a negligible amount of dye can be detected outside the dialysis bag. (see Figure 4).
(37) 8. Lifmn, J. them. Phys., 40,3706 (1964).
20 1
INTERACTION OF PYRONINE-G WITH POLY (STYRENESULFONIC ACID) system is obtained as the highest root of the equation
V ( x ) V ( x )- 1 = 0
(2) 88
where
U(x) = V ( x ) = qaXax-'
+
. *
X,
=
mF, = mFlqzm-2(q1v))m-1
9
=
i=l
q1qa2Xa2x-2
no/($ - qo)
(3)
+ . .. +
qlm-l(qaXa)mx-m,C (qrnqm-1.
*
m
m= 1
. qlqaXax-l)'-m
j=m
(4)
By assuming only first- and second-neighbor interactions, eq 4 reduces to
m
X,
=
saying v = qaXax-l, one obtains the following expression
where F1 = X1 is the fraction of monomer dye bound to the polyelectrolyte chain =
1
+
For q 2 = 1, eq 7 reduces to eq 13 of ref 19, where ql = k is the so-called staking coefficient. The fractions of monomer and dimer dye are
XI
=
Fi = (1 - qiqzv)2/ 11
+
-
q1qZ)v
Xz = 2F2
- qlqZ(q1 =
q1q~)v~l
2Flqiv
(84 (8b)
m- 1
mF, = 1
(10)
To apply Lifson's treatment to our data we had to make some simplifying assumptions. First, we corrected the experimental values of optical density for the presumed amount of free dye.40 From the graphs of Figure 3A we assumed the following value for the optical density of aggregate bound dye at 550 mp E3
P/D
(9)
where, of course
m
qm-1.
The fraction of the dye molecules in the mth aggregate stateagis (Figure 5)
=
0.110
This corresponds to a concentration of free dye a t D I P = 1 of 0.046 X mol/l. Assuming a linear dependence of cfreeon DIP the optical densities of all graphs of Figure 3B have been corrected by subtracting the absorption of the free dye, and the data have been normalized to a dye concentration of 1.45 mol/l. The true D I P ratios have also been computed using only the concentration of the bound dye. All results for two significant wavelengths are given in Table I. The optical densities of monomer bound dye a t 560 and 517 mp have been found by graphical extrapolation a t DIP = 0 of the experimental data E 1 = 0.552 a t X 560 mp and E 1 = 0.214 at X 517 mp. The optical densities of the aggregate bound dye at the same wavelengths were Ea = 0.095 a t X 560 mp and E 3 = 0.180 at X 517 mp. A more drastic but reasonable assumption was to take the same optical density for dimer and aggregate at X 560 mp: E 2 = Based on these assumpE3 = 0.095 a t X = 560 mp. tions, it was possible to calculate a set of values for the fraction of monomer bound dye
Xi
= F1 =
- 0.095 0.552 - 0.095 A660
A6e0being the corrected optical densities at 560 mp given in Table I. At 517 mp a value for the dimer optical density has been taken giving the best agreement between the F 1 computed at this wavelength and those computed a t 560 mp in the range of D / P values where the
0.8
'.
(38) The same expressions have been used here as in Lifson's paper: pa is the partition
0.2
0.4
x,
0.8
0.8
Figure 5. Fraction of dimer as a function of the fraction of monomer for different values of the second-neighbor interaction parameter QZ, computed through eq 6, 7, and 8.
function of a desorbed state site; pa is the partition function of asite in an absorbed state; Xa is the absolute activity of the the dye in the solution, Furthermore, it is assumed that neighboring adsorbed molecules interact each other such that a pair of first neighbors has a partition function of pairwise interaction 41, a pair of second neighbors has a partition function QZ, and so on up to the mth order of neighborhood. (39) It can be easily seen that the fractions X m are functions only of FI and pz and that they do depend on ql only through the dependence of FIon QI (Figure 5 ) . (40) The dialysis data (Figure 4) cannot be used for this purpose b e cause we are dealing with a complicated Donnan equilibrium and the concentration of free dye inside the dialysis bags is certainly less than that outside. Volume 74, Number 1 January 8,1970
202
NOTES
The experimental data of X I and Xz have been comI pared with the values given by eq 6-8. The best
Figure 6. Comparison of experimental monomer and dimer fractions with the values computed through eq 6, 7, and 8 for q1 = 5.5 and q 2 = 0.26.
amount of aggregates higher than dimers is negligible: = 0.355 a t 517 mp. Using these data and eq 11 the distribution of the different species was computed through the equation
agreement has been obtained for the following values of the q constants: q1 = 5.5 and q 2 = 0.26. The graph of Figure 6 shows that the agreement between experimental and statistical data is very good for the X I and reasonably good for the X z values. A low value of qz indicates a particular stability of dimers with respect to higher order aggregates; the fraction of dimer molecules is in fact much higher than that expected in the absence of second-neighbor interactions (q, = 1) (see Figure 5). The agreement of the experimental results with the statistical model seems to us of some interest because generally statistical treatments do not apply easily to the binding of small molecules because of the too many factors implied in the phenomenon. The good agreement obtained in our case, even with the limitation of several simplifying assumptions, by using only two binding parameters should be a good point in favor of the actual possibilities for a statistical treatment of the binding of dyes to polymers.
E,
A617 = 0.214Xi
+ O.355Xz +
0.180(1 - XI - X,)
(12)
Acknowledgment. The authors wish to thank Miss Paola Bianconi for her help during the experimental work.
NOTES
Rates of Mercapto Proton Exchange of Mercaptoacetic Acid in Acetic Acid'
by Jerry F. Whidby and Donald E. Leyden2 Department of Chemistry, University of Georgia, Athens, Georgia 90601 (Received March $0, 1969)
Recently there has been a great interest in the utilization of high-resolution and pulse nuclear magnetic resonance for the study of protolysis kinetics. For the great part this work has concentrated upon the exchange of amines and carboxylic acids in a variety of solvent~.*-~An interesting and important acidic functional group which has received less study is the mercapto g r o ~ p . ~The , ~ importance of this functional group in compounds of chemical and biological interest The Journal of Physical Chemistry
led us to investigate the nature of the protolysis kinetics of a representative compound. Because of its own importance and the similarity between some other important compounds, mercaptoacetic acid was chosen. Other mercapto compounds which have been studied include 2-mercaptoethanol in aqueous solutionE and (1) This investigation was supported by Public Health Service Research Grant GM-13935 from the National Institutes of Health. (2) Author to whom inquires should be addressed. (3) W. F. Reynolds and T. Schaefer, Can. J . Chem., 42,2641 (1964). (4) M. Cocivera, J . Amer. Chem. SOC.,88, 672, 677 (1966). (5) M. S. Puar and E. Grunwald, ibid., 89,4403 (1967). (6) E. Grunwald and E. K. Ralph, 111, ibid., 89,4405 (1967). (7) E. Grunwald and M. S. Puar, ibid., 89, 6842 (1967). (8) M. M. Kreevoy, D. S. Sappenfield, and W. Schwabacher, J . Phys. Chem., 69, 2287 (1965). (9) I. P. Gragerov, V. K. Pogorrelyi, and A. I. Brodskii, Dokl. Akad. Nauk SSSR, 178,880 (1968).
