Local Diffusion and Concentration of IgG near Planar Membranes

J. Phys. Chem. B 2002, 106, 2365-2371

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Local Diffusion and Concentration of IgG near Planar Membranes: Measurement by Total Internal Reflection with Fluorescence Correlation Spectroscopy Tammy E. Starr† and Nancy L. Thompson* Department of Chemistry, Campus Box 3290, UniVersity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290 ReceiVed: July 12, 2001; In Final Form: December 2, 2001

The local diffusion coefficients and concentrations of a fluorescently labeled, monoclonal IgG in the solution adjacent to substrate-supported phospholipid bilayers have been measured by using total internal reflection with fluorescence correlation spectroscopy (TIR-FCS). In these measurements, the evanescent depth was ≈1000 Å. TIR-FCS data were obtained as a function of membrane composition, pH, and ionic strength. Best-fits to previously determined theoretical forms provided measures of the average local IgG concentrations and diffusion coefficients. These values were compared to the bulk concentrations and diffusion coefficients, measured far from the membrane, by using FCS with a focused beam. The bulk diffusion coefficient was also measured with dynamic light scattering. The IgG concentrations close to the membrane surface were not significantly different from those in the bulk. However, the local diffusion coefficients, as measured by TIRFCS, were faster at low ionic strength and slower at high ionic strength. The observed ionic strength dependence of the local diffusion coefficient was not observed in the bulk. This work describes the first measurement of protein concentration and diffusion close to model membrane surfaces by using TIR-FCS. Future investigations with smaller evanescent wave depths, obtained by using high refractive index substrates, are expected to provide much more information about the physical dynamics of proteins in close proximity to membranes. This information should contribute significantly to the current picture of the physical factors that govern interactions between soluble ligands and membrane-associated receptors.

Introduction Numerous biochemical processes are mediated by the interactions between soluble ligands and membrane-associated receptors. Previous studies have shown that the association/ dissociation kinetics of ligands in solution with receptors in natural or model membranes are often not adequately explained as simple, reversible, bimolecular reactions between point particles.1-18 The observed nonideality is frequently described by formulating models in which the number of discrete states is increased.19 Commonly postulated models include the presence of multiple ligand or receptor types, receptor-receptor or ligand-ligand interactions, multivalent receptor-ligand binding, or ligand-induced conformational changes in the receptors. Other formulations describe the system as containing a continuum of different bound states.20-23 Another approach has been based on the consequences of the coupled two-dimensional/threedimensional reaction geometry.24-26 The role of transport in the reactions between soluble ligands and membrane receptors, including rebinding effects27-30 as well as rotational mobility and orientational effects,31,32 has also been evaluated. Absent from these attempts to formulate physical descriptions of the complexity of ligand-receptor interactions is an experimental survey of ligand translational mobility and concentration very close to the membrane. It is plausible that effects such as those arising from electrostatic fields33-35 or hydrodynamic interactions36-39 will cause local nonideal translational diffusion * To whom correspondence should be addressed. Tel: (919)962-0328. Fax: (919)966-3675. E-mail: [email protected]. † Present address: Cellomics, Inc., 635 William Pitt Way, Pittsburgh, PA 15238.

as well as changes in the local ligand concentration. If such effects occur, they could play major roles in the mechanisms and kinetics of ligand-receptor interactions. One method of directly measuring ligand concentrations and mobilities very close to membrane surfaces is to use total internal reflection with fluorescence correlation spectroscopy (TIR-FCS). In this method, a laser beam is internally reflected at the interface of a planar surface and an aqueous medium. The internal reflection generates an evanescent field that penetrates into the aqueous medium and excites fluorescence from molecules in solution very close to the surface. The fluorescence arising from a small surface-adjacent volume, defined by the depth of the evanescent field along with an image-plane aperture, is monitored and fluctuates as molecules move into and out of the volume. The shape and magnitude of the normalized autocorrelation function of the temporal fluorescence fluctuations provide information about the local translational mobilities and concentrations of the fluorescent molecules. The theoretical basis for using TIR-FCS to examine surface dynamics, including specific association/dissociation as well as translational diffusion in solution adjacent to the surface, has been established.40,41 However, only a limited number of experimental TIR-FCS studies have thus far been carried out. The nonspecific binding of tetramethylrhodamine-labeled immunoglobulin or insulin to serum albumin-coated fused silica42 and the concentration and reversible adsorption kinetics of rhodamine 6G to C-18-modified silica surfaces43,44 have been examined. In the work described herein, the translational diffusion and local concentration of fluorescently labeled,

10.1021/jp012689f CCC: $22.00 © 2002 American Chemical Society Published on Web 02/06/2002

2366 J. Phys. Chem. B, Vol. 106, No. 9, 2002

Starr and Thompson and R ) D/d2 is the rate for diffusion with coefficient D through the depth of the evanescent intensity. The function G(τ) in eq 1 decays monotonically with time. The time at which G(τ) equals one-half of its initial value is 3.3 R-1. For traditional FCS measurements in solution with a focused spot, the fluorescence fluctuation autocorrelation function is given approximately by46

G(τ) )

Figure 1. Schematic of TIR-FCS. A laser beam traveling from a higher refractive index medium n1 into a lower refractive index medium n2 is totally internally reflected when the incidence angle θ is greater than the critical angle, and an evanescent field is created that penetrates a depth d into the lower refractive index medium. A sample volume is defined by this depth along with a circular aperture placed at an intermediate image plane of a microscope, corresponding to a region with radius h in the sample plane. Fluorescently labeled molecules (A) diffuse into and out of the sample volume with coefficient D, creating fluctuations in the fluorescence collected from the sample volume.

monoclonal IgG adjacent to planar-supported model membranes were measured by using TIR-FCS. The diffusion coefficients and local concentrations of the protein were characterized as a function of the membrane composition as well as the pH and ionic strength of the solution. The local diffusion coefficients and concentrations measured by TIR-FCS were compared to those measured far from the membrane by FCS with a focused laser beam and in the absence of the membrane by dynamic light scattering. Theoretical Background The application of TIR-FCS to fluorescently labeled molecules diffusing in solution near surfaces has been theoretically considered.41 In this technique, a laser beam is internally reflected at a substrate/solution interface and creates an evanescent intensity that penetrates a depth d into the solution adjacent to the surface (Figure 1).45 In an experimental situation in which the interface of a fused silica substrate (n1 ) 1.467) and an aqueous solution (n2 ) 1.334) is illuminated by a 488 nm laser line at θ ≈ 70°, d ≈ 1000 Å. The evanescent intensity along with a small circular aperture placed at an intermediate image plane of a microscope, corresponding to a radius h in the sample plane, defines an observation volume. Individual fluorescent molecules in solution diffuse into and out of the defined observation volume. Their motion causes the measured fluorescence to fluctuate with time. These fluctuations are defined as the difference between the instantaneous fluorescence intensity and its average value. The fluctuations are autocorrelated to obtain information about the diffusion and concentration of fluorescent molecules in the observation volume. When the length of the sample volume along the surface (h) is much greater than the evanescent depth (d), the theoretical form of the TIR-FCS fluorescence fluctuation autocorrelation function is41

G(τ) ) (1/2N) {(1 - 2Rτ ) exp(Rτ ) erfc[(Rτ)1/2] + 2 (Rτ/π)1/2} (1) where N ) πh2dA is the average number of fluorescent molecules in the observed volume, A is the local concentration,

1 1 ‚ 2N 1 + τ/τD

(2)

where N is the average number of molecules in the sample volume, τD ) s2/4D, and s is the 1/e2 radius of the Gaussianshaped spot at the focus. This function is applicable when the depth of the sample volume along the direction of beam propagation is significantly larger than s. Methods and Materials IgG Preparation. anti-Dinitrophenyl (DNP) IgG1 was obtained from the hybridoma 1B711 (American Type Culture Collection, Rockville, MD).47 Hybridomas were maintained in culture, and 1B711 IgG was purified from cell supernatants by affinity chromatography with DNP-conjugated human serum albumin. The IgG was eluted with dinitrophenylglycine (DNPG), which was later removed by extensive dialysis followed by ion exchange chromatography. Each liter of supernatant yielded approximately 10-15 mg of antibody as determined spectrophotometrically by assuming that the molar absorptivity at 280 nm was 1.4 mL mg-1 cm-1. IgG was fluorescently labeled (A-) using the Alexa Fluor 488 Protein Labeling Kit (Molecular Probes, Inc., Eugene, OR) and then dialyzed into the appropriate sample buffer. The concentration of A-IgG and the molar ratio of Alexa Fluor 488 to IgG (1.9-2 dyes/protein) were determined spectrophotometrically according to the manufacturer’s protocol. The isoelectric points of IgG and A-IgG were measured by isoelectric focusing (BioRad, Hercules, CA) to be 6.9 and 6.5, respectively. Fluorescently labeled antibodies were clarified by air ultracentrifugation (130 000g, 30 min) (Beckman Airfuge, Fullerton, CA) immediately before use. Phospholipid Vesicles. Small unilamellar vesicles were prepared from mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero3-ethylphosphocholine (POethPC), 1-palmitoyl-2-oleoyl-snglycero-3-[phospho-RAC-(1-glycerol)] (POPG), or 1-palmitoyl2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) (Avanti Polar Lipids, Birmingham, AL). Vesicles were prepared by tip sonication of 2 mM suspensions of POPC and 0 or 5 mol % of POethPC, POPG, or POPS in water as previously described.47,48 The fluorescent lipid 1-acyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol4-yl) aminododecanoyl]-sn-glycero-3-phosphocholine (NBDPC) was included in vesicles (2 mol %) intended for photobleaching recovery experiments. Vesicle suspensions were clarified by air ultracentrifugation (130 000g, 30 min) immediately before use. Substrate-Supported Phospholipid Bilayers. Substratesupported planar phospholipid bilayers were formed as previously described.48 Fused silica substrates were cleaned extensively by boiling in detergent (Lot 08778, ICN, Aurora, OH), bath sonicating, rinsing thoroughly with deionized water, and drying at 160 °C. Substrates were cleaned in an argon ion plasma cleaner (15 min, 25 °C) (PDC-3XG, Harrick Scientific, Ossining, NY). Planar bilayers were formed by applying 45 µL of a vesicle suspension to a fused silica substrate (30 min, 25 °C), reapplying

IgG near Planar Membranes 45 µL of the vesicle suspension (1 h, 25 °C), and rinsing with 3 mL of either Tris (5 or 25 mM; pH 5.0, 6.4, 7.2, 8.0, or 9.0; and 0 or 225 mM NaCl), N-(2-hydroxyethyl)piperazine-N′ethanesulfonic acid (Hepes, 5 or 25 mM with 0 or 0.1 mM ethylenediaminetetraacetic acid (EDTA) and 0 or 0.2 µM CaCl2; pH 6.5, 7.0, 7.5, 8.0, or 8.5), or sodium phosphate (5 or 25 mM; pH 6.5, 7.0, 7.5, 8.0, or 8.5) buffer. The samples for use in TIR-FCS or FCS were treated with 400 µL of 30 nM A-IgG in the appropriate buffer. Fluorescence Microscopy. Fluorescence pattern photobleaching recovery (FPPR), TIR-FCS, and traditional FCS measurements were carried out on an instrument consisting of an argon ion laser (Innova 90-3; Coherent, Palo Alto, CA), an inverted microscope (Zeiss Axiovert 35), and a single-photon counting photomultiplier (RCA C31034A, Lancaster, PA). All experiments were carried out at 25 °C using the 488 nm line of the laser. The experimental parameters for FPPR have been described previously.48-50 For TIR-FCS measurements, the laser beam was s-polarized while incident on the fused silica/aqueous interface and generated an evanescent field polarized parallel to the interface. The incidence angle θ was 71° as determined by measuring the optical path of the laser beam. The refractive indices of the Tris buffers were measured using an Atago 1T model DTM-1 refractometer (Tokyo, Japan) with a sodium lamp at 25 °C. The refractive indices were independent of pH, and the average values for 5 mM Tris, 25 mM Tris, and 25 mM Tris with 225 mM NaCl were 1.3341, 1.3350, and 1.3372, respectively. A positive change in refractive index of 0.003 with 225 mM NaCl agrees well with literature values.51 For λ ) 4880 Å and θ ) 71°, these values of n2 imply evanescent wave depths of d ≈ 1020, 1030, and 1050 Å, respectively. The 1/e2 widths of the elliptically shaped evanescent illumination in the sample plane were ≈30 and ≈100 µm. For traditional FCS measurements, the laser beam was focused in the protein solution approximately 20 µm from the bilayer surface to form a small Gaussian-shaped illumination with a radius on the order of s ≈ 1 µm. In both TIR-FCS and focused beam FCS experiments, a pinhole with a radius of 50 µm placed at an internal image plane defined an area with a radius of h ≈ 1.2 µm when projected onto the sample plane. The fluorescence arising from the volume defined by the excitation light and the pinhole was collected through a 60×, 1.4 N.A. objective. The fluorescence signal was autocorrelated by a PC-based correlator board (model 5000/E, ALV). Autocorrelation functions were obtained within 2-10 min using incident laser intensities of 7-27 µW/µm2 for TIRFCS experiments or approximately 1-5 mW/µm2 for FCS experiments using the focused beam. The resulting evanescent intensities at the interface were greater by a factor of ≈2.6.45 Average blank signals were measured from samples containing buffer adjacent to supported bilayers. Data Analysis. TIR-FCS autocorrelation functions were fit to eq 1 plus an arbitrary constant G∞,62 and the free parameters were R, N, and G∞. The diffusion coefficient D of molecules near the membrane in solution was calculated as D ) Rd2. Autocorrelation functions measured using the focused beam were fit to eq 2 plus an arbitrary constant G∞,62 and the free parameters were N, τD, and G∞. All values of N were background-corrected by multiplying the best-fit value by the factor 〈F〉2/〈S〉2, where 〈F〉 ) 〈S〉 - 〈B〉 was the average fluorescence calculated by subtracting the average measured blank signal 〈B〉 from the average measured total signal 〈S〉.46 Dynamic Light Scattering (DLS). DLS experiments were carried out using a custom instrument.52 Samples were passed

J. Phys. Chem. B, Vol. 106, No. 9, 2002 2367 through 0.02 µm filters into dust-free cells, which were 9 mm culture tubes. An argon ion laser (Innova 90-6; Coherent, Palo Alto, CA) operating at 514.5 nm and 150 mW was used as the light source. The experiments were carried out with an IgG concentration of 1.3 µM and at angles ranging from 30-90°. Data were collected and processed using a PC-based correlator board (model 5000/E, ALV). Correlation functions were collected in runs of approximately 15 s, which is approximately 4 orders of magnitude longer than the decay time. Intensity traces were used to discriminate runs that were affected by occasional dust events. Data were analyzed with the ALV software by using the method of cumulants and by a constrained regularization method similar to CONTIN. Results Planar Membranes. Because the processes by which planarsupported membranes form are not yet well-understood, it was necessary to test the different membrane compositions and solution conditions to ensure adequate bilayer formation. Phospholipid bilayers composed of POPC with or without 5 mol % charged phospholipids were deposited onto planar fusedsilica substrates by allowing sonicated phospholipid vesicles to adsorb to and fuse at the surfaces as previously described.47,48 In addition to variations in the phospholipid compositions, the pH of the buffers in the volumes adjacent to the bilayers ranged from 5.0 to 9.0, and the buffer compositions were 5 or 25 mM Tris, 25 mM Tris with 225 mM NaCl, 5 or 25 mM sodium phosphate, or varying concentrations of Hepes in the presence or absence of EDTA and CaCl2. The translational mobility of the fluorescent lipid NBD-PC in the bilayers was monitored by FPPR as described previously to evaluate bilayer integrity.48-50 Supported phospholipid bilayers consisting of pure POPC or POPC with 5 mol % POethPC, POPS, or POPG were fluidlike and appeared uniformly fluorescent within optical resolution over the entire range of Tris buffer compositions. Total fractional mobilities were g95%, and diffusion coefficients were comparable to those observed previously.48,53 In sodium phosphate buffer, adequate membrane formation occurred only for POPC, POPC:POethPC, and POPC:POPG. POPC and POPC:POethPC membranes formed in 5 mM Hepes with 0.15 mM EDTA, but POPC:POPG membranes formed only for Hepes concentrations of at least 10 mM with the calcium concentration equal to 0.2 µM. POPC:POPS membranes did not adequately form for any attempted combination of Hepes, EDTA, and calcium over the entire pH range. Diffusion Coefficients and Local Concentrations of A-IgG Adjacent to Planar Membranes. The diffusion coefficients and local concentrations of A-IgG near planar membranes were measured by TIR-FCS over a broad range of membrane compositions containing zwitterionic, cationic, and anionic phospholipids, each for varying pH (5.0-9.0) and buffer compositions (5 mM Tris, 25 mM Tris, or 25 mM Tris with 225 mM NaCl). When viewed with evanescent illumination through a 1.4 N. A. objective, all samples exhibited visually detectable fluorescence fluctuations that appeared as twinkles against a uniform background. TIR-FCS was also carried out for blank samples for each combination of bilayer and buffer compositions, and there was no measurable correlation function in any case. Fluorescence fluctuations were autocorrelated for 2-10 min. The obtained G(τ) was then fit to eq 1 plus an arbitrary constant, G∞.62 The best-fit values of N, after correction for background, were in the range of 3-8 molecules. The best-fit values of R were between 3 and 6 ms-1, corresponding to values of D

2368 J. Phys. Chem. B, Vol. 106, No. 9, 2002

Figure 2. Representative TIR-FCS autocorrelation function. The data are for 30 nM A-IgG in 25 mM Tris with 225 mM NaCl at pH 6.4 adjacent to a POPC membrane. The fluorescence signal was monitored for 540 s. The average signal 〈S〉 was 9.55 kHz, and the average background intensity 〈B〉 was 0.09 kHz. The best-fit of G(τ) to eq 1 plus a constant G∞ gave N ) 7.57 molecules, R ) 4.06 ms-1, and G∞ ) -0.0073. The background-corrected value of N is 7.43.

ranging from 32 to 61 µm2/s. A typical fluorescence fluctuation autocorrelation function and its best-fit to the theoretical form are shown in Figure 2. To ensure that the TIR-FCS apparatus was responding appropriately to changes in the samples and to identify optimum conditions for further experiments, initial measurements were taken over a range of A-IgG solution concentrations and excitation intensities. As shown in Figure 3, N increased approximately linearly with increasing solution concentrations of A-IgG, and D did not change in the examined concentration range (0.5-100 nM). In addition, both N and D remained constant for incident laser intensities e20 µW/µm2. Photoartifacts (most likely photobleaching, triplet state involvement, or blinking) became apparent at higher intensities as indicated by an increase in D and a decrease in N. Thus, all remaining

Starr and Thompson measurements were carried out for incident intensities equal to 7.2-18.8 µW/µm2. As expected, the average fluorescence 〈F〉 increased approximately linearly with increasing A-IgG concentration, increasing excitation intensity, and increasing N (0.3 e N e 35), and D was independent of 〈F〉 (data not shown). Measurements were taken using pinholes with radii of 50 or 100 µm. The measured diffusion coefficient did not depend on the pinhole size, and N increased by a factor of 2.9 when the pinhole radius increased from 50 to 100 µm. Once the optimum concentrations and other experimental conditions were identified, the diffusion coefficients and local concentrations of A-IgG near supported bilayers containing zwitterionic, anionic, or cationic phospholipids were measured for different buffer compositions (pH 5.0-9.0; 5 mM Tris, 25 mM Tris, or 25 mM Tris with 225 mM NaCl). The bilayer compositions were 100 mol % POPC, or 95 mol % POPC with 5 mol % of POethPC (cationic), POPG (anionic), or POPS (anionic). The measured values of N were plotted as a function of pH and buffer concentration for each bilayer type. The relationship between the local concentration of A-IgG, pH, and buffer concentration was approximately identical for all bilayer compositions.63 The local concentration of A-IgG was approximately constant over the pH range of 5.0 to 9.0 at high ionic strength with an average of N ) 6.1 ( 0.2 molecules in the sample volume (Figure 4). At lower ionic strengths, the local concentration decreased with decreasing pH (9.0 to 6.0), by ≈30%, for all membrane types. The diffusion coefficients close to membrane surfaces were calculated from the best-fit values of R (D ) Rd2) and plotted vs pH and buffer composition for each type of bilayer. The measured, local diffusion coefficients were highest in 5 mM Tris and lowest in 25 mM Tris with 225 mM NaCl and were independent of pH and bilayer composition. Average diffusion coefficients over all samples at the high, intermediate, and low

Figure 3. Calibration measurements. Calibration measurements were carried out on samples consisting of 0.5-100 nM A-IgG in 25 mM Tris, 225 mM NaCl, pH 7.2, adjacent to POPC bilayers. N (a and b) and D (c and d) are plotted over ranges of protein concentrations (a and c) and intensities (b and d). The incident intensity in parts a and c is 11 µW/µm2. The concentration of A-IgG in parts b and d is 20 nM. The average values (lines) were (b) N ) 3.49 molecules, (c) D ) 39.1 µm2/s, and (d) D ) 42.5 µm2/s.

IgG near Planar Membranes

Figure 4. Local concentration of A-IgG near planar membranes. N is plotted as a function of pH for 30 nM A-IgG in 25 mM Tris with 225 mM NaCl adjacent to planar bilayers. Values are averages over all four bilayer types obtained from 40 to 45 autocorrelation functions measured from 8 to 9 different samples, and the error bars represent standard deviations of the means. The average value (line) was 6.1 molecules.

J. Phys. Chem. B, Vol. 106, No. 9, 2002 2369 to eq 2 plus an arbitrary constant. At high ionic strength, the A-IgG concentration as determined by the measured value of N did not change with pH. At low ionic strengths (5 or 25 mM Tris), the A-IgG concentration decreased by ≈45% with decreasing pH. The decay times (τD), and thus the diffusion coefficients, did not significantly change with pH or ionic strength. Diffusion of IgG in Bulk Solution. The diffusion coefficients of IgG in Tris-buffered solutions at varying salt and pH conditions (see Materials and Methods) were measured by DLS. The measured diffusion coefficient of 1.3 µM IgG at 25° did not change with pH or with ionic strength in the range evaluated, except for two conditions (pH 5.0, 5 or 25 mM Tris, without NaCl). The value of D averaged over the remaining pH and ionic strength conditions was 47.2 ( 0.1 µm2/s, which is in agreement with that reported previously.54 Population distribution profiles for all samples except for two (see above) indicated only one peak. Consistently, the measured D varied only slightly with the observation angle, and the plots of the decay rates of the DLS autocorrelation functions vs the squared scattering vector magnitude were highly linear. These results suggest that the IgG in Tris buffer is stable and monodisperse over the pH range of 6.4-9.0 for all three ionic strength values and at pH 5.0 for 25 mM Tris with 225 mM NaCl. On the contrary, DLS data suggested that the IgG at pH 5.0 and low buffer concentrations is susceptible to aggregation as indicated by the population distribution profile as well as slower measured values of D. Therefore, these two buffer conditions were not examined with TIR-FCS. Discussion

Figure 5. Local diffusion coefficients near membranes. The data are for bilayers composed of 95 mol % POPC with 5 mol % POethPC, and the buffer compositions were 5 mM Tris (triangle), 25 mM Tris (circle), and 25 mM Tris with 225 mM NaCl (square). Values are averages obtained from 10 to 15 autocorrelation functions measured from 2 to 3 different samples, and the error bars represent standard deviations of the means. The average values of D (line) were 51 µm2/s (5 mM Tris), 45 µm2/s (25 mM Tris), and 40 µm2/s (25 mM Tris with 225 mM NaCl). Values of D obtained from TIR-FCS data collected adjacent to the other three types of bilayers were identical and are not shown.

ionic strengths were 38 ( 3, 45 ( 5, and 52 ( 5 µm2/s, respectively (Figure 5). To ensure that the measured values of N and D were not dependent on the buffer type, TIR-FCS measurements were also carried out in buffers containing low concentrations of sodium phosphate (5 or 25 mM, pH 6.5-8.5). Planar bilayers containing 5 mol % POPS did not form well in these buffers, so measurements were made only for bilayers composed of POPC, POPC:POPG, and POPC:POethPC. TIR-FCS measurements were also carried out in Hepes buffer (10 mM with 0.3 mM EDTA, pH 6.5-8.5) for POPC and POPC:POethPC bilayers. The dependence of the local A-IgG concentration on pH was identical to that observed for Tris-buffered (5 or 25 mM Tris) solutions for all bilayer conditions in both sodium phosphate and Hepes/EDTA. The local diffusion coefficient of A-IgG decreased significantly with increasing ionic strength in these buffered solutions as well. Diffusion Coefficients and Concentrations of A-IgG Far From Planar Membranes. The characteristics of 3 nM A-IgG and 3 nM A-IgG with 27 nM IgG far from the membrane (∼20 µm) were examined in Tris-buffered solutions by using FCS with a focused laser beam. The values of N and τD were calculated from the best-fits of these autocorrelation functions

The data shown in Figures 2-5 demonstrate the feasibility of using TIR-FCS to measure the average local concentrations and translational mobilities of fluorescent ligands near substratesupported planar membranes. The signal-to-noise ratio of the obtained fluorescence fluctuation autocorrelation functions is remarkably high, in part, because of the small observation volume, which is a consequence of the thin evanescent depth. In addition, the average background signal resulting from total internal reflection illumination is very low as compared to the average signal (≈1-2%). The measured autocorrelation functions G(τ) are fit very well by the theoretical form describing translational diffusion through the evanescent intensity (eq 1). The measured values of the local concentration of ligand, N, and the diffusion coefficient, D, are consistent with expectations. Assuming a concentration of 30 nM, sample volume radius of 1.2 µm, and d ) 1040 Å, approximately 8-9 molecules would be expected in the observation volume. The measured value of N ) 6.1 is reasonably consistent with this prediction. Diffusion coefficients in the range of 38-52 µm2/s are consistent with published values for IgG in aqueous solutions at room temperature54 and with those measured using DLS. The observed decrease in the local concentration at low ionic strengths and low pH was completely accounted for by using FCS with a focused laser beam to measure the bulk concentrations in the mounted samples far from the membrane (≈20 µm). These measurements indicated that significant adsorption to surfaces occurred during sample preparation at the low ionic strengths and pH values. The measured values of the local diffusion coefficient were independent of the pH and membrane charge but changed significantly with the ionic strength. Diffusion coefficients measured at low ionic strengths were in agreement with values measured using DLS, whereas diffusion coefficients measured in the presence of 225 mM NaCl were

2370 J. Phys. Chem. B, Vol. 106, No. 9, 2002 slower by approximately 35%. The decrease in D was not observed in the bulk, either by DLS or by FCS with a focused beam. A possible explanation for the observed lower diffusion coefficients in the presence of high salt concentrations is aggregation of the labeled IgG, but it is highly unlikely that protein aggregates would form more readily at high as compared to low ionic strength. Another possible, but unlikely, explanation is the presence of rapid, reversible adsorption of the protein to the membranes. However, adsorption is more likely to occur at lower ionic strengths and would therefore result in decreased apparent diffusion coefficients at low rather than high ionic strengths. In addition, adsorption would produce concurrent, higher measured values of N as well as a change in the shape of the measured autocorrelation functions,41 and neither of these effects was observed. Previous measurements of protein diffusion in solution by DLS55-61 have shown that diffusion becomes slower with increasing ionic strength, as observed here with TIR-FCS, but the effects are prominent only at much higher protein concentrations (≈10 mg/mL). Therefore, it appears as though the membrane surfaces amplify this trend, making it apparent even at very low protein concentrations (0.0045 mg/mL). The observations in solution have been attributed to a combination of electrostatic and hydrodynamic effects. Finally, the addition of 225 mM NaCl results in a slight (3%) increase in the solvent viscosity, which would subsequently decrease the diffusion coefficient. The presence of the surface may amplify this effect as well by creating local viscosity gradients or changes in the local water structure. It should be emphasized that the values of D obtained by fitting to eq 1 are apparent local diffusion coefficients that are averaged over the depth of the evanescent intensity and may include local, heterogeneous motions that are not purely diffusive in nature. TIR-FCS has been used to measure the local concentration and diffusion of fluorescently labeled IgG adjacent to planar phospholipid membranes for a large variety of membrane and solvent compositions. Even with an evanescent intensity depth of d = 1000 Å, an ionic strength dependence of the average diffusion coefficient of the IgG close to planar membranes was detected. In a previous paper,48 we have demonstrated methods for depositing phospholipid bilayers on substrates with very high refractive indices (=2.5). By using different incidence angles on both fused silica and these high-index substrates (TiO2 and SrTiO3), evanescent intensity depths ranging from =200 to 1100 Å can be generated. Thus, future depth-dependent TIR-FCS measurements are expected to provide significantly more insight into the dynamics of ligands adjacent to membranes. This insight should increase our understanding of the physical factors governing ligand-receptor reaction kinetics. Acknowledgment. We thank Noah Allen for setting up the FCS with the focused beam experiments, Randy Cush for taking the DLS measurements, Dr. Paul Russo for permission to use the DLS apparatus, and Julie Bryant for helping with preliminary fluorescence photobleaching recovery measurements. This work was funded by NSF Grant MCB-9728116. References and Notes (1) Pisarchick, M. L.; Gesty, D.; Thomson, N. L. Biophys. J. 1992, 63, 215. (2) Pearce, K. H.; Hiskey, R. G.; Thompson, N. L. Biochemistry 1992, 31, 5983. (3) Hsieh, H. V.; Thompson, N. L. Biochemistry 1995, 34, 12481. (4) Mayo, K. H.; Nunez, M.; Burke, C.; Starbuck, C.; Lauffenburger, D.; Savage, C. R. J. Biol. Chem. 1989, 264, 17838.

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J. Phys. Chem. B, Vol. 106, No. 9, 2002 2371 (61) Kuehner, D. E.; Heyer, C.; Ra¨msch, C.; Fornefeld, U. M.; Blanch, H. W.; Prausnitz, J. M. Biophys. J. 1997, 73, 3211. (62) The constant G∞ accounts for contributions to G(τ) from slow fluctuations in the average fluorescence arising from processes far from the time range of interest. G∞ was always e0.10 G(0) in magnitude. (63) A small and possibly significant trend in N was observed as a function of the membrane composition. At high ionic strength, the average values of N were 6.6 (POPC/PoethPC), 6.5 (POPC), 5.8 (POPC/POPS), and 6.0 (POPC/POPG). In addition, at low ionic strength, N decreased more significantly with pH for negatively rather than positively charged membranes. These trends suggest the presence of a small amount of nonspecific, electrostatic interactions between the IgG and the membrane surfaces.