J . Chem. SOC., Furaduy Trans. I , 1981,77, 3049-3053

Solvent Isotope Effect in the Monomer-Dimer Equilibrium of Methylene Blue

B Y SANDRO L. FORNILI* A N D GIUSEPPE S G R O I

Istituto di Fisica dell’universita, Gruppo Nazionale Struttura della Materia, Consiglio Nazionale delle Ricerche, Via Archirafi, 36, 901 23 Palermo, Italy

A N D VINCENZO I z z o

Istituto Nazionale di Fisica Nucleare, Via Archirafi, 36, 90123 Palermo, Italy

Received 30th December, 1980

So-called stacking forces, of particular interest in the structural stability of nucleic acids, have been studied in a simple model system of planar, non-hydrogen-bonding dye molecules (Methylene Blue). The dimerization process has been studied by spectral analyses at different concentrations and temperatures in D,O as well as in H,O. Computer-aided data-processing techniques have been used to obtain detailed information on the dimerization process.

Results show that the high positive (hydrophobic) contribution to the ‘unitary’ entropy term due to solute-solvent interaction changes appreciably upon H-D substitution. This illustrates the role of hydrophobic interactions in dimerization (stacking) processes.

Methylene Blue (MB) is a well-known cationic dye whose planar molecules associate to form face-to-face d i m e r ~ l - ~ in dilute aqueous solutions and higher aggregates with increasing dye con~ent ra t ion .~~

A body of evidence supports the hypothesis that interactions depending upon molecular aggregation are responsible for the concentration-dependent changes in optical spectra (metachromasy) of dyes in aqueous solutions.

Self-association of simple molecules like MB or Acridine Orange makes them good model systems for the study of so-called stacking force^^-^ which play an important role in the conformational stability of nucleic acids.*

A number of interactions can favour ‘~tacking’ .~ Among them are hydrophobic interactions, which are more generally a unique source of organization for macro- molecular

Studies of self-association of MB5 and of other dyes’ have shown the role of hydrophobic interactions in these processes and stimulated interest in modulating these interactions by means of H-D substitution. The absence of hydrogen-bond donors in MB rules out possible effects due to changes in hydrogen-bond strength caused I I H-D substitution and makes MB a particularly useful system for the study of isotopic modulation of hydrophic interactions.

The present paper reports the results of a spectrophotometric study of MB dimerization at various temperatures for diluted MB solutions in H,O and in D20 .

3049

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3050 SOLVENT ISOTOPE EFFECT I N METHYLENE BLUE

E X P E R I M E N T A L Water from a Millipore Super Q system was used. Deuterium oxide (99.8% D,O) was

obtained from Prochem Ltd. Methylene Blue Chloride and n-propanol (R.G.) were purchased from Merck. Following three recrystallizations from ethanol the dye was dried overnight at 100 O C and held in a desiccator. The molar extinction coefficient at 25 "C at the band maximum in ethanol (A = 654 nm) was 9.4 x lo4 dm3 mol-l cm-I and 8.1 x l O4 dm3 rnol-' cm-l at the absorption maximum (2 = 664nm) for the most dilute aqueous solution we used (4.9 x 13, l4 Solutions were prepared by dissolving thoroughly ground and dried powder in H,O and D,O at lop3 mol dm-3 and shaking for ca. 24 h at 30 "C. Exposure to light was avoided. Samples for spectrophotometric measurements were prepared by suitable dilutions of the above mother solutions.

The highest concentration values in H,O and D 2 0 were determined by weight. All the other dye concentrations were determined by absorbance measurements in 3.2 mol dm-3 HC1 at 745nm.I4 Optical absorbance measurements were carried out on a Cary 118C double spectrophotometer using 0.1, 0.2 and 1 cm stoppered cuvettes. The reference was H 2 0 or D20 as required. Sample and reference holders were thermostatted within 0.1 "C. Temperature was measured with a platinum thermometer and a precision bridge, providing an overall accurxy better than 5 x lo-, O C . Amicrocomputer-based system15controlled theacquisition ofabsorbance values from the digital output of the spectrophotometer at wavelength intervals of 2.5nm. Spectra were analysed in terms of monomer-dimer equilibrium following a critical evaluation of the possibility of a sizeable concentration of higher aggregates as discussed in the next section. Computer-aided fitting proceduresl6Y l7 were adopted to this purpose. Curves H and D in fig. 1 show the quality of fit obtained. Slight experimental errors in the concentration values upon dilution from mother solutions (less than 1%) were corrected by making use of the known existence of an isosbestic point at ca. 1 = 620nm.

mol dm-3), in good agreement with previously reported

R E S U L T S A N D D I S C U S S I O N

Spectra were studied at six temperatures ( 5 , 10, 15, 20, 25 and 30°C) for each of five MB concentrations in water (0.49, 0.92, 1.4, 1.9 and 4.2 x lop5 mol dm-3) and six concentrations in D,O (0.38, 1.2, 1.7, 2.2, 3.7 and 5.1 x mol dm-3).

10 j

P

2 . UJ

0 550 650 550 650

h/nm FIG. 1 .-Curves H and D show experimental (dots) and calculated (continuous lines) spectra of Methylene Blue solutions 4.2 x moldmP3 in H,O and 5.1 x 10-5mol dmPJ in D,O, respectively, at 25OC. Calculated spectra of monomers (m' and m) and dimers (d' and d) at 5 and 3OoC, respectively, are also

reported for solutions in H,O (A) and D,O (B).

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S. L. F O R N I L I , G. S G R O I A N D V. I Z Z O 305 1 Monomer and dimer spectra must be evaluated in order to calculate dimerization

constants. This evaluation requires caution, since it can be affected by the presence of higher aggregates, despite the existence of generally well-defined isosbestics.l8 To ascertain this point without making use of more strict and rigorous analyses,la. l9 we shall provisionally assume that monomer-dimer equilibrium only is of relevance under our conditions,23 16, 20* 21 then we shall compare the temperature dependence of our calculated monomer spectrum with that of a dye spectrum in alcoholic solution, where monomers only can be assumed to be present.20t22323 Finally, we shall see that the temperature dependence of the calculated ' dimer ' spectrum is incompatible with a sizeable presence of higher aggregates.

In fig. 1 (A) the calculated monomer and dimer spectra for MB solutions in H 2 0 are reported for 5 and 3OOC. Analogous results for solutions in D 2 0 are shown in fig. 1 (B). The general features of these spectra (two branches for dimers and a shoulder for monomers) are similar to those reported in the literature for aqueous solutions of MB16 and of other dyes.'*$ 20, 2 4 9 25 For monomers in the H 2 0 case, both maximum heights and spectral areas (as integrated in the 500-750 nm range) decrease when going from 5 to 3OoC, by 5.6 and 1.5%, respectively. These values become 4.7 and 2.1% in the case of D20. This temperatre dependence could be either real or an artifact resulting from the basic assumption contained in our method (no relevant presence of trimers). In order to ascertain this point we measured the absorbance of 9.8 x 10-6moldm-3 MB solution in n-propanol at 5 and 31 OC. Beer's law was found to hold at least up to a tenfold increase in dye concentration, in agreement with previous findings.20, 2 2 v 23 The absorbance maximum and the integrated area value, corrected for the thermal expansion, are lower at 5OC than those at 3OoC by 5.4 and 0.9 %, respectively. This temperature dependence is very similar to that reported above for calculated monomer spectra in H20.

Absorbance peaks and integrated areas of the calculated dimer spectra exhibit the opposite temperature dependence: at 5 OC they are for H,O lower than those at 30 OC by 2.6 and lo%, respectively (1.4 and 10.3% for D20). This temperature dependence could hardly be attributed to the existence of trimers and higher aggregates, since fig.

TI" C

2 5 15 5 I I I 1 I I

9.5 i t

I i

I I I I I I

3.2 3.4 3.6 103 KIT

FIG. 2.-Plots of the logarithm of the dimerization constant, K. against the inverse of the absolute temperature for Methylene Blue solutions in H,O (m) and in D,O (a).

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3052 S O L V E N T ISOTOPE EFFECT I N METHYLENE B L U E

1 shows that most of the spectra changes occur in the 630-680 nm region, while trimers are expected to alter mostly the shorter-wavelength region of the spectrum.49

The temperature dependence of the dimerization constant K for MB solutions in H,O and D,O is shown in fig. 2. We observe that: (i) the plot is linear for both H,O and D,O. This confirms the results reported by Mukerjee and Gosh5 for H,O and extends them to D20. (ii) There is a definite isotope effect on monomer-dimer equilibrium favouring dimer formation in D,O. This parallels previous findings about isotope effects in micelle formation.26

Using the relationship AGO = - RT In K between free-energy change AGO and the equilibrium constant for dimer formation, we obtained by linear regression analysis the enthalpy and entropy changes reported in table 1. Our results for dimerization

TABLE 1 .-THERMODYNAMIC QUANTITITES RELATED TO THE DIMERIZATION OF METHY LENE BLUE IN H,O AND D,O FOR THE TEMPERTURE RANGE 5-3OoC

AHoa ASob ASZb

H,OC D,OC H,Od

- 6.7 - 5.4 2.6 - 6.2 - 3.4 4.6 - 5.9 -4.5 3.5

a In kcal mol-l; bin e.u.; Cpresent work; ddata from ref ( 5 ) .

constants in H,O agree well with those obtained at a single temperature from spectrophotometric measurements (5.9 x lo3 dm3 mol-1 at 25 "C) by Bergmann and O'KonskilG and (6.4 x lo3 dm3 mol-1 at 20 "C) by Ballard and Park.,' The value also determined spectrophotometrically by Braswel14 (2 x lo3 dm3 mol-1 at 30 "C) is different, but this author used a larger range of dye concentrations where the presence of higher aggregates is relevant. Mukerjee and Gosh5 used a non-spectrophotometric method at three temperatures and obtained for A H " and AS" values reported in table 1 which differ from ours by 13 and IS%, respectively.

From data shown in fig. 2 and in table 1 we see that within the range of temperatures explored, dimerization in D,O appears to be increased mainly as a consequence of isotopic modulation of the entropy term. From table 1 we compute the ratio R of the percentage isotopic alterations of A S o and AH"

R = [A(AS")/EF]/[A(AH">/KfHF] 2 6,

where We now recall that dimerization causes a decrease in the number of molecules and

therefore AS" contains a term related to the mixing entropy. This term has to be subtracted from AS" to give the 'unitary' entropy change,1°i2* AS:, which is positive both for H,O and D,O solvents. However, negative contributions to AS: exist, owing to charge interaction in the dimer and to loss of rotational and translational entropy in dimer formation. To account for the reported positive AS': values therefore, these negative contributions must be overcome by at large enough positive contribution deriving from interaction with the so1vent.j

A further notable increase of this positive contribution is caused by H-D isotopic substitution in the solvent, as table 1 shows. The size of this isotope effect could hardly be attributable to the isotopic alteration of the static dielectric constant and of its temperature derivative, since these are both of the order 29 of less than 1 "/o. This isotope

and A H " are average values.

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S. L. F O R N I L I , G. S G R O I A N D V. I Z Z O 3053 effect must therefore originate from the isotopic modulation of the same (hydrophobic) interaction which is responsible for the positive sign of AS: in both H,O and D,O. In this respect we note that isotope modulations of entropy terms of exactly the same size (ca. 2 e.u.) have been observed in uncharged polysaccharide i.e. conditions such that differences in the static dielectric constants of H,O and D,O are irrelevant. The sign of the isotope modulation of the entropy term involved in the dinierization process is also the same as that for the melting of ice I. In other words, it looks as if dimerization involves a process (loosely) analogous to the ‘melting’ of some water molecules. This is similar to what has been observed for the establishment of helical order in the previously mentioned case of a poly~accharide~~ and in a polynucleotide system.31 The special interest of the present finding, however, lies in the fact that no hydrogen bonding occurs here.5 Further work is in progress along these lines.

We thank all members of our GNSM group at the University of Palermo for use- ful discussions and Mr M. Lapis, Mr G. Lapis, Mr R. Nicotra, Mr s. Francofonte, Ms S. Nicosia and Ms A. La Franca for technical help.

General indirect support from CRRNSM and INFN is also acknowledged.

D. G. Duff and C. H. Giles, in Water, A Comprehensire Treatise, ed F. Franks (Plenum Press, New York, 1975), vol. IV, chap. 3. E. Rabinovitch and L. F. Epstein, J . Am. Chem. Soc., 1941, 43, 69. D. J. Blears and S. S. Danyluk, J. Am. Chem. Soc., 1966, 88. 1084. E. Braswell, J . Phys. Chem., 1968, 72, 2477. P. Mukerjee and A. K. Gosh, J . Am. Chem. Soc., 1970, 92, 6419. B. H. Robinson, A. Loffler and G. Schwarz, J. Chem. Soc., Furaday Trans. 1, 1973, 39, 56. G . G. Hammes, Adv. Protein Chem., 1968, 23, 1. H . Edelhoch and J. C. Osborne Jr, Adz. Protein Chenz., 19’76, 30, 183. E. Coates, J . Soc. Dyers Colour, 1969, 85, 355.

”’ W. Kauzmann, Adv. Protein Chem., 1959, 14, 1. l 1 C. Tanford, Science, 1978, 200, 1012. l P A. Ben-Naim, Hydrophobic Interactions (Plenum Press. New York. 1980). l 3 P. Mukerjee and A. K . Gosh, J . Phys. Chem., 1963, 67, 193. l 4 P. Mukerjee and A. K, Gosh, J . Am. Chem. Soc., 1970, 92, 6403. l 5

l 6 K. Bergmann and C. T. O’Konski, J . Phys. Chem., 1963, 67, 2169. l i M. E. Lam and D. M. Neville Jr, J . Phys. Chem., 1965, 69, 3872. l 8 C. A. Brignoli and H. De Voe, J. Phys. Chem., 1978, 82, 2570.

R. M. Wallace and S. M. Katz, J . Phys. Chem., 1964, 48, 3890. 2o W. West and S. Pearce, J . Phys. Chem., 1965, 69. 1894. 2 1 W Spencer and J. R. Sutter, J . Phys. Chem., 1979, 83, 1573. L 2 M. J. Blandamer, J. A. Brivati, M. F. Fox, M. C. R. Symons and G. S. P. Verma, Trans. Faraday Soc.,

2J J. Ferguson and A. W. H. Mau, Aust. J . Chem. 1973, 26, 1617. 24 W. H. J. Stork, G . J. M. Lippits and M. Mandel, J . Phys. Chem., 1972, 76, 1772. L 5 L. P. Gianneschi, A. Cont and T. Kurucsev, J . Chem. Soc., Faraday Trans. 2, 1977, 73, 664. 26 P. Mukerjee, P. Kapauan and H. G. Meyer, J . Phys. Chem., 1966, 70. 783. 27 R. E. Ballard and C. H. Park, J . Chem. Sac. A , 1970, 1340. 28 R. W. Gurney, Ionic Processes in Solution (McGraw-Hill, New York, 1953; Dover, New York, 1962). 2q D. Eisenberg and W. Kauzmann, The Structure and Properties of’ Water (Clarendon Press, Oxford,

30 G . Vento, M. U. Palma and P. L. Indovina, J . Chem. Phys., 1979, 70, 2848. 31 A. Cupane, E. Vitrano, P. L. San Biagio, F. Madonia and M. U. Palma, Nucleic Acids Research, 1980,

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(PAPER O/ 1990)

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