Determination of the Porphyrin Orientation Distribution in Langmuir

ionization mass spectrometry analysis of urinary porphyrins. Jinhua Li , Wan Chan , Zongwei Cai. ELECTROPHORESIS 2009 30 (10), 1790-1797 ...
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Determination of the Porphyrin Orientation Distribution in Langmuir Monolayers by Polarized Epifluorescence A. Tronin,*,† J. Strzalka,† X. Chen,‡,§ P. L. Dutton,‡ and J. K. Blasie† Chemistry Department, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received July 3, 2000. In Final Form: September 18, 2000 A new technique for the determination of the orientation distribution of a porphyrin within a Langmuir monolayer, in terms of the mean tilt angle relative to the monolayer normal and the width, is described. The technique utilizes the measurement of polarized fluorescence, excited with the electric field both parallel and perpendicular to the monolayer plane. The main difference between this technique and the existing ones [Fraaije et al., Biophys. J. 1990, 57, 965; Edmiston et al., J. Am. Chem. Soc. 1997, 119, 560; Bos et al., Biophys. J. 1995, 68, 2573.] is that the fluorophores are excited directly by the incident beam rather than an evanescent field, which greatly facilitates its application to Langmuir monolayers at the air-water interface. The technique was applied to the study of two systems with different porphyrin orientations: a Langmuir monolayer of a dihelical synthetic peptide BBC16 containing Zn(II)protoporphyrinIX and a monolayer of tetrakis (N-methyl-4-pyridil) porphyne electrostatically bound to the Langmuir monolayer of dipalmitoylphosphatidic acid. The results for the mean tilt angle are in good agreement with other relevant data in the literature for both systems. It is also shown that both systems exhibit rather narrow distributions, which makes them attractive for biophysical studies.

1. Introduction The Langmuir technique makes possible the assembly of amphiphilic molecules at the air-water interface and in some cases, the control of their orientation with respect to the normal to the interface. The possibility of changing this orientation may be even stronger in the case of protein and protein containing Langmuir monolayer films. The distribution of hydrophobic/hydrophilic areas on the surface of a protein molecule can be rather complex. Sitedirected mutagenesis allows some control of that distribution, but the control can be much greater in the case of synthetic peptides.1 As a result, the orientation of a natural or synthetic peptide molecule at the air-water interface can be different depending on external parameters such as surface pressure, subphase pH, etc. Of particular interest to fundamental biophysical studies and biotechnology applications are monolayer films of oriented natural or synthetic proteins containing functional groups. One such functional group of interest is the metalloporphyrin, a major effector of biological oxidation-reduction reactions. Methods are required for manufacturing the oriented functionality in monolayer films, and for their characterization. One of few relatively easy experimental approaches to assessing porphyrin orientation in thin monolayer films is a polarized fluorescence technique. In this technique, the orientation of the porphyrin is investigated by measuring the polarization of the fluorescence excited by an electric field directed normal to and along the film surface. The main advantage of fluorescence measurements over other linear optical techniques is that fluorescence, a two photon process, makes it possible to * Corresponding author, e-mail [email protected]. † Department of Chemistry. ‡ Department of Biochemistry and Biophysics. § Present address: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St. #292, Cambridge, Massachusetts 02138. (1) Chen, X.; Moser, C. C.; Pilloud, D. L.; Gibney, B. R.; Dutton, P. L. J. Phys. Chem. 1999, 103, 9029.

determine not only the mean orientation angle of the porphyrin with respect to the normal to the monolayer plane, but also the width of the orientational distribution.2,3 This feature is of particular importance for biological applications because protein films are usually not well oriented. In many cases knowledge of the mean angle alone has no useful meaning, since for a very broad distribution, the width itself may be more important than the mean value. For many applications, on the other hand, even a rough estimate of the distribution width is important, making it possible to characterize the quality of the film. In one version of the polarized fluorescence measurement, the fluorophores are excited by an evanescent field that appears upon total internal reflection. This technique is referred to as total internal reflection fluorescence (TIRF) and had been successfully used to study the adsorption of cytochrome c3-5 and methyl pyridinium porphyrins6 on solid substrates. Excitation by the evanescent field is very useful in the case where the substrates are immersed in a solution containing the same fluorophore. Since the evanescent field decays rapidly with distance from the surface, only a very small fraction of the dissolved fluorophores are excited and their fluorescence is relatively low with regard to that of the surface bound species. A similar approach can be realized for the study of Langmuir monolayers at an air-water interface with direct excitation by a beam incident on the interface from the air. In this case, the excitation beam propagates deep into the water. However, Langmuir monolayers are composed of amphiphilic molecules whose concentration in bulk solution is usually vanishingly small. In this paper, (2) Edmiston, P. L.; Lee, J. E.; Wood, L. L.; Saavedra, S. S. J. Phys. Chem. 1996, 100, 775. (3) Fraaije, J. G. E. M.; Kleijn, M.; van der Graaf, M.; Dijt, J. C. Biophys. J. 1990, 57, 965. (4) Edmiston, P. L.; Lee, J. E.; Cheng, S.-S.; Saavedra, S. S. J. Am. Chem. Soc. 1997, 119, 560. (5) Bos, M. A.; Kleijn, M. Biophys. J. 1995, 68, 2573. (6) Wienke, J.; Kleima, F. J.; Koehorst, R. B. M.; Schaafsma, T. J. Thin Solid Films 1996, 279, 87.

10.1021/la000935t CCC: $19.00 © 2000 American Chemical Society Published on Web 11/17/2000

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vectors by boldface characters and scalars by lightface characters. The angle between the absorbing and emitting dipoles (δ), which depends on the excitation/emission wavelengths, porphyrin environment, etc., can be determined from measurements of the anisotropy of fluorescence in solution.10 The fluorescence intensity from the porphyrin is a sum of the intensities from each dipole:

I ) E12 + E22

(1)

The far field of a dipole ν is given by

E ) p[(νr)r - ν]

(2)

where p denotes dipole polarization and r is the unit vector in the direction of observation.11 In this study we use normal collection with low aperture, thus r ) (0,0,1). Substitution into eq 2 gives Figure 1. Porphyrin ring orientation. In ZnPPIX, the two mutually perpendicular adsorbing dipoles, µ1 and µ2, are equivalent. So are the two emitting dipoles, ν1 and ν2. δ is the angle between corresponding absorbing and emitting dipoles. XYZ is the laboratory frame, Z is normal to the film surface. X′Y′Z′ is the porphyrin frame, Z′ is perpendicular to the ring. Ring orientation in a film is given by three angles: θ: tilt (between Z and Z′); R: precession (between X and X′); and φ; rotation (between X′ and µ1).

E ) p(νx,νy,0)

(3)

Both x and y components of the fluorescence can be measured separately with a polarizer in the detection path. Polarization is the scalar product of the excitation field vector A and the absorbing dipole µk,

pk ) (A µk)

(4)

we will provide a detailed description of this technique and report the results of its application to the study of two systems with different porphyrin orientations: Langmuir monolayers of a dihelical synthetic peptide BBC16 containing Zn(II)protoporphyrinIX and mixed monolayers of dipalmitoylphosphatidic acid (DPPA), methyl palmitate (PME), and tetrakis (N-methyl-4-pyridil) porphyne (TMPyP).

When using the incident beam directly, one can excite fluorescence either by s-polarization, which is perpendicular to the plane of incidence and contains a y component only, or by p-polarization, which is parallel to the plane of incidence and contains x and z components, thus

2. Fluorescence from a Surface Bound Porphyrin

Successive substitution of eq 5 into eqs 4 and 3 gives

General formulas for the TIRF intensity from a transition dipole in the case of high aperture collection were obtained in ref 7 and the case of fluorescence from the conjugated porphyrin ring were considered in ref 8. In the case of direct excitation by the incident beam, the formulas are essentially the same and could be used with proper expressions for excitation field intensities. However, in the case of low aperture collection, which is used in the present study, a straightforward derivation of the porphyrin fluorescence is less complicated and the resulting formulas bear more apparent physical sense, which can be useful in the treatment of experimental data. This detailed derivation is given below. Let us consider a porphyrin bound to the air-water interface. The porphyrin is arbitrarily oriented with respect to the laboratory frame (Figure 1). The laboratory frame XYZ is oriented so that the Z-axis is normal to the interface. This axis is also the direction of fluorescence collection. Plane XZ is the plane of incidence for the excitation beam. Both the free-base porphyrin and the ZnPPIX used in this study can be represented by two pairs of mutually perpendicular dipoles: absorbing, µ1 and µ2, and emitting, ν1 and ν2 (see insert in Figure 1). By symmetry, the absorbing and emitting dipoles have equal strength, so µ1 ) µ2 and ν1 ) ν2.9 Here and below we denote (7) Burghardt, T. P.; Thompson, N. L. Biophys. J. 1984, 46, 729. (8) Bos, M. A.; Kleijn, M. Biophys. J. 1995, 68, 2566. (9) Gounterman, M. In The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1978; Vol. 3, p 11.

As ) (0, Ay, 0)

Ap ) (Ax, 0, Az)

(5)

Isx′k ) (Ay µyk)2(νxk)2 Isy′k ) (Ay µyk)2(νyk)2 Ipx′k ) (Ax µxk + Az µzk)2(νxk)2 Ipy′k ) (Ax µxk + Az µzk)2(νyk)2

(6)

where k ) 1,2 denotes dipole number. To get the explicit dependence of the fluorescence intensity as a function of the porphyrin orientational angles, the dipole components should be written in the laboratory reference frame. This can be done easily by using three rotations, which provides the transfer from the porphyrin reference frame into the laboratory frame. They are: (1) Rotation around the Z-axis by the angle R (precession). The corresponding matrix is

(

cosR -sin R 0 R(R) ) sin R cosR 0 0 0 1

)

(2) Rotation around the X′ axis by the angle θ (tilt). The corresponding matrix is (10) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, Plenum Press: New York, 1983; Chapter 4. (11) Born, M.; Wolf, E. Principles of Optics, 2nd ed.; Pergamon Press: New York, 1964; Chapter 4.

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(

1 0 0 R(θ) ) 0 cosθ -sinθ 0 sinθ cosθ

}

Tronin et al.

)

3 1 Isy ) CA2y t(1 + I4) + (u + q)I2 8 4

[

(3) Rotation around the Z′ axis by the angle φ (pure rotation). The corresponding matrix is

(

)

cosR -sin φ 0 R(φ) ) sin φ cosφ 0 0 0 1

C Ipy ) A2z [u(1 - I2) + t(I2 - I4)] + 2 1 3 CA2x t(1 + I4) + (u - q)I2 8 4

[

Isx )

The total transformation is given by the matrix product

T ) R(R)R(θ)R(φ)

]

CA2y

]

1 3 t(1 + I4) + (u - q)I2 8 4

[

]

C Ipx ) A2z [u(1 - I2) + t(I2 - I4)] + 2 1 3 CA2x t(1 + I4) + (u - q)I2 8 4

]

[

(7)

(12)

where In the porphyrin reference frame, the absorbing dipoles are

t ) sin2 φ sin2(φ + δ) + cos2 φ cos2(φ + δ) q ) sin φ sin(φ + δ)cosφ cos(φ + δ)

µ1 ) (µ,0,0)

u ) sin2 φ cos2(φ + δ) + cos2 φ sin2(φ + δ)

(8)

µ2 )(0,µ,0)

and

Hence in the laboratory frame

I2(θm,σ) ) µ1 ) T(R,θ,φ)(µ,0,0) (9)

ν1 ) T(R,θ,φ + δ)(ν,0,0)

(10)

ν2 ) T(R,θ,φ + δ)(0,ν,0) Successive substitution of eq 7 into eqs 9 and 10 and into eqs 6 and 1 gives the explicit dependence of porphyrin fluorescence on orientation angles. Langmuir monolayer films are axially symmetric on the macroscopic level (scale of the acquisition area), which means that the fluorescence intensities obtained should be completely averaged over the precession angle R. We also allow some distribution over the tilt angle θ. We assume this distribution to be Gaussian; thus the intensities should be averaged over θ as well,

Ialm(θm,σ,φ)

)

(

(

∫0πexp -

2π

)

(θ - θm)2

2σ2 (θ - θm)2 2σ2

∫0

)

sinθ∂θ∂R

I4(θm,σ) )

)

(θ - θm)2

2σ2 (θ - θm)2

)

2σ2

π

The emitting dipoles can be expressed in the same way. The only difference is that the rotation angle φ should be substituted by φ + δ:

∫02π∫0πIlm exp -

(

∫0 exp π

µ2 )T(R,θ,φ)(0,µ,0)

(

∫0πcos2 θ exp -

(

;

sin θ∂θ

(

cos4 θ exp -

∫0πexp -

sin θ∂θ

2σ2 (θ - θm)2 2σ2

(11)

where l designates excitation modes (l ) s,p), m denotes the emitted polarization (m ) x,y), θm is the mean tilt angle, and σ is the distribution width. Applying eq 11 and performing all substitutions we finally get:

)

sin θ∂θ

sin θ∂θ

C is a constant that incorporates all common factors such as excitation power, fluorescence yield, detector sensitivity, etc. Let us designate

3 1 G| | ) t(1 + I4) + (u + q)I2 8 4 1 3 G|⊥ ) t(1 + I4) + (u - q)I2 8 4

(13)

1 G⊥| ) [u(1 - I2) + t(I2 - I4)] 2 The physical meaning of the coefficients in eq 13 is obvious. G|| || and G||⊥ give the intensity of the fluorescence excited by the electric field, which is parallel to the interface and detected with the polarizer either parallel or perpendicular to the excitation. G⊥|| gives the intensity of the fluorescence excited by the field, which is perpendicular to the interface. Due to the axial symmetry, G⊥|| should not depend on polarizer orientation in the detection path. Substituting eq 13 into eq 12 we get:

Isy ) CA2y G| |

sinθ∂θ

)

(θ - θm)2

Ipy ) CA2z G⊥| + CA2x G|⊥ Isx ) CA2y G|⊥ Ipx ) CA2z G⊥| + CA2x G| |

}

(14)

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It is readily seen that the structure of the eq 14 is very general; it describes the excited radiance of any system with axial symmetry regardless of the physical nature of the radiance. The physical nature of the radiance for a particular system determines only the form of the coefficients in eq 13. The fact that we arrived at the form which has proper symmetry starting from the fundamental electromagnetic theory provides some proof of the correctness of the derivation. There is another useful relation that can derived from eq 14. Expressing G|| || and G||⊥ from the 1st and 3rd equations of eq 14, substituting them into 2nd and 4th, and then subtracting the 2nd from the 4th we obtain:

A2y A2x

)

(Isy - Isx) (Ipx - Ipy)

(15)

Both sides of eq 15 contain measured values only. The left-hand side contains excitation intensities whose ratio can be measured by directing the excitation beam to a photodetector, and the right-hand side contains the ratio of measured fluorescent intensities, which eliminates the unknown coefficient C. Thus eq 15 can be used as a check of the experimental setup. It will not hold if the detector has an inherent polarization dependence or if the background is not treated properly. When the fluorophores are attached to the interface from above, the excitation field is the sum of the incident and the reflected waves at the interface, As ) Asi(1 + Rs), Ap ) Api(1 + Rp), where Rs,p denotes the reflection coefficient for s- and p-polarization, and subscript i indicates the incident beam. When the fluorophores are in the subphase, the excitation field is just the refracted wave, As ) AsiTs, Ap ) ApiTp, where Ts, Tp are transmission coefficients. Special care should be taken when the fluorophores are embedded into a thin surface layer with refractive index different from that of the air and water. One can, of course, write the exact wave amplitudes for the three media system, which are given, for example, in ref 11. However, in the case of very thin films, dfilm, λ, and low optical contrast, nfilm ≈ nwater, one can neglect the high order reflections in the film and assume that the tangential components (x,y) of the electric field in the film are the same as in the water, and the normal component Azfilm ) Azwaternwater/nfilm, where n is the refractive index. These assumptions follow directly from the boundary conditions. It is necessary to point out that the correction for the surface layer may be very important. As will be clear further on, the determination of the porphyrin tilt angle to some extent consists of a comparison of the overall fluorescence intensity excited by y and z components of the electric field. “Standing” porphyrins (in which the porphyrin plane is perpendicular to the monolayer plane) are more excited by the z component than by the y component, while “lying” porphyrins (in which the porphyrin plane is parallel to the monolayer plane) are not excited by the z component at all. Thus, the correct value for the z and y components should be used. Polarization of the surface layer material results in a significant change of the z component within it. In the case of a protein with typical refraction index of about 1.5, the z intensity in the film is (nwater/nfilm)2 ) (1.33/1.5)2 ) 0.8 of that in the water. Taking into account the considerations given above, one can write the expressions for the excitation field:

As ) Asi Ts (0,1,0), Ap ) Api Tp (cosφ, 0, sinφ water/film) where φ is the angle of refraction in water. If there is no

film and the porphyrins are dissolved in water, nfilm ) nwater . When the porphyrins are in air, one could use the same formula with nfilm ) nair , however special care should be taken for fluorescence lifetime correction due to radiant losses into more dense media.12 Equation 12 represents a nonlinear system over unknowns θm, σ, and φ. We solved this problem by fitting the calculated intensities to the measured ones. To eliminate the unknown coefficient C, each intensity was divided by Isy + Isx. For proper weighting, the experimental accuracy of each intensity was used. As was pointed out in ref 8, the system in eq 12 contains two order parameters for the Gaussian orientation distribution, which makes possible the determination of both the mean tilt angle and the distribution width. In our notation these order parameters are I2 and I4, and a minimization procedure essentially solves the system of equations:

I2 ) const1 I4 ) const2

(16)

Each of the equations in eq 16 represent, of course, a curve in the (θm, σ) plane. To find out how well eq 16 is determined, let us consider these curves for different values of const1 and const2. The overlay of the curves is shown in Figure 2. The step of const1 and const2 is the same and equals 0.1. (Note that I2 and I4 can vary in the range 0 < I2, I4 < 1). However, the density of the curves is not uniform; namely it is higher at either small or large tilt angles and small distribution widths, θm < 30° or θm > 70° with σ < 20°, and it becomes more sparse if θm is around 50°-60°, or σ is higher than 40° for any θm. The uncertainty of θm and σ is larger in the regions where curves are less dense. In such regions, small errors in I2 and I4 result in big shifts of the curves in the (θm,σ) plane, and the solution for (θm,σ) is, by definition, a point of intersection of the curves representing eq 16. This behavior shows that: (1) It is impossible to determine the distribution width in the range of 55° < θm < 60° because the lines I2 and I4 are sparse and almost parallel to the σ axis. This range is close to the magic angle of linear dichroism measurements of a porphyrin ring. Theoretically, two order parameters have different magic angles; however they are close to each other and may be not distinguishable in practice. (2) It is almost impossible to determine the mean tilt angle in the case of a broad distribution. This is understandable, since with increasing distribution width, the mean angle gets less determined per se. These qualitative considerations provide a general idea of the limitations of this technique. In practice, the limitations are affected by many parameters, primarily by experimental errors, and the overall accuracy should be evaluated in each particular case. Furthermore, the order parameters I2 and I4 are calculated on the assumption that the orientation distribution is Gaussian. If the distribution is very different from that, the uncertainties might be larger. In some cases, such as bimodal orientation for example, the model may not be working at all. In such cases the maximum-entropy approach, which does not impose a priori any shape of distribution, may be fruitful.5 3. Experimental Setup The experimental setup is shown in Figure 3. The light beam with λ ) 514 nm from an Ar laser was steered to (12) Hellen, E. H.; Axelrod, D. JOSA B. 1987, 4, 337.

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Figure 2. Lines for I2 ) const1 (thin), I4 ) const2 (thick), where const1 and const2 are uniformly sampled within the range of possible values for I2, I4, 0 < const1, const2 < 1. It is clearly seen that lines are less dense in the region 50° < θm < 60° for all distribution width values and for σ > 40° for all tilt angle values. The accuracy of θm and σ determination is related to the curves’ density, namely it is lower in the region where the density is lower.

replaced by a sanded glass surface and the cutoff filter was withdrawn from the detection path. The accuracy of the crossed polarizers position was better that 15′, and the accuracy of the absolute polarizer angle reading was better that 30′. Fluorescence was observed with a CCD camera (TE/CCD-512-TK by Princeton Instruments) through a F ) 28 mm lens (Nikon) and the collection angle was less than 5°. The camera was cooled to -50 °C. A CCD has the advantage of actually imaging the illuminated spot, making it possible to cut off the stray light, significantly reducing the background. This feature is of great help in view of the very weak fluorescent signals from the single monolayer specimens. 4. Langmuir Monolayers Figure 3. Experimental setup. See text for details.

the water surface at an angle of incidence of 60°. This value was chosen to maximize the z component of the excitation field, which is necessary to probe the component of the porphyrin transition dipoles normal to the surface. The laser output power was 10 mW. Before striking the surface, the beam passed through an expander, a quarter wave compensator (Melles-Griot) and a Glan-Thompson polarizing prism (Melles-Griot). The expander was used to increase the area of the beam footprint up to ∼3 cm2 in order to increase the overall fluorescence output signal. The compensator was used to produce circularly polarizated light before the linear polarizer, so that As ) Ap after the polarizer. The detection path was perpendicular to the water surface and contained a cutoff filter and a dichroic sheet polarizer. The wavelength cutoff was 570 nm (Melles-Griot) for ZnPPIXBBC16 monolayers and 650 nm (Melles-Griot) for TMPyP monolayers. Polarizers were aligned by zeroing the light scattering from a rough surface, similar to the standard procedure used to line up common fluorimeters.10 For this purpose water was

To test the technique, we applied it to two different systems, namely mixed Langmuir monolayers of DPPA, TMPyP, and PME and Langmuir monolayers of ZnPPIX containing synthetic peptide, so-called BBC16. These systems have been thoroughly studied previously.13-15 (In ref 13 dimirystoyl phosphatidic acid (DMPA) was used instead of DPPA. These two compounds are functionally identical for the purpose of TMPyP orientation.) For TMPyP containing monolayers, the porphyrin orientation has been shown to be parallel to the interface by direct measurement of the linear dichroism and some indirect observations such as compression isotherm behavior and spectroscopic observations in the visible regime.13 For BBC16 monolayers, there is a sufficient amount of structural data for the peptide14 and X-ray reflectivity data15 on the Langmuir monolayers, which strongly (13) Martin, M. T.; Prieto, I.; Camacho, L.; Moebius, D. Langmuir 1996, 122, 6554. (14) Gibney, B. R.; Rabanal, F.; Skalicky, J. J.; Wand, A. J.; Dutton, P. L. J. Am. Chem. Soc. 1999, 121, 4952. (15) Strzalka, J.; Chen, X.; Moser, C.; Dutton, P. L.; Ocko, B. M.; Blasie, J. K. Langmuir, in press.

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which gives the angle between the dipoles δ ) 35°. For the ZnPPIXBBC16, with the same excitation and emission at 595 nm, the corresponding values were found to be 0.26 and 28°. These values of δ were used for the θm and σ determination. Figure 4. Orientation of the dihelical synthetic peptide ZnPPIXBBC16 at the air-water interface. The peptide consists of two parallel R-helices, which provide bis-his coordination for ZnPPIX at the 10 and 24 positions, although only the 24 position is ligated here. Each helix is covalently bonded to a palmitoyl (C16) chain to increase the peptide’s amphiphilicity. At low surface pressure, both R-helices lie in the same plane parallel to the water surface, based on X-ray reflectivity.

support an orientation of the porphyrin plane perpendicular to the plane of these monolayers. In the first system, the porphyrin orientation is provided by electrostatic binding of TMPyP, which bears 4 positive charges, to the negatively charged polar headgroups of the DPPA monolayer. When the surface charge density of the DPPA monolayer matches that of the underlying TMPyP monolayer, the TMPyP molecules attach lying flat on the headgroup surface of the DPPA monolayer. The surface density of the DPPA monolayer is adjusted to the proper value of 1 negative charge per 0.8 nm2 by mixing DPPA with neutral PME. As was shown in ref 13, this surface density match is achieved for the molar proportion of TMPyP/DPPA/PME ) 1:4:16. BBC16 is a synthetic peptide designed de novo as a model for intramolecular electron transfer. The synthetic unit is an R-helical 31mer with a palmitoyl (C16) chain bonded to the N-terminal cysteine to increase the peptide’s amphiphilicity. The units dimerize via a disulfide bridge between two cysteines (R-S-S-R) to form two bis-his metalloporphyrin binding sites at sequence positions 10 and 24. In practice, however, the metalloporphyrin affinity to the first site is low possibly due to interference from the palmitoyl chain, and it is possible to bind porphyrin in the 24-position only. Details of the peptide design and structure can be found elsewhere.14,16 X-ray reflectivity studies of BBC16 in Langmuir monolayers at the airwater interface show that at low surface pressure (π < 25 mN/m), both R-helices are lying in the same plane parallel to the interface.15 In view of the structure, this means that the plane of the porphyrin is perpendicular to the water surface (see Figure 4). Thus, these two systems make up two extreme cases of porphyrin orientation with respect to the air-water interface. 5. Measurement of the Angle between the Excitation and Emission Dipoles The angle between the excitation and emission dipoles for ZnPPIX BBC16 and TMPyP was measured by the fluorescence anisotropy technique.10 The Hitachi F-2000 fluorometer with Glan-Thompson polarizers in the excitation and emission paths was used. Polarization dependent sensitivity of the detection was corrected by measuring the fluorescence with horizontal excitation (notation as in ref 10). For the TMPyP, a 1 mM solution in DMSO was dissolved in glycerol; for the ZnPPIXBBC16, a 0.05 mM water solution was dissolved in glycerol. The final concentration for the both fluorophores in glycerol was 3 nM. The samples were cooled to -20 °C. For the TMPyP, with the excitation at 514 nm and the emission at 655 nm, the fluorescence anisotropy was measured to be 0.2026, (16) Robertson, D. E.; Farid, R. S.; Moser, C. C.; Urbauer, J. L.; Mulholland, J. E.; Pidikiti, R.; Lear, J. P.; Wand, A. J.; De Grado, W. F.; Dutton, P. L. Nature 1994, 368, 425.

6. Materials and Methods TMPyP, DPPA, and PME were purchased from Sigma and used without further purification. ZnPPIX was purchased from Porphyrin Products, Inc. BBC16 was synthesized according to ref 14. Binding of ZnPPIX to BBC16 was performed by adding the equal molar amount of 2.7 mM solution of ZnPPIX in DMSO to the 0.1 mM solution of BBC16 in 1 mM phosphate buffer with 10 mM sodium chloride, pH 8, in accordance with ref 17. The concentration of the peptide solution was determined spectroscopically by measuring the absorption at λ ) 280 nm.16,17 The titration of ZnPPIX was monitored spectroscopically by observing the red shift of the Soret absorption band from 404 nm for free ZnPPIX to 427 nm, which corresponds to the metalloporphyrin in bis-his coordination. The resulting solution was used for spreading the Langmuir monolayer. Spreading solutions of TMPyP, DPPA, and PME were of 1 mg/mL concentration in a 3:1 chloroform/methanol mixture. The Langmuir trough was a custom trough from Riegler & Kirstein GmbH. A monolayer of TMPyP was prepared according to ref 13. Spreading solutions of TMPyP, DPPA, and PME in molar proportion 1:4:16 of the solutes were deposited onto the surface of pure water. After spreading the monolayer was left for 10 min and then compressed to the area corresponding to 3.20 nm2 per TMPyP molecule. This area corresponds, in turn, to the largest cross section of the molecule. Immediately after compression, the surface pressure reached the value of 40 mN/m and then decreased to 36 mN/m over 5 min and stabilized at that value. (Note that we used the constant area regime.) To prepare the monolayer of ZnPPIXBBC16, a technique described in ref 15 was used. The subphase was 1 mM Tris buffer, pH 7.8. After deposition, the monolayer was left for 15 min and then compressed to the surface pressure of 5 mN/m. To acquire the background signal for the fluorescence measurements, monolayers of apoBBC16 (i.e., BBC16 without bound metalloporphyrin) and of the DPPA and PME mixture prepared otherwise identically were used. The acquisition time for each fluorescence intensity measurement was 5 s. Intensities were repeatedly acquired in the order Isx, Ipx, Ipy, Isy, Isy, Ipy, Ipx, Isx, etc, to get sufficient statistics and to eliminate the influence of porphyrin photobleaching.4 7. Results and Discussion Fluorescence intensities for the ZnPPIX BBC16 monolayer are shown in Figure 5. The measured intensities satisfy the condition in eq 15 within experimental error, which to some extent proves the correctness of the data acquisition procedure. One can see good agreement between the experimental and calculated intensities. The calculated intensities correspond to orientation parameters θm ) 90°, σ ) 2°; and the corresponding calculated Gaussian orientational distribution is shown in Figure 6 by the thin line. To investigate how errors in the measured intensities affect the accuracy of the determination of orientation angles, an error propagation procedure should be applied. However, standard procedures do not work well in this case because the system of equations in eq 12 (17) Sharp, R. E.; Diers, J. R.; Bocian, D. F.; Dutton, P. L. J. Am. Chem. Soc. 1998, 120, 7103.

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Figure 5. Fluorescence intensity from a Langmuir monolayer of ZnPPIXBBC16. Experimental errors are 4%. Discrepancies between experimental and calculated values are within the errors.

Figure 6. Porphyrin orientation distribution for Langmuir monolayers of ZnPPIXBBC16 (thin line) and TMPyP (thick line).

is substantially nonlinear relative to θm and σ. As a result of this nonlinearity, θm and σ are highly coupled and their errors are interrelated. To circumvent this problem, we

analyze a suitable target function. This function is by definition the sum of the squared discrepancies between measured and calculated intensities. This means that all

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Figure 7. Uncertainty of the θm, σ determination for Langmuir monolayers of TMPyP and ZnPPIXBBC16. The regions of possible θm, σ values are indicated by hatching (vertical for TMPyP, horizontal for ZnPPIXBBC16). Uncertainty is due to errors in the measurement of the fluorescence intensities.

Figure 8. Fluorescence intensities for a Langmuir monolayer of TMPyP. Experimental errors are 2%. Discrepancies between experimental and calculated values are within the errors.

values of the orientation angles, which produce discrepancies lower than experimental errors, are experimentally indistinguishable. The loci of θm and σ that satisfy this condition are enclosed by the intersection of the target

function with the plane:

Z ) (Errsx2 + Errsy2 + Errpx2 + Errpy2)

(17)

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The corresponding region is shown in Figure 7 by horizontal hatching. As can be seen from the figure, the ranges of possible solutions for the orientation angles are 82° < θm < 90° and 0° < σ < 9°. Coupling between θm and σ causes a distinct shift of possible θm toward 90°. Fluorescence intensities for the TMPyP monolayer are shown in Figure 8. Again, one can see good agreement between experimental and calculated intensities. Comparison of Figures 5 and 8 shows that the TMPyP monolayer is less excited by the p-polarization, since the respective intensities are much lower for TMPyP than for ZnPPIX BBC16. This is due to the orientation of TMPyP being approximately parallel to the interface. The calculated intensities for TMPyP correspond to orientation parameters θm ) 0°, σ ) 15°, and the corresponding orientational distribution is shown in Figure 6 by the thick line. A section of the target function is shown in Figure 7 by vertical hatching. One can see a stronger coupling between θm and σ in this case than in the case of the ZnPPIX BBC16 monolayer. The range of possible orientation angles is broader, namely for tilt angles 0° < θm < 26° and distribution widths 0 < σ < 23°. However, the shape of the section is such that lower mean angles can be valid only with broader distribution widths and vice versa. The measured porphyrin tilt angles are in very good agreement with our general understanding of both systems used in this study. They also agree with the experimental data obtained in refs 13 and 15. Linear dichroism measurement of the first system13 shows that the TMPyP molecules are oriented parallel to the surface. For the BBC16, the X-ray reflectivity results show15 that the R-helices are oriented parallel to the surface, which, in

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turn, gives a perpendicular orientation of ZnPPIX (see Figure 4). The calculated intensities agree very well with measured intensities for both systems, and the condition in eq 15 is satisfied for both systems as well. All these facts demonstrate the applicability of the mathematical model and the correctness of the technique in general. The fluorescence measurements show rather good orientation of the fluorophores within the Langmuir films, namely for ZnPPIXBBC16 the distribution width is less than 9°, and for TMPyP it is less than 23°. The accuracy of the determination of the orientation distribution is reasonable; however, it should be noted that both porphyrin orientations we used were highly favorable for orientation distribution determination. In the case of θm ≈ 50-60° or σ > 30-40°, one should expect worse results. In summary, we can say that polarized epifluorescence measurement can be used to estimate the orientation distribution of porphyrin contained within single monolayers, thus providing information about both the mean tilt angle and distribution width. In subsequent papers we will show some results of how the orientation of ZnPPIXBBC16 in Langmuir monolayer films depends on the surface pressure and present a comparison between Langmuir and Langmuir-Blodgett films of the peptide, studied by polarized epifluorescence and X-ray reflectivity techniques. Acknowledgment. This work was supported by the NIH grants GM33525 and GM 41048 and the NSF/ MRSEC grant DMR96-32598. LA000935T