Temperature-Driven Adsorption and Desorption of Proteins at Solid

Article pubs.acs.org/Langmuir

Temperature-Driven Adsorption and Desorption of Proteins at Solid−Liquid Interfaces Irena Kiesel,* Michael Paulus, Julia Nase, Sebastian Tiemeyer, Christian Sternemann, Karin Rüster, Florian J. Wirkert, Kolja Mende, Thomas Büning, and Metin Tolan* Fakultät Physik/DELTA, Technische Universität Dortmund, 44221 Dortmund, Germany ABSTRACT: The heat-induced desorption and adsorption of the proteins lysozyme, ribonuclease A, bovine serum albumin, and fibronectin at protein layers was investigated in two different environments: pure buffer and protein solution. Using two different environments allows us to distinguish between thermodynamic and kinetic mechanisms in the adsorption process. We observed a desorption in buffer and an adsorption in protein solution, depending upon protein properties, such as size, stability, and charge. We conclude that the desorption in buffer is mainly influenced by the mobility of the proteins at the interface, while the adsorption in protein solution is driven by conformational changes and, thereby, a gain in entropy. These results are relevant for controlling biofilm formation at solid−liquid interfaces.

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where ΔH denotes the (internal) enthalpy change, T is the temperature, and ΔS is the entropy change. ΔH includes, e.g., the enthalpy of formation of the protein, Coulomb interactions between the proteins and between the surface and the protein, and van der Waals interactions. Although ΔH is positive, e.g., because of a repulsive interaction between the proteins, the entropy-driven adsorption of proteins with a rising temperature can occur. The entropy S can be increased by conformational changes, the hydrophobic effect, or changes of the hydration shell of the protein. The adsorption of proteins at interfaces increases the conformational entropy.16 The kinetic approach is based on the mobility of proteins in solution and at the surface, which increases with the rising temperature. The interactions between the proteins and the interface dominate the mobility of the proteins. The attraction between the protein and surfaces can be changed by electrostatic interactions, alteration of the contact surface caused by conformational changes, or the size of different proteins. The variation of the temperature, which affects the mobility and conformation of the proteins, gives new insight into adsorption and desorption mechanisms, particularly with respect to kinetically and thermodynamically driven processes. In the past, proteins at solid−liquid interfaces were investigated by neutron and X-ray reflectivity.7,17,18 X-ray reflectometry (XRR)3,19 is used to investigate the interfacial structure of these systems, allowing for in situ measurements in liquid environments with high resolution. The volume fraction profiles and adsorbed amounts depending upon the temperature and environment are discussed.

INTRODUCTION

Protein adsorption initiates the formation of biofilms, which are important for medical applications, such as the biocompatibility of implants or the functionality of sensor surfaces. In other fields, e.g., in the food industry, biofilm growth needs to be prevented. Thus, a control of the first step of biofilm formation, the adsorption of proteins onto surfaces,1,2 is crucial. To reach this goal, a general understanding of the behavior of proteins at solid−liquid interfaces is needed. Different parameters affect proteins at interfaces, such as the protein concentration,3 pH,3,4 pressure,5,6 ionic strength,4 hydrophobic effect,7 and van der Waals forces between proteins and the subsurfaces.8 The present study deals with the effect of the temperature on different adsorbed proteins at interfaces. The influence of heat on proteins in bulk solution was investigated in many studies, e.g., the denaturation of proteins in solution with differential scanning calorimetry9−11 and smallangle X-ray scattering.12−15 Thus far, only a few studies dealt with temperature-induced adsorption and desorption processes of proteins at the solid−liquid interface. Jackler et al.16 have investigated the heat-induced behavior of lysozyme in protein solution and observed heat-induced adsorption. However, most experiments with proteins at interfaces were performed in a protein solution that serves as a protein reservoir, which makes it impossible to disentangle the impact of the temperature on proteins at the interface and in the solution. To separate these effects, we investigated for the first time the influence of the temperature on protein layers at solid−liquid interfaces in different environments, namely, in pure buffer and protein solution, which permits the separation between thermodynamic and kinetic adsorption and desorption mechanisms. Thermodynamically, the change of the Gibbs free energy ΔG of the system is described by

Received: December 27, 2013 Revised: February 6, 2014 Published: February 11, 2014

(1)

ΔG = ΔH − T ΔS © 2014 American Chemical Society

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dx.doi.org/10.1021/la404884a | Langmuir 2014, 30, 2077−2083

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Table 1. Specifications of the Proteinsa protein

lysozyme

RNase A

BSA

molar mass, M (g/mol) mass, m (×10−20, g) molar volume, V (cm3/mol) volume, v (Å3) specific volume, ν (cm3/g) number of electrons electron density, ρprot (e−/Å3) PDB file isoelectric point, pI dimensions (Å3) shape amino acids Tdenat at pH 7, solution (°C)

14300 2.37 9348 15520 0.654 7628 0.49 2LYZ >1122 30 × 30 × 45 elliptical 129 7126

13600 2.26 8685 14420 0.639 7288 0.51 1FS3 9.323 22 × 28 × 38 elliptical 124 6827

66300 11.01 42390 70380 0.639 58234 0.83 3V03 4.822 80 × 69 × 3025 heart-shaped 589 579

fibronectin 440000 73.05 150575 250000 0.342 145266 0.47 5.224 20 × 20 × 1200 elongated, elliptical 2477 6511

a

The volumes, number of electrons, and resulting electron densities were calculated using the software Chimera.28,29 The volume of BSA was calculated without hydrogen atoms. The volume of fibronectin was estimated by assuming an elongated shape with a long axis of 120 nm.30

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EXPERIMENTAL SECTION

The substrates, undoped and polished silicon wafers with a roughness of 3 Å, were provided by Wacker Siltronic (Burghausen, Germany). They were cut into pieces of 10 × 19 mm2, rinsed and hydrophilized in a solution of NH4OH and H2O2 (RCA cleaning20), and stored in deionized water until usage. A native SiO2 layer with a thickness of 5− 10 Å and terminated with hydroxyl groups is formed at the surface. The proteins were purchased from Sigma-Aldrich (Taufenkirchen, Germany) as lyophilized powders and used without further purification. Four different proteins were studied: lysozyme from hen egg white, ribonuclease A (RNase A) from bovine pancreas, bovine serum albumin (BSA), and fibronectin from bovine plasma. For the phosphate buffer, H2PO4Na and HPO4Na2, were solved in Milli-Q water, resulting in a concentration of 0.01 mol/L and a pH value of 6.8. To prevent the formation of air bubbles at the solid−liquid interface when heating, the buffer was degassed under vacuum. A protein concentration of 1 mg/mL for lysozyme, RNase A, and BSA and 0.1 mg/mL for fibronectin was used. Table 1 summarizes the protein properties. The isoelectric point pI indicates that lysozyme and RNase A are positively charged at pH 6.8, whereas BSA and fibronectin are negatively charged. Furthermore, the native silicon dioxide surface of the substrate in aqueous solution is negatively charged at this pH.21 The substrates were stored for at least 60 min in the protein solution before used in the XRR experiments. Previous studies showed that the adsorption process is in equilibrium after 60 min.3 In the first experimental series, all measurements were performed with the substrate remaining in protein solution. For the second series, the wafer with a protein layer was transferred into a pure phosphate buffer solution. During the experiment, the sample and surrounding solution were heated. The temperature was adjusted via a LakeShore 330 temperature controller, using a Pt100 temperature sensor in the liquid phase. The temperature range was chosen between 25 and 90 °C, with a stability of ΔT = ±1 °C. The denaturation temperature Tdenat for all proteins in bulk solution (see Table 1) is in this temperature range. X-ray reflectivity measurements were performed at the synchrotron light source DELTA (Dortmund, Germany) at beamline BL9.19 The photon energy was set to 27 keV, which allows for the penetration of the water phase and reduces the beam damage of the proteins.31,32 Reflectometry gives access to the electron density profile ρ(z) perpendicular to the sample surface depending upon the distance over the sample surface z, whereas other methods, such as quartz crystal microbalance,33 ellipsometry,33 or surface plasmon resonance,34 provide only the adsorbed amount without a detailed resolution of the interfacial structure. For XRR, the incoming beam is specularly reflected at an angle αi, as shown schematically in Figure 1, so that the wave vector transfer q has only a component qz perpendicular to the surface and is calculated with the wavelength λ as

Figure 1. X-ray reflectivity scheme. qz =

4π sin(αi) λ

(2)

The reflectivity of the surface is given by35 R = RF

1 Δρbulk

∫

dρ(z) iqzz e dz dz

2

(3)

with the electron density contrast between the substrate and the subphase Δρbulk and the Fresnel reflectivity RF of an ideal flat substrate. By modeling the electron density profile, the reflectivity is simulated and the best fit can be obtained by the least-squares method. Furthermore, the volume fraction profile ϕ(z) provides the volume fraction of the protein at the height z. It can be obtained by

ϕ(z) =

ρprot (z) − ρsubstr (z) ρprot − ρH O 2

(4)

with the electron density profile of the pure substrate ρsubstr(z), the electron density profile of the substrate with protein layer ρprot(z), the average electron density of water ρH2O = 0.33 e−/Å3 and the average electron density of the pure protein ρprot, as given in Table 1. The adsorbed amount per area Γ can then be calculated as

Γ=

m v

∫ ϕ(z) dz

(5)

with the molar mass m and the molar volume v of the protein. The raw data were background-corrected and normalized to the intensity of an ideally flat substrate RF to highlight the small changes of the curves. The reflectivity curves are refined using the effective density model36 with the Parratt algorithm.37 Figure 2a displays the XRR data of a pure silicon wafer with a native oxide layer in buffer as reference and a lysozyme layer at a silica surface in pure buffer at different temperatures. The fits to the data are shown as solid lines. The obtained electron density profiles are shown in Figure 2b. The scheme behind the electron density profiles illustrates the underlying model with a protein layer. Using eq 4, the volume fraction profiles are 2078


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Figure 2. (a) Reflectivities of lysozyme at the silica interface in pure buffer at different temperatures. Two measurements were sequentially performed at 84 °C. For clarity, the reflectivities are shifted vertically by a constant value. Solid lines correspond to fits of the data. (b) Electron density profiles obtained from the XRR data of the pure substrate and with the lysozyme layer in buffer at different temperatures.

Figure 3. Volume fraction profiles of (a) lysozyme, (b) RNase A, (c) BSA, and (d) fibronectin in buffer solution at different temperatures. The graphs indicated with the same temperature are sequentially measured. The c after 25 °C indicates the measurement after heating.

of lysozyme (Figure 3a) have a fwhm of ≈32 Å, which corresponds to the size of the protein. With an increasing temperature, the layer thickness does not change significantly, whereas the coverage decreases, showing that the adsorbed amount per surface area becomes smaller. A similar trend was observed for RNase A (Figure 3b). The maximum of the volume fraction for BSA decreases abruptly between 30 and 40 °C, whereas the thickness remains nearly constant (Figure 3c). In contrast, the volume fraction profile of fibronectin shows a different behavior. At 25 °C, two different regions can be distinguished, with thicknesses of 20 and 160 Å (Figure 3d). Close to the silica surface, the coverage Θ decreases with an increasing temperature up to 70 °C, but the 160 Å layer does not change. For higher temperatures, the protein layer collapses abruptly to a thin region with increased electron density. This small layer remains stable even if the temperature is reduced to 25 °C again. Protein Solution. The second heating series was performed in protein solution. The resulting volume fraction profiles as

calculated and plotted in Figures 3 and 4. The maximum value of the volume fraction profiles indicates the coverage Θ of the surface with adsorbed proteins. The coverage describes the ratio of the maximum area that is covered with proteins within one layer to the total exposed area. The broadness of the profiles expresses the layer thickness. The full width at half-maximum (fwhm) is smaller than the real thickness of the protein layer, because long-range tails might extend into the solution. The electron density of the proteins in Table 1, which is needed for the determination of the adsorbed amount Γ, is calculated from Protein Data Bank (PDB) files38 with the software Chimera.28,29,39 This works well for the smaller proteins lysozyme and RNase A; however, the calculation is more complex for the proteins BSA and fibronectin because of the absence of complete PDB files in the case of fibronectin and missing hydrogen in the PDB file of BSA. Therefore, the absolute values of the calculated volume fraction profiles and adsorbed amounts are only approximations for BSA and fibronectin.

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RESULTS Pure Buffer. The volume fraction profiles of the proteins in pure buffer are shown in Figure 3. The volume fraction profiles 2079


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Figure 4. Volume fraction profiles of (a) lysozyme, (b) RNase A, (c) BSA, and (d) fibronectin in protein solution at different temperatures. The graphs indicated with the same temperature are sequentially measured. The c after 25 °C indicates the measurement after heating.

Figure 5. Adsorbed protein amount per area of the substrate (eq 5): (a) lysozyme, (b) RNase A, (c) BSA, and (d) fibronectin. The scales are not the same. Error bars are smaller than the symbol size (except for BSA). Full symbols correspond to protein solution, and empty symbols correspond to buffer solution. On panel d, the diamond symbols correspond to measurements at room temperature after heating. Points at the same temperature are measured sequentially. Gray areas refer to the temperature above the denaturation temperature Td, at which the proteins unfold in solution. The lines are guides to the eye.

temperature is kept constant above the denaturation temperature at 80 °C, whereas the layer thickness increases further with time. On the time scale of our experiment, no saturation of the layer growth was observed. A similar trend of an increasing layer thickness below the denaturation temperature of RNase A Td,rna = 68 °C was observed. (Figure 4b). The coverage decreases between 70 and 80 °C and then stays constant, while the layer thickness growth is enhanced above the denaturation

obtained from the XRR data are summarized in Figure 4. The volume fraction profile of lysozyme at 25 °C has a fwhm of 43 Å (Figure 4a). The maximum value of the volume fraction profile is similar to that observed in pure buffer. Below the denaturation temperature of lysozyme, Td,lys = 71 °C, the layer becomes thicker, whereas the coverage Θ decreases between 50 and 68 °C, which is already close to the denaturation temperature. The coverage remains constant when the 2080


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°C a strong increase of the layer thickness and the adsorbed amount similar to our results, which means that a multilayer is formed. In the study, the increased adsorption below the bulk denaturation temperature of Td,lys = 71 °C is caused by an entropy increase. The entropy gain is explained by small conformational changes at the interface.16 The stronger protein adsorption at 80 °C is explained by “dehydration of hydrophobic protein residues at high temperatures”.16,42 At this temperature, the proteins unfold in solution; thus, the hydrophobic residues extend into solution. Because of the hydrophobic effect, the adsorption of these proteins is preferred. Further adsorption with time indicates that the system is not in equilibrium in the time scale of our experiment. The trends for RNase A are similar to those observed for lysozyme. These hard2,43 proteins remain mobile because of a small contact area between the protein and substrate. Entropic effects lead to increasing layer growth in protein solution. For BSA in buffer, a decrease of the adsorbed amount is observed between 40 and 60 °C. This indicates a weak desorption of proteins. BSA is, in comparison to the hard proteins lysozyme and RNase A, a soft protein with a smaller structural stability.2,8,43 This leads to an explanation for the weaker desorption: BSA denatures at the interface and adapts the shape to the surface. Thus, the contact area between the protein and surface is increased, so that the mobility of BSA is suppressed and further desorption is prevented. In BSA solution, further protein adsorption is observed above 40 °C, similar to lysozyme and RNase A. This adsorption can also be explained by increasing conformational entropy. The weaker adsorption compared to the other proteins is caused by repulsive electrostatic forces between BSA and the wafer surface, which are both negatively charged at pH 7. It is well-known that fibronectin has two main conformations.30,44 The observed layer thicknesses are in good agreement with the two different conformations of fibronectin, a globular conformation with a diameter of 160−350 Å and an elongated shape with a diameter of 20 Å and a flexible length of 1200−1500 Å. Al-Jawad et al.45 detected only the elongated shape adsorbed at silicon dioxide surfaces with neutron reflectometry. The different shapes seem to play a key role in the adsorption behavior of fibronectin at the solid−liquid interface. With an increasing temperature, the volume fraction of the elongated form decreases in buffer solution and increases in protein solution. Together with a nearly constant adsorbed amount, this result indicates a conformational change from the elongated shape to the elliptical shape at the interface. The behavior of fibronectin is similar to the other proteins up to 70 °C: desorption is observed in buffer solution, whereas further proteins adsorb in protein solution. The behavior of fibronectin at 80 °C in buffer solution is particularly interesting: the whole layer collapses to a thin layer with high density. This effect can be explained by a total unfolding at the interface with partial desorption, a phenomenon that was not observed for the other proteins and also not in protein solution. In protein solution, a continuous replenishment of proteins from solution to the interface can lead to a stabilization of proteins close to each other at the interface. Also, the observed desorption at 80 °C in protein solution was unique for fibronectin. In both cases, pure buffer and protein solution, the volume fraction profiles after cooling are similar to that at 80 °C. No reversibility of the effects is observed. A schematic model of the heat-induced desorption and adsorption is shown in Figure 6.

temperature. For BSA (Figure 4c), the volume fraction profiles show no change below the denaturation temperature Td,bsa = 57 °C. At 60 °C, the layer thickness increases as well as the maximum volume fraction. The layer grows slightly further with an increasing temperature. In the case of fibronectin at 25 °C, the volume fraction profile displays again two different regions at the interface (Figure 4d). At 80 °C, the volume fraction of the 20 Å thick layer at the interface decreases, similar to the situation in buffer solution at lower temperatures. In contrast to the previous result, the volume fraction of the thick layer increases until a temperature of 70 °C, which is slightly above the denaturation temperature Td,fib = 65 °C, is reached. Further heating to 80 °C induces a slight decrease of the maximum value, but the layer thickness remains nearly constant. Cooling to 25 °C has again no effect on the volume fraction profile. Adsorbed Amounts. The adsorbed amount of proteins per area is determined using equation 5 and presented in Figure 5. The error of the adsorbed amount is estimated by repeated measurements to ±0.15 mg/m2. The adsorbed amount decreases for all four protein layers in buffer solution (open circles). This trend is less clear for BSA (Figure 5c) than for lysozyme (Figure 5a) and RNase A (Figure 5b). An abrupt decrease in the adsorbed amount of fibronectin (Figure 5d) is observed above the denaturation temperature. Cooling has no effect on the adsorbed amount of fibronectin. An opposite trend is observed for the protein layers in protein solution (filled circles), where the adsorbed amount increases with the temperature. The layer growth is enhanced above the denaturation temperature. The adsorbed amount of fibronectin increases further in time at 60 °C, which is below the denaturation temperature, and decreases at higher temperatures. The amount is again unchanged when the solution is cooled from 80 to 25 °C.

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DISCUSSION In buffer and protein solution, the maximum value of the volume fraction profile of lysozyme (≈45%) is nearly half of the close packing value. The model of a random sequential adsorption1,40 leads to a coverage of 54%, which is close to the value that we determined experimentally. The fwhm thickness of the lysozyme layer in buffer solution with 32 Å, together with the roughness of 10 Å, indicates the adsorption of one monolayer in end-on conformation.3,8,16,18 The adsorbed amount decreases with a higher temperature, whereas the layer thickness remains almost constant. These two observations lead to the assumption that the proteins desorb from the surface with an increasing temperature in pure buffer solution. The desorption of lysozyme can be explained kinetically by a higher mobility of proteins at the interface with an increasing temperature. Tie et al.41 have reported a history-dependent protein desorption of fibronectin, cytochrome c, and lysozyme at a Si(Ti)O2 surface. Their experiments indicate an equilibrium state 10 min after the protein solution is replaced by buffer. Our measurements were at the earliest started 10 min after the solution exchange. However, to exclude a time-dependent desorption, we checked an adsorbed lysozyme layer in pure buffer every few hours up to 18 h after preparation at room temperature (data not shown here). No significant change was found. Jackler et al.16 also investigated the temperature behavior of lysozyme at the silica/water interface in protein solution at pH 7 with a concentration of 0.1 mg/mL. They observed at 80 2081


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authors acknowledge the German Federal Ministry of Education and Research (BMBF) (Project 05K10PEC), NRW Forschungschule “Forschung mit Synchrotronstrahlung in den Nano- und Biowissenschaften” and German Research Foundation (DFG) (TO 169/17-1) for financial support. This work is supported by the Cluster of Excellence RESOLV (EXC 1069) funded by the DFG.

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(1) Rabe, M.; Verdes, D.; Seeger, S. Understanding protein adsorption phenomena at solid surfaces. Adv. Colloid Interface Sci. 2011, 162, 87−106. (2) Norde, W. My voyage of discovery to proteins in flatland ... and beyond. Colloids Surf., B 2008, 61, 1−9. (3) Evers, F.; Shokuie, K.; Paulus, M.; Sternemann, C.; Czeslik, C.; Tolan, M. Exploring the interfacial structure of protein adsorbates and the kinetics of protein adsorption: An in situ high-energy X-ray reflectivity study. Langmuir 2008, 24, 10216−10221. (4) Höök, F.; Rodahl, M.; Kasemo, B.; Brzezinski, P. Structural changes in hemoglobin during adsorption to solid surfaces: Effects of pH, ionic strength, and ligand binding. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 12271−12276. (5) Koo, J.; Erlkamp, M.; Grobelny, S.; Steitz, R.; Czeslik, C. Pressure-induced protein adsorption at aqueous−solid interfaces. Langmuir 2013, 29, 8025−8030. (6) Wirkert, F. J.; Paulus, M.; Nase, J.; Möller, J.; Kujawski, S.; Sternemann, C.; Tolan, M. X-ray reflectivity measurements of liquid/ solid interfaces under high hydrostatic pressure conditions. J. Synchrotron Radiat. 2014, 21, 76−81. (7) Lu, J.; Su, T.; Thirtle, P.; Thomas, R.; Rennie, A.; Cubitt, R. The denaturation of lysozyme layers adsorbed at the hydrophobic solid/ liquid surface studied by neutron reflection. J. Colloid Interface Sci. 1998, 206, 212−223. (8) Hähl, H.; Evers, F.; Grandthyll, S.; Paulus, M.; Sternemann, C.; Loskill, P.; Lessel, M.; Hüsecken, A. K.; Brenner, T.; Tolan, M.; Jacobs, K. Subsurface influence on the structure of protein adsorbates revealed by in situ X-ray reflectivity. Langmuir 2012, 28, 7747−7756. (9) Norde, W.; Giacomelli, C. E. BSA structural changes during homomolecular exchange between the adsorbed and the dissolved states. J. Biotechnol. 2000, 79, 259−268. (10) Pace, C. N.; Grimsley, G. R.; Thomas, S. T.; Makhatadze, G. I. Heat capacity change for ribonuclease A folding. Protein Sci. 1999, 8, 1500. (11) Koteliansky, V. E.; Glukhova, M. A.; Benjamin, M. V.; Smirnov, V. N.; Filimonov, V. V.; Zalite, O. M.; Venyaminov, S. Y. A study of the structure of fibronectin. Eur. J. Biochem. 1981, 119, 619−624. (12) Arai, S.; Hirai, M. Reversibility and hierarchy of thermal transition of hen egg-white lysozyme studied by small-angle X-ray scattering. Biophys. J. 1999, 76, 2192−2197. (13) Sosnick, T. R.; Trewhella, J. Denatured states of ribonuclease A have compact dimensions and residual secondary structure. Biochemistry 1992, 31, 8329−8335. (14) Ravindra, R.; Winter, R. On the temperature−pressure freeenergy landscape of proteins. ChemPhysChem 2003, 4, 359−365. (15) Panick, G.; Herberhold, H.; Sun, Z.; Winter, R. Heat, cold and pressure induced denaturation of proteins. Spectroscopy (N. Y., NY, U. S.) 2003, 17, 367−376. (16) Jackler, G.; Steitz, R.; Czeslik, C. Effect of temperature on the adsorption of lysozyme at the silica/water interface studied by optical and neutron reflectometry. Langmuir 2002, 18, 6565−6570. (17) Lu, J. R.; Zhao, X.; Yaseen, M. Protein adsorption studied by neutron reflection. Curr. Opin. Colloid Interface Sci. 2007, 12, 9−16. (18) Richter, A. G.; Kuzmenko, I. Using in situ X-ray reflectivity to study protein adsorption on hydrophilic and hydrophobic surfaces: Benefits and limitations. Langmuir 2013, 29, 5167−5180. (19) Paulus, M.; Lietz, D.; Sternemann, C.; Shokuie, K.; Evers, F.; Tolan, M.; Czeslik, C.; Winter, R. An access to buried interfaces: The

Figure 6. Model for the heat-induced behavior of different proteins: (a) lysozyme, (b) RNase A, (c) BSA, and (d) fibronectin, at the solid− liquid interface in pure buffer and protein solution. The dotted lines around the proteins illustrate conformational changes.

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CONCLUSION We investigated the heat-induced desorption and adsorption of different proteins (lysozyme, RNase A, BSA, and fibronectin) at the solid−liquid interface of a silicon wafer with a native dioxide layer in different environments, namely, pure buffer solution and protein solution. We are able to distinguish between kinetically and thermodynamically driven processes. A partial desorption of proteins in buffer solution and further adsorption in protein solution was observed. These trends are more or less pronounced depending upon the protein properties, especially the conformational stability but also the charge. The desorption in buffer can be explained by kinetic aspects and the mobility of proteins at the interface. Hard proteins stay mobile at the interface. For soft proteins, such as BSA, it is likely that they denature at the interface, leading to a higher contact area between protein and interface, which prevents heat-induced desorption. The adsorption in protein solution below the denaturation temperatures is induced by an entropy gain caused by conformational changes. The increased adsorption above the denaturation temperature is caused by the unfolding of the proteins in solution. The behavior of fibronectin seems to be determined mainly by two different stable conformations: an elongated shape and a globular shape. For fibronectin, a total denaturation at the interface was observed in buffer at the denaturation temperature. In contrast, the denaturation seems to be prevented in protein solution by a stabilization of adsorbed proteins close to each other. In summary, the adsorption process is mainly driven by conformational changes or unfolding above the denaturation temperature and an entropy gain, obscured by individual properties of the proteins, whereas the desorption process is strongly influenced by kinetics.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the DELTA machine group for providing synchrotron radiation and Wacker Siltronic (Burghausen, Germany) for supplying high-quality silicon wafers. The 2082


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Temperature-Driven Adsorption and Desorption of Proteins at Solid

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