Langmuir 1996, 12, 4025-4032
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Molecular Orientation in Heme Protein Films Adsorbed to Hydrophilic and Hydrophobic Glass Surfaces John E. Lee and S. Scott Saavedra* Department of Chemistry, University of Arizona, Tucson, Arizona 85721 Received March 15, 1996. In Final Form: June 18, 1996X Due to the heterogeneous distribution of chemical functionalities present on the surface of most proteins, adsorption to solid materials of differing surface chemistry may produce different bound molecular orientations. Differences in molecular orientation may in turn produce differences in adsorbed biofunction, which has important implications for fabrication of protein-based molecular devices. Aspects of this topic were addressed here by investigating molecular orientation in submonolayer to monolayer thick films of myoglobin (Mb) and cytochrome c (cyt c) adsorbed to hydrophilic and hydrophobic glass substrates. Orientation was determined by measuring the mean tilt angle of the heme moiety in protein films supported on a planar integrated optical waveguide. The results show (i) mean molecular orientation in monolayer films of both Mb and cyt c on both substrates is anisotropic rather than random (ii) molecular orientation in monolayer cyt c films is dependent on the wettability of the substrate and (iii) on both substrates, molecular orientation in submonolayer Mb films is substantially different than that in monolayer films.
Introduction Nonspecific protein adsorption is a ubiquitous process that occurs when virtually any synthetic material is brought into contact with a solution of dissolved proteins.1 Understanding and controlling it is fundamentally important to successful development of synthetic biomaterials and implantable chemical sensing devices.2 When compared to binding between a receptor and its ligand, nonspecific adsorption is generally thought to be a less localized interaction involving a larger fraction of the amino acids in a protein. The larger contact region may be due to the several types of noncovalent interactions that are possible between the surface of the material and the distribution of chemical moieties on the external surface of the protein. If the substrate surface itself is chemically microheterogeneous, the subset of amino acids involved in adsorption may differ substantially among individual protein molecules. The net result of these effects may be a film in which the orientation of adsorbed molecules is geometrically random. However, when one mode of interaction has a relatively high affinity compared to competing modes, and the contact region on the protein is localized, the adsorbed molecules may be anisotropically oriented.3 If the attraction/repulsion geometry is substantially different at the surface of a second adsorbent having chemical properties that differ significantly from the first, the mean molecular orientations on the two adsorbents may differ significantly. Experimentally observed differences in biofunctional activity and surface coverage among protein-surface combinations have been frequently attributed to differences in molecular orientation.4-11 For example, the * Corresponding author: Ph, (520) 621-9761 FAX (520) 6218407
[email protected] X Abstract published in Advance ACS Abstracts, August 1, 1996. (1) (a) Andrade, J. D.; Hlady, V. Adv. Polym. Sci. 1986, 79, 1-63. (b) Norde, W. Adv. Colloid Interace Sci. 1986, 25, 267-340. (2) Brash, J. L.; Horbett, T. A. In Proteins at Interfaces II; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995; pp 1-23. (3) (a) Darst, S. A.; Ribi, H. O.; Pierce, D. W.; Kornberg, R. D. J. Mol. Biol. 1988, 203, 269-273. (b) Furuno, T.; Sasabe, H. Biophys. J. 1993, 65, 1714-1717. (4) (a) Lin, J. N.; Andrade, J. D.; Chang, I.-N. J. Immunol. Methods 1989, 125, 67-77. (b) Chang, I.-N.; Herron, J. N. Langmuir 1995, 11, 2083-2089. (5) Lee,C.-S.; Belfort, G. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 83928396.
S0743-7463(96)00253-3 CCC: $12.00
orientation of an immobilized protein molecule may affect its ability to participate in electron transfer6 or bind to a dissolved ligand.4 However, systematic investigation of relationships between adsorbent surface chemistry and molecular orientation in protein films, and consequent effects on biofunction, has been hampered by the experimental difficulty of measuring orientation in protein films. When the shape of a protein molecule is highly asymmetric (e.g., IgG), molecular orientation can be indirectly inferred from measurements of optical thickness, using either ellipsometry, surface plasmon resonance, or guided wave perturbation.12-14 If the protein contains a chromophore that can be employed as a probe, the use of polarized spectroscopic techniques is an alternate approach.15-21 However, when the sample thickness is less than a few monolayers, spectral methods can be problematic with respect to instrumental sensitivity.20 Some progress has been made using Raman scattering, total internal reflection fluorescence (TIRF) and absorbance linear dichroism (LD) methods applied to films of small heme proteins. Saavedra and Reichert15 demonstrated that absorbance LD could be measured in a hydrated submonolayer of adsorbed myoglobin using the planar integrated optical waveguide attenuated total reflection (IOW-ATR) geometry. Walker et al.16 measured the mean tilt angle of the heme in reduced cyt c adsorbed to a silicon oxynitride planar IOW. Bohn and co-workers (6) (a) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 1847-1849. (b) Cullison, J. K.; Hawkridge, F. M.; Nakashima, N.; Yoshikawa, S. Langmuir 1994, 10, 877-882. (c) Collinson, M.; Bowden, E. F. Anal. Chem. 1992, 64, 1470-1476. (7) Hamachi, I.; Noda, S.; Kunitake, T. J. Am. Chem. Soc. 1991, 113, 9625-9630. (8) Iuliano D. J.; Saavedra, S. S.; Truskey, G. A. J. Biomed. Mater. Res. 1993, 27, 1103-1113. (9) Darst, S. A.; Robertson, C. R.; Berzofsky, J. A. Biophys. J. 1988, 53, 533-539. (10) Alarie, J. P.; Sepaniak, M. J.; Vo-Dinh, T. Anal. Chim. Acta 1990, 229, 169-176. (11) Lin, J.-N.; Chang, I.-N.; Andrade, J. D.; Herron, J. N.; Christensen, D. A. J. Chromatogr. 1991, 542, 41-54. (12) Spinke, J.; Liley, M.; Guder, H.-J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821-1825. (13) Herron, J. N.; Mu¨ller, W.; Paudler, M.; Riegler, H.; Ringsdorf, H.; Suci, P. A. Langmuir 1992, 8, 1413-1416. (14) Nellen, Ph. M.; Tiefenthaler, K.; Lukosz, W. Sens. Actuators 1988, 15, 285-295. (15) Saavedra, S. S.; Reichert, W. M. Langmuir 1991, 7, 995-999. (16) Walker, D. S.; Hellinga, H. W; Saavedra, S. S.; Reichert, W. M. J. Phys. Chem. 1993, 97, 10217-10222.
© 1996 American Chemical Society
4026 Langmuir, Vol. 12, No. 16, 1996
Lee and Saavedra
determined mean heme orientation in dried films of cyt b5 mutants covalently attached via disulfide bonding to glass planar waveguides.17 Lee and Saavedra compared heme orientation in hydrated ferricyt c adsorbed to hydrophobic and hydrophilic glass IOW surfaces.18 Using TIRF anisotropy, Kleijn and co-workers19 measured heme orientation in films of metal-free cyt c adsorbed to glass and tin oxide substrates. Macdonald and Smith21 used surface-enhanced resonance Raman spectroscopy to examine cyt c adsorbed to citrate-coated silver and found that heme orientation was influenced by protein surface coverage. Here we expand on initial work15,16,18 with an investigation of molecular orientation in myoglobin (Mb) films adsorbed to hydrophobic and hydrophilic glass surfaces. A particular focus is the effect of protein surface coverage, which our studies show exerts a considerable influence on orientation. Theory The theory for determining mean molecular orientation in hydrated heme protein films using IOW-ATR measurements of linear dichroism has been described previously18 and is briefly reviewed here. Lightguiding behavior in an IOW can be modeled as a process of repeated total reflection at the boundaries between the waveguiding layer and the adjacent, lower index media (the ray optics approximation.22 The reflection density per centimeter of beam propagation along the IOW-superstrate interface is given by
N/D ) (2d2 tan θi + ∆21 + ∆23)-1
(1)
where N is the number of reflections, D is the distance in cm, d2 is the IOW thickness, ∆21 and ∆23 are the GoosHa¨nchen shifts at the IOW-superstrate and IOWsubstrate interfaces, respectively, and θi is the angle of total reflection for the particular mode. Expressions for ∆21 and ∆23 for a step-index, asymmetric planar IOW are given in ref 22. The superstrate medium consists of an adsorbed protein film in contact with a dilute solution of dissolved protein. The intrinsic heme, which absorbs light at visible wavelengths, causes attenuated total reflection of the propagating mode. When the absorption of evanescent energy per reflection in both the adsorbed film and the bulk solution are relatively weak, the total absorbance per reflection, At/N, is given by
At/N ) Ab/N + Af/N ) bcb(Ie/Ii)dp + fcf(Ie/Ii)df
(2)
where the b and f subscripts refer to bulk solution and the thin film, respectively, and c are the molar absorptivity and concentration of the heme, respectively, Ie/Ii is the interfacial transmitted intensity per unit incident beam intensity, dp is the penetration depth of the evanescent wave, and df is film thickness. Expressions for Ie/Ii for TE and TM polarized light and dp are given in ref 22. As (17) (a) Stayton, P. S.; Ollinger, J. M.; Jiang, M.; Bohn, P. W.; Sligar, S. G. J. Am. Chem. Soc. 1992, 114, 9298-9299. (b) Hong, H.-G.; Bohn, P. W.; Sligar, S. G. Anal. Chem. 1993, 65, 1635-1638. (18) Lee, J. E; Saavedra, S. S. In Proteins at Interfaces II; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995; pp 269-279. (19) (a) Fraaije, J. G. E. M.; Kleijn, J. M.; van der Graaf, M.; Dijt, J. C. Biophys. J. 1990, 57, 965-975. (b) Bos, M. A.; Kleijn, J. M. Biophys. J. 1995, 68, 2573-2579. (20) Pachence, J. M.; Amador, S.; Maniara, G.; Vanderkooi, J.; Dutton, P. L.; Blasie, J. K. Biophys. J. 1990, 58, 379-389. (21) Macdonald, I. D. G.; Smith, W. G. Langmuir 1996, 12, 706-713. (22) Saavedra, S. S.; Reichert, W. M. Anal. Chem. 1990, 62, 22512256.
Figure 1. Schematic of the laboratory coordinate system, defined by the x, y, and z axes, in a linear dichroic IOW-ATR experiment. The light is propagating along the x-axis, and the origin is at the point of reflection in the waveguide (x - y) plane. The electric dipole transitions µ1 and µ2 of the porphyrin are perpendicular to one another in the molecular plane (the x′ - z′ plane), which lies at an angle θ from the waveguide surface normal (the z-axis) within the near surface region probed by the evanescent wave.
discussed in detail elsewhere,23,24 eq 2 is subject to the assumptions that (i) df < 0.1dp, which is valid for a monolayer of small protein molecules and (ii) (Ie/Ii)TE and (Ie/Ii)TM are not affected by the presence of the protein film. The validity of (ii) has been addressed theoretically24 and experimentally15 and is a good approximation when the protein film is thinner than 0.1dp and has a refractive index that differs from the index of the pure bulk solution (i.e., water) by e10%. Both of these conditions are reasonable assumptions for the protein films examined in this paper. Figure 1 diagrams the geometry of a linear dichroic ATR experiment in which the orientation of a proteinbound heme moiety at the IOW surface is determined. The two electric dipole transitions, µ1 and µ2, lie orthogonal to one another in the molecular plane of the heme. The quantity θ is the polar angle between the molecular plane and the z-axis (which is normal to the IOW plane). Two approximations are made to simplify the analysis: (i) The dipole transitions are circularly polarized in the molecular plane.25 (ii) The orientation distribution is azimuthally symmetric (uniaxial).19a Under these conditions, the relationship between the mean tilt angle of an ensemble of molecules (θµ) and the dichroic ratio of thin film absorbances (F) is
F)
Af,TM At,TM - Ab,TM ) ) Af,TE At,TE - Ab,TE
[
NTM E2x +
]
2E2z cos2 θµ 1 + sin2 θµ
[NTEE2y]-1 (3)
where the TE and TM subscripts refer to the pair of modes under comparison, N is the total number of reflections over which the measurement is made in each mode, and E2x , E2y , and E2z are the squared electric field amplitudes of the evanescent wave along the x-, y-, and z-axes.18,19a Experimental Section Materials. Silicon oxynitride (SiOxNy) waveguides on fused silica substrates (25 × 75 × 1 mm) were fabricated at the Microelectronics Center of North Carolina in RTP, NC.26 Waveguides were approximately 1.2-1.5 µm thick with refractive indices ranging from 1.52 to 1.56 and supported two to three guided modes. Dichlorodimethylsilane (DDS, 99%), zinc pro(23) Harrick, N. J. Internal Reflection Spectroscopy, 2nd ed.; Harrick Scientific: New York, 1979. (24) Reichert, W. M. Crit. Rev. Biocompat. 1989, 5, 173-205. (25) Hofrichter, J.; Eaton, W. A. Annu. Rev. Biophys. Bioeng. 1976, 5, 511-560. (26) Walker, D. S.; Reichert, W. M.; Berry, C. J. Appl. Spectrosc. 1992, 46, 1437-1441.
Molecular Orientation in Heme Protein Films toporphyrin IX, and bovine hemin were purchased from Aldrich and used as received. Ferricytochrome c (cyt c, 99%) and metmyoglobin (Mb, 95-100%), both from horse heart, were obtained from Sigma. Protein solutions were prepared in a 50 mM phosphate buffer (pH 7.2, 100 mM NaCl) and purified on a Sephadex G-25 gel filtration column. Protein concentrations were determined using molar absorptivities of 9730 M-1 cm-1 (514.5 nm) for Mb15 and 8770 M-1 cm-1 (514.5 nm) for cyt c.27 Glass beads (3 ( 0.3 mm diameter) used in determinations of protein surface coverage were purchased from Baxter. Surface Preparation. Waveguides were cleaned by soaking in an 80 °C chromic acid bath for 20 min. The waveguides were then rinsed extensively with deionized (Type I Reagent Grade) water, rehydrated in a 1 M nitric acid bath for 2 h, rinsed again with deionized water, blown dry with nitrogen, and dried for 2 h at 80 °C. This treatment produced a hydrophilic surface. To obtain hydrophobic surfaces, cleaned waveguides were silanized in 2% DDS (v/v) in dry toluene for 2 h. Dry nitrogen was bubbled into the solution during the silanization process, which was performed in a nitrogen-filled glovebag. Waveguides were then rinsed sequentially in toluene, toluene/ethanol, ethanol, ethanol/ water, and water, blown dry with nitrogen, and dried for 2 h at 80 °C. The same procedures were used to prepare hydrophilic and hydrophobic glass beads and fused silica slides. Static water contact angles on waveguide surfaces were measured using a digital CCD camera. Droplets (20 µL) were applied to a waveguide surface, allowed to settle for 10-20 s, and photographed through a 50 mm camera lens. An image analysis software package (IPLab, Signal Analytics, Vienna, VA) was used to determine the contact angle from the digital image. The angles on hydrophilic and hydrophobic waveguide surfaces were 10 ( 6.1° and 88 ( 1.2°, respectively. Linear Dichroism Measurements. The waveguide was sealed in a liquid flow cell with an integral SF6 coupling prism (Karl Lambrecht) and a rear window that allowed the waveguide to be viewed through the substrate.28 The flow cell was mounted on a computer-controlled rotary stage (New England Affiliated Technologies), which allowed the flow cell to be rotated relative to the stationary 514.5 nm beam from a Coherent Innova 70 argon ion laser. Light was launched into the highest order mode of the waveguide that was supported in both TE and TM polarizations (either m ) 1 or m ) 2). The incident power was typically 2-3 mW. Input polarization was selected using a combination of Glan laser polarizers and a half-wave Fresnel rhomb. The guided mode “streak” visible in the waveguide was photographed through the flow cell window using a thermoelectrically cooled, slow scan CCD camera (Princeton Instruments) oriented normal to the waveguide plane and fitted with a 50 mm camera lens. At least three photographs were recorded in each polarization (TE and TM) for subsequent averaging. Attenuation curves were generated by plotting the logarithm of the vertically averaged pixel intensity in the image of the streak against horizontal propagation distance. Curves were fit by least squares regression to log[I(x)] ) Rx + C, where I(x) is the average pixel intensity as a function of distance D, x is the propagation distance in centimeters, R is the loss coefficient in cm-1, and C is a constant. Experiments were initiated by injecting a buffer solution into the flow cell and allowing it to stand for an equilibration period of 30 min. Images of the guided mode streak were then acquired in both TE and TM polarizations. The protein solution was then injected followed by an adsorption period (usually 30 min), after which images of the guided mode streak were again acquired in both polarizations. The slopes of the attenuation curves generated from images with only buffer in the flow cell were R0,TE and R0,TM, the respective TE and TM intrinsic loss coefficients. With protein present in the flow cell, the slopes of the attenuation curves were RT,TE and RT,TM, the total loss coefficients due to both the intrinsic propagation loss and absorbance by bulk dissolved and adsorbed protein. The loss coefficients due solely to bulk dissolved protein, Rb,TE and Rb,TM, were calculated from knowledge of the physical parameters of the waveguide, as described in refs 18 and 22. The loss coefficients due solely to adsorbed protein, (27) Margoliash, E.; Frohwirt, N. Biochem. J. 1959, 71, 570-572. (28) Saavedra, S. S.; Reichert, W. M. Appl. Spectrosc. 1990, 44, 14201423.
Langmuir, Vol. 12, No. 16, 1996 4027 Rf,TE and Rf,TM, were then recovered by difference (i.e., Rf,TM ) RT,TM - R0,TM - Rb,TM). Since Rf is proportional to Af, the thin film absorbance, the dichroic ratio F is given by the ratio of the thin film loss coefficients (F ) [Rf,TM/Rf,TE]). Loss coefficients were measured as a function of the waveguide surface chemistry, the bulk protein concentration, the duration of the adsorption period, and the residence time of the protein on the surface. Protein Surface Coverages. Surface coverages of adsorbed Mb were determined using the pyridine hemochrome assay described by Hong et al.17b To increase the surface area relative to planar waveguides, hydrophilic and hydrophobic glass beads were used as substrates. Mb was incubated with glass beads for 30 min using the same bulk protein concentration and ratio of solution volume to surface area that was used in the corresponding molecular orientation experiments. Following incubation and subsequent rinsing with phosphate buffer, the heme groups were dissociated from the adsorbed Mb film using a 1:1: 3.5 (v/v) mixture of pyridine, 0.5 M NaOH, and phosphate buffer. Absorbance of the pyridine hemochrome solution was measured within 10 min in a 1 cm path length cell and converted to Mb concentration using a molar absorptivity of 61 360 M-1 cm-1 at 400 nm (our measurement) for Mb dissolved in the hemochrome assay solvent. Surface coverages were calculated by ratioing the amount of desorbed heme to the aggregate surface area of the glass beads, under the following assumptions: (i) The stoichiometric ratio of desorbed heme to adsorbed protein was 1:1 (i.e., the desorption of heme in the pyridine hemochrome solution was quantitative). (ii) The post-adsorption rinse in buffer did not remove heme from the glass beads.29 (iii) The surface of the glass beads was molecularly smooth. Cyt c surface coverages were determined in a similar manner, except that since the heme in cyt c is covalently bound to the protein, the procedure to effect desorption was different. Following a 30 min adsorption period (under conditions equivalent to those used for planar substrates) and subsequent rinsing with phosphate buffer, 10 ml of a 2% (v/v) PCC-54 surfactant solution (Pierce) was added to the vial containing the beads. The beads were then sonicated in this solution for approximately 1 h. After the beads were separated from the solution, absorbance was measured at 406 nm. The concentration of desorbed cyt c in the solution, and hence the protein surface coverage, was calculated using a molar absorptivity of 122 000 M-1 cm-1 at 406 nm (our measurement) for cyt c dissolved in 2% PCC-54. Analogous to the Mb measurements, the following assumptions were made: (i) The glass beads were molecularly smooth. (ii) The postadsorption rinse in buffer did not remove adsorbed protein. (iii) The desorption of adsorbed cyt c in 2% PCC-54 was quantitative. The latter assumption was assessed by measuring the fluorescence emission intensity from zinc-substituted cyt c30 adsorbed to hydrophilic glass beads. After sonication in 2% PCC-54, adsorbed cyt c could not be detected above background emission when the beads were examined under an epifluorescence microscope, from which we estimate that the desorption efficiency was at least 90%. Fluorescence Anisotropy Measurements on Zinc-Substituted Myoglobin. Steady-state emission anisotropy measurements of Mb films adsorbed to hydrophilic and hydrophobic fused silica slides were also performed. Since the Fe atom in native Mb strongly quenches the heme fluorescence, zincsubstituted Mb (ZnMb) was used for these measurements. Apomyoglobin (apoMb) was prepared as previously described.31a The viability of the preparation was assessed by measuring the extent of binding to bovine hemin. Hemin (0.4 mM) was added to 0.2 mM apoMb (280 ) 15 800 M-1 cm-1) at various hemin/ apoMb concentration ratios to give a final volume of 6 mL in the (29) This assumption is probably valid even if the protein adsorption process caused dissociation of the heme from Mb. Since heme is nearly insoluble in pH 7 buffer, it would likely remain adsorbed at the solidliquid interface even if it was not associated with the adsorbed protein in a “native” fashion. (30) Zinc-substituted cyt c was prepared by removing the native Fe3+ and replacing it with Zn2+, as described in Robinson, A. B.; Kamen, M. D. In Structure and Function of Cytochromes; Okunuki, K., Kamen, M. D., Sekuzu, I., Eds.; University Park Press: Baltimore, MD, 1968; pp 383-387. (31) (a) Edmiston, P. L.; Wambolt, C. L.; Smith, M. K.; Saavedra, S. S. J. Colloid Interface Sci. 1994, 163, 395-406. (b) Hamachi, I.; Fujimura, H.; Kunitake, T. Chem. Lett. 1993, 1551-1553.
4028 Langmuir, Vol. 12, No. 16, 1996
Figure 2. Loss coefficients measured using TE polarized light (Rf,TE) as a function of time while Mb (squares) and cyt c (circles) solutions were incubated with hydrophilic waveguide surfaces. The bulk protein concentrations were 35 µM in both cases. Data were corrected for absorbance of bulk dissolved protein and intrinsic waveguide propagation losses and therefore represent absorbance due only to adsorbed protein. phosphate buffer. The extent of the reaction was monitored by measuring the increase in absorbance at 408 nm. The results yielded a binding ratio of 1:1, consistent with full activity of the apoMb preparation. ZnMb was prepared by adding a 2-fold molar excess of zinc protoporphyrin IX (ZnPP) to the purified apoMb solution, followed by dialysis against phosphate buffer to remove excess ZnPP. The absorbance spectrum of the ZnMb product matched the spectrum published in ref 31b. Fluorescence measurements on adsorbed ZnMb films were performed in a TIRF geometry using procedures and instrumentation described in ref 32. Briefly, samples were excited using the 582 nm output of a Coherent 599 dye laser, in either TE or TM polarization. Fluorescence emission was collected normal to the substrate by a microscope objective, directed through a 635 nm bandpass filter (635DF55, Omega Optical), and detected with a liquid nitrogen cooled CCD camera. Adsorbed ZnMb films of relatively low surface coverage were prepared by incubating surfaces with a solution of 5 µM ZnMb. Higher surface coverage films were prepared by incubating surfaces with a solution that contained a 1:10 molar ratio of ZnMb:Mb with a total protein concentration of 35 µM. Diluting the ZnMb with Mb prevented energy transfer between adsorbed ZnMb molecules, which would depolarize emission and invalidate the results of the experiments. Surfaces were incubated with protein solutions for 30 min and rinsed with 50 mM phosphate buffer prior to measurement. The intrinsic anisotropy (ro) of ZnMb was measured at room temperature using a Spex Fluorolog fluorometer at excitation and emission wavelengths of 582 and 646 nm, respectively. The polarization bias of the instrument was measured at the same wavelengths using a dilute solution of rhodamine B in ethanol. At a concentration of 4 µM in 97% glycerol, ro of ZnMb was 0.118, from which a mean angle of 43° between the absorption and emission dipoles is computed.33 The value of 0.118 in a right angle geometry corresponds to a ro of -0.0742 in a total internal reflection geometry.34
Results and Discussion Adsorption Kinetics and Surface Coverages. Initial experiments were conducted to determine the incubation time required to reach a steady-state condition in the protein adsorption process. This was performed by injecting a protein solution into the waveguide flow cell and measuring the thin film loss coefficient (Rf) as a function of incubation time. In Figure 2 are plotted Rf,TE values for Mb and cyt c adsorption to hydrophilic (32) (a) Phimphivong, S.; Ko¨lchens, S.; Edmiston, P. L.; Saavedra, S. S. Anal. Chim. Acta. 1995, 307, 403-417. (b) Edmiston, P. L.; Wood, L. L.; Lee, J. E.; Saavedra, S. S. J. Phys. Chem. 1996, 100, 775-784. (33) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983; Chapter 5. (34) Excitation is polarized along the y or z axes, and emission is detected along the z axis (see Figure 1). Edmiston, P. L.; Lee, J. E.; Cheng, S.-S.; Saavedra, S. S. Manuscript in preparation.
Lee and Saavedra
waveguide surfaces from solutions containing 30-35 µM protein. In both cases, a plateau was observed within a 30 min incubation period; thereafter, relatively slight increases in adsorbed amount were observed up to 360 min. Equivalent experiments were performed on hydrophobic surfaces and showed that adsorption also reached a nearly steady state condition within 30 min of incubation (data not shown). It was also necessary to determine if the mean orientation of the heme changed after the amount of adsorbed protein reached a steady state. This was addressed by measuring the dichroic ratio, F, as a function of time. For incubation periods ranging from 30 to g400 min, there were no statistically significant changes in F for Mb and cyt c adsorbed to either hydrophilic or hydrophobic waveguides from solutions having a bulk protein concentration of 35 µM (data not shown). Thus the mean orientation of the protein-bound heme groups did not change over this period (note that F is related to the mean tilt angle, θµ, by eq 3). Since both the adsorbed amount and the mean orientation of both proteins on both surfaces were essentially constant after 30 min, a 30 min incubation time was selected for all further experiments. The IOW-ATR measurements described in the preceding paragraphs were performed without flushing the bulk dissolved protein solution from the flow cell. To investigate possible effects of “loosely” adsorbed protein, loss coefficients were also measured after flushing the cell with buffer. For Mb adsorbed for 30 min to hydrophilic glass at a bulk concentration of 35 µM, the loss coefficients decreased about 17% upon flushing but the dichroic ratio was unchanged. On hydrophobic glass, loss coefficients decreased slightly (6-11%) upon flushing the flow cell, but again, the dichroic ratio was constant. Thus, since removal of “loosely” adsorbed protein had no effect on the dichroic ratio, measurements of mean molecular orientation in the presence of bulk dissolved protein (i.e., without flushing the flow cell) were also unaffected. The effect of flushing bulk protein on the dichroic ratio of adsorbed of cyt c was not examined. Step adsorption isotherms were measured for Mb on each surface. A series of protein solutions of progressively increasing concentration was injected into the waveguide flow cell, and loss coefficients due to adsorbed Mb were measured after a 30 min incubation time for each concentration. The results are plotted in Figure 3. On the hydrophilic waveguide, the plateau region of the isotherm was reached at a bulk concentration of approximately 30-35 µM. On the hydrophobic waveguide surface, the plateau in the isotherm was reached at a lower Mb concentration, approximately 15 µM. The maximum loss coefficient on the hydrophobic surface (0.17 cm-1) was approximately 2-fold greater than that on the hydrophilic surface (0.08 cm-1). To facilitate a more quantitative comparison between the hydrophilic and hydrophobic surfaces, the Mb isotherm data were fit to the Langmuir adsorption model
θ)
R ) Rmax
KaCb 1 + KaCb
(4)
using a nonlinear curve fitting algorithm. In eq 4, θ is the ratio of occupied adsorption sites to total sites, Ka is the adsorption equilibrium constant, Cb is the protein molarity approximating its activity in solution, and R/Rmax is the loss coefficient normalized to the highest measured loss coefficient in each data set. The estimates obtained for Ka on the hydrophilic and hydrophobic surfaces were (8.2 ( 0.80) × 104 M-1 and (4.1 ( 0.79) × 105 M-1, respectively. These estimated values
Molecular Orientation in Heme Protein Films
Langmuir, Vol. 12, No. 16, 1996 4029 Table 1. Mean Heme Tilt Angle Data for Mb and Cyt c Adsorbed at High Bulk Protein Concentration
Figure 3. Mb adsorption isotherms on hydrophilic (A) and hydrophobic (B) waveguides. Loss coefficients using TE polarized light (Rf,TE) were measured as a function of bulk Mb concentration after a 30 min incubation period and are plotted as circles. The solid lines are the best fit of the data to the Langmuir adsorption model (see text).
were then used in eq 4 to calculate the solid lines plotted in Figure 3, representing the best fit of the experimental data to the Langmuir model. Despite the apparent goodness of fit, it is unlikely that Mb adsorption to these surfaces obeys ideal Langmuir behavior. Specifically, the inherent assumptions that only one type of surface site is present, that lateral interactions between adsorbed molecules are absent, and that the adsorption process is reversible are probably not valid. The calculated Ka values should therefore be treated as empirical parameters that enable the adsorption isotherms to be compared. The 5-fold difference in Ka values indicates that Mb binds more strongly to the hydrophobic surface. This result is consistent with a large body of evidence showing that the extent of protein adsorption to a surface can be correlated with the hydrophobicity of the surface.1,2 The difference in loss coefficients in the plateau regions of each isotherm suggests that the Mb surface coverage is greater on the hydrophobic surface than on the hydrophilic surface. However, this conclusion assumes that the molar absorptivities of the protein on the two surfaces are equal, which may not be true if differences in orientation and/or conformation exist. To resolve this issue, Mb surface coverages were determined independently using a pyridine hemochrome assay.17b The measurements were performed using a bulk protein concentration of 35 µM and an incubation time of 30 min. The surface coverage was 1.3 × 10-11 mol/cm2 on the hydrophilic glass beads and 2.1 × 10-11 mol/cm2 on the hydrophobic glass beads. From the crystallographic
protein
IOW surface
bulk protein concentration (µM)
tilt angle (deg)
Mb Mb cyt c cyt c
hydrophilic hydrophobic hydrophilic hydrophobic
35 30-35 35 35
45 ( 3 (n ) 3) 43 ( 5 (n ) 5) 17 ( 2 (n ) 2) 48 ( 3 (n ) 3)
dimensions of Mb (25 × 35 × 45 Å),35 a surface coverage ranging from 1.0 × 10-11 to 1.9 × 10-11 mol/cm2, depending on orientation, is equivalent to one monolayer, assuming that no “spreading” of the protein occurs due to adsorptioninduced conformational changes. Therefore, in the plateau region of the two isotherms plotted in Figure 3, the Mb surface coverage is slightly less than one monolayer on the hydrophilic surface and approximately 1.5 monolayers on the hydrophobic surface (using the geometrically random value of 1.5 × 10-11 mol/cm2 as the monolayer surface coverage). Adsorption isotherms for cyt c were not measured. Instead, a surfactant desorption assay was used to determine surface coverages under conditions identical to those used for Mb surface coverage measurements (bulk protein concentration of 35 µM; 30 min incubation time). On hydrophilic glass, the surface coverage was 2.9 × 10-11 mol/cm2, whereas on hydrophobic glass it was 1.7 × 10-11 mol/cm2. From the crystallographic dimensions of cyt c (25 × 25 × 37 Å)35 these data correspond to 1.3 monolayers and 0.8 monolayers respectively (using the geometrically random value of 2.2 × 10-11 mol/cm2 as the monolayer surface coverage). The surface coverage determinations for Mb and cyt c should be considered approximate. The standard error of the measurement is about 25%. A potentially greater source of uncertainty is the numerous assumptions involved. Specifically, the mean surface area occupied by an adsorbed protein molecule, the desorption efficiency of the heme from adsorbed Mb, and the surface roughness of the beads are unknown. Furthermore, to correlate measurements of proteins adsorbed on SiOxNy waveguides with surface coverage measurements on soda lime glass beads, one must assume that the adsorption behavior of Mb and cyt c on the two glass types is similar and that the surface roughness of the two materials is also similar. Despite these uncertainties, the surface coverage measurements were useful for designing and interpreting the results of the molecular orientation experiments on Mb and cyt c films. Molecular Orientation at High Surface Coverage. Molecular orientation was initially measured in adsorbed heme films under conditions that produced approximately monolayer surface coverage. In these experiments, a 3035 µM protein solution was injected into the waveguide flow cell and incubated for 30 min, and the dichroic ratio was measured. Mean heme tilt angles were calculated using eq 3. Results are summarized in Table 1 for both Mb and cyt c. For Mb, the tilt angles were 45 ( 3° and 43 ( 5° on the hydrophilic and hydrophobic waveguide surfaces, respectively. Thus in both cases, the mean orientation of the heme plane was nearly equidistant between the surface normal and the waveguide plane. However, these results do not imply that there is no preferential ordering of the heme groups in the adsorbed Mb films. Both angles are statistically distinct from 35°, which is the mean angle that would be measured for an isotropic distribution of heme orientations. Thus the (35) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117-6123.
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adsorption process appears to be geometrically anisotropic on both surfaces. On the other hand, the lack of a statistical difference between the tilt angles on the two surfaces is somewhat surprising. The possibility that these measurements are systematically biased must therefore be considered. This possibility is diminished by considering the data for cyt c. On hydrophilic waveguide surfaces, the mean tilt angle was 17 ( 2° from the surface normal, whereas on hydrophobic waveguide surfaces, the mean tilt angle was 48 ( 3°.36 Considering the Mb and cyt c data in tandem, it is apparent that (i) the mean heme orientation was nonrandom for both proteins adsorbed to each of the two surfaces (ii) the molecular orientation distribution in an adsorbed cyt c film can be manipulated by altering the wettability of the adsorbent surface and (iii) the statistically equivalent mean heme orientations in Mb films adsorbed to the two surfaces is real (i.e., is not an experimental artifact). The discussion of a physical basis for these observations must be prefaced by noting two limitations inherent in the experiment. For a protein of known crystal structure such as Mb, the geometric relationship between the heme plane and the three-dimensional protein structure is known. However, it cannot be assumed that this relationship is maintained upon adsorption to a solid surface. An adsorption-induced conformational change may alter the spatial relationship between the heme plane and the structure of the polypeptide matrix that surrounds it. Second, since the heme dipole transitions are rotationally degenerate about an axis normal to the heme plane, a given tilt angle measurement is consistent with numerous possible mean orientations of a heme-containing protein molecule adsorbed to a substrate surface. Thus even if the protein conformation is not altered upon adsorption, the mean orientation of the protein molecules cannot be determined using IOW-ATR linear dichroism measurements. Consequently, there are at least two possible explanations for the difference in tilt angle in cyt c films on hydrophilic and hydrophobic surfaces: (i) Cyt c is adsorbed in different geometric orientations. This implies that there are at least two structurally distinct contact regions on the surface of the protein that interact with the two chemically distinct adsorbent surfaces. The presence of distinct contact regions can be inferred from the asymmetric distribution of charged groups on the surface of cyt c at neutral pH, which has been well documented.37 Electrostatic interactions may therefore be a major factor in determining molecular orientation in cyt c films on these surfaces. More specifically, derivatizing the glass surface with DDS probably decreases the electrostatic attraction of cyt c to bare glass, which is negatively charged at neutral pH. (ii) Cyt c interacts with both adsorbent surfaces via the same contact region (or regions). In this case, the observed difference in mean tilt angle can be attributed to a substantial difference in the structure of the adsorbed protein in the vicinity of the bound heme. Different protein structures on the two surfaces would be likely caused by differences in the extent of conformational changes induced by adsorption. At this time, we cannot distinguish between differences in mean molecular orientation, conformation, or a combination of the two. In contrast, the lack of a difference in the tilt angles in the Mb films adsorbed to the two surfaces may be associated with a lack of electrostatic interactions. Since (36) These data are slightly revised from the preliminary measurements that were reported in an earlier publication.18 (37) Koppenol, W. H.; Margoliash, E. J. Biol. Chem. 1982, 257, 44264437.
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the experiments were performed at pH 7.2 and the pI of Mb is 7.0,35 the net charge on the protein molecule was nearly zero. Furthermore, from reported differences in the efficiency of forming multilayer films of Mb and cyt c by electrostatic adsorption to oppositely charged polyions,35,38 it appears that the distribution of acidic and basic amino acid residues is more homogeneous over the surface of Mb than over that of cyt c. This was confirmed by qualitatively comparing the crystal structures of the two proteins using the program Insight II (Biosym Technologies, San Diego, CA). At neutral pH, the amino acid distribution on the surface of cyt c is markedly asymmetric, as described by Koppenol and Margoliash.37 A cluster of cationic residues is located on the protein face where the heme edge is exposed to solvent. Rotating the protein about 150° from the exposed heme edge reveals a face that contains a relatively high density of anionic residues. In contrast, the distribution of charged residues over the surface of Mb is relatively uniform at neutral pH. The absence of charged faces suggests that, in contrast to cyt c, a strong electrostatic attraction between Mb and bare glass was lacking, and thus other types of forces probably dominate the adsorption process. Furthermore, this uniformity suggests that differences in adsorbent surface chemistry will exert less influence on molecular orientation in adsorbed Mb films in comparison to adsorbed cyt c films. Molecular Orientation at Low Surface Coverage. Heme orientation in adsorbed Mb films was also examined as a function of surface coverage, which was adjusted by varying the bulk protein concentration. A series of Mb solutions of progressively increasing concentration was injected into the waveguide flow cell, and dichroic ratios were measured after a 30 min incubation time for each concentration. The mean heme tilt angle is plotted as a function of bulk concentration in Figure 4 for films adsorbed on hydrophilic (two data sets) and hydrophobic (one data set) waveguides. At bulk concentrations of e5 µM, the tilt angle on the hydrophilic surface was about 70° (Figure 4a). As the bulk concentration was increased, the tilt angle in the film decreased sharply to a final value of approximately 45° at bulk concentrations g30 µM. Thus the final value is equivalent to the results obtained when hydrophilic waveguides were incubated directly with 3035 µM Mb solutions (Table 1). A substantial difference in mean heme orientation was also observed when Mb was adsorbed to a hydrophobic waveguide at low and high bulk concentrations (Figure 4b). However, in this case the tilt angle in the film was approximately 20° at the lowest concentration and increased sharply to a final value of approximately 45° at bulk Mb concentrations g20 µM. Again, this final value is equivalent to the results obtained when hydrophobic waveguides were incubated directly with 30-35 µM Mb solutions (Table 1). The existence of a substantial difference in mean molecular orientation at low and high bulk Mb concentrations was independently assessed by measuring the steady-state fluorescence anisotropy of adsorbed ZnMb. (The emission anisotropy of a thin film of fluorescent dipoles is related to their macroscopic orientational order.32b On the hydrophilic surface, the anisotropy values were 0.0733 ( 0.0050 and -0.1355 ( 0.0245 at bulk concentrations of 5 and 35 µM, respectively. The corresponding anisotropy values on the hydrophobic surface were -0.0609 ( 0.0466 and -0.1533 ( 0.0161 (n ) 3), respectively. These differences are consistent with the trends in the data plotted in Figure 4 and confirm the (38) Personal communication, Dr. Yuri Lvov, Supermolecules Project, Kurume, Japan.
Molecular Orientation in Heme Protein Films
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Lateral interactions between adsorbed molecules are a plausible explanation for the difference in tilt angles between Mb films of low and high surface coverage shown in Figure 4. At low surface coverage, lateral interactions between neighboring molecules should be few or absent. The orientation of an adsorbed Mb molecule should therefore be largely dictated by the nature of the interactions between the surfaces of the protein and the adsorbent. At higher surfaces coverages, the molecules are more closely packed, and the orientation should be a function of both the protein-adsorbent interactions and the protein-protein interactions. Thus in this scenario, adsorption adjacent to an occupied site (or sites) produces a different molecular orientation (or conformation) than adsorption at a site where none of the adjacent sites are occupied. The frequency of collisions between protein molecules and the adsorbent surface may also play a role. At low bulk concentrations, adsorbed molecules may have sufficient time to undergo conformational changes, leading to a larger occupied surface area per molecule. At high bulk concentrations, the presence of protein molecules in adjacent sites may restrict this conformational “spreading” and produce a different molecular orientation. This scenario was proposed by Morrisey39 and subsequently others40 to explain differences in adsorbed protein surface coverage as a function of bulk concentration. Conclusions
Figure 4. Mean heme tilt angle in Mb films adsorbed on hydrophilic (A) and hydrophobic (B) waveguides, measured as a function of bulk protein concentration after a 30 min incubation period. In part A, two independent sets of data are plotted as circles and squares.
dependence of adsorbed molecular orientation on bulk protein concentration. These observations prompt the question: At what surface coverage is the mean orientation of Mb substantially different from that at monolayer coverage? Due to sensitivity limitations of the pyridine hemochrome assay, surface coverages were not measured below the plateau region of the isotherms in Figure 3 (where high bulk concentrations produced approximately monolayer surface coverages). However, surface coverages at low bulk concentrations can be estimated by ratioing the loss coefficients to those measured at high bulk concentrations. The loss coefficients in the plateau regions of the isotherms on hydrophilic and hydrophobic glass were 0.080 and 0.17 cm-1, respectively. Assuming these values correspond to a one monolayer of protein, then at bulk Mb concentrations e5 µM, the surface coverage was about 0.2 monolayer on both the hydrophilic and hydrophobic waveguide surfaces. Thus at about 20% of a monolayer, the mean heme tilt angle was substantially greater (hydrophilic surface) or less (hydrophobic surface) than the tilt angle at 50-100% of monolayer surface coverage. Our direct observation of orientation dependence on surface coverage is not without precedent. In a very recent paper,21 Macdonald and Smith reported using surfaceenhanced resonance Raman spectroscopy to examine cyt c adsorbed to citrate-coated silver. They observed a difference of about 5° in the mean heme tilt angle between low and high surface coverage films. An influence of bulk solution pH on protein molecular orientation was also noted.
The results presented here illustrate three points: (i) the mean orientation of the heme plane in “nonspecifically” adsorbed films of both Mb and cyt c are anisotropic rather than random; (ii) molecular orientation in a protein film can be manipulated by changing the chemical properties of the adsorbent surface (for cyt c); (iii) molecular orientation in a protein film can be influenced by surface coverage (for Mb). These behaviors have been hypothesized by many others1,2 and proposed for example to explain (i) the effect of pH on the specific activity of antibodies adsorbed to DDS-treated glass,4 (ii) temporal changes in the activity of ribonuclease A adsorbed to mica,5 and (iii) differences in the binding affinity of anti-Mb antibodies to dissolved and adsorbed Mb.3 However, the molecular orientation data reported here provide direct evidence for these phenomena, which to date have been difficult to obtain despite the voluminous body of work published on protein adsorption behavior. These results also suggest some future directions. (1) The occurrence of protein conformational changes that may be induced upon adsorption should be addressed in tandem with measurements of molecular orientation. Absorbance spectroscopy can be used to address this issue, since the visible band shapes and intensities of a heme protein are sensitive to ligand binding, the oxidation state of the iron, and the conformation of the polypeptide in the vicinity of the porphyrin.41 Mendes et al.42 recently described a broad-band incoupling device that allows measurement of visible ATR spectra of a hydrated heme protein monolayer on a single mode planar IOW. This approach should assist in future efforts to investigate structural and functional characteristics of heme protein films on dielectric surfaces. (39) Morrissey, B. W. Ann. N.Y. Acad. Sci. 1977, 28, 50-64. (40) Jo¨nsson, U.; Lundstro¨m, I.; Ro¨nnberg, I. J. Colloid Interface Sci. 1987, 117, 127-138. (41) See, for example: Smith, D. W.; Williams, R. J. P. Struct. Bonding (Berlin) 1970, 7, 1-45. (42) Mendes, S.; Li, L.; Burke, J.; Lee, J. E.; Dunphy, D. R.; Saavedra, S. S. Langmuir 1996, 12, 3374.
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(2) Linear dichroism measurements provide only the mean tilt angle of the heme plane. No information about the distribution of tilt angles about the mean is available. The distribution can be recovered by measuring both fluorescence emission anisotropy and linear dichroism on the same (or equivalent) film(s).32b Employing this approach should make it possible to distinguish between molecular assemblies that exhibit equivalent mean tilt angles but differ in the distribution of tilt angles. Investigations of orientation distributions in heme protein
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films will be reported from our laboratory in the near future. Acknowledgment. We thank Paul Edmiston for assistance in performing the fluorescence anisotropy measurements. This work was supported by the National Science Foundation (Grant CHE-9403896) and the National Institutes of Health (Grants R03 RR08097 and R29 GM50299). LA960253Z
