J. Phys. Chem. 1996, 100, 16749-16753
16749
Characterization of the Interactions between Lysozyme and n-Alkyltrimethylammonium Bromides by Zeta Potential Measurements Vı´ctor Mosquera, Juan Manuel Ruso, Gerardo Prieto, and Fe´ lix Sarmiento* Grupo de Fı´sica de Coloides y Polı´meros, Departamentos de Fı´sica Aplicada y de Fı´sica de la Materia Condensada, Facultad de Fı´sica, UniVersidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain ReceiVed: March 27, 1996; In Final Form: July 8, 1996X
A detailed study of the adsorption of the n-alkyltrimethylammonium bromides (CnTAB) to lysozyme by measurements of the zeta potential (ζ-potential) has been realized as a function of concentration and pH. While at pH 3.2 the ζ-potential of lysozyme remains positive in the presence of surfactants, at pH 7 and 10 the alkylammonium ions affect the ζ-potential causing a change in the neighbourhood of the point of zero charge (pzc) from negative to positive values. From the pzc we have calculated Gibbs energies of adsorption and compared them with Gibbs energies of binding determined by equilibrium dialysis. The results are similar, showing that the ζ-potential technique can be useful in a study of the interactions of proteins with amphiphilic ligands.
2. Experimental Section
1. Introduction The proteins undergo changes in their natural state by the action of surfactants, which are used as adsorbates in order to control the hydrophobic-hydrophilic character of protein surface.1 Lysozyme is a small protein (molecular mass 14 603) with 18 cationic amino acid residues (six lysyl including one N-terminal, 11 arginyl and one histidyl) and 12 anionic residues (two glutamyl, nine aspartyl, and one leucyl C-terminal).2 Numerous studies on the interaction between surfactant and lysozyme have been reported, especially concerning anionic surfactants.3-12 These studies have shown that the interactions involve the ionic binding to the cationic sites and that further binding occurs by hydrophobic cooperative interactions. However, there have been few studies on the interactions between cationic surfactants and lysozyme, demonstrating that cationic surfactants inhibit the enzyme as a consequence of the interaction.13,14 We have recently reported a thermodynamic study of the interaction of a range of CnTAB (n ) 8, 10, 12, 14) with lysozyme at pH 10.0015 showing that the energy of interaction is dominated by the unfolding process. This study was performed by a combination of equilibrium dialysis and microcalorimetry experimental methods. Equilibrium dialysis permits us to obtain Gibbs energies of binding by a theoretical analysis of the isotherms of binding protein-surfactant but the experimental method is complex and time consuming. Thus, we have designed a zeta potential (ζ-potential) experimental study of the adsorption of CnTAB onto lysozyme at different pHs. The ζ-potential measurement provides information about the following properties of the materials: the electrical double layer at the surface interface, the surface charge density, and the identification of the surface chemical component of the particles.16 Also, the Gibbs energies of adsorption can be calculated from the pzc. This information, which we can compare with the Gibbs energies of binding, is important to understand the conformational changes of the macromolecule and the character of protein surface. * To whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, September 15, 1996.
S0022-3654(96)00927-6 CCC: $12.00
Materials. Lysozyme from chicken egg white (product No. L-6876, 48 000 units per mg) was obtained from Sigma Chemical Co. n-Octyl-, n-decyl-, n-dodecyl-, n-tetradecyl-, and n-hexadecyltrimethylammonium bromides, product numbers 8610, 12144, 3676, 10294, and 8594 respectively, were from Lancaster MTM Research Chemicals Ltd. Three buffers were used: glycine (50 mmol dm-3)-sodium hydroxide pH 10.00, phosphate (50 mmol dm-3) pH 7.00, and glycine (50 mmol dm-3)-hydrochloric acid pH 3.20. All other materials were of analytical grade and solutions were made in doubly distilled water. All measurements were below the literature values of the critical micelle concentrations (cmc) of the surfactants at each pH.17-19 Adsorption of Surfactants by Lysozyme. To obtain a complete adsorption onto lysozyme, alı´quots of 2.5 cm3 of the protein solution of concentration 1.25 × 10-3 kg dm-3 were placed in a test tube and equilibrated with 2.5 cm3 of surfactant solution covering the required range of concentration for over a week at room temperature. Equilibrium Dialysis. The binding of the n-dodecyltrimethylammonium bromide (C12TAB) to lysozyme was measured by equilibrium dialysis in which 2 cm3 aliquots of lysozyme solution of concentration 1.25 × 10-3 kg dm-3 were placed in dialysis bags and equilibrated with 2 cm3 of the C12TAB solution at pH 10.00 covering the required concentration range for over 96 h at 25 °C. The free surfactant concentrations at equilibrium were assayed using the Orange II dye method20 with reference to standard curves. Each data point on the binding isotherms corresponds to an individual dialysis sample and the curve was made up of three to four batches of separate dialysis experiments to cover the required range of free surfactant concentration. To obtain the Gibbs energies per surfactant bound (∆GVj) the isotherm was fitted to the second degree polynomial
Vj ) a + b(log[S]f) + c(log[S]f)2
(1)
where Vj is the average number molecules bound per monomer lysozyme molecule, [S]f, the free surfactant concentration and the parameters a ) 1238.93 ( 0.03, b ) 689.18 ( 0.02, and c © 1996 American Chemical Society
16750 J. Phys. Chem., Vol. 100, No. 41, 1996
Mosquera et al.
Figure 1. ζ-potential as a function of concentration of n-octyltrimethylammonium bromide at different pH: (9) pH 3.20; (b) pH 7.00; (2) pH 10.00.
Figure 2. ζ-potential as a function of concentration of n-decyltrimethylammonium bromide at different pH: (9) pH 3.20; (b) pH 7.00; (2) pH 10.00.
) 95.97 ( 0.03. With the polynomial we calculated the Wymann binding potentials (π) as a function of Vj from the equation21
π ) 2.303RT ∫(log[S] )f Vj Vjd(log[S]f) (log[S] )
f Vj)0
(2)
π ) 2.303RT{a(log[S]f) + b(log[S]f)2 + c(log[S]f)3} (3) The equilibrium constants (K) as a function of Vj are calculated from the equation
π ) RT ln(1 + K[S] fVj)
(4)
and hence ∆GVj as a function Vj from
∆GVj )
-RT ln K Vj
(5)
Zeta Potential Measurements. Zeta potentials were obtained with a Zetamaster Model 5002 (Malvern Instruments, England) by taking the average of (at least) five measurements at stationary level. The cell used was a 5 mm × 2 mm rectangular quartz capillary. The calculation of zeta potential is realized by application of the Smoluchowski equation22
ζ ) ηνe/E
Figure 3. ζ-potential as a function of concentration of n-dodecyltrimethylammonium bromide at different pH; (9) pH 3.20; (b) pH 7.00; (2) pH 10.00.
(6)
where η and are the viscosity and permittivity of medium, respectively, E is the applied electric field corrected for cell geometry, and νe is the electrophoretic velocity. The Smoluchowski equation is applicable only for particles with a Debye length, κ-1, much smaller than the mean radius of curvature of the particles.23-26 The reciprocal Debye length κ for charged particles in solution is given by
κ2 ) 2n0e2/kT
(7)
where n0 is the ionic concentration, e is the charge, and k is the Boltzmann constant. In the case of lysozyme, whose structure is known by X-ray diffraction,27 the Debye length obtained was approximately 10-4 a (a is particle radius) so it is possible to use the Smoluchowski equation. 3. Results and Discussion ζ-potentials against surfactant concentration for the adsorption lysozyme-CnTAB at different pHs are plotted in Figures 1-5.
Figure 4. ζ-potential as a function of concentration of n-tetradecyltrimethylammonium bromide at different pH: (9) pH 3.20; (b) pH 7.00; (2) pH 10.00.
At pH 3.20 the ζ-potential is always positive and practically independent of CnTAB. At pH 7.00 and 10.00 the ζ-potential changes in the neighborhood of the pzc from negative to positive values. These results confirm the fact that at pH 3.20 the protein is protonized and hence there is no electrostatic interaction between the anionic residues and the head of surfactant. Negative values of ζ-potential for pH 7.00 and 10.00 could
Interactions between Lysozyme and CnTAB
J. Phys. Chem., Vol. 100, No. 41, 1996 16751
Figure 5. ζ-potential as a function of concentration of n-hexadecyltrimethylammonium bromide at different pH: (9) pH 3.20; (b) pH 7.00; (2) pH 10.00.
Figure 6. Variation of the concentration of n-alkyltrimethylammonium bromides at zero ζ-potential as a function of alkyl chain length: (b) pH 7.00; (2) pH 10.00.
correspond to electrostatic and hydrophobic interactions. When the point of zero ζ-potential is reached the interaction is hydrophobic and the hydrocarbon chain of surfactant plays the most important role. Another important point to note is that the adsorption occurs at sufficient density to cause reversal of the sign of the ζ-potential at concentrations far below the cmc values. For the examination of the effects of surfactants on the lysozyme surface we have applied the treatment of Grahame of the double layer,28 based on original concept of Stern.29 The adsorption density of surfactants at the plane distance δ out from the surface is of the form
function of alkyl chain length of ionic surfactants.17,19,32 These linear relations implies that (Γδ+)0/2r is independent of chain length and the electrostatic contribution to the energy of adsorption is located entirely within the polar charged head of surfactant ion and is not affected by hydrocarbon chain association. Gibbs Energies of Adsorption. In agreement with Ottewill et al.33 we distinguish between the surface charges before addition of the surfactant
Γδ+ ) 2rc exp(-Wδ/kT)
(8)
where r is the radius of the adsorbed ion, c the surfactant concentration, and Wδ the work to bring the ion from the bulk of the solution up to the plane δ. The work Wδ can be divided into electrostatic and chemical work terms, so that for adsorption
Wδ ) Z+eψδ - Φ
(9)
where Z+ is the valence, e the electronic charge, ψδ the potential at plane δ, and Φ the van der Waals energy in units of kT associated with removal of the alkyl chain from its aqueous environment. If the alkyl chain has n carbon atoms, assuming that
Φ/kT ) nΦ′/kT
(11)
where c ) c0 at ψδ ) 0. In Figure 6 the concentration c0 at which the ζ-potential is reduced to zero as a function of the alkyl chain length (nc) of CnTAB is plotted. The linear relation between log c0 and n as predicted by eq 11 is verified. The slopes of these lines in terms of the van der Waals cohesive energy per CH2 group are 0.68kT for pH 7.0 and 0.73kT for pH 10.0 which are in reasonable agreement with values determined by Somasundaran et al.30 for alkylammonium acetate, with values of Φ′ determined from solubility data31 or from our studies of micelle formation as a
(12)
where σ0 is the charge per unit area on the surface, σi is the charge density of the ion, and σd is the charge density in the diffuse layer and the surface charges after adsorption
σ0 + σi + σd ) 0
(13)
where the eqs 12 and 13 have been written using the electroneutrality condition. Assuming that the surfactant adsorption did not affect the potential determining ions (so that σ0 ) σ00)
σi - σi0 ) σ0d - σd ) ∆σd
(14)
the Stern equation29 of the adsorption can be used in the form
-∆σd ) ∆σi )
(10)
then Φ′ is the van der Waals energy of interaction per CH2 group between adjacent chains of adsorbed surfactants molecules. If after association of the alkyl chains the potential is reduced to zero, the combination of the eqs 8, 9, and 10 gives
ln c0 ) -n(Φ′/kT) - ln(Γδ+)0 - ln 2r
σ00 + σi0 + σ0d ) 0
k1c 1 + k2c
(15)
where
k1 ) ZeN1k2
(16)
0 /kT)/55.6 k2 ) exp(-∆Gads
(17)
(The factor 55.6 converts concentration in mole L-1 into mole fraction for aqueous solution.) From the ζ vs log c curves and using the Ottewill and Watanabe equation34 at the pzc we have
( ) dζ d log c
[
]
0D(1 + κa)ζ0 ) 2.303ζ -1 aN1ze ζ)0 0
[
]
azeN1 1 -1 ) k2 c0 0Dζ0(1 + κa)
(18)
(19)
16752 J. Phys. Chem., Vol. 100, No. 41, 1996
Mosquera et al.
Figure 7. Binding isotherm for the binding of n-dodecyltrimethylammonium bromide to lysozyme (concentration 1.25 × 10-3 kg dm-3) at 25 °C, pH 10.0.
Figure 8. Gibbs energy of binding of n-dodecyltrimethylammonium bromide to lysozyme as a function of surfactants ions bound (Vj) at 25 °C, pH 10.00.
TABLE 1: Data for Adsorption of CnTAB on Lysozyme at pH 7.0
C8TAB C10TAB C12TAB C14TAB C16TAB
c0 (mmol L-1)
(dζ/d log c)ζ)0 (V)
K2 (L mol-1)
0 -∆Gads (kJ mol-1)
72.4 22.4 5.8 1.2 0.3
0.041 0.025 0.018 0.014 0.008
3.1 16.5 88.0 566.4 3547.3
11.3 15.0 18.7 22.8 26.8
TABLE 2: Data for Adsorption of CnTAB on Lysozyme at pH 10.0
C8TAB C10TAB C12TAB C14TAB C16TAB
c0 (mmol L-1)
(dζ/d log c)ζ)0 (V)
K2 (L mol-1)
0 -∆Gads (kJ mol-1)
58.9 17.0 5.5 0.5 0.2
0.031 0.033 0.016 0.015 0.008
5.0 16.4 106.2 1181.0 4858.1
12.4 15.0 19.1 24.4 27.5
where c0 is the surfactant concentration at the pzc, ζ0 is the zeta potential of the particles in the absence of surfactant, and D is the dielectric constant or relative permittivity (D ) /0, where 0 is the permitivity of free space). Equations 18 and 19 can be solved simultaneously using the experimental values of (dζ/d log c)ζ)0 and c0 to obtain values of N1 and k2. The results obtained are listed in Tables 1 and 2. We can compare with results of Gibbs energies of binding obtained from isotherms of binding as related in the Experimental Section. The binding isotherm for the binding of n-dodecyltrimethylammonium bromide to lysozyme is shown in Figure 7. The data relate to free C12TAB concentration below the cmc (for C12TAB log cmc ) -1.85) and are characteristic of cooperative binding. The Gibbs energies per n-dodecyltrimethylammonium ion bound ∆GVj as a function of Vj is shown in Figure 8. The curve tends to a limiting value of ∆GVj at high values of Vj of approximately -14 kJ mol-1 which is comparable within experimental error with that obtained by ζ-potential measurements and as we have previously related35 is independent of pH. 0 with the number of Figure 9 shows the variation of -∆Gads carbon atoms in the alkyl chain, nc, of TABs. This variation can be represented by a linear equation in the form 0 -∆Gads ) anc + b
(20)
with values of a of 1.92 ( 0.02 at pH 7 and 2.0 ( 0.3 at pH 10
Figure 9. Variation of the Gibbs energies of adsorption as a function of alkyl chain length: (b) pH 7.00; (2) pH 10.00.
and values of b of -4.1 ( 0.3 at pH 7 and -4.3 ( 0.4 at pH 10. The gradients of these lines represent the Gibbs energies of adsorption per CH2 group and the values derived here are of the same magnitude as found for the Gibbs energy change -∆G0hc per CH2 group of the unfolding of lysozyme in a hydrophobic environment.15 The contributions to the protein-surfactant interactions arise from the ionic binding of trimethylammonium head group to anionic amino acid residues, hydrophobic interactions of the n-alkyl chains, and the unfolding of the tertiary structure of the lysozyme. The charged amino acid residues are in the surface of native protein and hence accesible to surfactant so that the ionic interactions will not be markedly affected by the state of the unfolding of the tertiary structure. So our consideration of protein surface as the surface of adsorption is justified. The results show that the ζ-potential measurements can be a useful technique on the study of the protein interactions. Acknowledgment. The auhors thank the Xunta de Galicia for financial support. References and Notes (1) Jones, M. N.; Chapman, D. Micelles, Monolayers and Biomembranes; Wiley-Liss: New York, 1995. (2) Canfield, R. E.; Liu, A. K. J. Biol. Chem. 1965, 240, 2000. (3) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1979, 75, 1736. (4) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1980, 76, 654.
Interactions between Lysozyme and CnTAB (5) Fushima, K.; Murata, Y.; Nishikido, N.; Sugihara, G.; Tanaka, M. Bull. Chem.Soc. Jpn. 1981, 54, 3122. (6) Jones, M. N.; Manley, P.; Midgley, P. J. W. J. Colloid Interface Sci. 1981, 82, 257. (7) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1981, 77, 827. (8) Fukushima, K.; Murata, Y.; Sugihara, G.; Tanaka, M. Bull. Chem. Soc. Jpn.1982, 55, 1736. (9) Jones, M. N.; Manley, P. In Solution BehaViour of Surfactants; Mittal, K. L., Lindman, B., Eds.; Plenum Press: New York, 1983; Vol. 2, p 1403. (10) Jones M, N.; Manley, P.; Holt, A. Int. J. Biol. Macromol. 1984, 6, 65. (11) Jones, M. N.; Midgley, P. J. W. Biochemistry 1984, 219, 875. (12) Jones, M. N.; Brass, A. In Food Polymers, Gels and Colloids; Dickinson, E., Ed.; Royal Society of Chemistry Special Publication No. 82; Royal Society of Chemistry: Cambridge, U.K., 1991; p 65. (13) Hayashi, K.; Kugimiya, M.; Imoto, T.; Funatsu, M.; Bigelow, C. C. Biochemistry 1968, 7, 1461. (14) Hayashi, K.; Kugimiya, M.; Imoto, T.; Funatsu, M.; Bigelow, C. C. Biochemistry 1968, 7, 1467. (15) Jones, M. N.; Prieto, G.; del Rı´o, J. M.; Sarmiento, F. J. Chem. Soc., Faraday Trans. 1995, 91, 2805. (16) Lopes, V. C.; Hart, T. R. J. Vac. Sci. Technol. A 1987, 5, 174. (17) del Rio, J. M.; Pombo, C.; Prieto, G.; Mosquera, V.; Sarmiento, F. J. Colloid Interface Sci. 1995, 172, 137. (18) van Os, N. M.; Heak, J. R.; Ruper L. A. M. Physico-Chemical Properties of Selected Anionic, Cationic and Nonionic Surfactants; Elsevier: Amsterdam, The Netherlands, 1993. (19) del Rio, J. M.; Pombo, C.; Prieto, G.; Sarmiento, F.; Mosquera, V.; Jones, M. N. J. Chem Thermodyn. 1994, 26, 879.
J. Phys. Chem., Vol. 100, No. 41, 1996 16753 (20) Few, A. W.; Ottewill, R. H. J. Colloid Sci. 1956, 11, 34. (21) Wyman, J. J. J. Mol. Biol. 1965, 11, 631. (22) von Somoluchowski, M. In Handbuch der Electrizita¨ t und des Magnetismus; (Graetz) Barth: Leipzig, 1914; Vol. II, p 366. (23) Hunter, R. J. Zeta Potential in Colloid Science. Principles and Applications; Academic Press: New York, 1981. (24) Brinton, Jr. C. C.; Lauffer, M. A. In Electrophoresis; Bier, M., Ed.; Academic Press: New York, 1979; p 427. (25) James, M. A. In Surface and Colloid Science; Good, R. J., Stromberg, R. R., Eds.; Plenum: New York, 1979; Vol. II, p 121. (26) . Alexander, A. E.; Johnson, P. Colloid Science; Clarendon: Oxford, U.K., 1949; Vol. I. (27) Phillips, D. C. Sci. Am. 1966, 215, 78. (28) Grahame, D. C. Chem. ReV. 1947, 40, 441. (29) Stern, O. Z. Elektrochem. 1924, 30, 508. (30) Somasundaran, P.; Healy, T. W.; Fuerstenau, D. W. J. Phys. Chem. 1964, 68, 3562. (31) Ralston, A. W. Fatty Acids and Their DeriVatiVes; John Wiley and Sons: New York, 1948. (32) Prieto, G.; Paz Andrade, M. I.; Sarmiento, F. Colloids Surf. A 1994, 83, 57. (33) Ottewill, R. H.; Rastogi, M. C.; Watanabe, A. Trans. Faraday Soc. 1960, 56, 854. (34) Ottewill, R. H.; Watanabe, A. Kolloid-Z. 1960, 170, 132. (35) Sarmiento, F.; Prieto, G.; Jones, M. N. J. Chem. Soc, Faraday Trans. 1992, 88, 1003.
JP960927E
