Langmuir 2005, 21, 9017-9019
9017
In Deuterated Water the Unspecific Adsorption of Proteins Is Significantly Slowed Down: Results of an SPR Study Using Model Organic Surfaces Christian Grunwald,†,‡ Ju¨rgen Kuhlmann,‡ and Christof Wo¨ll*,† Physikalische Chemie I, Ruhr-Universita¨ t, Bochum, Germany, and Max-Planck-Institut fu¨ r Molekulare Physiologie, Dortmund, Germany Received June 14, 2005. In Final Form: August 10, 2005 The control of unspecific adsorption of proteins to natural and technical surfaces plays an important role in biology and also for many applications. Organic model surfaces, e.g., self-assembled monolayers, are often used to identify fundamental surface and/or protein properties that rule protein adsorption. Some techniques involved in biointerface research require the use of heavy water, e.g. neutron scattering techniques. Also in NMR studies D2O is the solvent of choice when focusing on biomolecular and hydration dynamics. So far several studies have been concerned with the characterization of the unspecific adsorption of proteins from normal water buffers. In the present work, we report a comparison of the unspecific protein adsorption from normal and heavy water buffers. So far it has been assumed that the surface kinetic of the unspecific adsorption is unaffected by the substitution of water by D2O. However, for the four proteins investigated here, this assumption does not hold. The ratio kH/kD of the adsorption rate constants of the different buffer conditions describe the strength of the isotope effect. We have measured ratios between 1.0 and 2.6, indicating that the adsorption kinetics are strongly affected by a H2O-D2O substitution.
Protein adsorption plays a crucial role, e.g., for biosensors, medical implants,1 and marine vessels.2 In analytical biochemistry, the goal is to achieve a specific adsorption of the target proteins but at the same time to avoid unspecific adsorption of other proteins. Unspecific adsorption may not only give rise to erroneous sensor responses but can also block receptor sites and thus deactivate a sensor device. There is a vast amount of literature on adsorption studies of proteins on natural and on model substrates. The latter, e.g., organic surfaces made by formation of self-assembled monolayers (SAMs) or well-defined inorganic surfaces such as silicon oxide, make quantitative and systematic investigations possible.3,4 A number of studies using a variety of different techniques4-6 have revealed that protein adsorption is a complex phenomenon with a complicated interplay of different factors (e.g., the precise structure of the H2O molecules at the solid/water interface7) which are not yet completely understood.3,6 Overall protein adsorption bears some similarity to the unfolding of proteins, whereas for the adsorption of proteins,8,9 a complex interplay between enthalpic and entropic contributions has been proposed.10-12 It has been demonstrated in the past that slight changes in the nature of the solvent, e.g., those introduced by deuteration, can provide valuable insights into the principles governing protein stability. In heavy water, D2O, * To whom correspondence should be addressed. E-mail:
[email protected]. † Ruhr-Universita ¨ t. ‡ Max-Planck-Institut fu ¨ r Molekulare Physiologie. (1) Schierholz, J. M.; Beuth, J. J. Hospital Infect. 2001, 49, 87-93. (2) Sever, M. J.; Weisser, J. T.; Monahan, J.; Wilker, J. J. Abstr. Papers Am. Chem. Soc. 2004, 227, U1298-U1298. (3) Czeslik, C. Z. Phys. Chem. 2004, 218, 771-801. (4) Ostuni, E.; Grzybowski, B. A.; Mrksich, M.; Roberts, C. S.; Whitesides, G. M. Langmuir 2003, 19, 1861-1872. (5) Seigel, R. R.; Harder, P.; Dahint, R.; Grunze, M.; Josse, F.; Mrksich, M.; Whitesides, G. M. Anal. Chem. 1997, 69, 3321-3328. (6) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9359-9366. (7) Schwendel, D.; Hayashi, T.; Dahint, R.; Pertsin, A.; Grunze, M.; Steitz, R.; Schreiber, F. Langmuir 2003, 19, 2284-2293.
D bonds are stronger than the corresponding H bonds between water molecules.13 As a result, protein unfolding temperatures are typically increased by several degrees and many works have reported a stabilization of the (folded) protein while at the same time the protein structure becomes more compact. Protein adsorption from heavy water buffers on solid substrates has to our knowledge not yet been studied, but it is commonly assumed that there are no major differences to adsorption from normal water. For example, in neutron scattering studies on protein adsorption at solid-liquid interfaces, generally deuterated water is used.12 When considering the effect of D2O on the unspecific adsorption of proteins on hydrophobic surfaces, one has to consider different effects (viscosity, enthalpy and entropy contributions) which go in different directions, and as a result, it is not straightforward to make predictions.14 Here we report the investigation of protein adsorption from normal and heavy water buffers (150 mM NaCl, 10 mM Hepes, pH 7.4 (H2O) and pD 7.8 (D2O)) on a hydrophobic surface using surface plasmon resonance (SPR) spectroscopy. The hydrophobic surface is fabricated using the self-assembly of octadecanethiol on an Au substrate. In previous work, SAMs (self-assembled monolayers) have been proven to be ideally suited to study the unspecific4,15 and specific16,17 adsorption of proteins on (8) Pertsin, A. J.; Hayashi, T.; Grunze, M. J. Phys. Chem. B 2002, 106, 12274-12281. (9) Kreuzer, H. J.; Wang, R. L. C.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 8384-8389. (10) Guzzi, R.; Sportelli, L.; La Rosa, C.; Milardi, D.; Grasso, D. J. Phys. Chem. B 1998, 102, 1021-1028. (11) Makhatadze, G. I.; Clore, G. M.; Gronenborn, A. M. Nature Struct. Biol. 1995, 2, 852-855. (12) Guzzi, R.; Arcangeli, C.; Bizzarri, A. R. Biophys. Chem. 1999, 82, 9-22. (13) Scheiner, S.; Cuma, M. J. Am. Chem. Soc. 1996, 118, 15111521. (14) Sasisanker, P.; Oleinikova, A.; Weingartner, H.; Ravindra, R.; Winter, R. Phys. Chem. Chem. Phys. 2004, 6, 1899-1905. (15) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf. B, Biointerfaces 1999, 15, 3-30. (16) Spinke, J.; Liley, M.; Schmitt, F. J.; Guder, H. J.; Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7012-7019.
10.1021/la0515930 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/23/2005
9018
Langmuir, Vol. 21, No. 20, 2005
Letters Table 1. Summary of Isotope Effect on Surface Adsorption. Kinetic Ratios kH/kD
Figure 1. Protein adsorption to octadecane thiol SAMs from normal water and from heavy water buffer measured by SPR (Biacore). Open symbols normal water buffer; closed symbols heavy water buffer. All proteins were diluted to a final concentration of 1 mg/mL18 and applied to the hydrophobic SAM surface from 0 to 900s. When not incubated with proteins the surface is rinsed with buffer. Flow rate was set to 5 µL per minute and temperature to 20 °C. Adsorption amplitude has been normalized to 100 RU at 900 s for better comparability.
organic surfaces. Four different proteins were chosen in the present work: streptavidin (SA), glutathione-Stransferase (GST), bovine serum albumin (BSA), and ribonuclease A (RNase). The SPR results reproduced in Figure 1 reveal, surprisingly, that three out of the four proteins studied here, BSA, GST and RNase, show a significantly slower adsorption kinetics in heavy water buffers. The amount of adsorbed protein (see the Supporting Information, Figure 2) does not show a major solvent isotope effect, with the exception of SA, where the amount of adsorbed protein is found to increase by a factor of 1.4 in D2O. Only the adsorption data for SA and RNase could be fitted satisfactorily by a monoexponential adsorption kinetics [y(t) ) y0 + a(1 - exp(-kt))], for the two other proteins, BSA and GST, a double-exponential function [y(t) ) y0 + a(1 - exp(-k1t)) + b(1 - exp(-k2t))] had to be used to yield a satisfactory fit of the experimental data. When changing from normal water buffer to heavy water buffer, the adsorption kinetics for BSA slows down approximately by a factor of 1.7 and by a factor of 2.6 for RNase (for details and error bars see Table 1). Although for GST the SPR data clearly reveal a slower adsorption kinetics in D2O, no kH/kD ratio could be calculated since the fit reveals the presence of at least two different time constants. Substituting water by D2O (17) Grunwald, C.; Eck, W.; Opitz, N.; Kuhlmann, J.; Wo¨ll, C. Phys. Chem. Chem. Phys. 2004, 6, 4358-4362.
protein
kH/s-1
kD/s-1
kinetic ratios kH/kD
streptavidin GST BSA RNase
0.0146 0.0023 0.1676 0.0369
0.0149 0.0028 0.0990 0.0144
1.0 ((0.2) n.a. (see text) 1.7 ((0.2) 2.6 ((0.5)
changes not only the kinetic constants but also the ratio of the corresponding amplitudes. These results indicate that for GST the isotope effect not only changes the kinetics but also the relative importance of the two different steps in the adsorption process. The large reduction in the kinetics seen for GST, BSA, and RNase in D2O is rather unexpected. In fact, such large effects of replacing normal water with deuterated water are generally not considered when analyzing neutron diffraction data obtained for protein adsorption from deuterated buffers.12 Although kinetic effects may not be important on the time-scale of the neutron experiments, the differences in protein coverage have to be considered (see supplement Figure 2). Note that the 20% larger viscosity of D2O19 cannot account (via a reduced diffusivity of the proteins) for the observed differences since in that case the changes should be approximately the same for all proteins. When trying to rationalize these surprisingly large changes in adsorption kinetics, one has to consider the different contributions to unspecific protein adsorption (see above). Since unspecific protein adsorption implies unfolding, the observed trend is consistent with the fact that in previous studies a general slow-down of protein unfolding in D2O has been observed.14,20,21 From the fact that the unfolding temperature is generally increased by a few degrees, it has been concluded that proteins are stabilized in heavy water,22-25 although in other studies a more detailed thermodynamic analysis has pointed toward a destabilization of proteins in heavy water.10,11 Protein flexibility in normal and heavy water buffers revealed kH/kD ratios from 1.0 to 1.8 indicating a more rigid and compact structure under D2O influence.22 These considerations indicate that the general slow-down in adsorption kinetics observed here results from the slower unfolding of the proteins at the hydrophobic SAM surface in the presence of D2O. Among the four proteins studied here RNase is the one with the most dramatic shift in adsorption rate. RNase is known to be one of the most stable proteins which is consistent with the fairly low amount of total adsorbed protein (see supplement). In heavy water, RNase is expected to adopt a more compact structure due to the now stronger hydrophobic interactions within the protein core.14 These hydrophobic forces stabilize the folded protein, and it thus appears plausible that the adsorption process is signifi(18) The proteins have been exposed to the corresponding solvent for several hours (e.g., overnight) before the solutions were used in the experiments. Based on literature data (Zhang et al., Protein Science 1995, 4, 804-814), we thus conclude that all accessible protons have been exchanged. (19) Horita, J.; Cole, D. R. Stable isotope partitioning in aqueous and hydrothermal systems to elevated temperatures. In Aqueous systems at elevated temperatures and pressures: Physical chemistry in water, steam and hydrothermal solutions; Palmer, D. A., Ferna´ndez-Prini, R., Harvey, A. H., Eds.; Elsevier: Amsterdam, 2004; pp 277-319. (20) Parker, M. J.; Clarke, A. R. Biochemistry 1997, 36, 5786-5794. (21) Svergun, D. I.; Richard, S.; Koch, M. H. J.; Sayers, Z.; Kuprin, S.; Zaccai, G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2267-2272. (22) Cioni, P.; Strambini, G. B. Biophys. J. 2002, 82, 3246-3253. (23) Chakrabarti, G.; Kim, S.; Gupta, M. L.; Barton, J. S.; Himes, R. H. Biochemistry 1999, 38, 3067-3072. (24) Bonnete, F.; Madern, D.; Zaccai, G. J. Mol. Biol. 1994, 244, 436447. (25) Kushner, D. J.; Baker, A.; Dunstall, T. G. Can. J. Physiol. Pharm. 1999, 77, 79-88.
Letters
cantly slowed. For SA, no difference in the kinetic rate constants was seen, but a significant change in the adsorbed mass occurs when switching from normal water buffers to heavy water buffers. The value of 204 ng/cm2 under normal water conditions is consistent with the presence of a full protein monolayer adsorbed on the CH3terminated SAM surface, but the mass adsorbed in the D2O buffer exceeds the equivalent of a protein monolayer by more than 40%. Since it has been previously reported that some proteins aggregate under the influence of D2O, it appears likely that the increase in mass loading is due to the adsorption of protein clusters.21 For BSA and GST, the data analysis reveals a more complicated adsorption behavior (see above). If we consider only the dominant exponential term, e.g., the term with the larger amplitude, we yield a kH/kD ratio of 1.7 for BSA (Table 1) which is less than the corresponding value for RNase but which nevertheless reveals a dramatic reduction in adsorption speed. For GST, unfortunately, this simplified analysis is found to be impossible. An accurate description of protein adsorption, of course, will have to go beyond the simplified model used for the considerations presented above and will require a more detailed description of protein topology and the particular interactions (hydrogen bonds between
Langmuir, Vol. 21, No. 20, 2005 9019
amino acid groups, etc.) present in the folded and, possibly, in a partially unfolded protein. Although the results reported here do not yet allow for the presentation of a unified picture describing the effect of heavy water on protein adsorption they demonstrate that, as in the case of protein unfolding dynamics in bulk water, experiments in deuterated buffers can provide important information on the mechanism behind unspecific protein adsorption on surfaces. In future experiments in particular the influence of temperature will be studied in order to unravel the importance of entropic contributions. Acknowledgment. The authors are grateful to Christine Nowak (MPI Dortmund) for supplying us with GST. We also acknowledge enlightening discussions with Dr. Rainer Dahint (Heidelberg) and Prof. Hermann Weinga¨rtner (Bochum). Supporting Information Available: Used materials, additional figures, and tables concerning protein surface kinetics. This material is available free of charge via the Internet at http://pubs.acs.org. LA0515930
