DNA−Surfactant Interactions: A Procedure for Determination Group

698

J. Phys. Chem. B 2008, 112, 698-702

DNA-Surfactant Interactions: A Procedure for Determination Group Contributions Elia Grueso and Francisco Sanchez* The Department of Physical Chemistry, UniVersity of SeVille, C/Profesor Garcıá Gonza´ lez s/n, 41012, SeVille, Spain ReceiVed: May 18, 2007; In Final Form: October 18, 2007

A procedure, based on the measurement of pyren-1-carboxyaldehyde fluorescence, has been used to obtain the free energy corresponding to the interaction of several surfactants of alkyltrimethylammonium-type RN(CH3)3+ and DNA. These free energies depend linearly on the number of carbon atoms in the tail of the surfactant. In this way, it was possible to determine the -CH2-/DNA interaction free energy as well as the free energy of interaction of DNA and the head group of the surfactants. According to the values of these free energies, the surfactant/DNA interaction is mainly electrostatic, for the surfactants used in this work. However, for long enough tails the free energy corresponding to the hydrophobic interaction can reach the same value as the electrostatic one. The procedure used in this work could be extended to the measurement of other group/DNA interactions.

Introduction DNA is a biological polyelectrolyte. Generally speaking, interaction of a polyelectrolyte with a surfactant of opposite charge sign involves the participation of electrostatic and nonelectrostatic interactions. Among the latter, hydrophobic interactions between the tails of the surfactants and the hydrophobic moieties of the polymers are particularly important because they can produce dramatic changes in the polymer structure. Thus, it is well known that monovalent and divalent cations do not condense DNA. However, monovalent alkylammonium cations that have sufficiently long aliphatic chains condense DNA efficiently.1 In this particular case of DNA, the complexation with surfactants has received interest because of the relation of these processes to DNA purification and, especially, in relation to gene transfer2 and gene therapy applications.3 Mostly, the studies on DNA/surfactant systems have been devoted to learning about the influence of surfactants on the conformational changes and the stability of DNA. However, there have been relatively few systematic studies of the binding free energies of surfactants (and other ligands) to DNA.1b,4 These studies are of interest with reference to the establishment of group contributions. The group contributions, in turn, are important because detailed information about this topic is fundamental in relation to the design of ligands able to perform a desired function.5 As a contribution to this field, we present here a procedure to measure the binding free energy of surfactants to DNA. Using this approach for different surfactants of the type RN(CH3)3+ (R ) CH3, C2H5, C4H9, C8H17, C12H25 (DTAB), C14H29 (TTAB), C16H33 (CTAB)), we have been able to perform a separation of the electrostatic and nonelectrostatic free energy of binding. Moreover, the nonelectrostatic free energy is linearly related to the number of carbon atoms in R, when this number is g4. In this way, the group contribution can be obtained easily. Our approach is based on the use of pyren-1-carboxyaldehyde as a probe for the binding of surfactants to DNA. As is well * To whom all correspondence should be addressed. Tel +34-954557175. Fax +34-954557174. E-mail: [email protected].

known, pyrene and its derivatives are good probes for sensing polarity changes.6 This is a consequence of the existence of two excited states of pyrenes that are close in energy, a luminescent state and a “dark” state, whose relative populations depends on the medium polarity.7 We observed that in the presence of DNA there is a change in the intensity of the emission of pyren-1-carboxyaldehyde.8 This fact permits us to measure the free energy of binding of the pyrene to DNA. In the presence of surfactants, as a consequence of their binding to DNA, the free energy of binding of pyrene derivative changes. The magnitude of this change depends on the association degree of the surfactants and DNA. This circumstance permits us to measure the free energy of binding of the surfactants to DNA. This approach is related to one employed by Bhattacharya et al. and Petrov et al.,9 although these authors do not calculate the surfactant/DNA binding free energy. Experimental Methods Materials. C2H5N(CH3)3+ and C4H9N(CH3)3+ were prepared and purified as described in refs 10 and 11, respectively. The counterion of the surfactants was always Br-. Calf thymus DNA was purchased from Pharmacia and used without further purification because preliminary experiments showed that purification does not produce any changes in the results of experiments. The average number of base pairs by DNA molecule is ca. 3000.12 Polynucleotide concentrations were determined spectrophotometrically from the molar absorptivity (6600 mol-1 dm3 cm-1 at 258 nm).13 The other reagents were all Anala. R. grade and were used as purchased. The water used in the preparation of solutions had a conductivity of less than 10-6 Sm-1. All of these solutions contained ethanol 8% (by weight). The presence of the alcohol was necessary in order to make the probe, pyren-1-carboxyaldehyde soluble. Alternatively, alcohols stabilize DNA and prevent denaturation,14 which could be favored by the absence of buffer or supporting electrolyte. Methods. (a) Fluorescence measurements were carried out in a spectrofluorimeter (Hitachi f-2500), interfaced to a PC for the reading and handling of the spectra, at 298.2 K. Intensity measurements were performed at [probe] ) 5 × 10-7 mol dm-3.

10.1021/jp0738457 CCC: $40.75 © 2008 American Chemical Society Published on Web 12/18/2007

DNA-Surfactant Interactions

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TABLE 1: Values of Kmax and ∆Gmax for the Binding of Pyren-1-carboxyaldehyde to DNA Corresponding to Different Concentrations of Surfactants

TABLE 2: Values of Solubility of Pyren-1-carboxyaldehyde Corresponding to Different Concentrations of Surfactants

105[CH3N(CH3)3+]/ mol dm-3

10-3Kmax/ mol-1dm3

∆Gmax/ kJ mol-1

105[CH3N(CH3)3+]/ mol dm-3

106 solubility/ mol dm-3

105[C2H5N(CH3)3+]/ mol dm-3


0 1 10 100 400 1000

177 170 158 122 80 59

-29.94 -29.84 -29.66 -29.02 -27.97 -27.22

105[C2H5N(CH3)3+]/ mol dm-3

10-3Kmax/ mol-1dm3

0 1 10 100 400 1000 10000

1.87 1.92 1.92 1.94 2.00 2.13 4.00

0 2 20 100 200 500 1000

1.87 2.02 2.05 2.19 2.37 2.89 3.77

∆Gmax/ kJ mol-1

0 2 20 100 200 500

177 165 151 98 88 77

-29.94 -29.76 -29.55 -28.47 -28.21 -27.88

105[C4H9N(CH3)3+]/ mol dm-3


105[C8H17N(CH3)3+]/ mol dm-3


105[C4H9N(CH3)3+]/ mol dm-3

10-3Kmax/ mol-1dm3

0 2 5 7 10

1.87 2.19 2.48 2.68 2.97

0 2 5 7 10

1.87 2.32 2.93 3.34 3.95

∆Gmax/ kJ mol-1

0 2 5 7 10

177 200 230 265 280

-29.94 -30.24 -30.59 -30.94 -31.08

105[C8H17N(CH3)3+]/ mol dm-3

10-3Kmax/ mol-1dm3

∆Gmax/ kJ mol-1

0 2 5 7 10

177 208 245 260 280

-29.94 -30.34 -30.74 -30.89 -31.08

105[C12H25N(CH3)3+]/ mol dm-3

10-3Kmax/ mol-1dm3

∆Gmax/ kJ mol-1

0.0 2.0 3.5 5.0 6.0 7.0

177 210 245 260 269 272

-29.94 -30.36 -30.74 -30.89 -30.98 -31.00

105[C14H29N(CH3)3+]/ mol dm-3

10-3Kmax/ mol-1dm3

∆Gmax/ kJ mol-1

0 1 2 3 4 5

177 216 233 253 265 270

-29.94 -30.43 -30.62 -30.82 -30.94 -30.98

105[C16H33N(CH3)3+]/ mol dm-3

10-3Kmax/ mol-1dm3

∆Gmax/ kJ mol-1

0.0 1.1 1.7 2.2 3.5 5.0

177 215 235 255 265 275

-29.94 -30.42 -30.64 -30.84 -30.94 -31.03

The excitation and emission wavelengths were 356 and 466 nm, respectively. It was checked that the results were independent of the excitation wavelength, provided that this was in the range from 300 to 425 nm. DNA concentrations ranged, for each surfactant concentration, from 10-5 mol dm-3 to 10-3 mol dm-3. The surfactant concentrations are given in Table 1.

106 106 5 + 10 [C12H25N(CH3)3 ]/ solubility/ 10 [C14H29N(CH3)3 ]/ solubility/ mol dm-3 mol dm-3 mol dm-3 mol dm-3 +

5

0.0 2.0 3.5 5.0 6.0 7.0

1.87 2.51 2.99 3.46 3.78 4.09

0 1 2 3 4 5

1.87 2.10 2.33 2.57 2.80 3.04

105[C16H33N(CH3)3+]/ mol dm-3


0.0 1.1 1.7 2.2 3.5 5.0

1.87 2.23 2.44 2.53 2.89 3.31

(b) The solubilities of the probe in solutions containing surfactants were measured by agitating a generous excess of solid with the appropriate solution in a thermostatted (298.2 K) vessel. After waiting a long time for undisolved solids to settle, an aliquot of the saturated solution was removed using a pre-thermostatted pipet and the solution was diluted as necessary. Concentrations were measured spectrofluorimetrically. Surfactant concentrations corresponding to solubility measurements are given in Table 2. (c) Viscosity measurements were carried out employing an Ostwald viscosimeter, calibrated with water and ethanol. Each value of the viscosity was the average of 10 measurements. Results and Discussion Pyrene and its derivatives are less emissive when they are bound to a DNA duplex. According to Nakamura et al. this circumstance arises from the intercalation of pyrene between the base pairs of the duplex.8 This behavior is illustrated clearly in Figure 1, which gives the intensity of emission of pyren-1carboxyaldehyde versus DNA concentration, for solutions without surfactant. Similar behavior, however, was also observed when the surfactants were added to the solutions. In these cases, experiments were performed at fixed concentrations of the probe and the given surfactant, and changing, as in the case of Figure 1, the DNA concentration. In all cases, it was found that the intensity, I, can be fitted to the equation

700 J. Phys. Chem. B, Vol. 112, No. 3, 2008

I)

I0 + IDNAK[DNA]

Grueso and Sanchez

(1a)

1 + K[DNA]

et K ) Kmax 1 + et

(1b)

[DNA] - h j

(1c)

t)

The meaning of these equations is the following: in the presence of DNA, pyrene is partially bound to it:

The free pyrene emits with intensity I0, and the bound pyrene with intensity IDNA, in such a way that the observed emission is the average given by eq 1a. Alternatively, eq 1b shows that the binding constant, K, depends on the relation [pyrene]/[DNA]. In fact, because a fixed [pyrene] was used in our experiments, K is only dependent on [DNA]. It is worth pointing out that this behavior has been observed in other cases when small ligands bind to DNA.15 According to eq 1b, K increases as [DNA] does, This implies that the binding of pyrene to DNA is anticooperative. The sigmoidal dependence of K on [DNA] is, in fact, frequently found for anticooperative processes.16 In eq 1b, Kmax represents the maximum (limiting) value of K. Finally, in eq 1c, h is the value of [DNA] for which K ) (1/ 2)Kmax and j is an adjustable parameter. Table 1 gives the values of Kmax corresponding to our experiments. From these values, those of ∆Gmax

∆Gmax ) -RT ln Kmax

(3)

were obtained (see Table 1). The values of ∆Gmax represent the affinity of pyrene for the binding to DNA relatiVe to the solutions where the free pyrene is present. Obviously, ∆Gmax cannot be compared directly because these solutions are different. However, solubility measurements in Table 2 permit us to compare ∆Gmax once they have been corrected, taking into account the differences in free energy of pyrene in the solutions due to the presence of surfactants. Thus, if S0 is the solubility in the absence of surfactant and S in the presence of a given concentration of the surfactant, this free energy, ∆G, is related to the activity coefficient of pyrene, and the latter to the solubility:

∆G ) RT ln γPy ) RT ln

S0 S

(4)

In this way, the ∆Gcorr max values appearing in Table 3 were obtained:

∆Gcorr max ) ∆Gmax - RT ln

S0 S

(5)

An interesting point concerning the data in Table 3 is that ∆ + Gcorr max increases for RN(CH3)3 with R ) CH3 and C2H5 when their concentrations do so. On the contrary, for RN(CH3)3+ when R ) C4H9, C8H17, C12H25, C14H29, and C16H33, ∆Gcorr max decreases, when their concentrations do so. According to these facts, it must be concluded that the head group of the surfactants and the tails modify DNA, upon binding. But this modification is different in the sense that the head induces a change in the

Figure 1. Intensity of emission of pyren-1-carboxyaldehyde vs DNA concentration, without surfactant.

TABLE 3: Values of Corrected Free Energy for the Binding Pyrenaldehyde to DNA Corresponding to Different Surfactants 105[CH3N(CH3)3+]/ mol dm-3

∆Gcorr max/ kJ mol-1

105[C2H5N(CH3)3+]/ mol dm-3


0 1 10 100 400 1000

-29.94 -29.92 -29.81 -29.00 -28.03 -27.57

0 2 20 100 200 500

-29.94 -29.84 -29.54 -28.80 -28.43 -28.23

105 [C4H9N(CH3)3+]/ mol dm-3


105[C8H17N(CH3)3+]/ mol dm-3


0 2 5 7 10

-29.94 -30.50 -31.19 -31.58 -32.10

0 2 5 7 10

-29.94 -30.82 -31.78 -32.27 -32.86

105[C12H25N(CH3)3+]/ mol dm-3


105[C14H29N(CH3)3+]/ mol dm-3


0.0 2.0 3.5 5.0 6.0 7.0

-29.94 -31.18 -31.85 -32.39 -32.70 -32.98

0 1 2 3 4 5

-29.94 -30.73 -31.21 -31.61 -31.94 -32.21

105[C16H33N(CH3)3+]/ mol dm-3


0.0 1.1 1.7 2.2 3.5 5.0

-29.94 -30.93 -31.32 -31.51 -32.01 -32.47

DNA that makes it less efficient than free DNA in order to bind pyrene. The tails, however, produce a change in the structure of DNA that makes it more efficient than free DNA to bind pyrene. This difference reflects the different character of interactions of DNA with the head and tails. Thus, N(CH3)4+ increases the melting temperature of DNA. On the contrary, the hydrophobic tails decrease this temperature, and more so for longer tails.17 The effects of long tail surfactants (R g C4H9) or short tail (R ) CH3, C2H5) surfactants can be interpreted in the following way. The effect of a given RN(CH3)3+ on DNA, for any R, is

DNA-Surfactant Interactions

J. Phys. Chem. B, Vol. 112, No. 3, 2008 701

Figure 2. Corrected maximum free energy, ∆Gcorr max, of the interaction pyren-1-carboxyaldehyde-surfactant vs [C2H5N(CH3)3+].

Figure 4. Free energy, ∆GSDNA, of DNA-surfactant interaction vs the number of carbon atoms of the alkyl chain of the surfactants (see eq 8).

tants changes, go in the opposite sense when dealing with long tail or short tail surfactants, as mentioned previously. S In any case, from the values of KDNA the free energy of the equilibrium given in eq 7 can be obtained:

∆GSDNA ) -RT ln KSDNA

Figure 3. Corrected maximum free energy, ∆Gcorr max, of the interaction pyren-1-carboxyaldehyde-surfactant vs [DTAB].

to induce a structural change in DNA. This change will be the result of the structural modifications due to the head group and tail. So two kinds of DNA will be in the solution, free and bound to surfactants, with different capacities to bind pyrene, in the sense that the corrected free energy of binding of the dye will be different for each kind of DNA. The observed free energy will be given by

∆Gcorr max )

S corr S (∆Gcorr max)DNA + KDNA(∆Gmax) DNA[S]

1 + KSDNA[S]

(6)

In this equation, (∆Gcorr max)DNA is the free energy of binding of pyrene to free DNA, that is, to DNA whithout surfactant. (∆ S Gcorr max) DNA it is the same but when the DNA binds the S is the equilibrium constant for the surfactant, S. Finally, KDNA binding of the surfactant to DNA: S KDNA

DNA + S y\z DNA/S

(7)

In other words, changes in ∆Gcorr max can be interpreted from a two-state model of the DNA-surfactant binding. Figures 2 and 3 correspond to the fits of data to eq 6 for RN(CH3)3+ with R ) C2H5 and C12H25, respectively. Similar fits are possible for other R. These figures show that changes in the corrected free energy, when the concentration of surfac-

(8)

These free energies are plotted in Figure 4, versus the number of carbon atoms in R. It is clear from the figure that there is a good linear relationship between the free energy of binding of the surfactant to DNA and the number of carbon atoms in R for n g 4. With reference to n ) 1 and 2, the free energy of binding deviates from the correlation. Moreover, the intercept (n ) 1) of the line corresponding to “long tail” surfactants would correspond to a free energy for N(CH3)4+ of -19.1 kJ mol-1, which is lower than the one found directly from the data corresponding to this ion, although it is not very different from this value, -15.4 kJ mol-1. These facts imply that, strictly speaking, the effect of the tails and the head group are not additives, although they are not far from being so. From the data in Figure 4, it is possible to have the contribution to the free energy of binding DNA-surfactant of each CH2 group. This contribution results in -0.36 kJ mol-1, which is close to the results of the Matulis et al. for RNH3+ surfactants.1b Alternatively, the contribution of the head is -19.1 kJ mol-1(extrapolated value at n ) 1, see Figure 4) in such a way that the major contribution to the binding free energy corresponds to the electrostatic interaction head-DNA. In fact, the tail interaction would be equal (or greater) than the head interaction for R g C33H67. To check our procedure, we used a second approach to measure the surfactant-DNA free energy of binding. We reasoned that if the surfactants produce conformational changes in the DNA then this would produce changes in the viscosity of the solutions containing them. For this reason, the viscosity of the solutions containing a fixed concentration of DNA and variable concentration of CTAB were measured. The results corresponding to a [DNA] ) 2 × 10-5mol dm-3 are given in Figure 5. We also performed measurements at other concentrations of DNA. In all cases, the results were similar to those in Figure 5. These results can be fitted to the equation

η)

ηDNA + KSDNA ηSDNA[S] 1 + KSDNA[S]

(9)

702 J. Phys. Chem. B, Vol. 112, No. 3, 2008

Grueso and Sanchez References and Notes

Figure 5. Viscosities of solutions DNA-CTAB vs [CTAB]. DNA concentration is constant and equal to 2 × 10-5mol dm-3. S which is similar to eq 6. From this equation, KDNA can also be obtained, and, from this, the free energy of binding of the surfactant to DNA. The average value (corresponding to the different concentrations of DNA) result is -26.4 kJ mol-1, which is close to the value obtained from fluorescence measurements, -24.9 kJ mol-1.

Conclusions In conclusion, from measurement of the fluorescence of a probe (pyren-1-carboxyaldehyde) and from viscosity measurements we have been able to obtain the free energy of binding of different surfactants to DNA. According to our results, the surfactants induce changes in the DNA structure. The changes are the consequence of the electrostatic interaction of the head group and the nonelectrostatic tail/DNA interaction. These changes seem to be different in the sense that electrostatic interaction of the head group produces changes in DNA that makes it less efficient to bind the pyrene probe. On the contrary, the tail interaction increases the binding of the probe to DNA. Moreover, head and tail interactions are additive for RN(CH3)3+ with R g C4H9 (but not for the smaller tails). This fact has permitted us to separate the group contributions for long tail surfactants. Acknowledgment. This work was financed by the D.I.G.Y.T. (CTQ-2005-01392/BQU) and the Consejerıá de Educacioń y Ciencia de la Junta de Andalucıá.

(1) (a) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 9951. (b) Matulis, D.; Rouzina, I.; Bloomfield, V. A. J. Am. Chem. Soc. 2002, 124, 7331. (c) Zhu, D. M.; Evans, R. K. Langmuir 2006, 22, 3735. (2) Kirby, A. J.; Camilleri, P.; Engberts, J. B. F. N.; Feiters, N. C.; Nolte, R. J. M.; Soderman, D.; Bergsma, M.; Bell, P. C.; Fielden, M. L.; Garcıá Rodriguez, C.; Guedat, P.; Kremer, A.; Mc Gregor, C.; Perrin, C.; Ronsin, G.; Van Eijk, M. C. P. Angew. Chem., Int. Ed. 2003, 42, 1448, and references therein. (3) Symietz, C.; Schneider, M.; Brezesinski, G.; Mo¨hwald, H. Macromolecules 2004, 37, 3865, and references therein. (4) (a) Chatterjee, R.; Chattoray, D. K. Biopolymers 1979, 18, 147. (b) Spink, C. H.; Chaires, J. B. Biochemistry 1999, 38, 496. (c) Zantl, R.; Baiai, L.; Artzner, F.; Sprenger, I.; Rapp, G.; Radler, J. O. J. Phys. Chem. B 1999, 103, 10300. (d) Barreleiro, P. C. A.; Olofsson, G.; Alexandridis, P. J. Phys. Chem. B 2000, 104, 7795. (e) Cardenas, M.; Nylander, T.; Thomas, R. K.; Lindman, B. Langmuir 2005, 21, 6495. (f) Jiang, N.; Li, P.; Wang, Y.; Wang, J.; Yan, H.; Thomas, R. K. J. Phys. Chem. B 2004, 108, 15385. (g) Jiang, N.; Wang, J.; Wang, Y.; Yan, H.; Thomas, R. K. J. Colloid. Interface. Sci. 2005, 284, 759. (h) Chen, X.; Wang, J.; Shen, N.; Liu, Y.; Li, L.; Liu, M.; Thomas, R. K. Langmuir 2002, 18, 6222. (i) Wang, C.; Li, X.; Wettig, S. D.; Badea, I.; Foldvari, M.; and Verreel, R. E. Phys. Chem. Chem. Phys. 2007, 9, 1616. (5) (a) Chaires, J. B.; Satyanarayana, S.; Suh, D.; Fokt, I.; Przwloha, T.; Pribe, W. Biochemistry 1999, 35, 2048. (b) Leal, C.; Bilalov, A.; Lindman, B. J. Phys. Chem. B 2006, 110, 17221. (6) See, for example, (a) Kalyanasundaram, K.; Thomas, J. K. J. Phys. Chem. 1977, 81, 2176. (b) Okamoto, A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820. (c) Kawamoto, T.; Hashidzume, A.; Morishima, Y. J. Colloid. Interface. Sci. 2005, 291, 537, and references in these papers. (7) Kalyanasundaram, K.; Thomas, J. K. J. Phys. Chem. 1977, 81, 2176. (8) Nakamura, M.; Fukunaga, Y.; Sasa, K.; Ohtoshi, Y.; Kanaori, K.; Hayashi, H.; Nakano, H.; Yamana, K. Nucleic Acids Res. 2005, 33, 5887. (9) (a) Bhattarcharya, S.; Mandal, S. S. Biochim. Biophys. Acta 1997, 1323, 29. (b) Petrov, A. I.; Khalil, D. N.; Kazaryan, R. L.; Savintsev, I. V.; Shukhorukov, B. I. Bioelectrochemistry 2002, 58, 75. (10) Grovenstein, E.; Blanchard, E. P.; Gordon, D. A.; Stevenson, R. W. J. Am. Chem. Soc. 1959, 81, 4842. (11) Smith, P. A.; Frank, S. J. Am. Chem. Soc. 1952, 74, 509. (12) Secco, F.; Venturini, M.; Lo´pez, M.; Pe´rez, P.; Prado, R.; Sańchez, F. Phys. Chem. Chem. Phys. 2001, 3, 4412-4417. (13) Felsendeld, G.; Hirschman, J. Mol. Biol. 1965, 13, 409. (14) Knight, J. D.; Adami, R. C. Int. J. Pharm. 2003, 264, 15. (15) Lo´pez-Cornejo, P.; Pe´rez, P.; Garcıá, F.; De la Vega, R.; and Sańchez, F. J. Am. Chem. Soc. 2002, 124, 5154. (16) Hammes, G. G. Thermodynamics and Kinetics for the Biological Sciences; Wiley-Interscience: New York, 2000; p 124. (17) Notice that although surfactants increase the melting point of DNA this increase is due to the polar head so that the melting point is lower in the presence of N(CH3)4+. Accordingly, the effect of the hydrophobic tails is to decrease the melting point of DNA. ( See table 2 in ref 9a).

DNA−Surfactant Interactions: A Procedure for Determination Group

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