J. Chem. Soc., Faraday Trans. I, 1983, 79, 1085-1090

Effects of Solvent on Stacking Interactions A Spectrophotometric Study of Methylene Blue Dimerization in Aqueous

Solutions of some Monohydric Alcohols

BY SANDRO L. FORNILI* A N D GIUSEPPE SGROI

Istituto di Fisica dell’universita, Gruppo Nazionale Struttura della Materia, Consiglio Nazionale delle Ricerche, Via Archirafi 36, 90123 Palermo, Italy

A N D VINCENZO I z z o

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

Received 5th May, 1982

A previous experimental study of the role of hydrophobic interactions on so-called stacking forces (of considerable interest in the conformational stability of nucleic acids) is here extended by considering alcohol solvent perturbation, as probed by dye self-association. The monomer-dimer equilibrium of Methylene Blue in aqueous solutions of methanol, ethanol, propan-1-01 and propan-2-01 has been studied using spectrophotometric techniques, microprocessor-based data acquisition and computer-aided data processing.

Results show that these alcohols mainly affect the dimerization entropy change. A linear enthalpy- entropy compensation is found, with a compensation temperature of ca. 220 K.

In aqueous solutions planar molecules of Methylene Blue (MB) self-associate to form face-to-face dimers and higher aggregates as the dye concentration is increa~ed.’.~ This feature makes MB and similar dye molecules good model systems for studying so-called stacking force^,^-^ which play an important role in stabilizing helical conformations of nucleic acids.’

Hydrophobic interactions are relevant in MB aggregat i~n.~ Also, as shown in the previous work of this series, H20-D20 substitution appreciably affects MB stacking, mainly by affecting the entropy term.* This agrees qualitatively with previous findings on native DNA denaturationg and quantitatively with results obtained for a poly- saccharide systemlo and for a polynucleotide system.ll Moreover, in the temperature range we used, a similar, although determined by different enthalpy and entropy changes, stabilizing effect of D 2 0 on the double-helix conformation of the oligo- nucleotide (dG-dC), has recently been found.12

Results of recent experiments aimed at ascertaining the role of ‘solvent hydro- phobicity ’ in isothermal DNA denaturation in the presence of monohydric alcohols13 caused us to investigate the influence of alcohols on MB self-association in order to obtain a quantitative thermodynamic insight of their effects on stacking.

In the present paper we report the results of a spectrophotometric study of MB dimerization at various temperatures in the presence of some monohydric alcohols. A linear enthalpy-entropy compensation is found, with compensation temperature z 220 K.

1085

Publ

ishe

d on

01

Janu

ary

1983

. Dow

nloa

ded

by T

empl

e U

nive

rsity

on

27/1

0/20

14 1

4:12

:00.

View Article Online / Journal Homepage / Table of Contents for this issue

1086 EFFECTS OF SOLVENT ON S T A C K I N G INTERACTIONS

E X P E R I M E N T A L Methylene Blue chloride was purchased from Merck and was used after three recrystallizations

from ethanol, as previously described.8 Dye solutions were prepared by diluting concentrated mother-solutions in suitable solvents and relative concentrations were determined by weighing. Dilutions were performed using plastic syringes (Becton-Dickinson, Ireland), rather than glass pipettes, to avoid dye adsorption. Water was from a Millipore Super Q system. Methanol, ethanol, propan-1-01 and propan-2-01 (RP, Carlo Erba, Milano) were used without any further purification. Spectrophotometric measurements, microcomputer-based data acquisition and computer-aided data processing were performed as described in ref. (8). For each dye concentration and each alcohol concentration, absorption spectra were measured successively from the lowest to the highest temperature used. The measurement of the spectrum at the lowest temperature was repeated to check that no irreversible reaction had occurred. In view of the low concentration of dye solutions, we assume that the activity coefficients are ca. 1. Isosbestic points have a variation of only 3 nm within the entire set of measurements. We prefer to use the dimerization constant, K, rather than the monomer and dimer spectra to represent the influence of the solvent on dye association, since it is statistically more stable, being determined by more experimental data.

RESULTS A N D DISCUSSION Absorption spectra in the wavelength range 500-750 nm were collected every 2.5

nm, at six temperatures (5, 10, 15, 20, 25 and 30 "C) and for various water + alcohol mixtures (methanol, 2 mol dm-3; ethanol, 1, 2 and 4 mol dm-3; propan-2-01, 2 mol dm-3; propan-1-01, 1 and 2 mol dm-3). For each alcohol concentration and for each temperature, at least five diluted MB solutions, at different dye concentrations, were used to evaluate the dimerization equilibrium constants from the monomer and dimer absorption spectra, as required by the data processing algorithm being l4 The range of dye concentrations (from 4 x to 2.3 x lop4 mol dm-3) was such that the monomer4imer equilibrium only was relevant, according to criteria previously discussed.8

The experimental results are shown in fig. 1 (A) for D20, H 2 0 , methanol, ethanol, propan-1-01 and propan-2-01 (all 2 mol dm-3) and in fig. 1 (B) for H 2 0 and ethanol (1, 2 and 4 mol dm-3). Data for H 2 0 and D 2 0 are taken from ref. (8).

The following observations are noted. (i) Alcohol cosolvents displace the MB monomer-dimer equilibrium toward monomers, the effect increasing with in- creasing alcohol concentrations and alkyl-group size. This result agrees with previous findings.15-ls (ii) In the temperature range and in all cases studied, a linear relationship appears to exist between the logarithm of the dimerization constant, K , and the inverse of the absolute temperature. Standard enthalpy and entropy changes for MB dimerization could thus be evaluated by linear least-squares regression according to the relationship A G e = -RTln K, between the standard free-energy change, AG*, and the equilibrium constant for dimerization. Enthalpy and entropy changes are reported in table 1. (iii) Solvent perturbation caused by alcohols used in the present work mainly affects the dimerization entropy term. Indeed, 4 mol dm-3 ethanol differs from water by 376% in the entropy change and only by 67% in the enthalpy change. This parallels what has been found for H,0-D20 isotopic substitution.8 (iv) The relative effects of these alcohols on MB dimerization parallel their relative influence on the functional stability of P-galacto~idasel~ and haemoglobin,20 on the confor- mational stability of haemoglobin,21 ribonuclease22 and and on iso- thermal DNA denaturation for low (up to ca. 4 rnol dm-3) alcohol concentrations.13 Moreover, our findings agree qualitatively with the relative destabilizing effects caused

Publ

ishe

d on

01

Janu

ary

1983

. Dow

nloa

ded

by T

empl

e U

nive

rsity

on

27/1

0/20

14 1

4:12

:00.

View Article Online

S. L. FORNILI , G. SGROI AND V. I Z Z O 1087

I I I I I I I I

8 -

6

FIG. 1.-Plot of the logarithm of the dimerization constant, K, against the inverse of the absolute temperature for Methylene Blue solutions in: (A) D,O (a), H,O (m), 2 mol dm-3 methanol (I), 2 rnol dm-3 ethanol (A), 2 mol dm-3 propan-2-01 (A) and 2 rnol dm-3 propan-1-01 (*); (B) H,O (m), 1 mol dmW3 ethanol (*), 2 rnol dm-3 ethanol (A) and 4 rnol dm-3 ethanol (+). Data for H,O and D,O are

taken from ref. (8). Variability of experimental data for K is within the dimensions of the symbols.

TABLE 1 .-STANDARD ENTHALPY AND ENTROPY CHANGES FOR DIMERIZATION OF METHYLENE BLUE IN THE PRESENCE OF SOME MONOHYDRIC ALCOHOLS FOR THE TEMPERATURE RANGE 5-30 OC

D,OC - 6.2 f 0.2 - 3.4f 0.5 H,OC - 6.7 k 0.2 - 5.4f 0.9 methanol, 2 mol dm-3 -8.3f0.2 - 12.7f0.8 ethanol, 1 mol dmP3 -8.4k0.1 - 12.5f0.3 ethanol, 2 mol dm-3 -8.7k0.1 - 14.8f0.3 ethanol, 4 mol dm-3 -11.2f0.1 -25.7f0.4 propan-2-01, 2 mol dm-3 - 20.5 f0.8 propan-1-01, 1 mol dm-3 -8.4k0.2 - 13.3f0.9 propan-1-01, 2 mol dm-3 - 10.4f0.2 -22.5f0.7

- 10.0 f 0.2

a In kcal mol-l (1 cal = 4.184 J); in e.u. (1 e.u. = 1 cal K-' mol-l); data from ref. (8).

by 10 mol dm-, methanol and 10 mol dm-3 ethanol on the double-helix conformation of the oligonucleotide (dG-dC),, but not with results obtained with 10 mol dm-3 propan-1-01, which shows a destabilizing effect greater than that of 10 mol dm-3 methanol but lower than that of 10 mol dm-, (In this respect, note that analogous findings have been reported for the effects that the same alcohols at about

36 FAR 1

Publ

ishe

d on

01

Janu

ary

1983

. Dow

nloa

ded

by T

empl

e U

nive

rsity

on

27/1

0/20

14 1

4:12

:00.

View Article Online

1088 EFFECTS OF SOLVENT O N S T A C K I N G INTERACTIONS

-3.5

- 4.0 - - I

0 E - $ -4.5 & Q

- 5.0

I 0 2 4 6 8

C/mol dm-3 FIG. 2.-Plot of standard free-energy change for Methylene Blue dimerization at 5 (lower set) and 30 O C

(upper set) against equivalent carbon concentration, C. Symbols as in fig. 1 with the addition of 1 mol dm-3 propan- 1-01 (0).

A

-30 - 20 -10 AS"1e.u.

- 5 -4.5 - 4 AG*/kcal mol-'

FIG. 3.-Plot of enthalpy change against entropy change (A) and against free-energy change at 15 O C (B) for Methylene Blue dimerization. Symbols as in fig. 1 and 2.

Publ

ishe

d on

01

Janu

ary

1983

. Dow

nloa

ded

by T

empl

e U

nive

rsity

on

27/1

0/20

14 1

4:12

:00.

View Article Online

S. L. FORNILI, G . SGROI AND V. I Z Z O 1089 the same concentrations cause on isothermal denaturation of DNA.13) (v) Propan- 1-01 has a stronger perturbation effect than propan-2-01 on MB dimerization as well as on isothermal DNA denaturation at low alcohol concentration,13 on B-galactosidase functional stability,19 on the functional and conformational stability of haemoglobin,209 21 and on the conformational stability of ribonuclease22 and~o l l agen .~~ This seems to rule out a direct correlation between the bulk dielectric constant and the dominant alcohol perturbation effects, since the dielectric constant of propan-2-01 is larger than that of propan-1-01.~~ This result agrees with previous findings.l59 l6,

To obtain a better insight into the relative influence of different alcohols and alcohol concentrations on MB dimerization, in fig. 2 we replot our data for 5 and 30 OC against the equivalent carbon concentrations corresponding to the different water + alcohol mixtures. Note that in the present case the relationship between the free-energy change and the equivalent carbon concentration is approximately linear, i.e. far from the functional relationship common to the functional stability of haemoglobin and of 8-galactosidase, and to the conformational stability of DNA13 and of ribonuclease,22 as affected by the same alcohols.

Further information can be obtained from our experimental results by plotting enthalpy against entropy changes as in fig. 3 (A). This suggests the existence of a linear enthalpy-entropy compensation.26 In fig. 3 (B) the same enthalpy data are reported against the free-energy changes determined at 15 OC. This was chosen as the experimental temperature closest to the harmonic mean of all temperatures used (17.2 “C). This second plot confirms that the compensation effect is not due to statistical error pr~pagat ion .~~’ 28 Straight linesin fig. 3 were determined by least-sqhares best-fitting of all data, except those for D20. A compensation temperature T, x 220 K can thus be evaluated. As a comment to this value, we observe that a number of cases with compensation temperatures ranging from 250 to 315 K have been reviewed by Lumry and Rajender26 and interpreted as consequent on the properties of liquid water. More recently, statistical analyses of the enthalpy-entropy compensation effect for the interaction of water with two a-chymotrypsin confo rme~s~~ have given a com- pensation temperature value of 433 f 22 K. The origin of this compensation effect has been attributed to a monolayer hydration mechanism.

Due to the limited range of temperatures used, the compensation temperature found here has to be taken with caution. Also, we may pose the question as to the mechanism(s) of enthalpy-entropy compensation shown by fig. 3. In a kinetic and spectroscopic study of thionine association in the presence of alcohols, experimental results were interpreted in terms of specific solvent-solute interactions.18 The present data, as shown in fig. 3, are suggestive of a more general thermodynamic interpretative framework. Within it, the effects on MB dimerization of all alcohols used in the present work, and of H20-D20 isotopic substitution, could involve one and the same kind of ‘structural alterations’ of the solvent, as probed (and partially affected) by dye stacking.

We thank all members of our GNSM group at the University of Palermo for useful discussions and Mr S. Francofonte, Mrs S. Nicosia and Mrs A. La Franca for technical assistance. General indirect support was provided by CRRNSM and INFN.

D. G. Duff and C. H. Giles, in Water, A Comprehensive Treatise, ed. F. Franks (Plenum Press, New York, 1975), vol. IV, chap. 3. E. Rabinovitch and L. F. Epstein, J. Am. Chem. SOC., 1941, 63, 69. E. Braswell, J. Phys. Chem., 1968, 72, 2477.

G. G. Hammes, Adv. Protein Chem., 1968, 23, I . .I P. Mukerjee and A. K. Gosh, J. Am. Chem. SOC., 1970, 92, 6419.

36-2

Publ

ishe

d on

01

Janu

ary

1983

. Dow

nloa

ded

by T

empl

e U

nive

rsity

on

27/1

0/20

14 1

4:12

:00.

View Article Online

1090 EFFECTS OF S O L V E N T O N S T A C K I N G I N T E R A C T I O N S

B. H. Robinson, A. Loffler and G. Schwartz, J. Chem. SOC., Faraday Trans. 1 , 1973, 69, 56. H. Edelhoch and J. C. Osborne Jr, Adu. Protein Chem., 1976, 30, 183. S. L. Fornili, G. Sgroi and V. Izzo, J. Chem. Soc., Faraday Trans. 1, 1981, 77, 3049. V. Izzo, S. L. Fornili and L. Cordone, Nucleic Acids Research, 1975, 2, 1805.

lo G. Vento, M. U. Palma and P. L. Indovina, J. Chem. Phys., 1979, 70, 2848. l1 A. Cupane, E. Vitrano, P. L. San Biagio, F. Madonia and M. U. Palma, Nucleic Acids Research, 1980,

l2 D. DePrisco Albergo, L. A. Marky, K. J. Breslauer and D. H. Turner, Biochemistry, 1981, 20, 1409. l3 L. Cordone, A. Cupane, M. A. Dolce, S. L. Fornili, V. Izzo, P. L. San Biagio, G. Sgroi and E. Vitrano,

in Developments in Biophysical Research, ed. A. Borsellino, P. Omodeo, R. Strom, A. Vecli and E. Wanke (Plenum Press, New York, 1980), p. 259.

8, 4283.

l4 M. E. Lam and D. M. Neville Jr, J. Phys. Chem., 1965, 69, 3872. l6 M. J. Blandamer, J. A. Brivati, M. F. Fox, M. C. R. Symons and G. S. P. Verma, Trans. Faraday SOC.,

l6 R. B. McKay and P. J. Hillson, Trans. Faraday SOC., 1967, 63, 777. 1967, 63, 1850.

J. Ferguson and A.W-H. Mau, Aust. J. Chem., 1973, 26, 1617. T. G. Dewey, P. S. Wilson and D. H. Turner, J. Am. Chem. SOC., 1978, 100, 4550. L. Cordone, V. Izzo, G. Sgroi and S. L. Fornili, Biopolymers, 1979, 18, 1965.

2o L. Cordone, A. Cupane, P. L. San Biagio and E. Vitrano, Biopolymers, 1981, 20, 39. A. Cupane, D. Giacomazza and L. Cordone, Biopolymers, 1982, 21, 1081.

22 P. Von Hippel and U. Y. Wong, J. Biol. Chem., 1965, 240, 3909. 23 J. Schnell, Arch. Biochem. Biophys., 1968, 127, 496. 24 D. DePiisco Albergo and D. H. Turner, Biochemistry, 1981, 20, 1413. 25 G. Akerloff, J. Am. Chem. Soc., 1932, 54, 4125. 26 R. Lumry and S. Rajender, Biopolymers, 1970, 9, 1125. 27 R. R. Krug, W. G. Hunter and R. A. Grieger, J. Phys. Chem., 1976,80, 2335. 28 R. R. Krug, W. G. Hunter and R. A. Grieger, J. Phys. Chem., 1976, 80, 2341. 2g M. Liischer, M. Ruegg and P. Schindler, Biochim. Biophys. Acta, 1978, 536, 27.

(PAPER 2/738)

Publ

ishe

d on

01

Janu

ary

1983

. Dow

nloa

ded

by T

empl

e U

nive

rsity

on

27/1

0/20

14 1

4:12

:00.

View Article Online