4679
J. Phys. Chem. 1992, 96, 4679-4685
Accordingly, the standard redox potential of EAM in polymeric solvents also is varied linearly with 1/X,, when X,, is not very small, and this is reflected into the CV wave potentials.
Figure 4. Plot of
vs l/Xn.
the basis of eq 19, eq 15 can be derived further into eq 20 (at least in PEO), where KR and KO are the K factors of reductive and Eo = El (RT/nF) In [SR(-)/So(m)] + ( R T / n F ) In [(I + K R / x n ) / ( l + KO/Xn)I (20) oxidative EAMs, respectively. &(a), So(-) are the EAM solubilities in the polymer with infinite large chains. Similarly, if X,, is large enough, the high-order expanding sections of eq 20 can be neglected, and it can be simplified into Eo = Eo'* K3/X,, (21) where E O s * = El + ( R T / n F ) In [SR(m)/SO(m)l K3 = (RT/nF)(KR - KO)
+
+
(13)
Ohno, H.; Wang, P. Polyym. Prepr. Jpn. 1990, 39, 542.
Results and Discussion The CV of hemin in various PEO solvents are shown in Figure 2. As seen from this figure, the redox potential and CV wave currents of EAM varied apparently with the change of X,. The larger the X,,, the weaker the ip, and the higher the CV wave potentials. Data treatment results of Figure 2 are plotted in Figures 3, and 4. According to them, In i , , varied with 1/X, linearly even a t low X,,.At 80 'C, In [(i,,)/pA] = -2.16 15.4/X,,. On the other hand, the plot of E , vs l/Xn has good linearity only when X,, is greater than about 15 and, EP/V = -0.025 - 0.46/Xn. The electrochemical behavior of hemin in PEO oligomers partly confirmed the theoretical considerations mentioned above. Apparently, eqs 1 and 2 need to be proved further by other experiments. Finally, it should be noted here, only EAM in amorphous polymer matrices has an electrochemical activity. Equations 5 and 19 are also only suitable to the same kind of systems. Therefore, all the conclusions described above are applicable only to amorphous polymeric solvents. Registry No. PEO,25322-68-3; LiCIO,, 7791-03-9; hemin, 1600913-5.
+
Dlffractlon Efficiency of Bacteriorhodopsln Films for Holography Containing Bacterlorhodopsln Wildtype BRW and Its Variants BRD,,, and BRDoeN Norbert Hampp,* Andreas Popp, Christoph Brauchle, University of Munich, Institute of Physical Chemistry, Sophienstrasse 1 1 , 0-8000 Munchen 2, Germany
and Dieter Oesterhelt Max-Planck-Institute for Biochemistry, A m Klopferspitz 18 A , 0-8033 Martinsried, Germany (Received: October 24, 1991; In Final Form: January 21, 1992)
-
-
Bacteriorhodopsin (BR) films made from purple membranes (PM) containing the wildtype form of BR or one of its variants with single amino acids exchanges Asp96 Asn or Asp85 Glu were compared with respect to their holographic properties. The dependence of the diffraction efficiency of BR films on the parameters pH, temperature, intensity, initial optical density, and spatial frequency was measured. Increasing the initial optical density of a BR material with a long lifetime of the M intermediate and a low steady-state concentration of all other intermediates, in particular the N and 0 state, yielded diffraction efficiencies of up to several percent for the technically important read-out wavelength 633 nm. Lowering the temperature of the BR films shifts the maximal diffraction efficiency to lower light intensities. The diffraction efficiency q of BR films is almost independent of the spatial frequency of the recorded grating in the range 150-1500 mm-l but decreases to about 80% of the maximal value at 2500 mm-'. Thin layers of PM suspension show essentially lower spatial resolution with strongly nonlinear behavior but could be used for holographic recording with less than 500 mm-I. The BR variant Asp96 Asn shows significantly improved optical properties compared with the wildtype form of BR and the variant Asp85 Glu. The highest diffraction efficiency of 7% at 633 nm was obtained with a BR-film containing this mutated bacteriorhodopsin at pH 8.0 which had an initial optical density of iOD = 5 at 568 nm and a thickness of 25 Mm.
-
+
Introduction Biological photochromic materials, like bacteriorhodopsin, phytochrome, and the visual pigments, have many interesting properties which challenge synthetic materials, such as high light sensitivity, photoreversibility, and stability. However, the possibility of engineering these materials toward the needs of a particular application has been lacking, and the low availability of the materials themselves has not allowed broad applications. Therefore none of the cited materials have been used in a technical application such as optical information processing up to now. In the case of the bacterial photochrome bacteriorhodopsin (BR) signifcant progress in solving these problems has been made during
the last few years. This molecule therefore might become a model system for the design and modification of photoactive materials by genetic methods. The photochromic retinal protein bacteriorhodopsin (BR) is contained within the purple membrane (PM) of Halobacterium halobium.' In the halobacterial cell BR acts as a light-driven proton pump. Upon illumination, protons are pumped from the cytoplasm through the halobacterial cell membrane to the outer medium and a transmembrane proton gradient is generated. Thereby, BR converts light energy into chemical energy. The (1) Oesterhelt, D.; Stoeckenius, W. Nulure (London) 1977, 233, 149.
0022-365419212096-4679%03.00/0 0 1992 American Chemical Society
4680 The Journal of Physical Chemistry, Vol. 96, No. 11. 1992
hvn
- msec
/
ti
'570
K-
0.5 psec
/
J600
/
\\f 1- '
O640
psec
f(
K590
N560
/.Isec
L550
MI
H' H' Asp 96 Asp 85 Figure 1. Scheme of the photochemical and thermal conversions of bacteriorhodopsin. The photointermediates are abbreviated by single letters. Index numbers indicate the absorption maxima of the intermediates. Asp85 and Asp96 are involved in the deprotonation (L M) and N) of the Schiff base. reprotonation ( M
-
-
structure and function of this membrane protein have been intensively investigated during the last 20 years. Reviews summarizing our current knowledge on the biochemical and photophysical properties of BR have been published.*~~ The initial B state, also called light-adapted BR if its chromophore has the all-trans, 15-anti configuration, shows maximal absorption at 570 nm (Figure 1). After the initial very fast and efficient photoreaction from B J which occurs in about 500 fs with a quantum yield q5 of about 0.64: the BR molecule passes through a sequence of intermediates2v5to the MI state in about 50 ps. Two different M forms, MI and MI1,with almost the same absorption maximum at about 410 nm, can be distinguished.6-8 The M states are about 160 nm blue-shifted from the B state, and the Schiff base is deprotonated. Aspartic acid 85 (Asp85) is involved in the deprotonation of the Schiff ba~e.~JODuring the reprotonation of the Schiff base which occurs in the MI1 N transition Asp96 serves as a proton donor. After reprotonation of Asp96 from the outer medium the retinal chromophore undergoes a configurational change during the N 0 transition from 1 3 4 s to all-trans. Finally, the B state is reached which differs from the M states not only in the configuration and protonation state of its chromophore but also in the protein conf0rmation.l' In the dark BR relaxes slowly to an equilibrium mixture of the B state and the D state which is characterized by a 13-cis,l5-syn chromophore configuration (dark-adapted).I2 Soon after the discovery of BR, suggestions were made for possible technical applications (e.g., refs 13 and 14) because BR
-
-
-
(2) Kouyama, T.; Kinositu, K.; Ikegami, A. Adv. Biophys. 1988, 24, 123. (3) Birge, R. R. Annu. Rev. Phys. Chem. 1990, 41, 683. (4) Tittor, J.; Oesterhelt, D. FEBS Lett. 1990, 263, 269. (5) Polland, H.-J.; Franz, M. A.; Zinth, W.; Kaiser, W.; Kolling, E.; Oesterhelt, D. Biophys. J . 1986, 49, 651. (6) Kriebel, A. N.; Gillbro, T.; Wild, U. P. Biochim. Biophys. Acta 1979, 546. 106. (7) Varo, G.; Lanyi, J. K. Biochemistry 1990, 29, 2241. (8) Varo, G.; Lanyi, J. K. Biophys. J . 1991, 59, 313. (9) Butt, H. J.; Fendler, K.; Bamberg, E.; Tittor. J.; Oesterhelt, D. EMBO J- . 19119 R I657 - - - - , -, - -- . . (10) Braiman, M. S.; Mogi, T.; Stern, L. J.; Khorana, H. G.; Rothschild, K. J. Biochemistry 1988, 27, 8516. (11) Trissl, H. W. Photochem. Photobiol. 1990, 51, 793. (12) Smith, S. 0.;Myers, A. B.; Pardoen, J. A.; Winkel, C.; Mulder, P. P. J.; Lugtenburg, J.; Mathies, R. Proc. Narl. Acad. Sci. U.S.A. 1984, 81, 2055. (13) Vsevolodov, N. N.; Ivanitskii, G. R.; Soskin, M. S.; Taranenko, V. B. Avtometriya 1986, 2, 41.
Hampp et al. is extremely stable toward thermal and photochemical degradation. One of the most attractive approaches is the use of BR in optical information recording and processing,'"16 especially in holographic applications, such as pattern recognitionl' and optical filtering.18 Furthermore, it has been proposed as material for artificial neural networks.19 BR films have already been demonstrated in practice as media for reversible holographic recording.20 However, the differing physical demands of various optical processing techniques cannot be fullfilled by wildtype BR (BRWT) alone. Variants of BR can be generated either by sitespecific mutation and expression of the modified bacterio-opsin gene in E . coli2I or by statistical mutations in the halobacterial genome and a selection procedure for functionally defective B R s . ~The ~ second approach, or a combination of both, seems to be the method of choice, since bacteriorhodopsin variants embedded in the purple membrane are obtained only for halobacteria. This is essential for BR's chemical and thermal stability. Some of these BR variants showed improved optical properties for holographic applications compared to BRWT.20 In this article the preparation and characterization of BR films containing BRwT or one of its variants for holographic recording is described, the influence of different parameters is reported and methods to optimize the holographic properties of BR-films have been devised. Besides BRw the two variants BRD8,E and BR" were examined. BRDssE differs from BRwT by the exchange of aspartic acid at position 85 (Asp = D) for glutamic acid (Glu = E). BR" contains an asparagine residue (Asn = N) at position 96 instead of aspartic acid. Both amino acids are involved in the deprotonation and reprotonation of the Schiff base during the p h o t o c y ~ l e . ~ *This ~ , ~systematic ~ experimental investigation of the diffraction efficiency of BR films containing wildtype and mutated bacteriorhodopsins with respect to its intensity, pH, and temperature dependencies characterizes the influence of physicochemical and genetic methods which allow the design of optimized BR media for various holographic applications.
Experimental Section Wildtype PM was isolated from halobacterial strain S9 by the procedure described in ref 24. PM variants were isolated from mutated Halobacterium halobium GRB strains. Generation and isolation of the variants BRDssEand BRD96Nare described in detail in refs 22 and 25. Glass substrates with a thickness of 1 mm were used for the preparation of BR films. They were cleaned with acetone and silanized twice with a solution of 3% dimethyldichlorosilane (DDS) in tetrachloromethane prior to use. Unbound DDS was removed by extensive washing with ethanol. Buffer stock solutions (100 mM) were prepared from citric acid (pH 3 . 9 , potassium phosphate (pH 6.5, pH 8.0),and sodium carbonate (pH 9.5) in doubly distilled water. Thin layers of PM suspension for holography containing 20 mM buffer and a final concentration of 1% glycerol were prepared by mixing the buffer stock solution of the desired pH value with PM suspension. These mixtures were filled in optically flat glass (14) Hampp, N.; Brluchle, C. In Photochromism: Molecules and System; Diirr, H., Bous-Laurent, H., Eds.; Elsevier: Amsterdam, 1990 Vol. XL, p 954. ( 1 5 ) Brauchle, C.; Hampp, N.; Oesterhelt, D. Adu. Mater. 1991, 3, 420. (16) Bazhenov, V. Y.; Soskin, M. S.; Taranenko, V. B.; Vasnetsov, M. V. In Optical Processing and Computing, Arsenault, A., Ed.; Academic Press: New York, 1989; p 103. (17) Hampp, N.; Thoma, R.; Oesterhelt, D.; Brauchle, C. Appl. Opr., in press. (18) Thoma, R.; Hampp, N.; Brauchle, C.; Oesterhelt, D. Opt. Lett. 1991, 16, 651. (19) Birge, R. R.; Fleitz, P. A.; Gross, R. B.; Izgi, J. C.; Lawrence A. F.; Stuart, J. A.; Tallent, J. R. Proc. IEEE Eng. Med. Biol. Soc. 1990, 12, 1788. (20) Hampp, N.; Brauchle, C.; Oesterhelt, D. Biophys. J . 1990, 58, 83. (21) D u m , R. J.; Hackett, N. R.; McCoy, J. M.; Chao, B. H.; Kimura, K.; Khorana, H. G. J . Biol. Chem. 1987, 262, 9246. (22) Soppa, J.; Oesterhelt, D. J . Biol. Chem. 1989, 264, 13043. (23) Miller, A.; Oesterhelt, D. Biochim. Biophys. Acta 1990, 1020, 57. (24) Oesterhelt, D.; Stoeckenius, W. Merhods Enzymol., 1974, 31, 667. (25) Soppa, J.; Otomo, J.; Straub, J.; Tittor, J.; Meeaen, S.; Oesterhelt, D. J . Biol. Chem. 1989, 264, 13049.
Diffraction Efficiency of Bacteriorhodopsin Films
The Journal of Physical Chemistry, Vol. 96, No. 11, 1992 4681
A, = 633 nm
A,
= 568 nm
Kr’ - laser
k, = r0 - l
z
/k,
=
T ~ . ’
O = N
Figure 3. Schematic model of the BR photocycle used for the interpretation of steady-state hologram efficiencies.
Figure 2. Holographic setup for the characterization of BR films. A planewave hologram is recorded with a Kr+ laser operating at hw = 568 nm and monitored by a probe beam from a HeNe laser (AR = 633 nm) which incidents at the first Bragg angle to the BR film. The intensities Iw, and Iw2of the recording beams are adjusted to equality by neutral density filters ND. The linear polarization of the reading and recording beams is directed perpendicular to the plane of the beams.
cuvettes (Type 20C G, Starna) with a thickness of 500 pmq BR films were formed between two glass substrates. Glycerol was added to the PM suspension in 10 mM of the desired buffer at a final concentration of 1% (V/V). These mixtures were sonified with a needle probe to destroy PM aggregates, filtered through a 5-rm disposable microfilter (Spartan 3 , Schleicher & &hall), pipetted onto the siliconized glass substrates, and stored overnight in a desiccator over CaCl, to remove water. The viscous PM material obtained was rapidly heated on a Peltier element to approximately 60 OC and then covered with a second glass. The material was sheared between the glass plates under pressure thereby forming the BR film. The glass substrates were glued together with poly(cyanoacry1ate) to prevent misalignment during use. The thickness of the BR films obtained by this procedure was approximately 25 pm. Absorption spectra of the different PM suspensions and the BR films were recorded with a spectral photometer (Uvikon 8 6 0 , Kontron) in the visible range. Since the maximal direct measurable absorption was OD = 3.5, higher values of absorption of BR films at 568 nm were calculated from a measurement at 6 3 3 nm and a conversion factor obtained from a PM suspension of lower optical density. Because shifts in the absorption spectrum occur during film formation, the accuracy of the values for optical densities at 568 nm higher than 3.5 is 0.3 OD units. For the measurement of the diffraction efficiency of BR films the holographic setup shown in Figure 2 was used. A beam from a Kr+ laser operating at wavelength Xw = 568 nm was divided by a beam splitter (BS) into two coherent beams which were overlapped at the BR sample at an angle of 2Ow. A plane wave hologram is formed in the BR film.26 The induced holographic grating is monitored by a second beam of wavelength XR = 6 3 3 nm emitted from a HeNe laser which incidents onto the BR film at Bragg angle OR. Alignment of the Gaussian recording beams and the probe beam (TEMoo mode) was controlled through a microscope objective. The two recording beams were adjusted to equal intensity by means of neutral density filters ND to obtain a contrast ratio of unity. The polarization of the writing and reading beams was perpendicular to the plane of the beams. The diffraction efficiency 7,which is defined as the ratio q = ZRD/ZR of the diffracted intensity ZRD and the incident intensity ZR of the read-out beam, was monitored by means of a calibrated powermeter. The spatial frequency v v = ( 2 sin Ow)/Xw (1)
*
of the holographic grating depends on the writing wavelength Xw and the angle 20w between the writing beams. For the determination of the spatial resolution of the BR media the angle 20w was varied between 5 and 90°. The temperature of the BR films (26) Brguchlc, C.; Burland, D. M. Angew. Chem., rnt. Ed. Engl. 1983, 22,
582.
was controlled by a thermostated sample holder. The limited beam diameters of the recording beams and the slightly differing diameters of recording and reading beams (A, = 568 nm, o.d., = 3 mm; XR = 6 3 3 nm, O.duR= 2 mm) were not mathematically compensated throughout all the experiments reported here. Classification of Holograms For a qualitative interpretation of the observations made in steady-state holography, the photocycle of BR (see Figure 1) can be reduced to the more simple model shown in Figure 3 , because the short-living intermediates J, K, and L have very low steadystate populations. The summed population of the MI and MI1 states is called M. The spatially varying intensity distribution Zw(x) in the interference pattern of the 568 nm writing beams (ZwI,Zw2) which is given for I,, = I,, by ZW(X) = 21w1(1 + cos 2TVX) (2) with x being the coordinate in the plane of the beams, induces a spatial modulation of the population distribution between the B, M, N, and 0 states in the BR film. The spatial modulation amplitudes of the absorption al(XRJw)and of the refractive index nl(XRJw) in a BR film of the thickness d at the reading wavelength XR dependent on the writing intensity Zw(x) is given by
where cI(XR) represents the molar absorption coefficient and Rl(XR) the molar refraction at the reading wavelength XR;26 6ci(Zw) represents the amplitude of the intensity-dependent concentration change of the ith intermediate, i.e., B, M, N, and 0 state. The spatial modulation of Zw(x)leads to modulations of the absorption u(XR,Zw) and the refractive index n(XR,Zw), which can be approximated by the expressions in eq 4 where a. and q,represent the average absorption and the average refractive index valid for a particular BR film and al and nl (eq 4) describe the amplitude of the periodic modulation of both parameters in the x direction. a(XR,ZW) = a0 al cos (2%xv) + ... n(XR,ZW)
=
+ + n1 cos (2?rxv) +
1 . .
(4)
The holograms investigated were of the transmission type. These are roughly classified into thin or thick holograms and into amplitude and/or phase holograms. Since the broad absorption bands of BR and its photointermediates cover almost the whole visible spectrum, holograms in BR films must be considered as a mixture of amplitude and phase hologram. The parameter Q’*’ is used to determine whether a hologram belongs to the class of thin or thick holograms, and the normalized grating modulation amplitude y2’ indicates whether a holographic grating falls into the two-wave or multiwave regimee2* These parameters are calculated by eq 5 where OR is the angle of incidence of the reading beam inside the grating. hRdV2 rnld Q’= 2%-no cos OR 7”(5) XR cos OR (27) Magnusson, R.; Gaylord, T. K. J . Opt. SOC.Am. 1978, 68, 809. (28) Kogelnik, H. Bell Sys?.Tech. J . 1969, 48, 2909.
4682 The Journal of Physical Chemistry, Vol. 96, No. 11, 1992
Hampp et al. suspension pH: 3 . 5 , 6.5, 9.5
TABLE I: Q and y Parameters Valid for the BR Films as a Function of the Thickness d of the Films and the Spatial Frequencies
filmisuspension pH 8.0
(mm-’)
Q’lr‘ d , bm
150 mm-I
1350 mm-’
2500 mm-I
25 500
1.610.12 3212.4
13310.14 266012.8
66610.20 1332014.0
‘Parameters used for calculation: Xw = 568 nm, XR = 633 nm, n, = 1.5, nl = 0.001, d = 25 pm for BR films, d = 500 pm for liquid films in glass cuvettes.
The Q’ parameters and y values derived for the BR films used in our experiments are summarized in Table I. For the calculation the Bragg angle was used for OR, the average refractive index no of PM was taken as 1.5 and a typical modulation of the refractive index of n, = 0.001 was assumed.14 Holographic gratings with Q’values 10 to the class of thick holograms. The Q’values for the BR films indicate that thick holograms occur in the BR films. The formula eq 6 derived by Kogelnik,Z8which tl = I R D / I R = (sin2 P
p = *XR
2.303 2d ’
600
700
800 400
560
660
760
800
500
600
700
800
a0
(6)
2.303 =a 0 7
describes the relation between the diffraction efficiency 7 = I m / I R and the modulation amplitudes of the refractive index n, and the absorption a , , the average absorption ao,and the thickness d of the grating can be applied for the description of BR films. The low y values of the BR films indicate that multiwave diffraction might occur. This correlates with the finding that with BR films at small recording angles higher diffraction orders are observed.
Absorption Spectra of BR Films The absorption spectra of the PM suspensions used for the preparation of the BR films and the spectra of the resulting BR films show clear differences. A selection of spectra is shown in Figure 4. The absorption maximum of BRwT shows almost no dependence on the pH value in the pH range 3.5-9.5.29 The maximum was found at 568 nm at all three pH values (Figure 4A). However, a strong pH dependence of the absorption was observed in BRDssEsamples (Figure 4c). At pH 3.5 and 6.5 the maximal absorption was at 6 11 and 6 12 nm, respectively, but at pH 6.5 a shoulder on the short-wavelength side of the band was observed. At pH 9.5 the absorption band shifted to shorter wavelengths and had its maximum at 537 nm. In the BRDBSE variant Asp85 is replaced by glutamic acid, which has a pK value different from aspartic acid. The pH-dependent protonation of the carboxylic group of glutamic acid causes the shift in the absorption spectrum. In the acidic range (pH = 3.5) the “blue” form and in the alkaline range (pH = 9.5) the “red” form are observed.30 The spectrum of pH = 6.5 corresponds to a mixture of both forms. BRD96Nbehaved similarly with regard to the pH dependence of its absorption to BRwT with an absorption maximum at 568 nm at all pH values examined (Figure 4E). Due to the increased lifetime of the M state at high pH values,z3 the absorption spectrum at pH 9.5 was measured after 10 min in the dark in the photometer. In the right column Figure 4 the spectra of PM suspensions having pH = 8.0 and BR films made therefrom are compared (Figure 4B,D,F). Except for BRDgsE the absorption maxima were not affected by the film formation procedure. BRDs5E showed a shift in the absorption maximum from 61 1 to 548 nm, which indicates that the effective pH value in the dried (29) Kimura. 1984, 40, 64 1.
500
+ sinh2 A ) exp(-2D)
mld A = *D = - a0d cos OR’ 2 cos OR’ cos OR a1 = a,-*
400
Y.:Ikenami. - A.: Stoeckenius. W. Photochem. Photobiol.
(30) Lanyi, J. K.; Tittor, J.; Varo, G.; Krippahl, G.; Oesterhelt, D. Biochim. Biophys. Acta 1992, 1099, 102.
400
wavelength [nm]
wavelength Inml
Figure 4. Absorption spectra of PM suspensions and BR films containing BRwT, BRDSsE, and BRD96N.On the left side the absorption spectra of PM suspensions in 100 mM buffer at the pH values pH 3.5 pH 6.5 (-), and pH 9.5 (---) containing BRwr, BRD~sE, and BRD96Nare shown. On the right side a comparison of the spectra recorded from PM-suspensions (---.) in 10 mM buffer at pH 8.0 and BR-films (---) obtained therefrom are shown for BRwT, BRDBSE, and BRD96N. (e-.),
films is more alkaline because of the lower proton availability and proton mobility in the film matrix compared to the initial BR suspension. Also, for the BRwTand BRD96Nfilms it is assumed that the effective pH in the film is more alkaline (pH > 8) than in the suspension. Since the spectrum of both is not pH sensitive in that range (see Figure 4A,E), no shift of the absorption maxima was observed. In all cases the absorption band of the films was somewhat narrower than in the corresponding suspension.
Diffraction Efficiency of BR Films in the Steady State In Figure 5A an overview is shown for the intensity dependence of the diffraction efficiency of BR-films which have varying pH values, BR variants, and initial optical densities. The given pH values correspond to the suspension used for the film preparation. Rather low diffraction efficiencies in the range 0.1-0.2% were observed for the BRDssEvariant at alkaline pH values, i.e., pH 8.0 and 9.5. At lower pH values the diffraction efficiency decreased below the detection limit of the experimental setup used. BR films containing BRwT showed a dependence of the maximal diffraction efficiency ~ ~ ( 6 3on3 the ) pH. The same was observed for BR films containing BRD9,N. In both cases, Le., BRwT and BRD96N,increasing the pH from 6.5 to 8.0 led to a significant increase of the diffraction efficiency. For the BRD96N films this change lowered the intensity needed to reach q,(633) by a factor of about 3. Almost no change was observed for the BRw films. A slightly increased diffraction efficiency was observed for BRw when the p H was further increased to pH 9.5 and then also the intensity was lowered where qmax(633)was obtained, but only by about 30%. BRD96Nfilms with p H 9.5 are not plotted in Figure 5A, since due to their extreme light sensitivity the intensity of the 633-nm readout beam significantly disturbs the holographic grating. In Figure 5B the dependence of the diffraction efficiency r ) on the spatial frequency of the recorded holograms is plotted which was measured at the intensities where ~ ~ ( 6 3was 3 ) obtained (see Figure SA). An almost constant diffraction efficiency was observed for spatial frequencies of 150-1 500 mm-’ for all BR-films.
Diffraction Efficiency of Bacteriorhodopsin Films
The Journal of Physical Chemistry, Vol. 96, No. 11, 1992 4683
intensity [mW/cm2]
iz
5
.,.-.---*---4
B -. * -. -. --.. .g .-. -. +.-.-.--5 - ----.-... .............................. \ 4-
a. D96N r-------.---"-
-*.
3-
E r
WT
" 2
%-.-Ad--
g1-
r
'c1
=..pt'
0
I
--
DIBE s::::::::t::...: I
intensitv [mW/cm*] Figure 6. Dependence of the diffraction efficiency on the initial optical density and the temperature of the BR-films. (A) Dependence of the maximal diffraction efficiency ~ ~ ~ ~ ( on 6 3the3 initial ) optical density iOD568at a recording angle of 2BW = 10'. Two series of BRD96Nfilms with pH 6.5 (-) and pH 8.0 (---), respectively, and the iOD568 values I = 2.6/IV = 2.5, I1 = 3.8/V, = 3.5 and I11 = 5.2/VI = 5.0 were analyzed. Increasing amounts of BR molecules, Le., higher iOD values, 3 3achieved. ) (b) Temperature dependence of enable higher ~ ~ ~ ~ ( to6 be ~ ~ ~ ( 6 of3 BR 3 ) films made from suspensions of pH = 8.0 containing BRwT with iOD568= 3.3 (-) and BRD96Nwith iOD568= 5.2 (---) at 10 OC (m), 21 O C (A), and 32 ' C ( 0 ) .Lowering the temperature leads to an increased M lifetime and ~ ~ ~ ~ ( is6 obtained 3 3 ) at lower light intensity.
Beyond this value a slight linear decrease was observed and about 80% of the ~ ~ ~ ( 6 was 3 3 found ) at 2500 mm-'. The results from Figure 5 demonstrate that the BRD96Nfilms are the best of the investigated materials using 633 nm as the readout wavelength. The maximal modulation amplitude in the holographic grating of the absorption coefficient a1 and of the refractive index nl depends on the total amount of photoactive material. For BRw and BR" films a significant increase of the diffraction efficiency was found when the pH was increased from 6.5 to 8.0. Therefore, the intensity-dependentdiffraction efficiency of BR-films of both pH values was analyzed for films with varying initial optical density. In Figure 6A the data obtained from BRDWNfilms at two different pH values with increasing initial optical density iOD, i.e., increasing amounts of BR molecules per volume, are shown. Increasing iOD values allow higher maximal diffraction efficiencies ~ ~ ( 6 3 to3 be ) achieved, but higher light intensities are necessary to reach it, since more BR molecules must be stimulated. The longer M-lifetime of the films with pH 8.0 (Figure 6A, broken line) has the result that qmax(633)is observed at lower intensities than with similar pH = 6.5 films. The maximal diffraction efficiencies of the pH = 8.0 films cannot be reached even at high light intensities with pH = 6.5 films. Therefore, other influences apart from the pure B/M modulation in the holographic grating must be considered. This means that BR" material at pH = 6.5 and 8.0 seem to be two different materials with respect to their holographic properties. Light intensities beyond the optimal value lead to a decrease in the diffraction efficiency of all the BR films since the holographic gratings are no longer sinusoidalZoand light is diffracted also in higher orders which are not considered here. A comparison with Figure SA where BRD96Nfilms with iOD values of 7.2 and 6.3 are shown indicates that for BRD96Nfilms further increased iODS8 values do not lead to higher efficiencies. The optimal iOD value is in the range iOD568= 5-6.
TABLE II: Temperature Dependence of the Lifetime T M of the M Intermediate in a BRW and a BRmN Film of pH = 8.0 rU. ms
10 21 38
600 290 83
1870
1050 600
The M lifetime in the BR film is important for its sensitivity, Le., the intensity needed to obtain maximal diffraction efficiency. Another possibility to modify the M lifetime apart from changing the pH is to cool the material. At -50 O C the thermal M decay is almost completely blocked.31 The mechanism for reprotonation differs between BR" and BRwT, because the internal proton donor is missing in BRD~,N. However, the temperature dependence of the decay should be similar for both BR types and therefore the intensity dependent diffraction effciency was analyzed for BR films made from BRW (iOD568 = 3.3, pH = 8.0) and B R D ~ (iOD ~ N = 5.2, pH = 8.0) in the range 10-38 OC (Figure 6B). The maximal diffraction efficiency vmax(633)reached with each film was almost independent of the temperature but was obtained at lower temperatures for lower light intensities. For both the BRwT and the B R D ~ ~ N films, changing the temperature from 38 to 10 O C lowered the light intensity needed to reach ~ ~ ( 6 3 by 3 )about the same factor of 7. In Table I1 the M lifetimes (measured at 410 nm) of the BR films at the temperature applied in the experiment are summarized. It is seen from Table I1 and Figure 6B that in general the increase in the M lifetime is correlated with a lower intensity required to reach ~ ~ ~ ~ ( 6 However, 3 3 ) . the temperature depen(31) Balashov, S. P.; Litvin, F. Biophysics 1981, 26, 566.
4684 The Journal of Physical Chemistry, Vol. 96,No. 11, 1992
Hampp et al. 2 mW/cm2
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spatial frequency [mm”] Figure 7. Intensity dependence of the diffraction efficiency of purple membrane suspensions on the pH value at room temperature. (A) PM suspensions of iODS68= 3.3 having pH 9.5 (O), pH 8.0 (A),and pH 6.5 (m) were measured in 500-pm-thick cuvettes at a writing angle of 28, = 20’. (B) The spatial resolution of PM suspensions is significantly lower than in BR films. Due to Brownian diffusion the diffraction efficiency shows a strong nonlinear dependence on the spatial frequency. The spatial frequency used for measuring the intensity dependence of the diffraction efficiency in (A) is indicated by an arrow.
dence of the M lifetime is different for BRwr and BRD96~?3 whereas the temperature dependence of the intensity required to reach q,(633) is about the same. This may reflect the fact that ~ ~ ( 6 3which 3 ) is related to the steady-state population difference in the holographic interference pattern depends not only on the M decay time. Suspensions of PM have been repeatedly used in the literature (e.g., ref 16) for holographic recording. Since the BR films containing BRwT or BRD96N showed a significant increase in the diffraction efficiency in correlation with the pH value of the suspensions which were used for film preparation, it was checked whether this effect was also observed in PM suspensions (Figure 7A) or only in BR films. Suspensions of BRw with pH 6.5 and 8.0 having an iODS8 = 3.3 showed diffraction efficiencies of about 0.2%,but suspensions with pH 9.5 reached values of up to 0.6%. Therefore the correlation between pH and diffraction efficiency is a property of the material and is not caused by the film formation procedure. A pH-dependent change in the photocycle of BR must be the reason for the observed diffraction efficiency increase. During water removal the pH value of the BR material tends to become more alkaline (see Figure 4), and therefore a pH-dependent increase in the diffraction efficiency apparently occurs at a somewhat lower pH value of 8.0 in the BR films. It should be mentioned here that one of the major differences between BR films and PM suspensions for holographic recording is their spatial resolution. The diffraction efficiency of BR films (see Figure 5B) was almost unchanged for spatial frequencies of 150-2500 mm-I. In PM suspensions (see Figure 7B) a strong nonlinear behavior of the diffraction efficiency on the spatial frequency was observed. Only at low frequencies (
