Desolvation of BSA–Ligand Complexes Measured Using the Quartz

Article pubs.acs.org/Langmuir

Desolvation of BSA−Ligand Complexes Measured Using the Quartz Crystal Microbalance and Dual Polarization Interferometer Theodore J. Zwang, Reena Patel, Malkiat S. Johal, and Cynthia R. Selassie* Chemistry Department, Pomona College, 645 North College Avenue, Claremont, California 91711, United States S Supporting Information *

ABSTRACT: By taking advantage of their unique difference in hydration sensitivity, we have shown that dual polarization interferometer (DPI) and quartz-crystal microbalance with dissipation monitoring (QCM-D) measurements can be used together to explore the degree of desolvation involved in the binding of small drug molecules to an immobilized bovine serum albumin film in real time. Results with DPI and QCM-D show significantly different mass values for three ligands of varying hydrophobicities that may be attributed to changes in the degree of hydration of the ligand−protein complexes in accordance with the physicochemical properties of the ligands. Furthermore, our data suggest that masses measured by QCM-D can be overwhelmed by changes in water content of ligand− protein, binary complexes, which has important consequences for future studies using mechanical resonators to study protein-binding events.

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magnitude.3 The total number of water molecules that are displaced is also important due to energy associated with solvation.4 We took advantage of the different solvent sensitivities of two instruments in order to create a new method for quantifying water displacement upon ligand binding with the hope that it will aid in understanding the importance of water in ligand binding. The quartz crystal microbalance with dissipation monitoring (QCM-D) and the dual polarization interferometer (DPI) both allow for the creation and observation of functional protein surfaces.5,6 Small-molecule binding and interactions with these proteins can be observed in real time and can reveal useful information about the binding event.7,8 QCM-D records the frequency change of an oscillating crystal when mass is adsorbed onto the surface. This frequency change can be transformed to give the mass of any bound molecules, including coupled solvent by using the Sauerbrey equation.9,10 The Sauerbrey equation is based on the assumption that the biofilm of interest is rigid; if the film is not rigid, then the relationship between mass and frequency breaks down.9,11 This is not a problem for the system presented in this paper because serum albumin deposits to form a surface that is rigid enough for the Sauerbrey equation to be applied with minimal error.12 However, in other less rigid systems with more energy dissipated the mass estimated by the Sauerbrey equation can be inaccurate. In such systems, it is more appropriate to apply a

INTRODUCTION Over the past few decades, significant work has gone into understanding how to rationally design drugs. Researchers have many new tools at their disposal that aid in this endeavor, such as the ability to generate 3D-structures of ligands and calculate their steric, electrostatic, and lipophilic properties.1,2 Unfortunately, attempting to predict interactions between these ligands and their target binding sites has proved difficult, and in many cases inaccurate, largely due to an insufficient understanding of the role that water plays.3 There are many different interactions that contribute to the favorability of complex formation. When possible, it helps to simplify these interactions to their free energy change so that the different interactions’ effects on affinity are comparable. Summing these together gives the free energy of binding (ΔG), which is an important quantity for drug design because it can be thought of as the net effect of all of these interactions added together, and it helps describe the favorability of complex formation. Thus, in order to have an accurate prediction of ligand−protein affinity, it is important to know the free energy change caused by the movement of water upon ligand binding.3 Unfortunately, determining this value can be difficult in part because of challenges ascertaining the number of water molecules present in a protein. X-ray crystallography is currently the major source of experimental data for determining the presence of water molecules within protein−ligand complexes. However, loosely bound or mobile water will not be identified by X-ray crystallography.4 The location of these water molecules is important because their displacement upon ligand binding as well as hydrogen bonding effects can increase or decrease ligand−protein affinity by several orders of © 2012 American Chemical Society

Received: September 28, 2011 Revised: May 21, 2012 Published: May 23, 2012 9616

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sensor consisting of two adjacent waveguides (AnaChip), and a CCD camera. Each sensor chip’s active surface (0.15 cm2) was unmodified silicon oxynitride with an average roughness of 5 nm. The fluidic system coupled to the instrument utilized standard HPLC components: an autosampler (Jasco AS-2055 Plus), a pump (Harvard Apparatus), and two three-way valves to direct flow into the two active channels of the chip. Internal temperature was fixed at 20 ± 0.1 °C. The two polarizations of light (transverse electric and magnetic) internally reflect through two paths in the waveguide, which produces an evanescent field and interference patterns that constitute the raw data output. Data were analyzed in AnaLight Explorer (Farfield Scientific) to calculate thickness, density, and mass values. Density and thickness values of the film were resolved simultaneously to less than 1 pg/mm2 and 10 pm, respectively. The BSA was deposited by flowing 200 μL of 10 mg/mL BSA in PBS across the surface of the DPI waveguide. Caffeine, salicylic acid, or desipramine was then flowed over the surface at 50 μL/min. Each drug was tested three times, and the data from each run were consistently reproducible. Details of DPI principles and operation can be found elsewhere.16,20

model that takes into account the viscoelasticity of the less-rigid layer, such as the Voigt model.13,14 DPI sends transverse electric and magnetic polarized light through a stacked waveguide. The top surface waveguide is exposed to experimental solutions with varying refractive indices, which cause the speed of light moving through it to change relative to a reference waveguide. Because the light enters both the experimental and reference waveguides in phase but moves through the experimental waveguide at a different speed, the light exits the two waveguides out of phase and produces interference fringes. Using classical optical theory, and the relationship between refractive index and density, it is possible to solve for the positions of the dark and light bands in terms of thickness and density. This information can be converted to a measurement of mass that is relatively insensitive to the hydration of the adsorbate, especially when the refractive index of the solvent remains unchanged. Further details on the theories underlying this instrument’s use can be found elsewhere.15−17 We have previously reported on the different solvent sensitivities of QCM-D and DPI,10 and other studies have applied the same theory by coupling QCM-D and ellipsometry.18 One benefit of employing DPI over ellipsometry is that an absolute determination of both thickness and refractive index is made within a single measurement, which can be used to provide an accurate measurement of adsorbed mass. In this study, advantage is taken of the unique ways that QCM and DPI differ in their measures of mass to create a novel method for investigating changes in desolvation of proteins. This is done using a model system of an immobilized, bovine serum albumin film and observing how the binding of extremely hydrophobic desipramine, the very hydrophilic salicylic acid, and the slightly hydrophilic caffeine induces desolvation.

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RESULTS AND DISCUSSION One might question whether adsorption of serum albumin onto a surface will denature it. Previous work by Freeman et al. suggest that serum albumin adsorbed onto a silicon oxynitride surface was not denatured by the surface21 because conformational changes of the BSA caused by cycling the solution pH are reversible which is characteristic of nondenatured globular frameworks.22,23 Our BSA surfaces were created in a similar manner and resulted in a surface density of 43 ± 4 ng/cm2, which compares well with Freeman et al.’s value of 42 ng/cm2 and suggests that our BSA surfaces are nondenatured as well.21 The DPI recorded an increase in mass of the adsorbed layer attributed to binding of the three ligands to the BSA binding site (Figure 1). Desipramine, caffeine, and salicylic acid adsorbed on BSA in the following amounts, respectively: 0.195, 0.134, and 0.065 nM/cm2. Figure 2 shows the

MATERIALS AND METHODS

Bovine serum albumin (BSA), caffeine, salicylic acid, and desipramine hydrochloride were obtained from Sigma-Aldrich and used as received. BSA was dissolved in phosphate buffered saline (PBS) at pH 7.4 that was obtained from Fischer. Stock solutions of 15 mM caffeine, 10 mM salicylic acid, and 5 mM desipramine were made in PBS at pH 7.4. Real-time frequency and dissipation data were collected using a QCM-D (E4, Q-Sense, Gothenberg, Sweden). Frequency data collected at the fifth harmonic was converted to mass using Sauerbrey’s equation9 to simplify comparison with DPI data. The QCM-D sensor, which is mounted in a 40 μL flow cell, consisted of an AT-cut piezoelectric quartz crystal disk coated with a gold electrode (100 nm thick) on the underside and an active surface layer of SiO2 (∼50 nm). The QCM-D sensor crystal (14 mm × 0.3 mm, active area of 0.2 cm2) is operated at a fundamental frequency of 4.95 MHz ± 50 kHz. Internal temperature was fixed at 20 ± 0.1 °C. All QCM-D crystals were optically polished with a root-mean-square roughness less than 3 nm. Crystals were decontaminated by UV/ozone treatment for 10 min, treated with 2 vol % Hellmanex solution (Hellma GmbH & Co.) for 15−30 min, rinsed with ultrapure water, blown dry with N2, and finally treated again with UV/ozone before use. Then the crystals were placed in the QCM-D flow cell, and a baseline was created using PBS. BSA was deposited, and then the binding of caffeine, salicylic acid, or desipramine was observed by flowing over the drug solutions over the sensor surface at a rate of 100 μL/min. Each drug was tested more than 8 times, and the data from each run were consistently reproducible. Once placed in the flow cell, PBS was used to rinse the sensor surface after each experimental solution. Details of QCM-D principles and operation can be found elsewhere.19 A DPI (AnaLight Flex Bio200, Farfield Scientific, Inc., Cheshire U.K.) was used to obtain the optical properties of the BSA-drug layers. The instrument utilizes a helium−neon laser (632.8 nm), a polarizer, a

Figure 1. Change in mass measured by DPI for adsorbed protein layer upon exposure to desipramine (dark gray), salicylic acid (light gray), and caffeine (black). Black dotted line indicates flow of drug solution. Gray dotted line indicates PBS rinse. The mass increase due to the drugs appears to be larger for higher molecular weight molecules. 9617

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QCM-D measures deposition of mass in such a way that it senses the total contribution of everything that attaches to the sensor surface. This measurement includes ligands and associated water in binding interactions of ligand−proteins. Figure 3 illustrates the changes in mass of the adsorbed protein

Figure 2. Change in density measured by DPI for adsorbed protein layer upon exposure to desipramine (dark gray), salicylic acid (light gray), and caffeine (black). Because water has a lower refractive index increment (1.00) than BSA (∼1.5) an increase in density is suggestive of dehydration of the protein layer, while a decrease in density is suggestive of increasing the hydration of the protein layer. Black dotted line indicates flow of drug solution. Gray dotted line indicates PBS rinse.

Figure 3. Change in mass measured by QCM-D for adsorbed protein layer upon exposure to desipramine (dark gray), salicylic acid (light gray), and caffeine (black). Black dotted line indicates flow of drug solution. Gray dotted line indicates PBS rinse.

corresponding changes in film density upon the binding of the three ligands to BSA. The change in density of the protein layer was calculated by using the differential refractive index of BSA compared to the aqueous buffer. The refractive index of the bulk aqueous buffer was calculated during the calibration to be n = 1.347. This value was used as a baseline for subsequent measurements of protein and ligand adsorption. This results in a thin film density that is essentially ignoring the mass contribution of water molecules, so the reported film density is lower than the real film density and may be called the differential density of BSA in PBS. This does not in any way alter the analysis of our results regarding desolvation upon ligand binding to these layers. Furthermore, our measurements, after analysis with AnaLight Explorer, report the differential density of BSA in PBS as 0.18 ± 0.01 g/cm3. Our result is in good agreement with a similar system, BSA in water, which has a differential density of 0.184 g/cm3.24 This supports the validity of the values reported herein by suggesting that the relationships that allow for the translation of refractive index into density are satisfied by this system. This is necessary for an accurate determination of thin film mass. See the Supporting Information for this raw density data. Since water is less dense than BSA, an increase in density suggests that the surface bound BSA−ligand complex is less solvated than the pure BSA layer. A decrease in density suggests enhanced hydration of the BSA−ligand complex. These changes in density correspond to greater values than would be expected for the desolvation of only the drug binding site. We ascribe this to the ligand causing a change in whole-protein hydration, possibly caused by ligandinduced conformational change. Previous work has shown that the conformational change of BSA on surfaces can result in a significant change in layer density.21,25

layer upon exposure to desipramine, caffeine, and salicylic acid. The decrease in mass is highest for the highly hydrophobic desipramine, which undergoes a significant contraction in mass when compared to the DPI-measured value (mQCM − mDPI = −195 ng/cm2). Surprisingly, the hydrophilic, ionized salicylate also undergoes a loss in mass on the order of −79 ng/cm2. Caffeine does not appear to significantly alter the solvation (mQCM − mDPI ≈ 0). See Table 1 for a comparison of values. One concern may be that the different surfaces used in DPI (silicon oxynitride) and QCM-D (silicon dioxide) may be causing the difference in measured mass, or may make the two protein layers incomparable to one another. To address this concern, we repeated these drug-binding experiments with three additional surfaces. Using gold surfaces, titanium dioxide surfaces, and EDC/NHS attachments to gold surfaces each resulted in nearly identical data when measured with QCM-D (data not shown). This suggests that the difference between the used DPI and QCM-D surfaces are inconsequential for our experiment and that the protein layers formed on each are comparable. Therefore, the mass discrepancy in the DPI and QCM-D measurements must be caused by the displacement of water that occurs when a ligand binds to bovine serum albumin (BSA). A calculation of the mass differences of the three drugs from the QCM-D and DPI data reveals an approximation of the number of water molecules displaced for each bound molecule of desipramine (−56), salicylic acid (−67), and caffeine (−0.4). Because of the low number of data points, and because a high degree of collinearity between hydrophobicity and molecular weight of these particular molecules (r = −0.908), a correlation between water loss and hydrophobicity or molecular weight was not tenable. Besides, it appears that salicylic acid is not an 9618

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Table 1. Mass Measurements and Physicochemical Properties of Ligands mass measurements in ng/cm2 ligand

DPI

QCM-D

Δm

ΔH2O (mol/mol)

desipramine

52 (0.195 nM/cm2) 26 (0.134 nM/cm2) 9 (0.065 nM/cm2)

−143

−195

25 −70

caffeine salicylic acid a

log Da

MW

−56

1.38

266.4

≈0

≈0

−0.07

194.2

−79

−67

−1.68

138.1

Distribution coefficient of the ligands at pH 7.4.

not appear to be hysteretic. Therefore, the magnitude of the mass response on QCM-D for similar systems should be carefully interpreted as being largely affected by the degree of solvation/desolvation. However, the measured change in mass of the system is still accurate. When water molecules rearrange and desorb from the protein, the viscoelastic attributes of the film change. Because the Sauerbrey equation is based on the assumption that any biofilm is rigid, if the film is not rigid, then the relationship between mass and frequency breaks down.9,11 This is not a problem for this system because serum albumin deposits to form a rigid surface,12 and the desolvation caused by the binding of the drugs tested in this work appears to make it more rigid, with the exception of caffeine. However, caffeine’s Sauerbrey mass was independent of the harmonic used to calculate it. Therefore, the change in dissipation even after the binding of drugs and desolvation are within the range that the Sauerbrey equation can be used.11 It is important to note that if one assumes that the change in hydration caused by each binding event is a constant value for the same type of molecule, kinetics measurements using a protein layer should be unaffected since the constant will be eliminated on division of the association rate by the dissociation rate to calculate affinity.

appropriate ligand for analyzing degrees of solvation/desolvation of a series of ligands because of its unusual, high affinity binding to a specific site that may differ from the desipraminebinding site.26 Serum albumin’s Sudlow binding site 1 is located in the core subdomain IIA and is predominantly hydrophobic in nature, although it does contain clusters of mostly basic, polar residues at the mouth and on the floor of the pocket.27,28 Thus, a hydrophobic drug such as desipramine at pH 7.4 (log D = 1.38) could localize in the hydrophobic pocket, resulting in complete desolvation of the ligand as well as the pocket, resulting in substantial loss of water molecules and thus a significant loss of mass. This loss of water will result in the BSA−ligand film having a larger density compared to pure BSA, as is observed in the case of desipramine (Figure 2). Because of its slightly polar nature, caffeine would not be expected to bind well in binding site 1 and water molecules in its environs and in the pocket would be retained with little or no net loss in mass. In fact, the BSA−caffeine complex results in an assembly with smallest density of all three ligands, consistent with loose binding (Figure 2). Salicylic acid’s log D value is −1.68 at pH 7.4, and thus one would expect measured mass to be higher in the QCM-D data, but this is not observed. Instead, a loss of water is observed. Salicylic acid’s component parts include a very hydrophobic phenyl ring and a carboxylate anion; the presence of these two structural moieties allows for maximal interaction of the phenyl group with Trp-214 in the hydrophobic pocket and strong electrostatic/hydrogen bond interactions with the protonated arginine residues lining the mouth of the binding site.29 The strength of these intermolecular interactions with BSA thus results in a reduced level of hydration and a net loss of mass. The magnitude of desolvation of a binding site depends on the physicochemical attributes of the ligand and the nature of the binding site.30 Because the hydrophilic salicylate and hydrophobic desipramine both resulted in a significant degree of desolvation that appears unrelated to their hydrophobicity, another possible explanation of our results is that water is not only being displaced from the ligand binding sites but also from reorganization of the protein layer. This would mean that the difference in measured mass was caused by desolvation of the protein including other regions outside of the binding pocket but in close proximity. This is plausible considering the reversible change in folding that has been observed when serum albumin binds heme31 as well as the ability of surface-bound BSA to undergo reversible folding induced by pH.21 The most surprising result is that the dehydration of BSA appears to be dominant over the ligand binding for determining the mass response of QCM-D. As is evidenced by the return of the protein’s density to pre-exposure values, the change in water content of BSA caused by small molecule binding does

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CONCLUSION DPI is insensitive to solvent, whereas QCM-D includes it in measurements of mass. This leads to a difference in the mass reported by these two instruments for the same binding events, the magnitude of which is dependent on the degree of dehydration. Our results demonstrate that by using these two instruments together on the same system, it is possible to measure changes in water content of a protein layer, BSA, when it binds drugs of varying hydrophobicity and/or size. More hydrophobic drugs bind tightly to BSA and displace bound water molecules, thus resulting in the overall QCM-D mass being substantially less than the DPI mass. More hydrophilic drugs do not displace water molecules as easily as their hydrophobic counterparts; however, both appear to desolvate BSA possibly due to a binding-induced conformational change. These results suggest that the overwhelming affect that the degree of hydration has on QCM-D measurements of mass may dramatically alter the interpretation of some proteinbinding data using this instrument. The two approaches in tandem allow for the determination of the degree of desolvation of a ligand in real time.

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ASSOCIATED CONTENT

S Supporting Information *

Figure of DPI data illustrating raw changes in density of adsorbed protein layers on exposure to desipramine, salicylic 9619

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acid, and caffeine; figures describing frequency and dissipation changes in the 3rd, 5th, 7th, and 9th harmonics upon binding of desipramine, caffeine, and salicylic acid to BSA-coated SiO2 surfaces on QCM-D. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; fax (909) 607-7726. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank T. J. Lane for his help in conducting DPI measurements. This research is funded in part by the Gordon and Betty Moore Foundation. This work was also supported in part by a grant from HHMI.

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