Spontaneous Reconfiguration of Adsorbed Lysozyme Layers

Nov 15, 1996 - with a pH-Sensitive Fluorophore. Julie L. Robeson† and Robert D. Tilton*. Department of Chemical Engineering and Colloids, Polymers a...
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6104

Langmuir 1996, 12, 6104-6113

Spontaneous Reconfiguration of Adsorbed Lysozyme Layers Observed by Total Internal Reflection Fluorescence with a pH-Sensitive Fluorophore Julie L. Robeson† and Robert D. Tilton* Department of Chemical Engineering and Colloids, Polymers and Surfaces Program, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890 Received May 14, 1996X By conjugating proteins with a common pH-sensitive fluorescent label, fluorescein isothiocyanate (FITC), and controlling the ionic strength, we provide a means to decrease the characteristic length scale of the total internal reflection fluorescence (TIRF) technique by two orders of magnitude. The usual characteristic length scale for TIRF is an optical length, specifically the evanescent wave penetration depth (on the order of 100 nm). In our experiments the penetration depth is replaced by the Debye screening length as the characteristic length scale. This is readily controlled to match the dimensions of an adsorbed protein layer (on the order of 1 nm). We achieve this length scale reduction by coupling the well-known pH-sensitivity of fluorescence emission by FITC-labeled proteins with the variation of electrostatic potential near a negatively charged surface. Using this fine-resolution TIRF capability in combination with scanning angle reflectometry, we find that lateral repulsions induce a dramatic reconfiguration of adsorbed lysozyme layers on negatively charged silica surfaces. This occurs as the surface concentration approaches the jamming limit for random sequential adsorption. The reconfiguration evidently optimizes electrostatic interactions in the adsorbed layer and decreases the effective excluded area per lysozyme. The decrease in effective excluded area allows adsorption to continue beyond the jamming limit to ultimately attain a hexagonal close packed monolayer of horizontally oriented lysozyme molecules. The adsorption kinetics switch abruptly from being transport-limited to surface-limited after the reconfiguration.

Introduction The biochemical and physical properties of adsorbed protein layers influence technologies as diverse as biocompatible materials, affinity chromatography, solid phase immunoassays, immobilized enzyme reactors, biosensors, targeted drug delivery vehicles, enzyme-enhanced laundry detergents, and supported cell culture. In any situation involving proteins at interfaces, it is the structure of the adsorbed layer that governs biochemical function, whether that be enzymatic catalysis or specific receptorligand binding. Here we define adsorbed layer structure as the distribution of protein conformational states, orientations relative to the surface, and any supramolecular assembly. There is evidence that adsorption may alter the conformation of some proteins, while others resist significant conformational change.1-5 The “hard protein” versus “soft protein” categorization scheme has proven useful in relating properties such as thermodynamic stability and adiabatic compressibility to a protein’s tendency to experience major surface-induced conformational change.1 “Major conformational change” is meant in the sense that configurational entropy changes contribute significantly to the free energy of adsorption. Such conformational changes would certainly affect the biochemistry of the adsorbed layer. Nevertheless, since many * To whom correspondence should be addressed. E-mail: tilton@ andrew.cmu.edu. Fax: (412) 268-7139. Telephone: (412) 268-1159. † Current address: NOVA Chemicals Inc., 400 Frankfort Road, Monaca, PA 15061. X Abstract published in Advance ACS Abstracts, November 15, 1996. (1) Arai, T.; Norde, W. Colloids Surf. 1990, 51, 1. (2) Kondo, A.; Higashitani, K. J. Colloid Interface Sci. 1992, 150, 344. (3) Elwing, H.; Nilsson, B.; Svensson, S.; Askendahl, A.; Nilsson, U. R.; Lundstro¨m, I. J. Colloid Interface Sci. 1988, 125, 139. (4) Darst, S. A.; Robertson, C. R.; Berzofsky, J. A. Biophys. J. 1988, 53, 533. (5) Blomberg, E.; Claesson, P. M.; Tilton, R. D. J. Colloid Interface Sci. 1994, 166, 427.

S0743-7463(96)00476-3 CCC: $12.00

proteins resist major conformational change upon adsorption, it is important to bear in mind that the biochemical function of an adsorbed protein can be diminished even in the absence of surface-induced conformational change. One example is if the protein orientation restricts access to the active site. The active site could be obstructed by the surface itself or by closely packed neighboring proteins. The effect of orientation on biochemical function has been demonstrated for the enzyme ribonuclease A adsorbed on mica.6 There are few techniques capable of resolving orientational changes in adsorbed protein layers. In this paper we present experimental means to decrease the characteristic length scale of the total internal reflection fluorescence (TIRF) technique7-11 by two orders of magnitude, thereby enabling it to resolve microscopic structural changes that occur within the adsorbed layer.12 We use this fine-resolution TIRF technique in combination with an independent scanning angle reflectometry technique13-15 to study the spontaneous reconfiguration of adsorbed protein layers caused by lateral repulsions between neighboring proteins. Specifically, we observe how crowding affects the average orientation in lysozyme layers adsorbed on negatively charged silica surfaces. This spontaneous reorientation within the adsorbed layer in (6) Lee, C. S.; Belfort, G. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 8392. (7) Lok, B. K.; Cheng., Y. L.; Robertson, C. R. J. Colloid Interface Sci. 1983, 91, 87. (8) Lok, B. K.; Cheng., Y. L.; Robertson, C. R. J. Colloid Interface Sci. 1983, 91, 104. (9) Axelrod, D. Annu. Rev. Biophys. Bioeng. 1984, 13, 247. (10) Hlady, V.; Reinecke, D. R.; Andrade, J. D. J. Colloid Interface Sci. 1986, 111, 555. (11) Rondelez, F.; Ausserre, D.; Hervet, H. Annu. Rev. Phys. Chem. 1987, 38, 317. (12) Robeson, J. L. Application of Total Internal Reflection Fluorescence to Probe Surface Diffusion and Orientation of Adsorbed Proteins. Ph.D. Dissertation, Carnegie Mellon University, 1995. (13) Mandenius, C. F.; Mosbach, K.; Welin, S.; Lundstro¨m, I. Anal. Biochem. 1986, 157, 282. (14) Schaaf, P.; De´jardin, P.; Schmitt, A. Langmuir 1987, 3, 1131. (15) Furst, E. M.; Pagac, E. S.; Tilton, R. D. Ind. Eng. Chem. Res. 1996, 35, 1566.

© 1996 American Chemical Society

Reconfiguration of Adsorbed Lysozyme Layers

turn controls the adsorption kinetics at high coverage. Since they alter the adsorption mechanism, it is important to consider such spontaneous effects in adsorption kinetic models. Evidence for a coverage-dependent lysozyme orientation has previously been obtained with mica surfaces in the surface force apparatus.16 Background TIRF owes its surface sensitivity to the evanescent wave produced at the point of total internal reflection at the solid-liquid interface. Since the intensity of the evanescent wave decays exponentially with distance normal to the surface, TIRF is ordinarily capable of distinguishing changes in fluorophore proximity to a surface over length scales dictated by the evanescent wave penetration depth. This is on the order of hundreds of nanometers in typical experimental applications and is much larger than the thickness of an adsorbed protein layer. TIRF has proven to be most useful in measuring protein and polymer adsorption, desorption, and competitive exchange kinetics, but details on much finer length scales are difficult to observe. This is still the case even if one systematically varies the angle of incidence to sample a range of penetration depths.11 Here we decrease the characteristic TIRF length scale from the evanescent wave penetration depth to the Debye screening length, κ-1. By controlling the solution ionic strength, the Debye length can be adjusted to match the thickness of an adsorbed protein layer (on the order of 1-10 nm). We achieve this length scale reduction by coupling the well-known pH sensitivity of fluorescence emission by fluorescein isothiocyanate (FITC)-labeled proteins17-19 with the variation of electrostatic potential near a negatively charged surface. Given a constant excitation wavelength, the fluorescence emission intensity of fluorescein-labeled proteins drops sharply as the pH is decreased between approximately pH 8 and 6. This is caused not by a decreased quantum yield but instead by a shift in the excitation spectrum to lower wavelengths as the molecule shifts among its prototropic forms.17 The basis of our current TIRF experiments is that the protonation state of the proteinbound fluorescein label is controlled by the local electrostatic potential, and this in turn controls its fluorescence emission intensity. Thus, proximity to a negatively charged surface favors protonation and diminishes the fluorescence emission intensity. Under appropriately controlled conditions, any relocation of the fluorophore within the electrostatic double layer adjacent to the surface will change the fluorescence intensity. The extent of that fluorescence response depends on the relative magnitude of the change in fluorophore position compared to the Debye length and is thus governed by the ionic strength. Fluorescein’s sensitivity to local electrostatic potential has been exploited previously in a solution phase assay of antigen-antibody binding by Lee et al.,20 and Rebar and Santore21 very recently published a similar TIRF application of this principle to probe the electrostatic potential within adsorbed fluorescein-labeled poly(ethylene oxide) layers. TIRF also has been used to examine the orientation of adsorbed cytochrome c.22 Rather than potential-sensitive emission, that measurement was based (16) Blomberg, E.; Claesson, P. M.; Fro¨berg, J. C.; Tilton, R. D. Langmuir 1994, 10, 2325. (17) Chen, R. Arch. Biochem. Biophys. 1969, 133, 263. (18) Klugerman, M. R. J. Immunol. 1966, 95, 1165. (19) Emmart, E. W. Arch. Biochem. Biophys. 1958, 73, 1. (20) Lee, C. S.; Huang, P. Y.; Ayres, D. M. Anal. Chem. 1991, 63, 464. (21) Rebar, V. A.; Santore, M. M. J. Colloid Interface Sci. 1996, 178, 29. (22) Bos, M. A.; Kleijn, J. M. Biophys. J. 1995, 68, 2566; 1995, 68, 2573.

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on overlap of the evanescent wave polarization vector and the transition dipole moment of the fluorescent porphyrin prosthetic group. In later sections of this paper we present fluorescence titrations to quantify the pH dependence of FITClysozyme fluorescence. We present the results of simple experiments with FITC-labeled bovine serum albumin (BSA) to confirm that the Debye length is an appropriate reference length scale for such TIRF experiments. In doing so we also help resolve an issue raised in an earlier TIRF study of FITC-BSA adsorption.23 The main focus of the current work is the presentation and interpretation of TIRF kinetics as lysozyme adsorbs to negatively charged silica surfaces. TIRF measurements are complemented by independent scanning angle reflectometry measurements of the lysozyme adsorption kinetics. The paper concludes with a suggestion for the mechanism of lysozyme reorientation as the adsorbed layer evolves. Theory The fluorescence behavior of the FITC-protein conjugate near neutral pH is dominated by the equilibrium between the monoanion (weakly fluorescent) and dianion (highly fluorescent) forms of fluorescein,24 represented as Ka

AH- 798 A2- + H+

(1)

If ni and µi are the stoichiometric coefficient and the electrochemical potential of species i, respectively, the dissociation constant associated with this process is25

( ) ( ∑i niµi

-

Ka ) exp

RT

) exp

)

s s s µH + µA2- - µAH-

RT

(2)

The superscript s emphasizes that the electrochemical potentials are for species at the surface. The electrochemical potentials of species AH- and A2-, both of which are confined to the surface region by virtue of the irreversible protein adsorption, are

µAs 2- ) RT ln[A2-]s - 2FΨ

(3)

s - s µAH - ) RT ln[AH ] - FΨ

(4)

where F is the Faraday constant and Ψ is the electric potential in the immediate vicinity of the fluorophore. Since the protons are freely mobile between the bulk (b) and surface regions, their electrochemical potential is simply

µsH+ ) µbH+ ) RT ln[H+]b

(5)

Substituting the electrochemical potentials eqs 3-5 into eq 2 yields

Ka )

[A2-]s[H+]b [AH-]s

(

exp -

FΨ RT

)

(6)

The degree of dissociation for the chemical equilibrium in eq 1, defined as (23) Cheng, Y. L.; Darst, S. A.; Robertson, C. R. J. Colloid Interface Sci. 1987, 118, 212. (24) Ygueribide, J. E.; Talavera, E.; Alvarez, J. M.; Quintero, B. Photochem. Photobiol. 1994, 60, 435. (25) Denbigh, K. The Principles of Chemical Equilibrium, 4th ed.; Cambridge University Press: Cambridge, 1981.

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x)1-

Robeson and Tilton

[AH-]s [AH-]s + [A2-]s

(7)

can therefore be related to the pH, the pKa, and the local electrostatic potential by

x)

10-pKa 10-pH exp(-FΨ/RT) + 10-pKa

(8)

To relate this degree of dissociation to the fluorophore’s position in the adsorbed layer, we need to describe the electric field within the adsorbed layer. This field is complicated by pronounced inhomogeneities in dielectric constant and a complex spatial distribution of neutral, acidic, and basic amino acid residues. Ideally, one would propose a detailed model for the structure of the adsorbed layer on the charged surface and solve the Poisson Boltzmann equation to describe the electric field. Unfortunately, detailed calculations of the electric field surrounding even an isolated protein adsorbed to a charged planar surface are computationally intensive.26,27 Here we make several simplifying assumptions intended to capture only the primary effect of local electrostatic potential on the fluorescence emission from FITC-labeled proteins adsorbed on charged dielectric surfaces. We neglect electric field distortions arising from inhomogeneities in dielectric constant, and we assume that the electric field is dominated by the negatively charged surface, ignoring the presence of ionized amino acid residues in the layer. We use the Gouy-Chapman model28 to approximate the decay of the electrostatic potential with increasing distance y between the fluorophore and the surface:

Ψ(y) ≈

( )

FΨ0 4RT exp(-κy) tanh F 4RT

(9)

where Ψ0 is the surface potential and F is the Faraday constant. This model, although certainly approximate, serves to capture the important decay of the negative electrostatic potential from the surface over distances comparable to the Debye length. (In the lysozyme experiments that follow, the adsorbed layer is approximately 60 vol % water at the point where the interesting fluorescence phenomena occur.) As expected, eqs 8 and 9 together indicate that the degree of dissociation increases with increasing fluorophore distance from a negatively charged surface. We will show in the Results section that the fluorescence emission intensity (F) of FITC-lysozyme conjugates is directly proportional to the degree of dissociation. Given that F ∝ x, the emission intensity from an adsorbed FITC-labeled protein is

F∝ 10-pKa exp(-y/dp) (10) FΨ0 -pH -pKa 10 exp -4 tanh exp(-κy) + 10 4RT

{

( )

}

when the fluorophore located at y is excited by an evanescent wave of penetration depth dp. The proportionality constant implied in relation 10 depends on instrumental factors such as detector efficiency, excitation intensity, etc. Since dp is generally two orders of (26) Yoon, B. J.; Lenhoff, A. M. J. Phys. Chem. 1992, 96, 3130. (27) Roth, C. M.; Lenhoff, A. M. Langmuir 1993, 9, 962. (28) Israelachvili, J. Intermolecular and Surface Forces, 2nd ed.; Academic Press: New York, 1991.

magnitude larger than the thickness of an adsorbed protein layer, the factor exp(-y/dp) is approximately unity and is insensitive to any changes in the thin adsorbed layer. Because the fluorophore is sensitive to the local electrostatic potential, any change in fluorophore position ∆y will produce a pronounced change in the TIRF signal provided κ∆y is at least of order unity. Thus, fine resolution can be lost at very low ionic strengths if κ-1 is much greater than the adsorbed layer thickness. The fine resolution might also be compromised at high ionic strengths by virtue of the correspondingly small surface potential for a fixed surface charge density. Although we have not experimentally explored this, “moderate” ionic strengths on the order of 1-10 mM would seem to be best suited for resolving reconfigurations in adsorbed protein layers. Experimental Section Buffers. Acetic acid/acetate buffers spanning the pH range 3.6-5.6 were prepared by mixing stock solutions of 0.2 M acetic acid (Fisher Scientific) and 0.2 M sodium acetate (Fisher Scientific). Buffers spanning the pH range 5.8-9.2 were prepared from mixtures of 0.05 M sodium borate decahydrate (Aldrich) and 0.1 M monobasic sodium phosphate (Aldrich). All TIRF experiments were conducted with pH 7.4 triethanolamine hydrochloride (TEA; Aldrich) buffers prepared with varying ionic strengths according to the protocol outlined by Bates.29 All TEA buffers were extremely stable. The pH of the least stable TEA buffer, 0.005 M ionic strength, changed by less than 0.05 pH units over a 2 week period. TEA buffers contain only 1:1 electrolyte. All buffers were prepared from deionized water further purified by the MilliQ Plus system (Millipore). Protein Labeling. We labeled bovine serum albumin (essentially fatty acid free, Sigma) by the procedure described previously.30 We reacted chicken egg lysozyme (Sigma) with FITC (Molecular Probes) in 0.10 M, pH 9.2 borate buffer for 1 h at room temperature in the dark. We reacted 0.4 mol of FITC per mole of lysozyme and kept the lysozyme concentration below 2 mg/mL. We then passed the reaction mixture through a 0.2 µm filter and into a Bio-Gel P6 (BioRad) column that was preequilibrated with pH 7.4, 0.005 M TEA buffer. The proteincontaining fraction of the column effluent was then dialyzed against a large excess of fresh TEA buffer for 3 h in a vigorously stirred vessel in an ice bath. Three hours of dialysis was sufficient to remove the unreacted FITC from the labeled lysozyme solution. We determined the average lysozyme-labeling ratio L by UVvis spectrophotometry using the following extinction coef278 278 ficients: lys ) 3.26 × 104 M-1 cm-1, FITC ) 1.82 × 104 M-1 cm-1, 500 4 -1 -1 and FITC ) 7.01 × 10 M cm . The lysozyme stock solutions had a labeling ratio of L ) 0.23 ( 0.05. For fluorescence and reflectometry measurements, we mixed the stock with unlabeled lysozyme to decrease the average number of labels per protein. We conducted differential scanning calorimetry analyses of labeled and unlabeled lysozyme samples in pH 7.4, 0.005 M TEA buffer using a MicroCal MC2 instrument at a scan rate of 60 °C/h. FITC labeling slightly decreased the denaturation peak temperature from 73 °C for unlabeled lysozyme to 72 °C for FITClysozyme. Peak areas were similar for the two materials. This indicates that the labeling procedure has little effect on the lysozyme conformation. Adsorption Substrates. TIRF experiments were conducted with polished fused silica microscope slides (ESCO). We cleaned these slides by sonicating in RBS detergent (Pierce) for 20 min, rinsing thoroughly with MilliQ water, and placing them in concentrated nitric acid (EM Science) for 30 min. They were then rinsed thoroughly with MilliQ water and soaked in fresh MilliQ water for 1 h. After another thorough water rinse, they were placed in 0.01 M sodium hydroxide (EM Science) solution for 30 min to increase the negative surface charge. They were finally rinsed thoroughly with MilliQ water, blown dry with a high-purity nitrogen jet, and dried under a heat lamp for 10 min. (29) Bates, R. G. Determination of pH Theory and Practice; Wiley: New York, 1964. (30) Robeson, J. L.; Tilton, R. D. Biophys. J. 1995, 68, 2145.

Reconfiguration of Adsorbed Lysozyme Layers

Langmuir, Vol. 12, No. 25, 1996 6107

The surfaces were completely wettable by water after this procedure. Slides could be cleaned and reused repeatedly with no signs of wear or changes in protein adsorption behavior. Reflectometry experiments were conducted with thermally oxidized optical grade silicon wafers (Lattice Materials). Before heat treatment, the wafers were cleaned by a slight modification of the procedure of Frank.31 We soaked the wafers in a saturated aqueous solution of potassium dichromate (Fisher Scientific) and 36 N sulfuric acid (EM Science) for 20 min at room temperature. The wafers were thoroughly rinsed with MilliQ water and then soaked in 6 M hydrochloric acid (Fisher Scientific) for 20 min and rinsed with MilliQ water again. They were then blown dry with a high-purity nitrogen jet and held under a heat lamp for 10 min to complete the drying. We then oxidized them at 1000 °C in air for 20 min to produce 20-30 nm thick oxide layers on the wafer surfaces. Before we used the thermally oxidized wafers in an experiment, we repeated the above cleaning procedure with an additional step of soaking in 0.01 M sodium hydroxide solution for 30 min at room temperature before the final MilliQ water rinse and drying. This left the surfaces completely wettable by water. Reproducible adsorption results were obtained with oxidized wafers that had been cleaned and reused repeatedly. Supported poly(methyl methacrylate) (PMMA; Polysciences) films were prepared on silanized disposable glass microscope slides by spin casting from toluene (Fisher Scientific) solution, following procedures described previously.30 Methods. The basic principles and procedural details of TIRF7,8,30 and of scanning angle reflectometry14,15 have been described extensively elsewhere in the literature. For this study we used the TIRF instrument described in refs 12 and 30. Proteins were adsorbed from solutions in laminar flow in a rectangular slit flow cell measuring 63.5 × 12.7 × 0.5 mm3. The scanning angle reflectometer was described in ref 15. Scanning angle reflectometry provides the optical average refractive index n and thickness d of an adsorbed layer. These optical properties are determined via nonlinear least squares regression of the reflectivity of p-polarized light as a function of the angle of incidence. Our measurements span a range of approximately 4° centered on the Brewster angle. The regression is based on a striated interface optical model that includes the effect of the oxide layer.15 The surface concentration Γ is calculated from n and d in a straightforward manner:14

Γ)

(n - n0)d dn/dc

(11)

where n0 is the refractive index of the aqueous solution and the refractive index increment dn/dc is 0.18 cm3/g for the protein. In the reflectometry experiments, as in the TIRF experiments, proteins were adsorbed from solutions in laminar flow. The reflectometry adsorption chamber is a rectangular slit flow cell measuring 24.7 × 12.7 × 1.45 mm3. All adsorption experiments were conducted at 30 °C. Although the fluorophore is not required for reflectometry, we adsorbed FITC-lysozyme (with the same average number of labels per protein as in the TIRF experiments) rather than unlabeled lysozyme in order to match the reflectometry and TIRF conditions as closely as possible. Since FITC does not absorb at the 632.8 nm wavelength of the He-Ne laser used for reflectometry, no absorbance correction is required to calculate the FITC-lysozyme surface concentration from scanning angle reflectometry data. Fluorescence emission spectra for labeled protein solutions were measured with a Perkin-Elmer Model LF5B luminescence spectrometer at a constant excitation wavelength, λex ) 488 nm, matching the argon ion laser line used in the TIRF experiments. To avoid concentration quenching and inner filter effects, the bound FITC concentration was kept below 7 × 10-7 M, corresponding to an absorbance less than 0.05 at the absorbance maximum (λ ) 500 nm). Emission spectra were scanned between 490 and 620 nm. FITC-lysozyme was dissolved in the acetate/ acetic acid buffers for the pH range 3.6-5.6 and in the borate/ phosphate buffers for the pH range 5.8-9.2. (31) Frank, B. Surfactant Self-Assembly near Contact Lines: Control of Advancing Surfactant Solutions. Ph.D. Dissertation, Carnegie Mellon University, 1995.

Figure 1. pH-dependent FITC-lysozyme fluorescence intensity normalized by the fluorescence intensity at pH 9.2, F/FpH)9.2 (b), which maps onto the degree of dissociation, x (solid curve), with a pKa of 6.2.

Results and Discussion Fluorescence Emission of FITC-Protein Conjugates. The strong pH dependence of FITC-lysozyme fluorescence is presented in Figure 1. Integrated emission spectra were directly proportional to the peak heights in all cases examined. Thus, we report the fluorescence emission intensity at each pH as the peak height normalized by the peak height obtained at pH 9.2. Although fluorescein is known to exist in at least four prototropic states with three distinct pKa values, when it is bound to lysozyme, its titration behavior is characteristic of a single pKa of 6.2. This single pKa behavior has been observed previously for FITC-labeled immunoglobulins.17 The excellent mapping of the fluorescence intensity onto the degree of dissociation (eq 7) in Figure 1 is the basis for eq 10. The fluorescence titration for FITC-BSA12 displayed a more gradual variation with pH than it did for FITClysozyme, but like FITC-lysozyme it did show a complete loss of fluorescence as the pH approached 3. In addition to the pH titrations, we also measured FITC-BSA and FITC-lysozyme fluorescence emission at pH 7.4 in TEA buffers ranging in ionic strength from 0.005 to 0.15 M. As observed previously for FITC-BSA in phosphate buffer,23 the ionic strength did not affect the fluorescence emission of either conjugate.12 TIRF of FITC-BSA Adsorbed to a Nonionic Surface. In a previous TIRF study of FITC-BSA adsorption to polydimethylsiloxane films, Cheng et al.23 noted that TIRF signals were quite sensitive to the ionic strength of the solution in spite of the insensitivity of dissolved FITCBSA fluorescence to changes in ionic strength. Here we show that this behavior is reproduced on another nonionic surface, PMMA. Moreover we show that the effect is caused by the overlap of the negative electric fields surrounding the adsorbed proteins at a pH in excess of the isoelectric point (iep ) 5 for BSA). The influence of one negatively charged protein on the electric field near a second protein obviously depends on the distance between their surfaces, r. The electric field near a BSAbound fluorophore becomes more negative for smaller values of κr, i.e., smaller separation and/or lower ionic strength. We adsorbed FITC-BSA onto PMMA surfaces from pH 7.4, 0.15 M TEA buffer in laminar slit flow at a wall shear

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Robeson and Tilton

Figure 2. TIRF intensity for an adsorbed layer of FITC-BSA on a PMMA surface, which decreases sharply with each stepwise decrease in ionic strength of the pH 7.4 TEA buffer bathing the surface. The dashed line is an extrapolation based on linear regression of the data from the initial rinse in 0.15 M TEA buffer. Data are shown from separate experiments using orthogonal incident beam polarizations.

Figure 3. TIRF intensity normalized by the intensity at 0.15 M ionic strength, F/F0.15M, which is more sensitive to changing ionic strength for the larger FITC-BSA surface concentration, Γ ) 1.5 mg/m2 (0, 4), than it is for the lower surface concentration, Γ ) 0. 5 mg/m2 (O). The buffers bathing the adsorbed layers are pH 7.4 TEA of varying ionic strength. No FITC-BSA is present in solution during these measurements.

rate of γ ) 102 s-1 until the TIRF signal became steady (approximately 1 h). We then rinsed the adsorbed layer with protein-free buffer of the same pH and ionic strength for 30 min to establish a baseline for the gradual desorption of FITC-BSA. At that point, we proceeded to rinse the adsorbed layer with a series of pH 7.4 TEA buffers of varying ionic strength, as shown in Figure 2 for ΓBSA ) 1.5 mg/m2. Each stepwise decrease in ionic strength (increasing κ-1) abruptly decreased the TIRF signal. Since the stepwise decreases in TIRF signal were rapidly reversible upon reversing the series of step changes in ionic strength, we conclude that the ionic strength changes decrease the TIRF signal by altering the FITC fluorescence behavior rather than simply accelerating FITC-BSA desorption. When we examine the final TIRF signal after the ionic strength cycle with respect to the linear extrapolation from the first extended rinse in 0.15 M TEA buffer, it is evident that the desorption rate was constant throughout the experiment. Note also that this behavior was independent of the incident laser beam polarization. The incident polarization is a concern because reorientation of the FITC fluorescence transition dipole relative to the direction of the incident electric field vector would alter its excitation efficiency. If the FITC labels had severely limited rotational mobility and the adsorbed BSA molecules were to reorient in response to changes in ionic strength, the fluorescence signal might then have changed merely due to the altered projection of the incident polarization vector on the transition dipole. Noting the similarity of TIRF results with different laser polarizations, it is evident that the observed TIRF behavior is not due to FITC-BSA reorientation in response to ionic strength changes. Besides varying κ-1, we also varied r in these experiments by adsorbing FITC-BSA to PMMA surfaces at two different surface concentrations, Γ ) 0.5 and 1.5 mg/m2. These are attained simply by adsorbing from solutions with different bulk FITC-BSA concentrations.32 If we represent the adsorbed BSA molecules as uniformly spaced disks whose area is the same as the largest projection of the molecule on the surface, these two surface concentra-

tions correspond to edge-to-edge separations of r ) 13 nm and r ) 3.7 nm, respectively. This assumes BSA has the same shape as human serum albumin, whose largest projection is approximated as an equilateral triangle 8 nm on a side.33 As shown in Figure 3, the TIRF signal is far less sensitive to ionic strength for the lower surface concentration, i.e., the larger average intermolecular separation. The TIRF response to independent changes in κ and r confirms that the ionic strength dependence of the TIRF signal originates in the overlap of negative electric fields surrounding neighboring BSA molecules and the consequent shift to less fluorescent prototropic forms of fluorescein. More important for what follows, this confirms that the Debye length is the characteristic length scale for electric field-induced changes in FITC-protein emission. Thus, by matching the Debye length to the size of a protein molecule, we gain extra information from TIRF experiments. FITC-Lysozyme Adsorption to Negatively Charged Silica. The surfaces in the experiments described above were prepared from nonionic polymers, and the electric field effects were dominated by the proximity of adsorbed, negatively charged proteins. In what follows, the protein lysozyme carries a net positive charge (iep ) pH 11) but is adsorbed to a negatively charged, ionized surface. Changes in fluorescence emission are now dominated by the proximity of the FITC to the negatively charged surface. Adsorption of FITC-lysozyme to a negatively charged silica surface from 10 µg/mL solutions in pH 7.4, 0.005 M TEA buffer at a wall shear rate of γ ) 32 s-1 produced the TIRF kinetics plotted in Figure 4. The sample contained an average of 0.04 FITC labels per protein, obtained by mixing the labeled stock solution (L ) 0.23 ( 0.05) with unlabeled lysozyme. The most prominent features in Figure 4 are the sharp overshoot at approximately 200 s, the subsequent minimum, and the second broad maximum. This behavior is independent of the incident beam polarization, as the slight differences between the two curves in Figure 4 corresponding to parallel and perpen-

(32) Tilton, R. D.; Robertson, C. R.; Gast, A. P. J. Colloid Interface Sci. 1990, 137, 192.

(33) He, X. M.; Carter, D. C. Nature 1992, 358, 209.

Reconfiguration of Adsorbed Lysozyme Layers

Langmuir, Vol. 12, No. 25, 1996 6109

Figure 4. TIRF intensity passing through a pronounced overshoot when FITC-lysozyme adsorbs to silica, followed by a minimum and a second maximum at later times. Time zero is the moment the FITC-lysozyme is introduced to the flow cell. FITC-lysozyme is present in solution at all times. Data are shown for two separate experiments using orthogonal incident beam polarizations.

dicular incident beam polarizations are well within the limits of variability for repeated experiments. We independently measured the kinetics of FITClysozyme adsorption in a series of reflectometry experiments in order to relate the TIRF kinetics to the instantaneous surface concentrations. We obtained adsorption kinetics by recording the reflectivity of parallel polarized light incident at the Brewster angle as a function of time, Rp(θB,t). We convert these time-dependent reflectivities to surface concentrations Γ(t) using the proportionality between Γ(t) and Rp(θB,t)1/2 - Rp(θB,0)1/2 , where Rp(θB,0) corresponds to the reflectivity of the bare oxide layer.15 We determine the proportionality constant by performing a full scanning angle reflectometry measurement Rp(θ) at the adsorption plateau to determine Γ(plateau) and relating it to Rp(θB, plateau)1/2 - Rp(θB,0)1/2. Each silicon wafer has an approximately 20 nm thick oxide layer. In that case, direct conversion from Rp(θB,t)1/2 - Rp(θB,0)1/2 to Γ(t) is valid to within approximately 2% for the full range of protein surface concentrations we measured (0-2.2 mg/m2). Since the flow cells used for reflectometry and TIRF are different, care must be taken in comparing kinetic data. If adsorption is transport limited, it is necessary to compare TIRF and reflectometry data obtained at matching wall shear rates, not flow rates. As shown in Figure 5, the initial steady-state adsorption rate measured in a series of reflectometry experiments obeys the well-known wall shear rate dependence expected for transport-limited adsorption in laminar slit flow:8

|

1 dΓ γ ) dt t)0 Γ(4/3)91/3 Dl

1/3

( )

DC

(12)

where C is the bulk concentration, l is the distance from the flow cell inlet to the observation point, and Γ(4/3) refers to the gamma function. The slope of the initial adsorption rate versus γ1/3 plot in Figure 5 is in quantitative agreement with eq 12 for the lysozyme diffusion coefficient D ) 1.2 × 10-6 cm2/s. Since the initial rate of adsorption is transport limited, it is acceptable to compare kinetics from reflectometry and TIRF experiments that use different flow cells provided the wall shear rates are equal.

Figure 5. γ1/3 dependence of the initial steady-state adsorption rate, dΓ/dt0, confirming that FITC-lysozyme adsorption at a concentration of 10 µg/mL in 0.005 M, pH 7.4 TEA buffer is transport limited over the relevant range of wall shear rates.

Figure 6. Superposition of the TIRF kinetics with the FITClysozyme adsorption kinetics measured independently via reflectometry (average of 0.04 FITC labels per protein in both cases), showing that the TIRF maxima are not caused by maxima in the surface concentration. Adsorption kinetics are transport limited until an abrupt change in rate occurs at approximately the same time as the sharp TIRF overshoot. The wall shear rate is 32 s-1 in both experiments shown.

Qualitatively, lysozyme behaved as expected for a hard protein undergoing primarily electrostatic adsorption with minimal conformational changes. Adsorption on silica was favored by low ionic strengths.12 Desorption was extremely slow upon rinsing the adsorbed layer with 0.005 M TEA buffer, but upon rinsing with 0.15 M TEA buffer, 90% of the adsorbed lysozyme rapidly desorbed. We compare TIRF kinetics and lysozyme adsorption kinetics at γ ) 32 s-1 in Figure 6. Clearly, the exceptional overshoots in the TIRF signal are not due to any overshoots in the FITC-lysozyme surface concentration. The reflectometry data indicate a nearly constant adsorption rate until approximately 1.7 mg/m2, at which point the adsorption kinetics become surface limited and eventually reach a plateau at 2.1 mg/m2. On average the final surface concentration was 2.2 mg/m2. In experiments conducted at several wall shear rates between 3 and 32 s-1, the transition from transport-limited to surface-limited ki-

6110 Langmuir, Vol. 12, No. 25, 1996

netics was quite distinct and occurred at surface concentrations between 1.3 and 1.7 mg/m2. Representing the lysozyme molecule as an ellipsoid with minor and major axes of 3 nm × 4.5 nm,34 the random parking (i.e., jamming) limit35 for lysozyme in a “horizontal” orientation is 1.3 mg/m2. The hexagonal close packing limit is 2.1 mg/m2 . For a “vertical” orientation, these limits are 1.9 and 3.1 mg/m2, respectively. Thus, the transition from transport-limited to surface-limited adsorption occurs near or above the random parking limit for random sequential adsorption (RSA). Of course the RSA model assumes irreversible adsorption and complete immobility of adsorbed species. The fact that FITClysozyme adsorbs beyond the RSA jamming limit suggests that it is mobile after adsorption. The first peak in the TIRF kinetics occurs at precisely the point where the adsorption mechanism becomes surface limited. Thus, just when packing constraints in the adsorbed layer start to dominate the adsorption mechanism, it appears that the adsorbed layer is reconfigured in a way that puts the fluorophores in closer proximity to the negatively charged surface. Alternative Explanations for the Overshoot. Before discussing the likely cause for this spontaneous layer restructuring, we first consider and eliminate plausible alternative explanations for the TIRF maxima: rotation of the fluorescence excitation dipole, concentration quenching, photobleaching by the incident laser beam, and competitive adsorption of labeled versus unlabeled lysozymes. The first alternative is ruled out by the similarity between TIRF experiments conducted using orthogonal incident beam polarizations (e.g. Figure 4). The second alternative, concentration quenching, refers to a decrease in the fluorescence quantum yield that can result if two FITC labels are placed in close proximity. We have shown previously that this can be an important factor in TIRF as well as fluorescence recovery after photobleaching (FRAP) experiments with adsorbed proteins.30 Previous surface force apparatus studies16,36 of lysozyme layers (on mica) suggested the possibility that lysozyme may form a partial bilayer, i.e., that some fraction of the adsorbed lysozyme molecules may in effect be dimerized. Should such surface dimers form during our TIRF experiments, concentration quenching would be a very real possibility. If concentration quenching were responsible for the TIRF overshoot, the magnitude of that overshoot, expressed as the ratio of the maximum TIRF intensity to the subsequent minimum Fmax/Fmin, should depend strongly on the probability that both members of a dimer are labeled. At such a low average labeling ratio as 0.04, the probability that a dimer contains two FITClabeled molecules is only L2 ) 0.0016. As we increase L from this low value, Fmax/Fmin should increase in proportion to L2. In fact, as we increased L from 0.04 to 0.15, corresponding to a 14-fold increase in the likelihood of forming a self-quenched dimer, Fmax/Fmin actually decreased slightly from 1.67 to 1.32. Furthermore, the ratio of Fmax to L was constant for all L. That is inconsistent with concentration quenching. These observations indicate that concentration quenching was not important in these experiments. We note that one effect of FITC protonation (as arising from a negative surface potential or low pH) is to shift the excitation spectrum to lower wavelengths without shifting the emission spectrum or decreasing the quantum yield. This shift would decrease (34) Blake, C. C. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.; Sarma, V. R. Proc. R. Soc. London 1967, 167, 365. (35) Vigil, R. D.; Ziff, R. M. J. Chem. Phys. 1989, 4, 2599. (36) Tilton, R. D.; Blomberg, E.; Claesson, P. M. Langmuir 1993, 9, 2102.

Robeson and Tilton

Figure 7. TIRF kinetics obtained with intermittent exposure to the monitoring beam, which closely match the TIRF kinetics with continuous monitoring beam exposure for FITC-lysozyme adsorption to silica from 10 µg/mL solutions in 0.005 M TEA buffer. Since we made no effort to synchronize the shutter and the data sampling frequency, extraneous points were occasionally recorded during shutter openings or closings.

the fluorescence overlap integral and minimize any tendency for concentration quenching. The third consideration, slow photobleaching by the monitoring laser beam, is always a concern in TIRF experiments. Its occurrence is checked readily by varying the exposure time to the incident beam. We varied the total exposure time by periodically opening and closing a shutter in the laser path. The TIRF kinetics traced out by this intermittent exposure to the laser beam are shown in Figure 7. The similarity of the TIRF kinetic data obtained with intermittent (Figure 7) and continuous (Figure 4) exposure indicates that photobleaching was not significant here. The fourth consideration is that competitive adsorption of labeled and unlabeled proteins could produce a maximum in a TIRF kinetic experiment if one species preferentially adsorbed. We modeled the adsorption kinetics with a modified Langmuir adsorption mechanism wherein labeled and unlabeled species were allowed to displace each other from the surface:

dθL ) kLCL(1 - θL - θU) - kdUCUθL + kdLCLθU (13) dt dθU ) kUCU(1 - θL - θU) + kdUCUθL - kdLCLθU (14) dt Here, the subscripts L and U represent labeled and unlabeled proteins respectively; θ is the surface coverage; C is the bulk protein concentration; kL and kU are adsorption rate constants; and kdL and kdU are the rate constants for labeled proteins displacing unlabeled proteins, and for unlabeled proteins displacing labeled proteins, respectively. An overshoot in the TIRF signal would result from an overshoot in θL. The kinetic calculations are summarized as follows: if kU g kL, a fluorescence overshoot occurs only if kdU > kdL (preferential displacement of labeled proteins by unlabeled proteins); if kU < kL, an overshoot occurs provided kdU * 0. Most importantly, the competitive adsorption model cannot predict a maximum followed by a minimum, and

Reconfiguration of Adsorbed Lysozyme Layers

Langmuir, Vol. 12, No. 25, 1996 6111

Figure 8. Coordinate system with the origin at the center of the circular cross section of an adsorbed cylinder with its axis parallel to the surface. y is the height of the fluorophore above the surface.

in all cases where an overshoot occurs, the ratio Fmax/L decreases with increasing L. In experiments conducted with L ) 0.04, 0.08, 0.12, and 0.15, the intensity-to-labeling ratio quotients Fmax/L for both the first and second TIRF maxima were constant for all L. These observations argue against competitive adsorption as the cause of the TIRF overshoots. Crowding-Induced Reconfiguration of the Adsorbed Layer. Relation 10 provides the TIRF signal for the case when the separation between the fluorophore and the charged surface is fixed at one particular value y. Given the possibility that lysozyme may be labeled at different sites on the molecule and the possibility that lysozyme molecules may sample a random set of orientations on the surface, the TIRF signal should be averaged over all possible fluorophore positions. For simplicity, we treat the lysozyme molecule as a cylinder of diameter D ) 3 nm lying with its axis parallel to the surface. Thus, the FITC may be positioned anywhere on the perimeter of the cylinder. With the coordinate system defined in Figure 8, the fluorophore distance from the surface is

y)

D (1 + sin θ) 2

(15)

Averaging the fluorescence intensity described by relation 10 over all possible fluorophore positions yields

∫

F ∝ 10-pKa

π/2

-π/2

×

[

]

-D(1 + sin θ) dθ 2dp FΨ0 κD (1 + sinθ) 10-pH exp -4 tanh exp 4RT 2 exp

{

( ) [

]}

+ 10-pKa

(16)

for the TIRF signal. The ionic strength in the TIRF experiments was 5 mM, corresponding to a Debye length of κ-1 ) 4.3 nm or κD ) 0.7. Figure 9 illustrates how an increasingly negative surface potential decreases the TIRF signal for the case when κD ) 0.7 and D/dp ) 0.015. Figure 9 includes plots for the rotationally averaged TIRF signal, the TIRF signal for the case when the FITC is held at its maximum possible height y ) D, and the TIRF signal for the case when the FITC is fixed in contact with the surface, y ) 0. Clearly, closer FITC proximity to the negatively charged surface decreases the TIRF signal for any negative surface potential. This effect is quite pronounced at surface potentials that should be expected for NaOH-treated silica surfaces at 5 mM ionic strength. The streaming potential measurements of Zorin et al.37 indicate that the surface charge density of silica is approximately 2300 Å2/charge. While this surface charge density certainly will depend on the

Figure 9. Variation of the TIRF intensity normalized by the intensity for a zero surface potential, F/FΨ)0, with changing surface potential depending on the position of the fluorophore. y ) 0 corresponds to the situation where the fluorophore is fixed in contact with the surface. y e D corresponds to the situation where the fluorophore is randomly distributed and the TIRF signal is averaged over all possible fluorophore positions on the circumference of a cylinder of diameter D. y ) D corresponds to the situation where the fluorophore is fixed at its largest possible separation, a distance D from the surface.

surface preparation method, it would produce a surface potential of -40 mV in the presence of a 5 mM solution of 1:1 electrolyte as calculated by the Grahame equation assuming a constant surface charge density.28 As may be seen in Figure 9, a change of the orientation distribution in the adsorbed layer would have a pronounced effect on the TIRF signal for surface potentials of this order. Before comparing the trends predicted by relation 16 with the TIRF data, we must re-emphasize that this proportionality is based on a simple model of a smoothly varying electric field near the surface, and it should therefore be regarded as semi-quantitative. Nevertheless, it is interesting to note that a reconfiguration of the adsorbed layer from a population of horizontal but randomly rotated proteins, denoted by y e D in Figure 9, to a preferred orientation that placed the FITC in contact with the surface, denoted by y ) 0, would decrease the TIRF signal by approximately a factor of 1.1-1.6 for Ψ0 values of -40 to -100 mV. This is consistent with the Fmax/Fmin ratios of approximately 1.5 we observed for the sharp TIRF overshoots. Since this overshoot coincided with the transition from transport-limited to surfacelimited adsorption kinetics, i.e., at the point where packing effects begin to dominate the adsorption mechanism, we conclude that lateral interactions between adsorbed lysozymes forced this reconfiguration. Protein labeling by FITC occurs at exposed lysine residues. Chicken egg lysozyme has six lysine residues, all of which are exposed on the surface of the protein. Since FITC can bind to any of these six sites, the precise orientation of the lysozyme molecule cannot be determined unambiguously with this system. We do note that four of the six lysines are located in the single largest patch of positive charge on the lysozyme surface. This may be visualized from the published X-ray crystallographic data38 maintained in the Brookhaven Protein Data Bank. This basic patch includes the lysine residues lys1, lys13, lys96, and lys97 in addition to arg14, his15, and arg128. It (37) Zorin, Z. M.; Churaev, N. V.; Esipova, N. E.; Sergeeva, I. P.; Sobolev, V. D.; Gasanov, E. K. J. Colloid Interface Sci. 1992, 152, 170. (38) Wilson, K. P.; Malcolm, B. A.; Matthews, B. W. J. Biol. Chem. 1992, 267, 10842.

6112 Langmuir, Vol. 12, No. 25, 1996

accounts for 7 of the total 17 basic amino acid residues in lysozyme. An orientation that placed this positive patch near the surface would thus place 4 of the 6 FITC labels in a region of large negative electrostatic potential, thereby decreasing the TIRF signal relative to that of a random orientation distribution. Proposed Adsorption Mechanism. Roth and Lenhoff27,39 have shown that the free energy of interaction between an isolated lysozyme molecule and a negatively charged surface depends significantly on orientation. If the adsorbed lysozyme molecules sampled a random orientation distribution, this would not maximize the electrostatic attraction to the surface. Neither would it minimize the electrostatic repulsion between neighboring lysozymes. These lateral repulsions become important at larger surface concentrations. An orientation that placed the large positively charged patch in close proximity to the surface would maximize the surface attraction and minimize lateral repulsions. Haggerty and Lenhoff’s calculations40 of the electrostatic potential contours surrounding the lysozyme molecule suggest that an orientation with the face containing the active site parallel to the surface decreases the electrostatic repulsion between neighboring lysozyme molecules in an ordered adsorbed array. This orientation even allows for favorable electrostatic intermolecular interactions along one axis of the array. Using scanning tunneling microscopy, they have in fact observed ordered arrays of lysozyme on graphite surfaces, consistent with their calculations. The orientation we suggest, with the largest positively charged patch toward the surface, does correspond to the orientation that Haggerty and Lenhoff suggest will improve the intermolecular electrostatic interactions. Note also that if the adsorbed lysozyme molecules in this preferred orientation were to assemble into a similar ordered array, the increased intermolecular separations between net positively charged proteins would also contribute to the decreased TIRF signal after the reconfiguration. Why does it seem to be necessary to approach the jamming limit before lysozyme assumes this orientation? At low coverages, the magnitude of the intralayer repulsive energy is small, and the free energy minimum may be quite shallow around the optimum orientation. The shallow free energy minimum would allow a broader distribution of orientations with respect to the surface. In contrast, as the surface concentration approaches the jamming limit where lateral repulsions become severe, the free energy well deepens and biases the orientation distribution more strongly toward the optimum. Assuming equal probabilities of FITC labeling at all six lysine residues, 2/3 of which are in the large positive patch, the average FITC height above the surface would then be strongly biased to smaller values. This favors the protonated state and decreases the TIRF signal. This mechanism is illustrated schematically in Figure 10. The TIRF kinetics display two maxima. The first, sharper overshoot is due to the rapid restructuring brought on by lateral repulsions at the jamming limit. Since electrostatic repulsions between the net positive charged proteins extend the effective excluded area per molecule beyond the boundary of its van der Waals surface, the effective size of an adsorbed protein depends on κ-1 and on the charge density on the exposed protein surface. Thus by sequestering the large positive patch against the negative surface, the crowding-induced reorientation would decrease the effective excluded area per lysozyme molecule, making room for more proteins to adsorb. (39) Roth, C. M.; Lenhoff, A. M. Langmuir 1995, 11, 3500. (40) Haggerty, L.; Lenhoff, A. M. Biophys. J. 1993, 64, 886.

Robeson and Tilton

Figure 10. Schematic illustration of the adsorption mechanism. At low coverages, lysozyme assumes a wide distribution of orientations. As the surface concentration increases, lateral electrostatic interactions force the adsorbed proteins into a narrow distribution of orientations that place the large positive patch in contact with the surface.

Because of the increase in the accessible surface area, the TIRF signal would pass through a minimum after the reconfiguration as additional molecules adsorb and add to the TIRF signal. Note that continued adsorption beyond the point of layer reconfiguration is clearly demonstrated in the reflectometry data in Figure 6. Those proteins that adsorb after the reconfiguration initially may fill available area in random orientations, depending on their trajectory from the bulk solution. Provided that those adsorbed proteins are mobile, they can reorient to assume a similar optimal orientation with the large positive patch against the surface. To further support this mechanism, note that the final lysozyme surface concentration was 2.2 mg/m2. This compares very well with the hexagonal close packing limit for horizontal lysozyme molecules (i.e., oriented with the largest positive patch against the surface). Since the reflectometry data indicate that the adsorption rate has become extremely small by the time of the second, broad TIRF maximum, this gradual reorientation to a less fluorescent state is what decreases the TIRF signal after the second maximum. We should also consider the possibility that the adsorbed lysozyme reconfiguration is not necessarily a reorientation. A conformational change that allowed the lysozyme molecules to relax to a “thinner” state on the surface would also place the fluorescein in closer proximity to the surface. Here we simply note that lysozyme is a rigid protein that is not expected to experience such a dramatic conformational change upon adsorption.1 Furthermore, it has been shown experimentally that adsorbed lysozyme layers (on mica) are incompressible, and their thickness closely matches the native dimensions of the lysozyme molecule.16 The layer reconfiguration at the jamming limit is more likely a reorientation rather than a conformational change. We note also that fluorescein may be quenched by free tryptophan in solution, with an expected decay length of 1.1-1.4 Å.41 While intramolecular tryptophan quenching

Reconfiguration of Adsorbed Lysozyme Layers

is unimportant in fluorescein-labeled lysozyme,41 it remains possible that if the layer reconfiguration were to place FITC labels within approximately 1 Å of an exposed tryptophan residue on a neighboring protein, the TIRF signal would also decrease due to tryptophan quenching. The TIRF overshoot is not a general feature of lysozyme adsorption. In addition to silica surfaces, we examined FITC-lysozyme adsorption to PMMA surfaces (data not shown). Compared to silica surfaces, adsorption was quite slow and produced no TIRF overshoots. This is not surprising, given the nonionic character of PMMA. Conclusions Exploiting the sensitivity of a familiar fluorophore to its state of protonation and hence the local electrostatic potential, we have decreased the characteristic length scale for TIRF experiments by two orders of magnitude, from the evanescent wave penetration depth to the Debye length. At moderate ionic strengths, this length scale is comparable to the dimensions of a protein molecule, so small changes in the adsorbed layer may be observed. Using this fine resolution TIRF capability, we have observed a packing-induced reconfiguration of adsorbed lysozyme layers on negatively charged silica surfaces. The combination of TIRF and reflectometry demonstrates that this reconfiguration coincides with the onset of surfacelimited adsorption and allows adsorption to proceed beyond what would otherwise be the jamming limit for random sequential adsorption. Consideration of lysozyme’s anisotropic structure and its known resistance to major surface-induced conformational change leads us to conclude that the reconfiguration is probably a reorientation that places the large positively charged patch in close proximity to the negatively charged surface. Accordingly, the plateau surface concentration corresponds to hexagonal close packing of horizontally oriented lysozyme molecules. The spontaneous, packing-induced reconfiguration is the most dramatic feature of the lysozyme adsorption mechanism. Such layer reconfigurations should be con(41) Periasamy, N.; Bicknese, S.; Verkman, A. S. Photochem. Photobiol. 1996, 63, 265.

Langmuir, Vol. 12, No. 25, 1996 6113

sidered in future models of protein adsorption kinetics when experimental evidence warrants the effort. The method presented here may prove to be quite useful in correlating time-dependent structural changes in adsorbed layers with observable time-dependent changes in the biochemical function of the adsorbed layer. The method does place certain requirements on the ionic strength and may therefore be inappropriate for certain applications involving very high or very low salt concentrations. Furthermore, the interpretation of experimental results may be difficult for randomly labeled proteins whose distributions of labeling sites are not as anisotropic as lysozyme. Site-specific labeling should prove to be quite useful in those cases. We conclude with a suggestion for the conduct of TIRF measurements. An important consequence of our observations is that external calibration is highly advisable when the magnitude of the TIRF signal is used to measure surface concentrations. Suitable external calibrations include reflectometry, ellipsometry and radiolabeling, for example. Internal calibrations39,42 previously have been used to translate TIRF signals to surface concentrations. These assume that the fluorescence emission properties of adsorbed species are the same as the bulk fluorescence properties and that they do not change over the course of the experiment. This study and others21,30 remind us that the fluorescence properties of adsorbed labeled macromolecules may be strong functions of the local environment and/or surface concentration. While internal calibrations may be used successfully in many cases, we recommend that extra precautionary measures should be taken to characterize the fluorescence properties of dissolved and adsorbed species. Acknowledgment. This work was supported in part by a DuPont Young Faculty Award and by a grant from The Whitaker Foundation. We thank Mike Domach for access to the differential scanning calorimeter and fluorescence spectrometer and Fred Lanni for several insightful conversations over the course of this study. LA960476P (42) Go¨lander, C. G.; Hlady, V.; Caldwell, K.; Andrade, J. D. Colloids Surf. 1990, 50, 113.