Protein Interactions with Self-Assembled Monolayers Presenting

Fairbank, R. W. P.; Xiang, Y.; Wirth, M. J. Anal. Chem. 1995, 67, 3879−3885. [ACS Full Text ACS Full Text ], [CAS]. (12) . Use of Methyl Spacers in ...
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Protein Interactions with Self-Assembled Monolayers Presenting Multimodal Ligands: A Surface Plasmon Resonance Study Srinavya Vutukuru, Sridhar R. Bethi, and Ravi S. Kane* Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180 ReceiVed July 18, 2006. In Final Form: September 10, 2006 This paper describes the use of surface plasmon resonance (SPR) spectroscopy and self-assembled monolayers (SAMs) to understand the characteristics of surfaces that promote the adsorption of proteins at high ionic strengths (high-salt conditions). We synthesized SAMs presenting different multimodal ligands and determined the influence of surface composition, solution composition, and the nature of the protein on the extent of protein adsorption onto the SAMs. Our results confirm that hydrophobic interactions can contribute significantly to protein adsorption under high-salt conditions. In particular, the extent of protein adsorption under high-salt conditions increased with increasing surface hydrophobicity. The extent of protein adsorption was also influenced by the solution composition and decreased with an increase in the chaotropicity of the anion. The combination of SPR and SAMs is well-suited for studying the interaction of proteins with complex surfaces of relevance to chromatography.

Introduction The objective of this study is to correlate the molecular scale structure of surfaces with their ability to promote the adsorption of proteins at high ionic strengths, conditions of increasing relevance for the separation of proteins by chromatography. Ionexchange chromatography offers cost-effective, rapid, and efficient separations and is widely used for the concentration and purification of proteins.1,2 The use of classical ion-exchange resins, however, is not always optimal, particularly under highsalt conditions.3,4 For instance, cell culture supernatants often have a high ionic strength and need to be diluted to ensure acceptable sample capacity (i.e., acceptable levels of protein adsorption onto the resin). The use of multimodal stationary phases, which use multiple chromatographic mechanisms simultaneously, may enable separation efficiency to be optimized.3,4 Multimodal chromatography enables direct capture of proteins at high-salt conditions, making it an attractive alternative to ionexchange chromatography. It is clear that a secondary interaction, in addition to electrostatic interactions, is required for protein binding to multimodal ligands under high-salt conditions. However, proteins are complex biomolecules, and studying their retention on a multimodal stationary phase in a chromatographic column may not provide a complete understanding of the fundamental physics of the purification process.5,6 Self-assembled monolayers (SAMs) provide excellent model surfaces for such studies. The terminal group of an ω-functionalized alkanethiol determines the properties of the interface between the SAM and the contacting liquid, and varying this group enables the molecular scale structure of surfaces * Corresponding author. E-mail: [email protected]. (1) Mazza, C.; Kundu, A.; Cramer, S. M. Biotechnol. Tech. 1998, 12, 137141. (2) Yamamoto, S.; Nakinishi, K.; Matsuno, R. Ion-Exchange Chromatography of Proteins, 2nd ed.; Marcel Dekker: New York, 1998; Vol. 43. (3) Johansson, B. L.; Belew, M.; Eriksson, S.; Glad, G.; Lind, O.; Maloisel, J. L.; Norrman, N. J. Chromatogr., A 2003, 1016, 21-33. (4) Johansson, B. L.; Belew, M.; Eriksson, S.; Glad, G.; Lind, O.; Maloisel, J. L.; Norrman, N. J. Chromatogr., A 2003, 1016, 35-49. (5) Barrett, D. A.; Power, G. M.; Hussain, M. A.; Pitfield, I. D.; Shaw, P. N.; Davies, M. C. J. Sep. Sci. 2005, 28, 483-491. (6) Pitiot, O.; Folley, L.; Vijayalakshmi, M. A. J. Chromatogr., B 2001, 758, 163-172.

to be correlated to the extent of protein adsorption.5,7-11 There have only been a few reports of the use of SAMs as potential12,13 or model5 chromatographic surfaces, and these reports did not involve studies of multimodal ligands. This work utilizes surface plasmon resonance spectroscopy to investigate the nature of the secondary interactions between proteins and SAMs presenting different multimodal ligands. In particular, we test the hypothesis that hydrophobic interactions contribute significantly to interactions between proteins and multimodal ligands. Experimental Section Materials. All materials and reagents were used as received. Lysozyme (chicken egg white; L6876) and cytochrome c (equine heart; C2506) were purchased from Sigma (St. Louis, MO). 11Mercaptoundecanoic acid, trityl chloride, di-isopropyl ethylamine, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, triethylamine, benzoyl chloride, isovaleryl chloride, acetyl chloride, triethylsilane, and lithium hydroxide were purchased from Aldrich (Allentown, PA). 6-Amino-2-tert-butoxycarbonylamino-hexanoic acid methyl ester (Boc-Lys-OMe)-acetate salt was purchased from Bachem Biosciences, Inc. (King of Prussia, PA). Glass cover slips (0.16-0.19 mm thick, No. 1.5) were obtained from Fisher Scientific (Agawam, MA). Synthesis. 2-Amino-6-(11-tritylsulfanyl-undecanoylamino)hexanoic Acid Methyl Ester (1). Compound 1 was synthesized as shown in Scheme 1. To a solution of 11-mercaptoundecanoic acid (1.0 g, 4.59 mmol) in toluene (30 mL) were added di-isopropyl ethylamine (2.4 mL, 13.76 mmol) and trityl chloride (2.56 g, 9.17 mmol). After 12 h, the reaction mixture was concentrated. The product was purified by silica gel chromatography using a mixture of ethyl acetate and hexane (1:9) as eluent to give 11-tritylsulfanyl-undecanoic acid as a white solid. (7) Bain, C. D.; Whitesides, G. M. Angew. Chem., Int. Ed. Engl. 1989, 28, 506-512. (8) Lahiri, J.; Isaacs, L.; Grzybowski, B.; Carbeck, J. D.; Whitesides, G. M. Langmuir 1999, 15, 7186-7198. (9) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. ReV. 2005, 105, 1103-1169. (10) Mrksich, M.; Whitesides, G. M. Annu. ReV. Biophys. Biomol. Struct. 1996, 25, 55-78. (11) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6, 87-96. (12) Fairbank, R. W. P.; Xiang, Y.; Wirth, M. J. Anal. Chem. 1995, 67, 38793885. (13) Wirth, M. J.; Fairbank, R. W. P.; Fatunmbi, H. O. Science 1997, 275, 44-47.

10.1021/la062093p CCC: $33.50 © 2006 American Chemical Society Published on Web 10/24/2006

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Scheme 1. Synthesis of Alkanethiolsa

lamino-6-(11-mercapto-undecanoylamino)-hexanoic acid methyl ester as a white solid. To a solution of this thiol (5.5 mg, 0.012 mmol) in THF (2 mL) was added a solution of lithium hydroxide (2.9 mg, 0.12 mmol) in water. The solution was stirred for 24 h and neutralized with HCl. The resulting acid 3a was lyophilized and used without further purification. 1H NMR (D2O, 500 MHz): δ 1.09-1.56 (m, 22H), 1.80-2.00 (d, 2H), 2.12-2.16 (s, 2H), 3.20-3.34 (d, 2H), 4.744.85 (m, 1H), 7.42-7.65 (m, 5H). HRMS-FAB: m/z 451.2631 ([M + H]+; calculated for C24H39N2O4S, 451.2630). A similar procedure was used to synthesize alkanethiols 3b and 3c (Scheme 1). 2-(3-Methyl-butyrylamino)-6-(11-mercapto-undecanoylamino)hexanoic Acid (3b). 1H NMR (D2O, 500 MHz): δ 0.73-0.82 (m, 6H), 1.09-1.73 (m, 22H), 1.98-2.08 (m, 4H), 2.23-2.29 (m, 1H), 2.49-2.55 (m, 2H), 3.41-3.48 (m, 2H), 3.96-4.04 (s, 1H), 4.554.61 (m, 1H). HRMS-FAB: m/z 431.2943 ([M + H]+; calculated for C22H43N2O4S, 431.2943). 2-Acetylamino-6-(11-mercapto-undecanoylamino)-hexanoic Acid (3c). 1H NMR (D2O, 500 MHz): δ 1.22-1.68 (m, 22H), 0.730.82 (m, 6H), 1.09-1.73 (m, 22H), 1.98-2.08 (m, 4H), 2.23-2.29 (m, 1H), 2.49-2.55 (m, 2H), 3.41-3.48 (m, 2H), 3.96-4.04 (s, 1H), 4.55-4.61 (m, 1H), 2.01-2.0 (s, 3H), 2.12-2.18 (s, 4H), 3.183.30 (br, 2H), 4.52-4.58 (s, 1H). HRMS-FAB: m/z 389.2474 ([M + H]+; calculated for C19H37N2O4S, 389.2474). Preparation of SAMs. Glass cover slips were cleaned by rinsing with benzene and methanol followed by sonication in acetone. Gold substrates for ellipsometric and contact angle measurements were prepared by evaporating 5 nm of titanium followed by 100 nm of gold onto cleaned glass cover slips. Self-assembled monolayers were formed by immersing the gold substrates in 2 mM aqueous solutions of the alkanethiols for 12 h. For SPR studies, commercially available untreated gold sensor chips from Biacore (SIA kit Au) were used. Contact Angle Measurements. The contact angle of water under air on SAMs was measured using a goniometer. Matrix Technologies Microelectrapipette was used to place and remove a water drop from the surface of the SAM. Reported contact angle values are the average of measurements taken at three different locations on each of two different samples. Ellipsometry. Ellipsometric measurements of the thickness of SAMs were carried out on a VASE ellipsometer (J. A. Woollam Co., Inc.; Lincoln, NE). The refractive indices for ambient air and SAM were assumed to be 1.0 and 1.45, respectively,14 and for each measurement, the incident angle was varied from 65° to 75°. Reported values are the average of measurements taken at three different locations on each of two different samples. Surface Plasmon Resonance Spectroscopy. The Biacore 3000 instrument was used for SPR studies. We used a 20 mM phosphate buffer at pH 6.8 for experiments under “low-salt” conditions, and a 20 mM phosphate buffer at pH 6.8 with 300 mM NaCl added for experiments under “high-salt” conditions. All of the buffers and samples were filtered and degassed prior to use. The flow-rate was set at 10 µL/min. The following protocol was used for measuring adsorption of protein: A solution of protein (2 mg/mL) in buffer was allowed to flow for 10 min, followed by a wash with buffer for 5 min and SDS (0.5% w/v) for 5 min.

a Reagents and conditions: (a) (i) trityl chloride, DIPEA, CH Cl , 2 2 room temperature; (ii) NHS, EDCI, CH2Cl2, room temperature; (iii) 6-amino-2-tert-butoxycarbonylamino-hexanoic ester-acetate salt, Et3N, CH2Cl2, room temperature; (iv) TFA-CH2Cl2 (1:4), 0 °C; (b) (i) RCOCl, Et3N, CH2Cl2, room temperature; (c) (i) TFA-CH2Cl2 (1:1), Et3SiH, room temperature; (ii) LiOH, THF-H2O (1:1), room temperature.

To a solution of 11-tritylsulfanyl-undecanoic acid (1.0 g, 2.17 mmol) in CH2Cl2 (15 mL) were added N-hydroxysuccinimide (0.52 g, 4.56 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl (0.50 g, 2.61 mmol). After 12 h, the reaction mixture was concentrated. The product was purified by silica gel chromatography using ethyl acetate-hexane (1:3) as eluent to give 11-tritylsulfanylundecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester as a white solid. To a solution of 6-amino-2-tert-butoxycarbonylamino-hexanoic acid methyl ester (0.36 g, 1.4 mmol) in CH2Cl2 (5 mL) was added a solution of 11-tritylsulfanyl-undecanoic acid 2,5-dioxo-pyrrolidin1-yl ester (0.7 g, 1.3 mmol) in CH2Cl2 (5 mL). After 12 h, the reaction mixture was concentrated. The product was purified by silica gel chromatography using methanol-dichloromethane (1:19) as the eluent to give 2-tert-butoxycarbonylamino-6-(11-tritylsulfanylundecanoylamino)-hexanoic acid methyl ester as a colorless liquid. To an ice-cooled solution of compound 2-tert-butoxycarbonylamino-6-(11-tritylsulfanyl-undecanoylamino)-hexanoic acid methyl ester (0.2 g) in CH2Cl2 (2 mL) was added trifluoroacetic acid (0.5 mL). After 2 h, the reaction mixture was concentrated. The product was purified by silica gel chromatography using methanoldichloromethane (1:9) as the eluent to give 1 as a clear liquid. 1H NMR (CDCl3, 500 MHz): δ 1.09-1.56 (m, 22H), 1.92-1.99 (s, 2H), 2.09-2.15 (t, 4H), 3.15-3.22 (s, 3H), 3.74-3.78 (s, 3H), 3.974.02 (s, 1H), 6.16-6.20 (s, 1H), 7.17-7.42 (m, 15H). 2-Benzoylamino-6-(11-tritylsulfanyl-undecanoylamino)-hexanoic Acid Methyl Ester (2a). To a solution of amine 1 (40 mg, 0.07 mmol) in CH2Cl2 (1 mL) were added triethylamine (0.02 mL, 0.12 mmol) and benzoyl chloride (0.02 mL, 0.2 mmol). After 12 h, the reaction mixture was concentrated. The product was purified by silica gel chromatography using methanol-dichloromethane (1:9) as eluent to give the benzoyl ester 2a as a viscous liquid. 1H NMR (CDCl3, 500 MHz): δ 1.09-1.56 (m, 22H), 2.09-2.15 (t, 4H), 3.15-3.22 (d, 2H), 3.74-3.78 (s, 3H), 4.78-4.84 (s, 1H), 6.937.02 (d, 1H), 7.17-7.64 (m, 20H). A similar procedure was used to synthesize 2b and 2c (Scheme 1). 2-(3-Methyl-butyrylamino)-6-(11-tritylsulfanyl-undecanoylamino)-hexanoic Acid Methyl Ester (2b). 1H NMR (CDCl3, 500 MHz): δ 0.94-0.98 (t, 6H), 1.22-1.74 (m, 22H), 1.80-1.89 (br, 1H), 2.10-2.22 (m, 4H), 2.49-2.55 (m, 2H), 3.21-3.28 (m, 2H), 3.73-3.76 (s, 3H), 4.55-4.61 (m, 1H), 5.68-5.72 (s, 1H), 6.186.22 (d, 1H), 7.16-7.42 (m, 15H). 2-Acetylamino-6-(11-tritylsulfanyl-undecanoylamino)-hexanoic Acid Methyl Ester (2c). 1H NMR (CDCl3, 500 MHz): δ 1.09-1.68 (m, 22H), 2.01-2.04 (s, 3H), 2.09-2.15 (m, 4H), 3.163.29 (br, 2H), 3.72-3.75 (s, 3H), 4.52-4.57 (m, 1H), 5.62-5.68 (s, 1H), 6.29-6.33 (d, 1H). 2-Benzoylamino-6-(11-mercapto-undecanoylamino)-hexanoic Acid (3a). To a solution of 2a (37 mg, 0.05 mmol) in CH2Cl2 (0.5 mL) were added triethylsilane (0.4 mL, 0.25 mmol) and trifluoroacetic acid (0.5 mL). After 12 h, the reaction mixture was concentrated. The product was purified by silica gel chromatography using methanol-dichloromethane (1:9) as the eluent to give 2-benzoy-

Results and Discussion Synthesis of Alkanethiols. We hypothesized that hydrophobic interactions could contribute significantly to the binding of proteins to multimodal ligands under high-salt conditions. One approach for assessing the role of hydrophobic interactions is to compare the extent of protein adsorption to surfaces varying in hydrophobicity. To that end, we synthesized alkanethiols presenting headgroups varying in their hydrophobicity (Figure 1). The structure of alkanethiol 3a was inspired by the structure of a previously (14) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12-20.

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Vutukuru et al. Table 1. Characterization of Multimodal Surfaces

a Log P values represent the values calculated for highlighted headgroups. b Contact angle of water under air. c Reported value is the mean of three readings taken on two different SAMs; uncertainty is the standard deviation of the readings. d Thickness of the SAM as estimated by Chem3D software package using MM2. The lengths were calculated as the sum of the Au-sulfur bond and the length of thiol in minimum energy conformation and were multiplied by cos 32°18 to account for the tilt angle of the thiol on gold.

Figure 1. Structure of ligands. Ligands 3a, 3b, and 3c present benzoyl, isovaleryl, and acetyl headgroups, respectively.

described multimodal ligand (17-5317; GE Healthcare BioSciences Corp., Piscataway, NJ); we note that Johansson and co-workers3,4 suggested that aromatic multimodal cationexchangers were optimal for protein binding under high-salt conditions. The alkanethiols were synthesized as shown in Scheme 1. The synthetic route started with the commercially available mercaptoundecanoic acid, which was first reacted with trityl chloride to protect the thiol group; the carboxylic acid group was subsequently converted to an activated NHS ester. This compound was allowed to react with Boc-protected lysine, followed by the deprotection of the Boc group. Subsequent reaction with acid chlorides followed by deprotection of the trityl group and hydrolysis of the methyl ester yielded compounds 3a-3c. Characterization of SAMs. SAMs were formed by immersing gold-coated glass cover slips for 12 h in 2 mM aqueous solutions of the alkanethiols. To characterize the SAMs, we first measured the contact angle of water under air on the surfaces (Table 1). The value of the contact angle is a useful indicator of surface hydrophobicity.15 SAMs formed from alkanethiol 3a exhibited (15) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464-3473.

the highest value of the contact angle of water under air, while the value was lowest for SAMs formed from alkanethiol 3c. We also used commercially available software (ChemDraw Ultra) to determine the octan-1-ol/water partition coefficients of the headgroups of the alkanethiols (Table 1).16,17 The value of this partition coefficient (P) is another useful indicator of the hydrophobicity of a group, with a greater value corresponding to a greater hydrophobicity. As seen in Table 1, the trend in the value of this partition coefficient is consistent with the trend in the value of the contact angle of water under air for these SAMs, confirming that we have designed surfaces varying in their hydrophobicity. Next, we measured the thickness of SAMs formed using alkanethiols 3a-3c by ellipsometry (Table 1). The values of ellipsometric thickness were in good agreement with the values estimated using commercially available software (Chem3D), taking into account the known angle of tilt of such alkanethiolate SAMs. Protein Adsorption on SAMs Presenting Multimodal Ligands. To test the influence of surface hydrophobicity on protein adsorption under high-salt conditions, we measured the extent of adsorption of lysozyme (2 mg/mL) on SAMs formed from alkanethiols 3a-3c. We used lysozyme in this work because it is a well-characterized (MW ) 14 kDa, pI ) 11.1)15 model protein commonly used in protein adsorption studies; it is positively charged under conditions of the experiment, therefore making it relevant for binding to “cation-exchangers”, and it has been used in previous studies of binding to multimodal resins.4 We measured the extent of protein adsorption on the SAMs using SPR spectroscopy.19-23 SPR is well-suited for these studies because it allows the label-free detection of binding events in real time. (16) Ghose, A. K.; Crippen, G. M. J. Comput. Chem. 1986, 7, 565-577. (17) Gombar, V. K.; Enslein, K. J. Chem. Inf. Comput. Sci. 1996, 36, 11271134. (18) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605-5620. (19) Jordan, C. E.; Frey, B. L.; Kornguth, S.; Corn, R. M. Langmuir 1994, 10, 3642-3648.

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Figure 3. Effect of salt chaotropicity on protein-ligand interactions. (A) SPR sensograms for binding of lysozyme (2 mg/mL) to SAMs presenting ligand 3a in the presence of buffer containing (a) 300 mM NaCl, (b) 300 mM NaNO3, and (c) 300 mM NaClO4. “Buffer”, “protein in buffer”, and “buffer” indicate the solutions that were allowed to flow over the surface of the sensor chip. (B) Amount of lysozyme adsorbed (RU) as a function of the Jones-Dole Bcoefficients for the anion.19

Figure 2. Effect of ligand chemistry on the adsorption of lysozyme to multimodal ligands. (A) Amount of lysozyme adsorbed (RU, 1000 RU ≈ 1 ng/mm2)24 under low-salt and high-salt conditions to multimodal SAMs presenting ligands 3a (black bars), 3b (white bars), and 3c (gray bars). (B) SPR sensograms for binding of lysozyme (2 mg/mL) to SAMs presenting ligands 3a, 3b, and 3c under highsalt conditions. “Buffer”, “protein in buffer”, and “buffer” indicate the solutions that were allowed to flow over the surface of the sensor chip. (C) Amount of lysozyme adsorbed (RU) under high-salt conditions as a function of log P; the values of log P were calculated with the ChemDraw Ultra software package using Crippen’s fragmentation method.

We observed a similar extent of protein adsorption on SAMs formed from alkanethiols 3a-3c under low-salt conditions (Figure 2A). Low-salt conditions are representative of classical ionexchange conditions, under which electrostatic interactions contribute significantly to protein adsorption.4,25 However, under high-salt conditions, where electrostatic interactions would be (20) Kane, R. S.; Deschatelets, P.; Whitesides, G. M. Langmuir 2003, 19, 2388-2391. (21) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 43834385. (22) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. Langmuir 1997, 13, 27492755. (23) Thiel, A. J.; Frutos, A. G.; Jordan, C. E.; Corn, R. M.; Smith, L. M. Anal. Chem. 1997, 69, 4948-4956. (24) Kwon, Y.; Han, Z. Z.; Karatan, E.; Mrksich, M.; Kay, B. K. Anal. Chem. 2004, 76, 5713-5720.

partially screened, there was a significant influence of surface chemistry on the extent of protein adsorption (Figure 2A and B). Moreover, the extent of adsorption of lysozyme on these three surfaces correlated with the surface hydrophobicity as measured by the partition coefficient (Figure 2C). Similar results were observed at higher protein concentrations (see Supporting Information Figure 1) and higher salt concentrations (see Supporting Information Figure 2). The increase in the amount of bound lysozyme with an increase in surface hydrophobicity is consistent with a role for hydrophobic interactions in mediating protein adsorption under high-salt conditions. Influence of Anion Chaotropicity on the Extent of Protein Adsorption. Having demonstrated a role for surface hydrophobicity in influencing protein adsorption under high-salt conditions, we next tested the influence of the composition of the solution.26,27 For this purpose, we systematically varied the composition of the solution, in particular, the chaotropicity of the salt anion, and measured the extent of adsorption of lysozyme to SAMs formed from alkanethiol 3a. We hypothesized that if hydrophobic interactions contribute significantly to the extent of protein binding to this multimodal ligand,4 we would observe a decrease in the extent of protein adsorption with an increase in the chaotropicity of the anion, because chaotropes are known to disrupt hydrophobic interactions.28 The Jones-Dole B-coefficient29,30 provides one quantitative measure of the chaotropicity of an anion. This parameter is a measure of the ion-water interaction and describes the change in viscosity with change in ion concentration. In general, chaotropes are weakly hydrated ions, exhibit smaller (25) Gallant, S. R.; Vunnum, S.; Cramer, S. M. J. Chromatogr., A 1996, 725, 295-314. (26) Tilton, R. D.; Robertson, C. R.; Gast, A. P. Langmuir 1991, 7, 27102718. (27) Wendorf, J. R.; Radke, C. J.; Blanch, H. W. Biotechnol. Bioeng. 2004, 87, 565-573. (28) Farrah, S. R.; Shah, D. O.; Ingram, L. O. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 1229-1232. (29) Collins, K. D. Biophys. J. 1997, 72, 65-76. (30) Marcus, M.; Marcus, Y. Ion Properties; Marcel Dekker: New York, 1997.

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Figure 4 shows the amount of lysozyme and cytochrome c adsorbed to SAM formed from 3a under low-salt and high-salt conditions. As seen in the figure, the amount of lysozyme and cytochrome c adsorbed is similar under low-salt conditions, where electrostatic interactions are likely to be dominant, consistent with the similar retention times of these proteins in ion-exchange chromatography. However, a greater adsorption of lysozyme is seen under high-salt conditions, again consistent with a role for hydrophobic interactions in mediating binding to multimodal ligands under these conditions.

Conclusions Figure 4. Effect of protein hydrophobicity on protein-ligand interactions. Amount of lysozyme (white bars) and horse cytochrome c (gray bars) adsorbed under low-salt and high-salt conditions to SAMs of multimodal ligand 3a.

changes in viscosity with concentration than strongly hydrated kosmotropes, and have negative B-coefficients.31 The value of the Jones-Dole B-coefficient decreases with increasing anion chaotropicity. Figure 3A shows SPR sensograms for the adsorption of lysozyme (2 mg/mL) to SAMs formed from 3a in the presence of buffers containing 300 mM sodium chloride, sodium nitrate, and sodium perchlorate. Figure 3B plots the amount of protein adsorbed as a function of the Jones-Dole B-coefficient of the anion. Consistent with our hypothesis, we observed a decrease in the extent of protein adsorption with an increase in anion chaotropicity, providing further evidence of the contribution of hydrophobic interactions to protein binding under high-salt conditions. Influence of the Hydrophobicity of the Protein on the Extent of Protein Adsorption. Finally, we tested the influence of the hydrophobicity of the protein on the extent of binding to multimodal ligands under high-salt conditions. For these experiments, both the surface composition and the composition of the solution were held constant. We selected two proteins for these experiments: lysozyme (MW ) 14 kDa, pI ) 11.1) and horse cytochrome c (MW ) 12.5 kDa, pI ) 10.1). These proteins have similar retention times in ion-exchange chromatography (pH 7.0, 20 mM phosphate buffer), where protein-adsorbent interactions are primarily electrostatic interactions (data not shown). However, the retention time is significantly greater for lysozyme in hydrophobic interaction chromatography (pH 7.0, 100 mM phosphate buffer with 4.2 M NaCl added), where proteinadsorbent interactions are primarily hydrophobic interactions.32 (31) Lindsay, J. P.; Clark, D. S.; Dordick, J. S. Biotechnol. Bioeng. 2004, 85, 553-560. (32) Xia, F.; Nagrath, D.; Garde, S.; Cramer, S. M. Biotechnol. Bioeng. 2004, 87, 354-363.

This work illustrates that the combination of SPR spectroscopy and SAMs is well-suited for studying the interaction of proteins with complex surfaces of relevance to chromatography. In particular, we have synthesized model surfaces for investigating the interactions of proteins with multimodal ligands. By carefully controlling the molecular-level structure of the surface, the composition of the solution, and the nature of the protein, our experiments confirm that hydrophobic interactions can contribute significantly to protein adsorption at high ionic strengths. These results provide an example of the advantages of this model system in elucidating the fundamental physics underlying the interaction of proteins with chromatographic resins. Our future work will exploit other advantages of the combination of SAMs and SPR spectroscopy, including the ability to control ligand density,8,33-35 to measure the kinetics of protein adsorption and desorption,36-39 and to screen for novel ligands that mediate binding under conditions of interest.18,40 Acknowledgment. This work was supported by a grant from NSF (BES-0418413). We thank Prof. S. M. Cramer and T. Yang, RPI, Troy, NY, for helpful discussions. Supporting Information Available: Data for protein adsorption at different protein and salt concentrations. This material is available free of charge via the Internet at http://pubs.acs.org. LA062093P (33) Chapman, R. G.; Ostuni, E.; Yan, L.; Whitesides, G. M. Langmuir 2000, 16, 6927-6936. (34) Folkers, J. P.; Laibinis, P. E.; Whitesides, G. M.; Deutch, J. J. Phys. Chem. 1994, 98, 563-571. (35) Ostuni, E.; Grzybowski, B. A.; Mrksich, M.; Roberts, C. S.; Whitesides, G. M. Langmuir 2003, 19, 1861-1872. (36) Brandani, P.; Stroeve, P. Macromolecules 2003, 36, 9502-9509. (37) Pesika, N. S.; Stebe, K. J.; Searson, P. C. Langmuir 2006, 22, 34743476. (38) Schlereth, D. D. J. Electroanal. Chem. 1999, 464, 198-207. (39) Silin, V.; Weetall, H.; Vanderah, D. J. J. Colloid Interface Sci. 1997, 185, 94-103. (40) Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.; Whitesides, G. M. J. Am. Chem. Soc. 2000, 122, 8303-8304.