A Fluorescent-Photochrome Method for the Quantitative Characterization of Solid Phase Antibody Orientation

Analytical Biochemistry 305, 121–134 (2002) doi:10.1006/abio.2002.5601

A Fluorescent-Photochrome Method for the Quantitative Characterization of Solid Phase Antibody Orientation Arti Ahluwalia,* Danilo De Rossi,* Giuseppe Giusto,* Oren Chen,† Vladislav Papper,† and Gertz I. Likhtenshtein† *Interdepartmental Research Center “E. Piaggio”, via Diotisalvi 2, 56126, Pisa, Italy; and †Department of Chemistry, Ben Gurion University of Negev, P.O. Box 653, 84105 Beersheba, Israel

Received May 14, 2001; published online May 9, 2002

A fluorescent-photochrome method of quantifying the orientation and surface density of solid phase antibodies is described. The method is based on measurements of quenching and rates of cis–trans photoisomerization and photodestruction of a stilbene-labeled hapten by a quencher in solution. These experimental parameters enable a quantitative description of the order of binding sites of antibodies immobilized on a surface and can be used to characterize the microviscosity and steric hindrance in the vicinity of the binding site. Furthermore, a theoretical method for the determination of the depth of immersion of the fluorescent label in a two-phase system was developed. The model exploits the concept of dynamic interactions and is based on the empirical dependence of parameters of static exchange interactions on distances between exchangeable centers. In the present work, anti-dinitrophenyl (DNP) antibodies and stilbene-labeled DNP were used to investigate three different protein immobilization methods: physical adsorption, covalent binding, and the Langmuir– Blodgett technique. © 2002 Elsevier Science (USA) Key Words: total internal reflection; immobilization; Langmuir–Blodgett; stilbene probe; photoisomerization; chromophore; fluorescence quenching; exchange interaction.

Solid phase proteins are utilized in several applications, such as in biosensors, biomaterials, and chromatography. Almost every application requires that the immobilized proteins perform a specific biochemical function, such as the recognition of antigens, substrates, or ligands. Although in most cases the proteins are randomly oriented, it would be advantageous, from the point of view of surface sensitivity and selectivity, for them to be oriented with their recognition sites 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

accessible. Several efforts have been made to orient proteins using covalent chemistry, in particular antibodies (1), enzymes (2), and cell adhesion ligands such as fibronectin (3). Genetic engineering techniques have also been utilized in an attempt to site-specifically attach reactive groups to proteins for oriented covalent immobilization (4). In most cases the activity of oriented solid phase proteins is compared with that of randomly oriented films (usually prepared by physical adsorption), and the activity is usually found to increased, thus providing an indirect means of verifying orientation. Although many physical methods for evaluating the degree of orientation exist, a standard and reliable method is still lacking, and as yet there are no detailed studies on the relative efficiency of various protein orientation methods. Among the techniques available for the characterization of the orientation of solid phase macromolecules are ellipsometry (5), circular dichroism, solid state NMR (6), coupled plasmon-waveguide resonance spectroscopy (7, 8), and polarized X-ray absorption fine structure spectroscopy (9). All these methods can give an estimate of the orientation of the molecule in question as a whole, but cannot provide information on specific areas, for example, on active sites in the case of enzymes and antibodies. The use of a combination of fluorescence labeling and the total internal reflection technique (TIRF) 1 can enable the characterization of an immobilized system by monitoring the kinetics of adsorption of the labeled molecule (5), as well as the fluorescence polarization or fluorescence quenching by a quencher in solution (usually iodide) (10, 11). 1

Abbreviations used: TIRF, total internal reflection technique; DNP, dinitrophenyl; StDNP, stilbene-labeled DNP; PA, physical adsorption; CB, covalent binding; LB, Langmuir–Blodgett; PBS, phosphate-buffered saline; IC, intersystem crossing; ET, electron transfer; ICHA, intersystem crossing transition by heavy atoms. 121

122

AHLUWALIA ET AL.

More sophisticated methods based on the measurement of the second- and fourth-rank order parameters using TIRF spectroscopy have also been used to investigate the distribution and orientation of fluorescent probes (12–14). These methods require special polarization rotator techniques and, therefore, are highly sensitive compared with steady-state fluorescent measurements. In order to use these methods, the experimental data must be processed using complex theoretical algorithms which must be specific to the fluorescent probe used. In this paper we present a simple and sensitive fluorescent-photochrome technique for evaluating the degree of orientation of antibody-binding sites based on the determination of the degree of accessibility of bound fluorescent antigens to a quencher. The technique is used to compare different protein immobilization methods. The fluorescent-photochrome method developed jointly by our two universities utilizes the principle of TIRF to excite the antigen dinitrophenyl (DNP) labeled with a stilbene derivative (StDNP). We used 4-(N-2,4dinitrophenylamino)-4⬘-(N,N⬘-dimethylamino)stilbene which has an enhanced fluorescence at 420 nm after irradiation at its absorption maximum wavelength at 350 nm. The light-induced reversible isomerization of the molecule proceeds from the lowest excited singlet state 1 t* through the twisted singlet intermediate 1 p* or, alternatively, by the intersystem crossing pathway via the biradical twisted triplet state 3 p*. Because the cis form is not fluorescent under the steady-state conditions of our experiment, measuring the rate of decrease of emitted fluorescence can monitor the process of photoisomerization. The fluorescence and photoisomerization of stilbenes has been investigated extensively in the past few decades (15, 16). Stilbene derivatives are relatively easy to synthesize and are generally thermally and chemically stable. Moreover, they possess absorption and fluorescence properties similar to typical biochemical probes that can be monitored by simple optics. In addition, the quantitative study of direct and sensitized photoisomerization of stilbenes opens up new possibilities for the measurement of rotational and translational diffusion, both of which depend on molecular dynamics and steric hindrance in the vicinity of the probe (17–19). The measurements were performed using TIRF, in which the antibody film lies at a reflecting boundary. The evanescent wave generated by total internal reflection at the interface is used to excite stilbene-labeled antigens bound to the antibody film. Upon the addition of KI to the measuring cell, the quenching of fluorescence and the rate of isomerization of stilbene can be monitored and correlated with the accessibility of the antibody-binding site. In fact, the initial intensity and rate of decrease of fluorescence depend on the

local environment of the chromophore and steric hindrance to isomerization and therefore on the orientation of the binding site with respect to the interface. Furthermore, the data on rate constants of dynamic quenching of fluorescence can also be used to estimate the depth of immersion of the stilbene label within the antibody layer. The TIRF system was used in two different configurations. In the first (Configuration I), antiDNP antibodies were immobilized on quartz slides and then exposed to StDNP in the measuring cell, whereas in the second system (Configuration II), antiDNP was first bound to its stilbene-labeled antigen and then immobilized onto the solid phase. Three different antibody immobilization techniques were examined: ● ● ●

physical adsorption (PA); covalent binding to a silane activated surface (CB); Langmuir–Blodgett (LB) films of antibodies (20, 21).

MATERIALS AND METHODS

Preparation of Reagents 4-(N-2,4-Dinitrophenylamino)-4⬘-(N,N⬘-dimethylamino)stilbene was prepared from trans-4-dimethylamino-4⬘-aminostilbene (18) and 2,4-dinitrochlorobenzene (Aldrich Chemicals Ltd.). Equivalent moles of both reagents were dissolved in a minimal volume of absolute ethanol. The resulting clear solution was left overnight. The precipitated brown crystals were collected by vacuum filtration and washed with water and petroleum ether (60 – 80°C). The crude product was recrystallized from chlorobenzene and then dried in a vacuum oven. The product was subjected to elemental and 1H NMR (200 MHz, CDCl 3) analyses, which proved its chemical purity and structural identity. Antibody Immobilization Polyclonal antiDNP antibodies from rabbit serum were purchased from Sigma (Milan, Italy). The specific antibody titer was 1:15. All other reagents were of analytical grade and freely available commercially. Milli-Q water was used for all experiments and, since oxygen is a potential quencher of stilbene, prior to use N 2 was bubbled through the water for several minutes to reduce the concentration of oxygen. Antibodies were immobilized on 2 ⫻ 5 cm 2 quartz slides, which were then coupled optically to the prism base in the TIRF setup. For Configuration II (see Solid Phase Fluorescent Measurements below), StDNP dissolved in DMSO was added to a solution of antiDNP in glycerol and water to give a final concentration of 1.5 mg/ml antibody, 10 ⫺5 M StDNP in 55% water, 43% glycerol, and 2% DMSO (henceforth referred to as the “the standard solvent mixture”). This solution was allowed to stand

FLUORESCENT-PHOTOCHROME FOR ANTIBODY ORIENTATION

for 24 h, after which it was dialyzed extensively against the solvent mixture and then again against phosphate-buffered saline (PBS). The dialyzed solution was then used as a source of antibodies bound with StDNP for immobilization on quartz slides. The water/ glycerol/DMSO composition of the solvent mixture was necessary because StDNP is virtually insoluble in water and the addition of glycerol increases the viscosity of the medium, thus increasing the quantum yield of stilbene and decreasing the kinetics of isomerization, allowing the isomerization process to be easily monitored. The immobilization methods have been described in detail in (20) and only a brief description is given here. Physical adsorption. Clean quartz slides were incubated overnight in 50 ␮g/ml of antiDNP antibody solution in PBS. The slides were rinsed in PBS before incorporation in the TIRF measuring cell. Covalent binding. Clean quartz slides were immersed in a 5% aminopropyltriethoxy solution in propanol and water (about 9:1 v/v) for an hour and then rinsed with propanol and dried under a vacuum. Subsequently, the silanized slides were placed in a 1% glutaraldehyde solution in PBS for an hour and then rinsed copiously with water. The slides were then incubated in 50 ␮g/ml of antiDNP solution in PBS overnight. Langmuir–Blodgett films. LB films were formed on a homebuilt trough specially designed for producing protein monolayers (22). Small drops of antiDNP solution (1.5 mg/ml) were applied to the surface of a phosphate-buffered subphase of pH 6.9. The film was compressed to a surface pressure of 20 mN/m and the antibody films were deposited on glass slides by the Langmuir–Schaeffer method (horizontal touching). For Configuration II antiDNP was previously incubated with StDNP and dialyzed as described and then spread on the gas–water interface. These three immobilization techniques were evaluated in a previous study (20) and shown to give rise to active surface densities of 41 ⫾ 5 ng/cm 2 for PA, 320 ⫾ 40 ng/cm 2 for CB, and 490 ⫾ 100 ng/cm 2 for LB. Fluorescence Measurements Liquid phase fluorescence measurements. An AmincoBowman SLM 4800 GREG-MM multifrequency phasemodulation spectrofluorimeter upgraded by ISS was used for measuring fluorescence intensity, kinetics of reversible trans– cis photoisomerization, and fluorescence polarization of free StDNP and the antiDNP– StDNP complex in solution. A phase-modulation fluorimeter ISS-GREG-90 with modulation frequencies between 20 and 190 MHz was employed for measurements of excited singlet state lifetime. The absorption spectrum of the samples was measured with a Packard

FIG. 1.

123

Schematic representation of the TIRF optical setup.

HP 89532A UV visible spectrophotometer. Analysis of the experimental data was performed using KaleidaGraph 3.0.5 (by Abelbeck software). Measurements were performed in the standard solvent mixture. The fluorescence polarization value and trans– cis isomerization rate constant of StDNP were measured, maintaining the StDNP concentration at 2 ␮M and varying the concentration of antiDNP from 0 to 2 ␮M. Fluorescence emission of StDNP was recorded at ␭ em ⫽ 438 nm after excitation near its absorption maximum at ␭ ex ⫽ 350 nm using a 16-nm slit-width for excitation and emission. All sample solutions were degassed with nitrogen before measurements. The kinetics of reversible trans– cis and cis–trans isomerization reactions of free StDNP were measured by irradiation of the sample at ␭ ex ⫽ 350 nm for 500 s and collection of the emission data at ␭ em ⫽ 438 nm. Then the sample was irradiated at a shorter wavelength of ␭ ex ⫽ 300 nm, where mainly the cis-isomer absorbs, while the emission data were collected again at ␭ em ⫽ 438 nm for an additional 500 s. The fluorescence lifetime of excited singlet state of free and immobilized StDNP was found to be 0.9 and 1.4 ns, respectively. TIRF setup. A schematic representation of the optical bench setup is shown in Fig. 1 and the system is similar to that described in (23). The components used were as follows: ● a 200-W He–Xe lamp (Spectral Energy, Hillsdale, NJ) with an emission peak at 360 nm; ● a bandpass filter (Corion, Franklin, MA) centered at 360 nm, with a bandwidth of 10 nm; ● a specially constructed measuring cell with inlet and outlet permitting laminar flow in the vicinity of the slide; ● a 70° fused quartz prism; ● quartz slides, upon which the antibodies are immobilized, in optical contact with the base of the prism; ● a monochromator set at 420 nm (Spex, NJ); ● an optical multichannel analyzer (Model 1461 EG&G, Princeton, NJ); ● a PC for data acquisition and processing.

124

AHLUWALIA ET AL.

FIG. 2. Antibody, antigen, and quencher interaction in Configuration I and Configuration II (see text).

Light incident on the base of the prism at an angle of 70° undergoes total internal reflection, giving rise to an evanescent wave in the solution. This is capable of exciting fluorescence in molecules within about a wavelength’s distance from the interface, and a small fraction of the total emitted fluorescence is collected by the detection system. Solid Phase Fluorescence Measurements All measurements were performed in the standard solvent mixture. As shown in Fig. 1, the quartz slide with immobilized antibodies made up one wall of the flow cell. The prism–slide–flow cell assembly was screwed in place and mounted on an optical stand such that the light from the lamp was incident on the sloping face of the prism. The cell was then filled with the solvent mixture and a background fluorescent was recorded. All experiments were triplicated. Two different types of configuration were used to assess the orientation of binding sites. In the first, Configuration I, antibodies were immobilized and then exposed to the fluorescent-chromophore labeled hapten. Excess antigens may remain on the surface due to nonspecific binding in this configuration. In the second, Configuration II, antibodies were bound to the hapten prior to immobilization. Configuration I. In the first series of experiments (Fig. 2), 2 ⫻ 10 ⫺5 M of StDNP was injected into the cell and allowed to react for half an hour. The cell was flushed again with the solvent mixture and increasing amounts of KI solution were injected successively into the cell following exposure to the UV source. The final concentrations of iodide ions in the cell ranged from 0.01 to 0.50 M. After waiting a few minutes to ensure that iodide ions diffused to the quartz surface, the shutter was opened and the fluorescence signal measured for about 60 s. Each series of measurements of a single slide took about 2–3 h because the back cis–trans isomerization of StDNP proceeds slowly in the dark, which makes it possible to recover the trans-isomer. Figure 2 illustrates the conceptual differences between Configurations I and II.

Configuration II. Experiments using Configuration II were carried out in a similar manner but without the addition of StDNP to the measuring cell, since the immobilized antibodies were already bound to StDNP (Fig. 2). After the antibody-coated quartz slides were mounted onto the prism and the flow-cell assembly, the cell was filled with the solvent mixture to measure background fluorescence. Iodide solutions were then injected into the flow cell and the fluorescence intensity was measured for 1 min for each iodide concentration. Each data point in the photoisomerization curves represents the total signal integrated over the central 600 (of a total of 1024) photodiodes of the optical multichannel analyzer. In both configurations, the photoisomerization rates for varying concentrations of KI were measured for three samples per immobilization method. Computation Computer simulation was performed on a Power Macintosh 4400 using the following software: PC-Model 5.03 (Serina Software, Bloomington), Ball-and-Stick 3.7 (Oxford, UK), and MacMimic 3.0 (In-Star Software, Lund, Sweden). The 2.9-Å resolution structure of the antiDNP-spin-labeled antibody Fab fragment with bound hapten (antiDNP/4-[5-(2-aminoethylamino)-2,4dinitrophenylamino]-2,2,6,6-tetramethyl-1-piperidinoxyl radical) determined by X-ray crystallography (24) was retrieved from the Brookhaven Protein Data Bank (PDB ID 1BAF, Brookhaven, NY). THEORETICAL GROUNDS

General Collisional quenching is described by the Stern– Volmer equation (25), I 0 /I ⫽ 1 ⫹ k q␶f 关Q兴 ⫽ 1 ⫹ K q关Q兴,

[1]

where [Q] is the concentration of quencher, I 0 and I are the fluorescence intensities in the absence and presence of a quencher, respectively, k q is the bimolecular quenching rate constant, ␶ f is the lifetime of the fluorophore in the absence of the quencher, and K q is the Stern–Volmer quenching constant, which is calculated from a plot of I 0 /I versus [Q]. The effect of interactions between excited and quenching molecules depends on the distance of closest approach of their centers (R 0 ) and, therefore, on the depth of immersion of the center in a matrix. Five main mechanisms of interaction between the excited singlet state of a chromophore and quencher complex are possible during an encounter: 1. singlet–singlet Foerster energy transfer;

125

FLUORESCENT-PHOTOCHROME FOR ANTIBODY ORIENTATION

2. singlet–triplet energy transfer; 3. electron exchange induced intersystem crossing (IC) between singlet and triplet states; 4. electron transfer between a chromophore and a quencher (ET); 5. catalysis of the intersystem crossing transition by heavy atoms (ICHA). In the case of quenching of stilbene fluorescence by iodide the first three mechanisms can be ruled out because the experimental data on quenching do not fit the most important requirement of mechanisms of energy transfer from the fluorophores excited singlet state. The last two mechanisms both involve delocalization of orbitals of interacting particles at distances exceeding van der Waals contacts. Therefore, we can expect that the rate constant of catalysis of the ICHA and the exchange mechanism of IC have a similar dependence on distance. According to the theory of exchange interactions (26, 27), the value of the rate constants of all exchange processes k IC, k TT, and k ET (subscripts refer to the intersystem crossing, triplet–triplet energy transfer, and electron transfer mechanisms, respectively) in solution is affected by the orbital overlaps of interacting particles, which are quantitatively characterized by both the values of the exchange integrals, J IC and J TT, and the resonance integral V ET. The values of exchange parameters are in turn related to the orbital overlap integral S.

k ET ⫽ 10 13 exp关⫺␤ ET共r ⫺ r V兲兴.

The exchange integrals characterize the degree of overlap of molecular orbitals containing the unpaired electrons. J IC,TT,ET values were estimated to be about 10 14 s ⫺1 at the van der Waals distance and decrease exponentially with increasing distance between spins in a vacuum or in homogeneous media (26, 27). The experimental relationship between exchange parameters (in s ⫺1) and the distance (r, in Å) between interacting centers can be approximated by the equation (28, 29) [2]

where r V is the distance at the van der Waals contact and ␤ is a constant. In the case of triplet–triplet energy transfer, for a system in which the centers do not belong to one single molecule and are separated by a homogeneous “nonconducting” medium (for example, in solvents consisting of molecules with saturated chemical bonds), ␤ TT is approximately 2.6 Å ⫺1. To a first approximation, spin exchange involves two orbitals with unpaired electrons, whereas four orbitals are involved in the triplet energy transfer process (orbitals of

[3]

Dynamic Exchange Interactions in Solution According to the theory of dynamic exchange interactions (31) the rate constant of exchange interaction can be expressed as k ex ⫽ P exk d ⫽

f g f nsk dJ 2 ␶ c2 , 共1 ⫹ J 2 ␶ c2兲

[4]

where k d is the rate constant of encounters in solution, P ex is the probability of exchange in the course of the life time (␶ c) of the encounter complex, f g is the geometric steric factor, f ns is the nuclear statistical factor, and J is the integral of corresponding interaction in the encounter complex. If J 2 ␶ c2 Ⰷ 1, k ex is not dependent on J (diffusion limited strong interaction), then d k ex ⫽ f g k d.

Static Exchange Interactions in Solids

k TT, J SE ⬇ 10 14 exp关⫺␤ 共r ⫺ r V兲兴,

the donor in the ground and exited states, and the acceptor in the ground and excited state). Taking this into consideration we can estimate ␤ SE for nonconducting media to be about 1.3 Å ⫺1. Since three orbitals are involved in the intersystem crossing under effect of a paramagnetic or a heavy atom, we can expect ␤ IC ⫽ ␤ ICHA ⫽ 2 Å ⫺1. According to empirical data collected in (28, 30), the relationship between the rate constant of electron transfer and the distance between donor and acceptor centers is

[5]

In the case of kinetic limited weak interactions, J 2␶ c2 Ⰶ 1, k ⫽ f gk dJ 2 ␶ c2. k ex

[6]

Substituting the definition of J from Eqs. [2] and [3] with ␤ ICHA and ␤ ET in Eq. [6] gives an equation for the rate constant, k ET, k ICHA ⫽ f g k d␶ c210 a exp共2关⫺␤ET,ICHA共R 0 ⫺ r V兲兴兲,

[7]

with a ⫽ 28 and 26 for of intersystem crossing under effect of a heavy atom and electron transfer, respectively. If the quenching of fluorescence or phosphorescence occurs by ET or ICHA mechanisms, the quenching rate constant k q ⫽ k ex. Depth of Immersion of a Chromophore The distance of closest approach R 0 derived from Eq. [7] can be taken as the depth of immersion of a fluorescent or phosphorescent chromophore in a biological matrix.

126

AHLUWALIA ET AL.

The ratio of the quenching rate constants for a chromophore– quencher pair in solution (diffusion limited), k qd, and after docking in a matrix (kinetic limited), k qk, is

If k 1 Ⰷ k 2 and [Q] Ⰶ 1/k 1 ␶ f, ␣ (Q) tends to ␣ 0 and the initial slope of the Stern–Volmer plot is 兵d共I 0 /I兲/d关Q兴其 0 ⫽ ␣ 0 k 1 ␶ f.

k qk /k qd ⫽ ␶ c210 a exp 2关⫺␤ 共R 0 ⫺ r V兲兴,

If k 1 Ⰷ k 2 and [Q] Ⰷ 1/k 1 ␶ f, ␣ (Q) tends to 0, and

and the depth of immersion of the center under investigation can be expressed as 共R 0 ⫺ r V兲 ⫽ 0.5 ␤ ⫺1 关ln共k qd /k qk兲 ⫹ ln共 ␶ c210 a 兲兴.

[9]

␤ ⫽ 2 and 1.3 Å ⫺1 and a ⫽ 28 and 26 for the intersystem crossing and electron transfer mechanisms, respectively. Equation [9] can be used to give an estimate of the depth of immersion of a fluorescent chromophore in a nonconductive matrix using experimentally measured values of the ratio k qd/k qk and known or estimated values for the lifetime of the encounter complex, ␶ c, and the parameter ␤. Fluorescence Quenching in a Two-Phase System In any solid phase system under investigation, fluorophores (native chromophores in proteins, fluorescence probes, etc.) can be immersed at different distances from the surface (10 and references therein). The characteristic feature of the Stern–Volmer plots in such circumstances is their downward curvature. Then in the case of two quenched targets with different quenching rate constants k 1 and k 2 , the reciprocal of the fractional steady state fluorescence (I 0 /I) is given by the Stern–Volmer equation modified as in the method described by Lakowicz (25). I 0 /I ⫽ 1 ⫹ 关 ␣ 共Q兲 k 1 ⫹ ␤ 共Q兲 k 2 兴 ␶ f 关Q兴,

[11]

where ␣ (Q) and ␤ (Q) are the fractions of fluorophores with quenching constants k 1 and k 2 , respectively. Both ␣ (Q) and ␤ (Q) are functions of the quencher concentration. Equation [11] is valid if the magnitude of the fluorescence lifetime for both fractions is similar. Considering that for each quencher concentration, the sum ␣ (Q) ⫹ ␤ (Q) ⫽ 1, we have I 0 /I ⫽ 1 ⫹ 关 ␣ 共Q兲 共k 1 ⫺ k 2 兲 ⫹ k 2 兴 ␶ f 关Q兴.

[12]

␣ (Q) can be written in terms of ␣ 0, the fraction of fluorophores with the rate constant k 1 in the absence of quencher, as ␣ 共Q兲 ⫽ ␣ 0

1 . 1 ⫹ k 1 ␶ f 关Q兴

[14]

[8]

[13]

I/I 0 ⫽ 1 ⫹ k 2 ␶ f 关Q兴.

[15]

Using Eqs. [12]–[15], the apparent rate constants ␣ 0 k 1 and ␣ 0 k 2 can be calculated from an experimental Stern–Volmer plot if ␶ f is known. RESULTS AND DISCUSSION

Computer-Assisted Molecular Modeling Computer-assisted molecular modeling was utilized to demonstrate the location of StDNP within the antiDNP-binding site. We critically examined the possible rotation of the stilbene aromatic fragments around the central single bond within the antibody binding site in the trans– cis photoisomerization process and its accessibility to the iodide ion quencher. The energetically accessible conformations of StDNP were searched using the GMMX subprogram of PCModel running on a Power Macintosh 7600/120 workstation. Using the MacMimic program it was established that due to the unsymmetrical 2,4-dinitro substitution pattern of the hapten aromatic ring, the trans and the cis StDNP conformers can dock in two possible modes within the DNP-binding site of the antibody molecule. According to the computer simulation, the twisting of the stilbene fragments in the excited state is not sterically hindered and there is free access of the octahedral iodide– hexahydrate complex to the stilbene moiety StDNP label in solution. However, the encounter probability between iodide ions and the bound StDNP label is greatly reduced as a result of steric hindrance by the antiDNP antibody. According to the model schematized in Fig. 3, a “geometric” steric factor of encounters (␰ ⫽ 0.185) was calculated. Taking into consideration the solvent “cage effect” for encounters in the diffusion limit (32) the “apparent” steric factor, ␰ app ⫽ (␰) 1/2 was estimated to be 0.43. This implies that the degree of quenching of bound StDNP is expected to be 2.3 times less than that of the free label in solution. The experimental data were obtained using polyclonal rabbit antiDNP antibodies, while the computerassisted molecular modeling utilized a structural model of the monoclonal antibody. Nevertheless, data on the antiDNP antibody binding sites obtained by physical methods indicate that there is a structural resemblance between the mono- and polyclonal proteins, as well as between different clones. For example,

FLUORESCENT-PHOTOCHROME FOR ANTIBODY ORIENTATION

127

FIG. 3. Model of a segment-like cavity of the antiDNP-binding site (A) and (B) possible accessibility between iodide ions and the free StDNP label (the whole sphere) relative to the bound label (the segment circumscribed between the two straight lines with an angle ␣ ⫽ 102°).

the 2,4-dinitrophenyl hapten undergoes similar characteristic spectral shifts of the absorption and circular dichroism spectra when bound specifically by monoand polyclonal antiDNP antibodies (33, 34). The analysis of NMR spectra of anti-dinitrophenyl-spin-label Fab fragments of monoclonal murine antibodies AN0 1–12 with a bound spin-label hapten shows tryptophan moieties in the vicinity of the hapten (34 –37). These finding were confirmed by an X-ray structural model of a Fab fragment from a different clone (AN02) which placed the DNP fragment in a pocket formed by the hypervariable loops with the DNP ring sandwiched between the indols rings of Trp91 L and Trp96 H (24). Furthermore, the investigation of the complex between spin-label DNP 12 monoclonal anti-dinitrophenyl antibodies indicated the simplicity of double difference NMR spectra and the amino acid sequence of the variable regions of different antibodies (36).

sented in Fig. 5. As shown, the enhancement of the polarization value with an increase of the StDNP/antiDNP concentration ratio, demonstrating the formation of the StDNP–antiDNP complex, does not drastically affect the photoisomerization rate of stilbene. The apparent binding constant K b for antiDNP–StDNP was found to be 1.5 ⫻ 10 6 M, which is close to the literature value of the constant K b ⫽ 2.8 ⫻ 10 6 M for binding of a spin-labeled derivative of DNP to monoclonal antiDNP antibodies (38). The Stern–Volmer plot for fluorescence quenching by iodide is found to be approximately linear up to I ⫺ ⫽ 0.06 M with a quenching constant K q ⫽ (1.8 ⫾ 0.07) M ⫺1. In conformity with the experimental value of the StDNP fluorescence lifetime of 0.9 ns, the quenching rate constant k qd ⫽ (2.0 ⫾ 0.03) 10 9 M ⫺1 s ⫺1. Further addition of the quencher led to deviation from linearity

StDNP–AntiDNP Complex in Solution The kinetics of reversible trans– cis and cis–trans photoisomerization of StDNP in solution, studied by fluorescence techniques, are shown in Fig. 4. The values of apparent rate constants of trans– cis and cis– trans photoisomerization were found to be k t3c ⫽ (9.2 ⫾ 0.1) ⫻ 10 ⫺3 s ⫺1 and k c3t ⫽ (5.1 ⫾ 0.1) ⫻ 10 ⫺3 s ⫺1, respectively. These values are very close to the values for free StDNP, indicating that the computer model demonstrating sterically free isomerization is qualitatively correct. Addition of antiDNP to the StDNP solution led to a gradual increase of the fluorescence polarization. The plots of the polarization value and photoisomerization rate constant versus the StDNP/antiDNP ratio are pre-

FIG. 4. Reversible trans– cis and cis–trans photoisomerization of the StDNP label (2 ⫻ 10 ⫺6 M) in “the standard solvent mixture.”

128

AHLUWALIA ET AL.

FIG. 5. Fluorescence polarization (left Y-axis) and photoisomerization rate constant (right Y-axis) of StDNP versus [antiDNP]/[StDNP] molar ratio in “the standard solvent mixture.”

cal behavior of the excited stilbene molecules may be fitted to two first-order exponential decay curves. The first is the reversible trans– cis photoisomerization of the excited stilbene molecules with an apparent rate constant, which is the sum of the positive forward (k t3c) and negative back (k c3t) rate constants. The second exponential decay appears after approximately 30% of photoconversion and is most probably a result of photodestructive processes. We define the degree of photoconversion as ␥ ⫽ 1 ⫺ I p/I 0 , where I 0 and I p are the experimental initial and final (for t ⫽ 45 s) fluorescence intensities at the steady-state approximations, respectively. According to the data on kinetics of the fluorescence decay, the LB films of antibodies are a fairly wellordered system. The degree of photoconversion, ␥, of StDNP at a KI concentration of 0.25 M is equal to 0.24

owing to photodestructive processes. Because the viscosity of the solvent mixture is about 6 cP for this solution, the k qd value is close to the rate constant in the diffusion limit. Taking into account the theoretically calculated value of the apparent steric factor ␰ app ⫽ 0.43 for the StDNP–antiDNP complex and the abovementioned rate constant of encounters in the solution, we calculated a theoretical bimolecular rate constant of fluorescence quenching of the complex by iodide ions to be k qd ⫽ (0.86 ⫾ 0.03) 10 9 M ⫺1 s ⫺1. Immobilized Systems Of the three immobilized systems, the LB method for solid phase immobilization of antibodies has been least investigated, although it has been shown that at a pressure of 20 mN/m and above, antibodies are densely packed and oriented in a random up– down manner at the gas–water interface (21, 39). Physical adsorption of antibodies on glass or quartz gives rise to low surface densities, antibody orientation is casual, and the adsorption may be reversible in the presence of detergents or chaotropic agents (20). Covalent binding produces higher surface densities, although values in the literature vary greatly. In general, surface phase antibodies may possess various orientations with respect to the surface and this may also vary with the immobilization method. Various theoretically possible arrangements of solid phase antibodies bound to a fluorescent probe and their corresponding Stern–Volmer plots are shown in Fig. 6. Configuration I. Figures 7 and 8 show, respectively, the decay of the StDNP fluorescence (I) and the experimental Stern–Volmer plots for quenching of the initial fluorescence (I 0 ) of the StDNP–antiDNP complex with iodide ions in Configuration I. The fluorescence decay kinetics which represent the photochemi-

FIG. 6. A. Possible arrangements of solid phase antibodies bound to a fluorescent antigen. (a) A totally ordered system; (b) a partially ordered system; (c) a completely disordered system. B. Predicted Stern–Volmer plots for (a) a totally ordered system; (b) a partially ordered system; (c) a completely disordered system.

FLUORESCENT-PHOTOCHROME FOR ANTIBODY ORIENTATION

129

FIG. 7. Fluorescence decay kinetics of StDNP bound to immobilized antiDNP measured in the presence of potassium iodide in Configuration I. (A) Physical adsorption; (B) covalent binding; (C) LB films.

and is largely independent of the quencher concentration. In the case of physical adsorption and covalent binding, which are less ordered, the value of ␥ is strongly dependent on the iodide concentration and in both cases attains values of around 0.9. The photode-

FIG. 8. Stern–Volmer plot for quenching fluorescence of StDNP bound to antiDNP immobilized by different methods (Configuration I).

struction rate of excited stilbene molecules was found to be much higher in antiDNP layers immobilized by covalent binding or physical adsorption techniques than in LB films (Fig. 7). As was shown in the section entitled StDNP–AntiDNP Complex in Solution (Fig. 3), in the StDNP–antiDNP complex, the stilbene molecule in the excited state is not restricted in twisting about the central double bond. However, the photoisomerization may be restricted by steric hindrances of immobilized antibodies. The apparent rate constants of trans– cis photoisomerization of StDNP are given in Table 1. According to the table, the isomerization rate constant is largely independent of the quencher concentration. The isomerization rates of antibodies immobilized by physical adsorption and covalent binding are almost double that of LB films, indicating an increase of microviscosity in the latter system. The Stern–Volmer plot was obtained by calculating the I 0 /I values at all [Q] for all three samples pertaining to each immobilization method, and the error bars represent their mean and standard deviations. As can be seen from Fig. 8, at quencher concentrations [Q] ⬍

130

AHLUWALIA ET AL. TABLE 1

Trans– cis Photoisomerization Rate Constants of StDNP Bound to Immobilized AntiDNP Antibodies (Configuration I) Trans–cis photoisomerization rate constant, k t3c (s ⫺1) KI concentration (M)

Physical adsorption

Covalent binding

LB films

0.010 0.075 0.100 0.250

0.121 ⫾ 0.005 0.092 ⫾ 0.004 0.095 ⫾ 0.004 0.091 ⫾ 0.004

0.099 ⫾ 0.004 0.113 ⫾ 0.005 0.082 ⫾ 0.004 0.087 ⫾ 0.004

0.055 ⫾ 0.002 0.068 ⫾ 0.003 0.059 ⫾ 0.002 0.057 ⫾ 0.002

0.06 M, the Stern–Volmer plot is approximately linear. Following (10, 25, 40) we can take this to imply that the fluorescent probe is quenched by dynamic mechanisms. The values of the apparent rate constants k app ⫽ ␣ 0 k d were calculated using Eq. [13] and approximating the initial slope to a straight line using a least squares fit, with ␶ f ⫽ 1.4 ns. k app values are 0.9 ⫾ 0.2. 10 9 M ⫺1 s ⫺1 for the LB system and 1.3 ⫾ 0.2. 10 9 M ⫺1 s ⫺1 for physical adsorption and covalent binding. The STDNP antibody in solution has a k qd ⫽ (0.86 ⫾ 0.03) 10 9 M ⫺1 s ⫺1 similar to that of LB films, indicating that they are more stable and less prone to photodegradation processes as shown in Fig. 7. At higher iodide concentrations, [Q] ⬎ 0.06, the Stern–Volmer plots for physically adsorbed and covalently bound films tend to curve slightly downward, whereas the LB data have a markedly positive slope in comparison. Stern–Volmer plots with slightly positive slopes are usually said to be related to the static or “quenching sphere of action” mechanisms (25). Nevertheless, given that the gradient is higher than expected in such cases, in the case of the LB film it is most probably caused by the photodegradation of a certain fraction of StDNP accessible to the solution phase, while fluorophores located inside the antibody matrix are protected and tend to retain their stability. Thus, in this series of experiments, the physically adsorbed and covalently bound films are characterized by fast fluorescence decay of the StDNP molecules. This means that in these films there is a large fraction of stilbene molecules that are bound nonspecifically to the quartz surface. These nonspecifically bound stilbene molecules are activated to photodestruction in the presence of iodide ions. LB films of antibodies restrict the trans– cis photoisomerization of a sizeable fraction of specifically bound stilbene molecules and prevent their photodestruction in the presence of iodide ions. Configuration II. In the second series of experiments, the StDNP–antiDNP complex was immobilized by PA, LB, and CB techniques as described under Materials and Methods. The degree of photodestruc-

tion, ␥, is markedly less than that in Configuration I and increases in the following order PA (␥ ⫽ 0.020) ⬎ CB (␥ ⫽ 0.14) ⬇ LB (␥ ⫽ 0.15) (Fig. 9). With respect to Configuration I, ␥ does not depend significantly on the quencher concentration, because there are no nonspecifically bound antigens on the surfaces. The experimental Stern–Volmer plots for fluorescence quenching of the StDNP–antiDNP complex with iodide ions, shown in Fig. 10, indicate that the dependence of fluorescence quenching on the iodide concentration is similar for the different immobilization techniques. They do not fit the Stern–Volmer equation modified by Lehrer (40) where only one fraction of fluorophore is accessible for quenching, and as a result (I 0 /I) is independent of [Q] at high quencher concentrations. The shape of the experimental Stern–Volmer plot in Fig. 10 corresponds qualitatively with the model in Section 3.5, which describes the different accessibilities of different fractions characterized by different quenching rate constants (Eqs. [11] and [12]). At low concentrations of the quencher (less than 0.1 M), the value of the apparent quenching constant was found by approximating the initial slope to a straight line using a least squares fit. Using Eq. [14] the quenching constant ( ␣ 0 K (Q)) was found to be in the range 1.2–1.8 ⫾ 0.2 M ⫺1. At higher concentrations of iodide ions (greater than 0.1 M), the data were again approximated to a straight line using the least squares method and the quenching constant calculated from Eq. [15]. The values are much lower: 0.04 – 0.07 ⫾ 0.02 M ⫺1. Taking into account the experimental fluorescence lifetime for StDNP when bound to the antibody (␶ f ⫽ 1.4 ns) and above-mentioned values of ␣ 0 K q(I), the apparent quenching rate constants ( ␣ 0 k q) were calculated (Table 2). At low concentrations of KI the apparent quenching rate constant ␣ 0 k q(I) is in the range (0.9 –1.3) 10 9 M ⫺1 s ⫺1, which is close to the value of k q (0.86 ⫾ 0.03) 10 9 M ⫺1 s ⫺1) expected for the free StDNP– antiDNP complex in solution (see StDNP–AntiDNP Complex in Solution). We can take this value of ␣ 0 k q to represent the rate constant for quencher interaction with a fraction of StDNP bound to immobilized antiDNP oriented with the binding site freely accessible. For this fraction of antibodies, quenching of the bound fluorescent-chromophores occurs in the diffusion limit ( ␣ 0 k q(I) ⬇ k qd) because they are exposed to the solution and can be easily approached by iodide ions. We call the fraction of oriented antibodies giving rise to this type of interaction Fraction I. At high concentrations of iodide (0.1– 0.5 M) the quenching process takes place in the kinetic limit because the rate constants (k qk ⫽ ((0.03– 0.04) ⫾ 0.02) 10 9 M ⫺1 s ⫺1) were found to be lower than k qd. These values can be taken to correspond to quenching rates for the fraction of randomly ordered antiDNP in which the

FLUORESCENT-PHOTOCHROME FOR ANTIBODY ORIENTATION

131

FIG. 9. Fluorescence decay kinetics of StDNP in the immobilized antibody–antigen complex, in Configuration II, measured in the presence of potassium iodide. (A) Physical adsorption; (B) covalent binding; (C) LB films.

StDNP molecules are more shielded from quenching by iodide ions, Fraction II. Values of depth of chromophore immersion (R 0 ⫺ r V) for different quenching mechanisms, calculated using Eq. [9] with an experimental ratio (k qd/ ␣ 0 k q(I)) and a lifetime of the encounter complex, ␶ c ⫽ 3 ⫻ 10 ⫺10 s, are also presented in Table 2. As shown in the table, (R 0 ⫺ r V) ⬎ 6 Å and ⬎7.0 Å for ICHA and ET quenching mechanisms, respectively. Given that the two quenching rate constants correspond, respectively, to the more oriented (Fraction I) and less oriented (Fraction II) portions of the immobilized layer, then the fluorescent intensity corresponding to the two segments of the Stern–Volmer plot (above and below 0.1 M) must be also proportional to the number of molecules in the two fractions. In Table 2, the relative weight of the fractions for different methods of immobilization in the second series of experiments are presented. As one can see from the table, the contribution of the more ordered fraction is about 63–70% and is slightly higher for the antibodies immobilized by the LB method. Thus, in the remaining 37 to 30% of the complexes, or Fraction II, the stilbene group is prevented from direct encounters with iodide ions by “a shield” of about 7.0 Å (Table 2). This observation confirms previous findings (21) which

indicate that LB films of antibodies are capable of furnishing high surface densities with at least 50% of the antibodies oriented with their binding sites directed away from the solid substrate. As reported in Table 3, the trans– cis photoisomerization rate constants are approximately equal for all systems under investigation in this series of experiments; therefore, the photoisomerization process in the immobilized systems does not encounter serious steric hindrance in the vicinity of the antibody-binding site. As with almost all solid phase analytical methods for investigating proteins, the techniques described here are unable to distinguish between molecules that are denatured and those which are “facing the wrong way.” This is particularly true for Configuration I because although nonspecific binding can be recognized by high rates of photodestruction, there is no way of distinguishing a nonreactive antibody from a randomly oriented one. As far as Configuration II is concerned, it is important to stress that the ordered and randomly oriented fractions of antibodies refer only to those receptors that are still capable of recognizing and binding to the antigen. Denatured or nonspecific antibodies do not contribute to the fluorescent signal.

132

AHLUWALIA ET AL.

FIG. 10. Stern–Volmer plot for quenching fluorescence of the StDNP–antiDNP complex for the different immobilization techniques in Configuration II.

CONCLUSION

A fluorescent-photochrome method of quantitative characterization of immobilized antibodies based on phenomena of fluorescence quenching of a labeled hapten in the binding site of immobilized antibodies is described. The method allows the measurement of depth of immersion of fluorescence chromophores in the object under investigation, their distribution in the object, and the rate of photoisomerization and degree of photodegradation. Together these experimental parameters characterize the degree of orientation of binding sites of antibodies immobilized onto a surface.

The proposed method was applied to study of a stilbene derivative of DNP (StDNP) bound to antiDNP antibodies (antiDNP). The antibodies were immobilized using three different methods, namely, physical adsorption, covalent binding, and via LB films. Iodide ions were used as the quencher. Two experimental configurations for analyzing the immobilized systems were developed. Configuration I involves immobilization of antiDNP followed by exposure of the antibody-binding site to StDNP, whereas in Configuration II, StDNP–antiDNP is prepared in solution and then is immobilized on a quartz slide. For all

TABLE 2

Results of StDNP Fluorescence Quenching after Binding to AntiDNP (Configuration II) (R 0 ⫺ R V) (Å)

LB b PA CB

␣ 0 k q (I), 10 9 M ⫺1 s ⫺1

␣ 0 k q (II), 10 9 M ⫺1 s ⫺1

ICHA

ET

␩ app a

0.9 ⫾ 0.2 1.3 ⫾ 0.2 0.9 ⫾ 0.2

0.04 ⫾ 0.02 0.04 ⫾ 0.02 0.03 ⫾ 0.15

5.5 ⫾ 1.2 5.5 ⫾ 1.2 6.0 ⫾ 1.3

7⫾2 7⫾2 7⫾2

0.70 ⫾ 0.20 0.67 ⫾ 0.20 0.63 ⫾ 0.20

Note. ␣ 0 k q (I) and ␣ 0 k q (II) are the apparent rate constants of quenching for iodide concentrations in the range 0 to 0.1 M (Fraction I) and 0.1 to 0.4 M (Fraction II), respectively. (R 0 ⫺ r V) is a depth of immersion of StDNP for quenching by mechanisms of intersystem crossing (ICHA) and electron transfer (ET), respectively, in Fraction II. The values were calculated using Eq. [9] with ␤ ICHA ⫽ 2 Å ⫺1 and ␤ ET ⫽ 1.3 Å ⫺1. ␩ app is the apparent fraction of StDNP bound to highly oriented antiDNP antibodies (corresponding to Fraction I). a ␩ app is defined as a ratio of fluorescence intensity from Fraction I to the fluorescence intensity at the highest concentration of the quencher [KI] ⫽ 0.5 M. b LB, PA, and CB are the methods of immobilization as given in the text.

FLUORESCENT-PHOTOCHROME FOR ANTIBODY ORIENTATION

133

ACKNOWLEDGMENTS

TABLE 3

Trans– cis Photoisomerization Rate Constants of StDNP Bound to AntiDNP (Configuration II) Trans–cis photoisomerization rate constant, k t3c (s ⫺1) Concentration of potassium iodide (M)

Physical adsorption

Covalent binding

LB films

0.010 0.050 0.075 0.100 0.250 0.500

0.075 ⫾ 0.005 0.071 ⫾ 0.005 — 0.077 ⫾ 0.005 0.079 ⫾ 0.005 0.067 ⫾ 0.005

0.083 ⫾ 0.006 0.091 ⫾ 0.006 0.067 ⫾ 0.005 — 0.069 ⫾ 0.005 —

0.063 ⫾ 0.004 0.070 ⫾ 0.005 0.070 ⫾ 0.005 0.077 ⫾ 0.005 0.079 ⫾ 0.005 0.071 ⫾ 0.005

The Ben-Gurion University group thanks the German–Israeli James Frank Program on Laser–Matter Interactions and the Harry Stern Applied Program for valuable financial support.

REFERENCES

samples the Stern–Volmer plots exhibit deviations from linearity, indicating that stilbene is not uniformly accessible to the quencher and, therefore, that the antibody-binding sites have different orientations relative to the solid phase. The resistance to photodestruction in the presence of iodide is different for the different immobilization methods. It was also observed that the rate constants of the trans– cis photoisomerization of StDNP molecules bound to immobilized antiDNP in Configuration II were found to be similar to those in solution, indicating that steric hindrance in the region of binding sites after immobilization is minimal. In Configuration I samples of StDNP bound to the antibody after immobilization using PA and CB undergo strong photodestruction in the presence of iodide, while LB films prevent the destruction. If formation of the complex preceded immobilization (as in Configuration II), the photodestructive process was damped in all cases. The phenomenon of photodestruction was attributed to the presence of nonspecifically bound StDNP. Analysis of the Stern–Volmer plots using the theory of the depth of immersion showed that in samples immobilized in Configuration II a fraction of the StDNP chromophores is easily available to the quencher (about 63–70% depending on the method of immobilization) and the remainder are embedded in the matrix at a depth of about 7 Å. The first fraction, which we called Fraction I, is related to the more highly ordered part of the immobilized system and corresponds to antibodies with their binding sites oriented away from the quartz surface. The theoretical considerations and experimental data presented in this work demonstrate that this fluorescent-photochrome method is suitable for the characterization of antibodies immobilized on solid surfaces. It can also be used for characterizing the physical parameters of a wide range of immobilized systems, such as proteins, enzymes, lipids, and polymer films.

1. Ahluwalia, A., Carra, M., De Rossi, D., Ristori, C., Tundo, P., and Bomben, A. (1994) Improvement of antibody surface density by orientation of reduced fragments. Thin Solid Films 247, 244 – 247. 2. Rao, S. V., Anderson, K. W., and Bachas, L. G. (1998) Oriented immobilization of proteins. Mikrochim. Acta 128(3– 4), 127–143. 3. Fitchmun, M. I., Falk Marzillier, J., Marshall, E., Cruz, G., Jones, J. C. R., and Quaranta, V. (1998) Mode of adsorption and orientation of an extracellular matrix protein affect its cell-adhesion-promoting activity. Anal. Biochem. 265(1), 1–7. 4. Huang, W., Wang, J. Q., Bhattacharyya, D., and Bachas, L. G. (1997) Improving the activity of immobilized subtilisin by sitespecific attachment to surfaces. Anal. Chem. 69(22), 4601– 4607. 5. Malmsten, M., Lassen, B., Holmberg, K., Thomas, V., and Quash, G. (1996) Effects of hydrophilization and immobilization on the interfacial behavior of immunoglobins. J. Colloid Interface Sci. 170, 70 –78. 6. Vogt, B., Ducarme, P., Schinzel, S., Brasseur, R., and Bechinger, B. (2000) The topology of lysin-containing amphipatic peptides in bilayers by circular dichroism, solid-state NMR, and molecular modeling. Biophys. J. 79, 2644 –2656. 7. Salamon, Z., and Tollin, G. (2001) Optical anisotropy in lipid bilayer membranes: Coupled plasmon waveguide resonance measurements of molecular orientation, polarizability, and shape. Biophys. J. 80, 1567. 8. Kneller, L. R., Edwards, A. M., Nordgren, C. E., Blasie, J. K., Berk, N. F., Krueger, S., and Majkrzak, C. F. (2001) Hydration state of single cytochrome c monolayer on soft interfaces via neutron interferometry. Biophys. J. 80, 2248 –2261. 9. Edwards, A. M., Zhang, K., Nordgren, C. E., and Blasie, K. (2000) Heme structure and orientation in single monolayers of cytochrome c on polar and nonpolar soft surfaces. Biophys. J. 79, 3105–3117. 10. Horsley, D., Herron, J., Hlady, V., and Andrade, J. D. (1991) Fluorescence quenching of adsorbed hen and human lysozyme. Langmuir 7, 218 –222. 11. Chang, I., and Herron, J. (1995) Orientation of acid-pretreated antibodies on hydrophobic dichlorodimethylsilane-treated silica surface. Langmuir 11, 2083–2089. 12. Bos, M. A., and Kleijn, J. M. (1995) Determination of the orientation distribution of adsorbed florophores using TIRF. I. Theory. Biophys. J. 68, 2566 –2573. 13. Bos, M. A., and Kleijn, J. M. (1995) Determination of the orientation distribution of adsorbed florophores using TIRF. II. Measurement on porphyrin and cytochrome c. Biophys. J. 68, 2573– 2579. 14. Yang, J., and Klein, J. M. (1999) Order in phospholipid Langmuir–Blodget layers and the effect of electrical potential of the substrate. Biophys. J. 76, 323–332. 15. Waldeck, D. H. (1991) Photoisomerization dynamics of stilbenes. Chem. Rev. 91, 415– 436. 16. Saltiel, J., and Sun, Y. P. (1990) in Photochromism—Molecules and Systems (Durr, H., and Bouas-Laurent, H., Eds.), pp. 64ff, Elsevier, Amsterdam. 17. Likhtenshtein, G. I., Bishara, R., Papper, V., Uzan, B., Fishov, I., Gill, D., and Parola, A. H. (1996) Novel fluorescence-photo-

134

18.

19.

20.

21.

22.

23.

24.

25. 26.

27. 28.

29.

AHLUWALIA ET AL.

chrome labeling method in the study of biomembrane dynamics. J. Biochem. Biophys. Methods 33, 117–133. Papper, V., Likhtenshtein, G. I., Pines, D., and Pines, E. (1997) Photophysical and photochemical characterization of para-substituted stilbenes. Photochem. Photobiol. A Chem. 111, 87–96. Papper, V., Likhtenshtein, G. I., Medvedeva, N., and Khoudyakov, D. V. (1999) Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals. Photochem. Photobiol. A Chem. 122, 79 – 85. Ahluwalia, A., De Rossi, D., Ristori, C., Schirone, A., and Serra, G. (1992) A comparative study of protein immobilization techniques for immunosensors. Biosensors Bioelectronics 7, 207–213. Ahluwalia, A., De Rossi, D., and Schirone, A. (1992) Antigen recognition properties of antibody monolayers. Thin Solid Films 210/211, 726 –728. Giovannini, E. (1995) Design and Construction of a Langmuir Trough for Deposition of Protein Monolayers. Graduate Thesis, University of Pisa, Department of Electronic Engineering. Celebre, M., Domenici, C., Francesconi, R., Ahluwalia, A., and Schirone, A. (1992) A comparative study of efficiencies of fiber optic and prism TIRF sensors. Meas. Sci. Technol. 3, 1166 –1170. Brunger, A. T., Leahy, D. J., Hynes, T. R., and Fox, R. O. (1991) 2.9 Å resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibodie Fab fragment with bound hapten. J. Mol. Biol. 221, 239 –256. Lakowicz, J. R. (1983) Principle of Fluorescence Spectroscopy. Plenum, New York. Zamaraev, K. I., Molin, Yu. N., and Salikhov, K. M. (1981) Spin Exchange. Theory and Physico-chemical Application. Springer, Berlin. Likhtenshtein, G. I. (1993) in Biophysical Labeling Methods in Molecular Biology, pp. 46–79, Cambridge Univ. Press, New York. Likhtenshtein, G. I. (1996) Role of orbital and dynamic factors in electron transfer in reaction center of photosynthetic systems. J. Photochem. Photobiol. A Chem. 96, 79 –92. Likhtenshtein, G. I. (2000) Depth of immersion of paramagnetic centers, in Magnetic Resonance in Biology (Berliner, L., Eaton,

30.

31.

32. 33.

34.

35.

36.

37.

38.

39.

40.

S., and Eaton, G., Eds.), pp. 309 –345. Kluwer Academic, Dordrecht. Moser, C. C., and Dutton, P. L. (1992) Engineering protein structure for electron transfer function in photosynthetic reaction centers. Biochem. Biophys. Acta 1101, 171–176. Salikhov, K. M., Doctorov, A. B., Molin, Yu. N., and Zamaraev, K. I. (1971) Spin relaxation of radicals and complexes upon encounters in solution. J. Magn. Reson. 5, 189 –196. Temkin, S. J., and Jacobson, B. J. (1984) Diffusion controlled of chemically anisotropic molecules. J. Chem. Phys. 88, 2679 –2685. Anglister, J., Bond, M. W., Frey, T., Leahy, D., Levitt, M., McConnel, H. M., Rule, G., Tomasello, J., and Whittaker, M. (1987) Contribution of tryptophane resudiues to the combining site of a monoclonal anti dinitrophenyl spin-label antibody. Biochemistry 26, 6058 – 6064. Dwek, R. A., Wain-Hobson, S., Dower, S., Getting, P., Sutton, B., Perkins, S. J., and Givol, D. (1977) Structure of an antibody combining site by magnetic resonance. Nature 226, 31–37. Leahy, D. J., Rule, G. S., Whittaker, M. M., and McConnell, H. M. (1988) Seqences of 12 monoclonal anti-dinitrophenyl spinlabel antibodies for NMR studies. Proc. Natl. Acad. Sci. USA 85, 3661–3665. Theriault, T. P., Leahy, D. J., Levitt, M., and McConnell, H. M. (1991) Structural and kinetic studies of the Fab fragment of a monoclonal Anti-spin label antibody by nuclear magnetic resonance. J. Mol. Biol. 221, 257–270. Little, J. R., and Eisen, H. N. (1967) Evidence for tryptophan in the active sites of antibodies to polynitrobenzenes. Biochemistry 6, 3119 –3125. Anglister, J., Frey, T., and McConnell, H. M. (1984) Magnetic resonance of a monoclonal anti-spin-label antibody. Biochemistry 23, 1138 –1142. Tronin, A., Dubrovsky, T., De Nitti, C., Erokhin, V., and Nicolini, C. (1994) Langmuir–Blodgett films of immunoglublin G: Ellipsometric study of the deposition process and of immunoglobulin avtivity. Thin Solid Films 238, 127–132. Lehrer, S. (1971) Solute perturbation of protein fluorescence compounds and lysozyme by iodide ions. Biochemistry 10, 3254 – 3263.