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Analysis of the SDS-Lysozyme Binding Isotherm Mitaben D. Lad,† Victoria M. Ledger,† Barbara Briggs,† Rebecca J. Green,*,† and Richard A. Frazier‡ Department of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom, and School of Food Biosciences, The University of Reading, P.O. Box 226, Whiteknights, Reading RG6 6AP, United Kingdom Received December 5, 2002. In Final Form: April 14, 2003 A scheme to describe SDS-lysozyme complex formation has been proposed on the basis of isothermal titration calorimetry (ITC) and FTIR spectroscopy data. ITC isotherms are convoluted and reveal a marked effect of both SDS and lysozyme concentration on the stoichiometry of the SDS-lysozyme complex. The binding isotherms have been described with the aid of FTIR spectroscopy in terms of changes in the lysozyme structure and the nature of the SDS binding. At low SDS concentrations, ITC isotherms feature an exothermic region that corresponds to specific electrostatic binding of SDS to positively charged amino acid residues on the lysozyme surface. This leads to charge neutralization of the complex and precipitation. The number of SDS molecules that bind specifically to lysozyme is approximately 8, as determined from our ITC isotherms, and is independent of lysozyme solution concentration. At high SDS concentrations, hydrophobic cooperative association dominates the binding process. Saturated binding stoichiometries as a molar ratio of SDS per molecule of lysozyme range from 220:1 to 80:1, depending on the lysozyme solution concentration. A limiting value of 78:1 has been calculated for lysozyme solution concentrations above 0.25 mM.
Introduction Proteins and surfactants are major components of a wide range of food, pharmaceutical, and cosmetic systems. Their interactions in solution and at interfaces play a key role in controlling the physical characteristics and the colloidal nature of such systems.1 In addition, as detergents, surfactants remove proteins from surfaces through adsorption to the surface and via electrostatic and/or hydrophobic binding to the protein. In the case of anionic surfactants, the interaction between proteins and surfactant is strong, often leading to the formation of large complexes and denaturation of the protein.2 Our recent work to investigate coadsorption from SDS and lysozyme solutions at air/water interfaces has led to a proposed adsorption mechanism that discusses the role of SDS-lysozyme complex formation.3 Clearly, the interaction between SDS and lysozyme in solution prior to adsorption as well as the interfacial interaction must play a role in the adsorption process. Our results suggested that the stoichiometry of the solution phase SDSlysozyme complex alters the relative surface affinities of the adsorbing species and therefore controls adsorption from the mixed solution. A mechanism for the adsorption process was proposed with reference to a suggested regime of the corresponding solution interactions and complex structure. The present study was initially undertaken with the aim of providing a thermodynamic basis of under* To whom correspondence should be addressed. Current address: School of Chemistry, The University of Reading, P.O. Box 224, Whiteknights, Reading RG6 6AD, United Kingdom. Fax: +44 118 378 6331. E-mail:
[email protected]. † University of Leicester. ‡ The University of Reading. (1) Ananthapadmanabhan, K. P. In Interactions of surfactants with polymers and proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993; p 320. (2) Goddard, E. D. In Interactions of surfactants with polymers and proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993; p 171. (3) Green, R. J.; Su, T. J.; Joy, H.; Lu, J. R. Langmuir 2000, 16, 5797.
standing for the solution phase SDS-lysozyme binding and, thus, with a view to defining the role that the interface has on initiating SDS-lysozyme interactions and conformational change. Isothermal titration calorimetry (ITC) and FTIR spectroscopy have been used in order to enable the investigation of the interaction and complex formation through evaluation of the SDS-lysozyme binding isotherm. Previous work has shown that complex formation between SDS and lysozyme can form structures with in excess of 100 SDS molecules binding per lysozyme molecule.1 For the interaction between proteins and anionic surfactant, such as SDS, complex formation appears to occur via two distinct steps. Initially, both electrostatic and hydrophobic interactions will occur as the sulfate headgroup of the surfactant binds to positively charged amino acid residues at the surface of the protein,4 while the surfactant hydrophobic chains interact with adjacent hydrophobic regions of the protein. Charge neutralization of the complex then leads to precipitation.5 SDS binding beyond neutralization leads to redissolution and is controlled by a cooperative hydrophobic interaction that results in protein structural change. There is, however, some uncertainty over the nature of the saturated complex, its stoichiometry and structure, and the precise mechanism of the binding and structural change within the complicated nonspecific binding region. Studies of strongly interacting surfactant-synthetic polyelectrolyte systems have also led to similar proposed mechanisms. Indeed, Chen et al.6,7 have investigated the formation of SDS complexes with polycations and amphiphilic polymers, using a surfactant selective membrane electrode to monitor free surfactant concentration, where they have (4) Nozaki, Y.; Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1974, 2249, 4452. (5) Muren, A. K.; Khan, A. Langmuir 1995, 11, 3636. (6) Chen, L.; Yu, S.; Kagami, Y.; Gong, J.; Osada, Y. Macromolecules 1998, 31, 787. (7) Yu, S. Y.; Hirata, M.; Chen, L.; Matsumoto, S.; Matsukata, M.; Gong, J. P.; Osoda, Y. Macromolecules 1996, 29, 8021.
10.1021/la0269560 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/10/2003
Analysis of the SDS-Lysozyme Binding Isotherm
clearly illustrated the presence of a two step interaction. Initially, SDS binds one-to-one to each of the charged binding sites on the polymer chain to form a complex that is insoluble in water. Upon further addition of SDS, the complex solubilizes, this step being highly dependent upon the hydrophobic regions of the polymer and the alkyl chain length of the surfactant. In the present study, ITC and FTIR spectroscopy have been used to probe both the low surfactant concentration region, where specific binding occurs, and the high surfactant concentration region, where nonspecific cooperative binding occurs. Recent studies using ITC to investigate polymer-surfactant interactions have demonstrated its ability to provide additional insight into the mode of binding and to determine the primary driving forces of interaction.8-11 In this study, ITC has been used to probe the complete SDS concentration range via a single titration experiment and has provided information regarding the energetics and mode of the SDS-lysozyme binding process. The complementary technique of FTIR spectroscopy has provided the corresponding structural information. Experimental Section Materials. Sodium dodecyl sulfate (SDS) was obtained from Polysciences Europe GmbH (Eppelheim, Germany) and has a molecular weight of 288 g mol-1. Hen egg white lysozyme (cat. no. L6876, 95% purity) was supplied as a lyophilized powder from Sigma (Poole, U.K.) and used as supplied. Lysozyme has a molecular weight of 14 300 g mol-1 and an isoelectric point at approximately pH 11. Thus, at pH 7, lysozyme has a net positive charge of 8.12 Microcalorimetry. A MicroCal Omega ITC instrument (Northampton, MA) was used to measure enthalpy changes associated with SDS-lysozyme interactions at 298 K. In a typical experiment, lysozyme solution was placed in the 1.4115 cm3 sample cell of the calorimeter and SDS solution was loaded into the injection syringe. Both solutions were prepared in phosphate buffer (pH 7, I ) 0.02) and were degassed before use. The reference cell was filled with deionized water. SDS solution was titrated into the sample cell as a sequence of 50 injections of 5 × 10-6 dm3 aliquots. The duration of each injection was 5 s, and the time delay (to allow equilibration) between successive injections was 180 s. The contents of the sample cell were stirred throughout the experiment at 400 rpm to ensure thorough mixing. Raw data were obtained as a plot of heating rate (µcal s-1) against time (min). These raw data were then integrated to obtain a plot of observed enthalpy change per mole of injected SDS (∆Hobs, kJ mol-1) against SDS concentration (mM) or molar ratio (SDS/ lysozyme). Control experiments included the titration of SDS into buffer, buffer into lysozyme, and buffer into buffer. The last two controls resulted in small and equal enthalpy changes for each successive injection of buffer and, therefore, were not further considered in the data analysis.13 The titration of SDS into buffer yielded enthalpy changes that are discussed later. FTIR Spectroscopy. A ThermoNicolet Nexus FTIR spectrometer (Madison, WI) with a deuterated triglyceride sulfate (DTGS) detector was used to collect the infrared spectra. The spectrometer was continually purged with dry air during the experiment to remove water vapor from the chamber. The solution FTIR spectra were recorded using a Specac Omni liquid cell fitted with CaF2 windows and a Mylar spacer giving a path length of (8) Dai, S.; Tam, C.; Li, L. Macromolecules 2001, 34, 7049. (9) Seng, W. P.; Tam, K. C.; Jenkins, R. D.; Bassett, D. R. Langmuir 2000, 16, 2151. (10) Singh, S. K.; Nilsson, S. J. Colloid Interface Sci. 1999, 213, 152. (11) Wangsakan, A.; Chinachoti, P.; McClements, D. J. J. Agric. Food Chem. 2001, 49, 5039. (12) Tanford, C.; Roxby, R. Biochemistry 1972, 11, 2192. (13) O’Brien, R.; Ladbury, J. E.; Chowdhry, B. Z. In Protein-Ligand Interactions: Hydrodynamics and Calorimetry; Harding, S. E., Chowdhry, B. Z., Eds.; Oxford University Press: Oxford, U.K., 2001; pp 263-286.
Langmuir, Vol. 19, No. 12, 2003 5099 6 µm. Data were collected as interferograms at 4 cm-1 resolution, of which 64 interferograms were collected and coadded. IR spectra were obtained by ratioing against a background single beam spectrum of the buffer solution. Finally, Fourier transform deconvolution was performed on the amide I peak to provide information about changes to protein secondary structure. For the purposes of this study, IR spectra of the amide I (1650 cm-1) and amide II (1540 cm-1) have been studied. The amide I peak is a composite peak due to primarily CdO bond stretching coupled with NsH bending and CsH stretching modes,14 and it is used to determine secondary structure changes. The amide II peak, which is due mostly to NsH bending modes, can be used to monitor protein unfolding if a deuterated solvent is used. The H-D exchange of labile hydrogens on the protein surface results in a shift in the position of the amide II peak to 1450 cm-1 but has very little effect on the amide I peak. For globular proteins such as lysozyme, a residual NsH amide II peak at 1540 cm-1 is observed due to the presence of nonexchanged labile hydrogens within the core of the folded protein and inaccessible to the solvent. Therefore, by monitoring the residual amide II peak, it is possible to observe protein unfolding transitions.15,16 The lysozyme/SDS solutions were made up in a deuterated buffer (pH 7, I ) 0.02 M) solution. D2O (99.9% deuterium, Aldrich) was used rather than H2O because water adsorbs strongly in the amide region of the spectra and in order to allow protein unfolding transitions to be studied. FTIR spectra were recorded for 0.25 mM lysozyme solutions with 0-20 mM SDS.
Results and Discussion As expected, titration of SDS into buffer leads to the observation of an inflection in the ITC isotherm plot of molar enthalpy change against SDS concentration (Figure 1a). The position of this inflection corresponds to the critical micellar concentration (cmc) for SDS under the conditions of this study.17,18 The initial enthalpic responses are endothermic and are due to demicellization that occurs upon dilution of the titrated SDS micellar solution in the calorimetry cell. At the point of inflection the cell solution concentration reaches the cmc of the surfactant and demicellization no longer occurs. For SDS, in a pH 7 buffered solution (I ) 0.02 M), the cmc occurs at a concentration of approximately 5 mM3, as is observed in Figure 1a (compared to a cmc of ∼8 mM in water). Figure 1b shows the corresponding ITC data for titration of SDS into lysozyme solutions of varying concentrations (0.03-0.25 mM). The magnitude of the observed molar enthalpy changes (∆Hobs) and the general shape of the isotherms are completely different from those recorded for SDS demicellization. Since the heat of demicellization effects are not mirrored in the protein-surfactant binding experiments, the ITC data shown in this paper have not been corrected by subtraction of the SDS-buffer control. For SDS titration into lysozyme solutions, the resulting ITC isotherm is highly convoluted, since the data are a composite of more than one type of interaction occurring sequentially within the cell. In the following discussion, and with the aid of FTIR spectra, binding isotherms are explained in terms of the nature of SDS-lysozyme binding interactions and the resultant complex structure. In the recent paper by Chatterjee et al.,19 the authors have discussed their ITC data for the interaction of SDS with lysozyme at 30 °C in terms of critical aggregation (14) Dong, A.; Prestrelski, S. J.; Allison, S.; Carpenter, J. F. J. Pharm. Sci. 1995, 84, 415. (15) Green, R. J.; Hopkinson, I.; Jones, R. A. L. Langmuir 1999, 15, 5102. (16) Ball, A.; Jones, R. A. L. Langmuir 1995, 11, 3542. (17) Blandamer, M. J.; Briggs, B.; Cullis, P. M.; Irlam, K. D.; Engberts, J. B. F. N.; Kevelam, J. J. Chem. Soc., Faraday Trans. 1998, 94, 259. (18) McClements, D. J. J. Agric. Food Chem. 2000, 48, 5604. (19) Chatterjee, A.; Moulik, S. P.; Majhi, P. R.; Sanyal, S. K. Biophys. Chem. 2002, 98, 313.
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Figure 1. ITC isotherm for SDS titration into (a) buffer (from 60 mM (b) and 100 mM ([) SDS injectant solutions) and (b) 0.03 (O), 0.05 (4), 0.077 (]), 0.1 (0), 0.15 (b), and 0.25 (2) mM lysozyme solutions.
Figure 2. Surface tension data for SDS in pH 7 buffer (b) and 0.01 g dm-3 lysozyme solution (×). The original data are discussed in ref 3.
concentrations and T1 and T2 transitions, as is the convention for weakly interacting polymer-surfactant systems.20-22 However, it has been recently shown from surface tension data of surfactant-polyelectrolyte systems that this usual method of explanation does not apply to strongly interacting systems.22 Indeed, the surface tension data for the SDS-lysozyme system (Figure 2) are in agreement with the trends observed by Staples et al.22 and do not exhibit classical T1/T2 behavior.3 In contrast to the classical surface tension behavior, these data exhibit an unusual increase in surface tension at intermediate SDS concentrations, which can be attributed to the changing nature of the SDS-lysozyme complex with increasing SDS concentration.3 Therefore, the present results are explained in terms of the structure of the SDSlysozyme complex rather than in terms of critical aggregation concentrations. The lysozyme concentration (20) Jones, M. N. J. Colloid Interface Sci. 1967, 23, 36. (21) Murata, M.; Arai, H. J. Colloid Interface Sci. 1973, 44, 475. (22) Staples, E.; Tucker, I.; Penfold, J.; Warren, N.; Thomas, R. K.; Taylor, D. J. F. Langmuir 2002, 18, 5147.
used in Figure 2 is much lower than that used in the present ITC experiments. However, as discussed later, the A-D transitions highlighted do appear to correspond to the A-D transitions indicated by the ITC data in Figure 7. The plots in Figure 1b represent a series of complicated data that suggest more than one type of binding interaction occurs. These data are a more complete representation of the binding isotherm than that identified by Chatterjee et al.19 especially with respect to the low SDS concentration region, where the present data clearly identify a large exothermic peak. As discussed below, this peak defines the initial specific binding of SDS to lysozyme. Our interpretation of the full SDS-lysozyme binding isotherm provides a more complete description of the modes of interaction that occur during formation of the saturated SDS-lysozyme complex than provided elsewhere. From what is known of the SDS-lysozyme interaction, it is reasonable to assume that the thermodynamic changes measured for the SDS-lysozyme interaction will also be coupled with structural unfolding and denaturation of the protein.23 Therefore, to aid the interpretation of ITC data, spectroscopic and observational (monitoring of solution cloud points) data are also considered. For clarity we have considered the interactions at low SDS concentration and high SDS concentration separately. Low SDS Concentration Region. At low SDS concentration (
