Photoselection studies of the P-800 band in the photoreaction center of Rhodospirillum rubrum

Polarization measurements of light-induced absorption changes in photoreaction center prepared from Rhodospirillum rubrum indicate that the 870 nm band is most likely due to a single transition dipole. The 800 nm band appears to be formed by transition dipoles with at least three different orientations. In photoreaction center from strain G9, none of the transition dipoles of the 800 nm band appears to form an angle larger than 70° with the 870 nm transition dipole.     

Rotational mobility of the photoreaction center in chromatophore membranes of Rhodospirillum rubrum

We investigated the rotational mobility of the photoreaction center in chromatophores of Rhodospirillum rubrum by studying the photoinduced linear dichroism of absorption changes at 865 nm. The study was carried out in suspensions of chromatophores treated with ferricyanide in order to bleach their antenna bacteriochlorophyll and thus minimize depolarization by energy transfer. Very little depolarization of the photoinduced absorbance change at 865 nm was observed at room temperature for chromatophores immersed in a highly viscous medium over the time range 0–10 ms following an exciting light flash. In the light of independent evidence for transmembrane arrangement of the photoreaction center, we conclude that the photoreaction center protein is immobilized in the chromatophore membrane for at least 10 ms.     

Prompt and delayed fluorescence in pigment-protein complexes of a green photosynthetic bacterium

Excitation spectra were measured at 4 K of bacteriochlorophyll a fluorescence in reaction center containing pigment-protein complexes obtained from the green photosynthetic bacterium Prosthecochloris aestuarii. Excitation spectra for the longest-wave emission (838 nm) showed bands of bacteriochlorophyll a, carotenoid, and of a pigment with absorption bands at 670, 438 and possibly near 420 nm, which is probably identical to an unidentified porphyrin described in the preceding paper (Swarthoff, T., Kramer, H.J.M. and Amesz, J. (1982) Biochim. Biophys. Acta 681, 354–358). At room temperature the longest-wave emission is stimulated by a magnetic field, which indicates that at least part of the emission is delayed fluorescence brought about by a reversal of the primary charge separation. Below about 150 K no stimulation was observed. The excitation spectra for short-wave emission (828 nm) were very similar to the absorption spectrum of the isolated antenna bacteriochlorophyll a-protein complex, and showed bands of bacteriochlorophyll a only. This indicates that two forms of the antenna protein exist that are spectroscopically similar: a soluble form that is released by treatment with guanidine hydrochloride and a bound form that remains attached to the reaction center complex. The bands of the antenna complexes were weak in the excitation spectra of the 838 nm fluorescence, which indicates that the efficiency of energy transfer to the reaction center complex is low.     

Evidence for monomeric bacteriochlorophyll in P800 of the photoreaction center from Rhodospirillum rubrum.

To find out whether weak or strong coupling exists between the bacteriochlorophyll molecules of the photoreaction center, the relative efficiency of energy transfer to P870 was measured at 795 nm and at 808 nm, at room temperature and at 77 degrees K. At room temperature, both relative efficiencies are close to 100%. However, at 77 degrees K, 795 nm light has a quantum efficiency of 76% and 808 nm light has an efficiency of 87%. These results confirm the fact that P800 is formed of at least one short wavelength component and one long wavelength component. Moreover, the short wavelength component is weakly coupled to both P870 and to the long wavelength component of P800. The conclusion is that the short wavelength component is due to monomeric bacteriochlorophyll. By comparison with other data, all four bacteriochlorophyll molecules of the photoreaction center are inferred to be monomeric.     

Differently oriented chlorophylls in Mesotaenium caldariorum detected by microphotometrical dichroism measurements in vivo

The chloroplasts of individual cells of Mesotaenium caldariorum were examined microphotometrically under non-polarized and polarized measuring light. The measurement with non-polarized light showed different absorption bands of the thylakoids depending on the position of their surface with respect to the incident light beam: in the edge position, the absorption bands lie at 672 nm, in the face position at 678 nm. From this difference in absorption maxima, we conclude that the molecules related to the sub-bands at the two wavelengths are oriented differently. The Q_y transition of the molecules which absorb light at 678 nm must be oriented parallel to the face of the thylakoids (fraction I), while that of the molecules absorbing at 672 nm is oriented perpendicular to the face (fraction II). Measurement with polarized light leads to the same conclusion that two fractions of differently oriented chlorophylls exist: In the edge position, a very large difference between E_∥ and E_⊥ (dichroism) was found in red light, with a maximum of E_∥ lying at 675 nm and a maximum of E_⊥ at 670 nm, with a shoulder at 650 nm. In the blue region, especially in the Soret band zone, the chloroplast showed a negative dichroism in the edge position, which changes over to positive values when the wavelength exceeds 450 nm. In the face position no dichroism in red or blue light could be detected. Comparison of the ‘edge position dichroism’ in red light with that in blue light justifies the supposition that the chlorin planes of the chlorophyll molecules may be oriented perpendicular or parallel to the thylakoid face, in the case of perpendicular orientation with the Q_y transitions of fraction II and the x-transitions (B_x, Q_x) of fraction I projecting out of the plane, and for parallel orientation with all transition moments lying parallel to the plane (fraction I). The relative dichroism, $\text{(E}{}_{\mathrm{\parallel }}\text{? E}{}_{\mathrm{\perp }}\text{)}\text{(E}{}_{\mathrm{\parallel }}\text{+ E}{}_{\mathrm{\perp }}\text{)}$, measured at the edge position amounts to 0.34 (i.e., 34% of the total absorption) at 680 nm. These data probably do not reflect the total quantity of oriented chlorophyll because from the opposite orientations of the Q_y transition moments of fraction I and II pigment a partial quenching of the measurable dichroism results. The red light absorption bands of the two chlorophylls oriented in an opposite manner (fractions I and II) correspond to the known bands of Photosystem I and II.     

Circular dichroism of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus

Circular dichroism (CD) spectra of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus exhibit three positive ellipticity bands between 240 and 300 nm (250, 283, and 292 nm), two negative bands at 327 and 480 nm, and a low-intensity positive band at 390 nm. The fractions of helix β-form, and unordered form of the enzyme are 8, 38, and 54%, respectively. The circular dichroic bands at 327 and 480 nm and a part of the positive bands at 292 and 390 nm are associated with enzyme activity. Significant changes in absorption and CD spectra of the enzyme were observed when the temperature of the enzyme preparation was increased to 47°C, coinciding with the sharp decrease in enzyme activity observed at this temperature.     

Energy transfer and bacteriochlorophyll fluorescence in purple bacteria at low temperature

Emission spectra of bacteriochlorophyll a fluorescence and absorption spectra of various purple bacteria were measured at temperatures between 295 and 4 K. For Rhodospirillum rubrum the relative yield of photochemistry was measured in the same temperature region. In agreement with earlier results, sharpening and shifts of absorption bands were observed upon cooling to 77 K. Below 77 K further sharpening occurred. In all species an absorption band was observed at 751-757 nm. The position of this band and its amplitude relative to the concentration of reaction centers indicate that this band is due to reaction center bacteriopheophytin. The main infrared absorption band of Rhodopseudomonas sphaeroides strain R26 is resolved in two bands at low temperature, which may suggest that there are two pigment-protein complexes in this species. Emission bands, like the absorption bands, shifted and sharpened upon cooling. The fluorescence yield remained constant or even decreased in some species between room temperature and 120 K, but showed an increased below 120 K. This increase was most pronounced in species, such as R. rubrum, which showed single banded emission spectra. In Chromatium vinosum three (835, 893 and 934 nm) and in Rps. sphaeroides two (888 and 909 nm) emission bands were observed at low temperature. The temperature dependence of the amplitudes of the short wavelength bands indicated the absence of a thermal equilibrium for the excitation energy distribution in C. vinosum and Rps. sphaeroides. In all species the increased in the yield was larger when all reaction centers were photochemically active than when the reaction centers were closed. In R. rubrum the increase in the fluorescence yield was accompanied by a decrease of the quantum yield of charge separation upon excitation of the antenna but not of the reaction center chlorophyll. Calculation of the F?rster resonance integral at various temperatures indicated that the increase in fluorescence yield and the decrease in the yield of photochemistry may be due to a decrease in the rate of energy transfer between antenna bacteriochlorophyll molecules. The energy transfer from carotenoids to bacteriochlorophyll was independent of the temperature in all species examined. The results are discussed in terms of existing models for energy transfer in the antenna pigment system.     

Anisotropy of the emission and absorption bands of spinach chloroplasts measured by fluorescence polarization and polarized excitation spectra at low temperature

Spectra of fluorescence polarization were measured between 4 and 120 K of spinach chloroplasts, oriented in a magnetic field. At least seven emission bands were observed. The well known bands near 685 nm (‘F-685’) and 735–740 nm (‘F-735’) and the band near 680 nm (‘F-680’) were strongly polarized parallel to the plane of the thylakoid membrane, whereas emission bands near 695 nm (‘F-695’), 710, 730–735 and 760 nm showed perpendicular polarization. Assuming perfect orientation of the thylakoid membranes, we calculated orientation angles of 64, 47 and 66.5° for the emission dipoles of F-685, F-695 and F-735, respectively, with respect to the normal of the membrane. Excitation spectra of F-695 and F-735 in polarized light at 4 K provided information about the orientation of the absorption dipoles of chlorophylls a and b. The spectra thus obtained were in very good agreement with the linear dichroism spectrum. Moreover, they allowed us to distinguish between the pigments associated with Photosystems I and Ii, which is not possible from measurement of linear dichroism alone. The results indicate that a high degree of orientation is not confined to the long-wave absorbing bands, but also bands at shorter wavelength show a clear anisotropy. The calculated orientations were in quantitative agreement with the hypothesis that F-685 and F-735 are associated with chlorophylls absorbing at 676 and 710–715 nm, respectively.     

Triplet-minus-singlet absorbance difference spectra of reaction centers of Rhodopseudomonas sphaeroides R-26 in the temperature range 24–290 K measured by Magneto-Optical Difference Spectroscopy (MODS)

The recently developed technique of Magneto-Optical Difference Spectroscopy (MODS) [10] has been applied to reaction centers (RC) of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26. Absorbance changes induced by a magnetic field are measured as a function of wavelength yielding the triplet-minus-singlet (T-S) absorbance difference spectrum. (T-S) spectra thus obtained have been measured from 24–290 K. Going from low to high temperature the (T-S) spectra show the following features:

1. A rapid decrease of positive absorption bands at 809 and 819 nm.
2. A slow appearance of a band shift at 798 nm.
3. A shift of the peak wavelength of the Q_y absorbance band of the primary donor P-860 from 992 to 861 nm, and of its Q_x band from 603 to 600 nm.

The spectra at 24, 66, 116, and 290 K have been analyzed by Gaussian deconvolution. The 800 nm region of the spectrum at 24 K can be decomposed in a combination of two band shifts and an appearing band. The temperature dependence of the spectra in this region is well explained by spectral broadening of the two shifting bands combined with a decrease in intensity of the appearing band when the temperature increases. The two shifting bands in the 800 nm region are identified as the two bands at 803 and 813 nm which together make up the 800 nm band in the absorption spectrum and are assigned to the two accessory RC bacteriochlorophylls (BChls). The band shift of the 813 nm pigment is appreciably larger than that of the 803 nm pigment. The appearing band at 808 nm is attributed to monomeric absorption of ³P-860, the triplet state being localized on one BChl. We find no evidence for admixture of a charge transfer (CT) state of ³P-860 with one of the accessory BChls at higher temperature.     

Linear dichroism and the orientation of reaction centers of Rhodopseudomonas sphaeroides in dried gelatin films

Aqueous mixtures of reaction centers of Rhodopseudomonas sphaeroides and gelatin were dried to form thin films. Following hydration, these films were stretched as much as two to three times their original length. Polarized absorption spectra showing linear dichroism were obtained for both unstretched and stretched films, with the planes and stretching axes of the films mounted in various geometries relative to the electric vector of the measuring beam. These data were analyzed in terms of the following model: Reaction centers possess an axis of symmetry that is fixed in relation to the reaction center structure. In unstretched films this axis is confined to the film plane and oriented at random within the plane. In stretched films the symmetry axis is aligned with the direction of stretching. In both preparations reaction centers are distributed randomly with respect to rotation about the axis of symmetry. The data are consistent with this model when the analysis acknowledges less than perfect orientation. For perfect orientation in a stretched film the model predicts uniaxial symmetry about the axis of stretching. The approach to this condition was examined with films stretched to different extents. Extrapolation yielded dichroic ratios for the ideal case of perfect orientation, and allowed calculation of the angles between the axis of symmetry and the various optical transition dipoles in the reaction center. This treatment included the two absorption bands of the bacteriochlorophyll ‘special pair’ (photochemical electron donor) in the Q_x region, at 600 and 630 nm, which we were able to resolve in light minus dark difference spectra.     

Dichroism of Photosensitive Pigment in Rhabdoms of the Crayfish Orconectes

Microspectrophotometric measurements of isolated crayfish rhabdoms illuminated transversely show that their photosensitive absorption exhibits a dichroic ratio of 2 in situ. The major absorption axis matches the axial direction of the closely parallel microvilli comprising the receptor organelle. Since these microvilli are regularly oriented transversely in about 24 layers, with the axes of the microvilli at 90° in alternate layers, transverse illumination of a properly oriented rhabdom displays alternate dichroic and isotropic bands. Because all the microvilli from any one cell share the same orientation, the layers of microvilli constitute two sets of orthogonal polarization analyzers when illuminated along the normal visual axis. Furthermore, since the dichroic ratio is 2 and transverse absorption in isotropic bands is the same as that in the minor absorbing axis of dichroic bands, the simplest explanation of the analyzer action is that the absorbing dipoles of the chromophores, as in rod and cone outer segments, lie parallel to the membrane surface but are otherwise randomly oriented. The rhabdom's functional dichroism thus arises from its specific fine structural geometry.     

Antenna organization and evidence for the function of a new antenna pigment species in the green photosynthetic bacterium Chloroflexus aurantiacus

Whole cells and isolated chlorosomes (antenna complex) of the green photosynthetic bacterium Chloroflexus aurantiacus have been studied by absorption spectroscopy (77 K and room temperature), fluorescence spectroscopy, circular dichroism, linear dichroism and electron spin resonance spectroscopy. The chlorosome absorption spectrum has maxima at 450 (contributed by carotenoids and bacteriochlorophyll (BChl) a Soret), 742 (BChl c) and 792 nm (BChl a) with intensity ratios of 20:25. The fluorescence emission spectrum has peaks at 748 and 802 nm when excitation is into either the 742 or 450 nm absorption bands, respectively. Whole cells have fluorescence peaks identical to those in chlorosomes with the addition of a major peak observed at 867 nm. The CD spectrum of isolated chlorosomes has an asymmetric-derivative-shaped CD centered at 739 nm suggestive of exciton interaction at least on the level of dimers. Linear dichroism of oriented chlorosomes shows preferential absorption at 742 nm of light polarized parallel to the long axis of the chlorosome. This implies that the transition dipoles are also oriented more or less parallel to the long axis of the chlorosome. Treatment with ferricyanide results in the appearance of a 2.3 G wide ESR spectrum at g 2.002. Whole cells grown under different light conditions exhibit different fluorescence behavior when absorption is normalized at 742 nm. Cells grown under low light conditions have higher fluorescence intensity at 748 nm and lower intensity at 802 nm than cells grown under high light conditions. These results indicate that the BChl c in chlorosomes is highly organized, and transfers energy from BChl c (742 nm) to a connector of baseplate BChl B792 (BChl a) presumably located in the chlorosome baseplate adjacent to the cytoplasmic membrane.     

Photodichroic studies of the photoreaction center from Rhodospirillum Rubrum. I. Attribution of P870 to two non parallel dipoles

A randomly oriented sample of photoreaction center prepared from Rhodospirillum rubrum was excited at 77 °K by an actinic linearly polarized light of 870 nm. Under such conditions, only those chromophores with components of their absorption dipoles oriented parallel to the polarization of the actinic light are bleached. The change in absorbance at 900 nm of this photoselected sample was observed while varying the angle of polarization of a weak measuring light. The polarization of the absorbance change was thus evaluated as 0.25.

This value is interpreted to mean that P₈₇₀ is attributable to two absorption dipoles forming an angle included between 35.75° and 90°. Comparison with the p value of 0.5 obtained on a similar preparation by polarization of fluorescence (Ebrey, T. G. and Clayton, R. K. (1969) Photochem. Photobiol. 10, 109–117) leads to the conclusion that either these two dipoles emit fluorescence without being coupled by singlet-singlet energy transfer or that only one of them is a fluorescence emitter in the absence of reversible singlet-singlet energy transfer.     

Titles of related papers in other sections

A method is described for isolation of the Rhodopseudomonas viridis reaction center complex free of altered, 685 nm absorbing pigment. This improved preparation contains two c-type cytochromes in the ratio P-960: cytochrome c-558: cytochrome c-553 of 1 : 2 : 2 to 3. The near infrared spectral forms of the reduced preparation are located at 790, 832, 846 and 987 nm at 77 K; the oxidized complex absorbs at 790, 808, 829 and approx. 1310 nm. The 790 nm band is attributed to bacteriophaeophytin b and the other absorbances to bacteriochlorophyll b. The visible absorption bands may be assigned to these pigments and to the cytochromes present and, probably, to a carotenoid. The presence of two bacteriochlorophyll b spectral forms in the P⁺-830 band suggests that exciton interactions occur among pigments in the oxidized, as well as the reduced, reaction center. Changes in the 790 and 544 nm bands upon illumination of the reaction center preparation at low redox potential may be indicative of a role for bacteriophaeophytin b in primary photochemical events.     

Properties of reaction centers of Rhodopseudomonas sphaeroides in dried gelatin films.: Linear dichroism and low temperature spectra

Methods of preparing dried gelatin films containing purified reaction centers of Rhodopseudomonas sphaeroides are described. The spectral properties of reaction centers in solution are essentially maintained in dried gelatin films. These films are uniform and have excellent optical properties, showing little particulate scattering at temperatures down to about 4K. Film contraction on cooling to 90K is less than 1% in linear dimension. Linear dichroism spectra are reported for films at room and low temperature. Reaction centers show a moderate amount of linear dichroism in unstretched gelatin films; the magnitude of the linear dichroism becomes much greater when the films are stretched. In stretched films, linear dichroic ratios ($\text{A}{}_{\mathrm{\parallel }}\text{A}{}_{\mathrm{\perp }}$; absorbance measured with electric vector parallel and perpendicular to stretching direction) between 1.7 and 2.2 were obtained for the 860 nm absorption band of the bacteriochlorophyll component that undergoes primary photooxidation. The relative polarizations of light-induced absorption changes of reaction centers in stretched films are similar to those reported by Vermeglio and Clayton ((1976) Biochim. Biophys. Acta 449, 500–515) and support their hypothesis that absorbance decreases, maximal near 860 and 810 nm, and an increase near 790 nm are associated with the respective disappearance and appearance of discrete bands characteristic of the reduced and oxidized bacteriochlorophyll dimer. This interpretation is also supported by the polarization of the absolute absorption spectrum near 810 and 860 nm. An absorption band near 540 nm, ascribed to the Q_x transitions of two molecules of bacteriopheophytin in the reaction center, is split at low temperatures into two bands having similar polarizations. This splitting is probably not due to exciton coupling of the two molecules, since excition theory predicts different polarizations.     

The photoreaction center of Rhodospirillum rubrum mutant strain F24.1

Rhodospirillum rubrum strain F24.1 is a spontaneous revertant of nonphototrophic mutant F24 derived from wild-type strain S1. Strain F24 shows no detectable photochemical activity and contains, at most, traces of the photoreaction center polypeptides. Strain F24.1 has a phototrophic growth rate close to that of the wild-type strain (Picorel, R., del Valle-Tascón, S. and Ramírez, J.M. (1977) Arch. Biophys. Biochem. 181, 665–670) but shows little photochemical activity. Light-induced absorbance changes in the near-infrared, photoinduced EPR signals and ferricyanide-elicited absorbance changes indicate that strain F24.1 has a photoreaction center content of 7–8% as compared to strain S1. Polyacrylamide gel electrophoresis of isolated F24.1 chromatophores shows the photoreaction center polypeptides to be present in amounts compatible with this value. Photoreaction center was prepared from strain F24.1 and showed no detectable difference with that of strain S1. It is concluded that strain F24.1 photosynthesis is due entirely to its residual 7–8% of typical photoreaction center.     

Spectroscopic properties of the reaction center and of the 47 kDa chlorophyll protein of Photosystem II

The D1-D2-cytochrome b-559 reaction center complex and the 47 kDa antenna chlorophyll protein isolated from spinach Photosystem II were characterized by means of low temperature absorption and fluorescence spectroscopy. The low temperature absorption spectrum of the D1-D2-cytochrome b-559 complex showed two bands in the Q_y region located at 670 and 680 nm. On the basis of its absorption maximum and orientation the latter component may be attributed at least in part to P-680, the primary electron donor of Photosystem II. The 47 kDa antenna complex showed absorption bands at 660, 668 and 677 nm and a minor component at 690 nm. The latter transition appeared to be associated with the characteristic low temperature 695 nm fluorescence band of Photosystem II. The 695 nm emission band was absent in the D1-D2 complex, which indicates that it does not originate from the reaction center pheophytin, as earlier proposed. The transition dipole responsible for the main fluorescence at 684 nm from this complex had a parallel orientation with respect to the membrane plane in the native structure. The reaction center preparation contains two spectrally distinct carotenoids with different orientations.     

Orientation of the chromophores in the reaction center of Rhodopseudomonas viridis. Comparison of low-temperature linear dichroism spectra with a model derived from X-ray crystallography

Linear dichroism (LD) and absorption (A) spectra of reaction centers from Rhodopseudomonas viridis included in the native chromatophores or reconstituted in planar aggregates have been recorded at 10 K. The samples were oriented in squeezed polyacrylamide gels and the primary donor P was in the reduced or (chemically) oxidized state. The LD spectra of reaction centers in these two states are in favor of a dimeric model of P in which excitonic coupling between the two non-parallel Q_Y transitions leads to a main transition at 990 nm (parallel to the membrane plane) and another one of smaller oscillator strength at 850 nm (tilted at approx. 60° out of the membrane plane). These assignments are in close agreement with the ones proposed in a previous LD study at room temperature (Paillotin, G., Verméglio, A. and Breton, J. (1979) Biochim. Biophys. Acta 545, 249–264). The main Q_X excitonic component of P has a broad absorption peaking at 620 nm and it corresponds to dipoles exhibiting the same orientation as those responsible for the 850 nm transition. On the basis of the present LD study and of CD data of chemically oxidized-minus-reduced reaction centers, we proposed that the minor Q_X excitonic component of P is oriented close to the membrane plane and absorbs around 660 nm. The two monomeric bacteriochlorophylls exhibit a positive LD for both their Q_Y transitions (unresolved at 834 nm) and their Q_X transitions (resolved at 600 and 607 nm), indicating that the planes of these molecules are only slightly tilted out of the membrane plane. The two bacteriopheophytins exhibit strong negative LD with identical LD/A values for their Q_Y transitions (resolved at 790 and 805 nm) and small positive LD for their Q_X transitions (resolved at 534 and 544 nm), demonstrating that these two molecules are strongly tilted out of the membrane plane with each of the Q_Y transitions tilted at approx. 50° out of that plane. A comparison of these LD data with the structural model derived from X-ray crystallography (Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H. (1984) J. Mol. Biol. 180, 385–398) clearly suggests that a good agreement exists between the results of the two techniques under the following conditions: (i) the C-2 symmetry axis of the reaction center runs along the membrane normal; (ii) excitonic coupling is present only in the primary donor special pair; and (iii) the direction of the optical transitions of the monomeric bacteriochlorophylls and of the bacteriopheophytins is not significantly perturbed by the interactions among the pigments. In addition, a carotenoid is detected in the isolated reaction center with an orientation rather perpendicular to the C-2 symmetry axis. Finally, a comparison of these data with similar ones obtained on the bacteriochlorophyll a-containing reaction center of Rhodopseudomonas sphaeroides 241 points towards a geometrical arrangement of the chromophores which is indistinguishable from the one observed in the reaction center of Rps. viridis.     

The Orientation of Rhodopsin and Other Pigments in Dry Films

When rhodopsin in a gelatin film is dried, the rhodopsin chromophores orient primarily in the plane of the film. When the film is wetted, the chromophores disorient. These changes are reversible. When rhodopsin in a wet film. is bleached in the presence of hydroxylamine and redried, the retinal oxime which results is oriented more perpendicularly to the plane of the film. These orientations in dry gelatin films resemble those in the disc membranes of rod outer segments. A variety of other proteins are similarly oriented in dry gelatin films: methemoglobin, cytochrome c, phycocyanin. Films of methemoglobin and cytochrome c display prominently the high Soret band near 410 nm when measured with unpolarized light passing through the face of the fim, but display no Soret band at all with light passing through the edge of the film. All of these orientations imply a large asymmetry of the protein micelles, perhaps conferred upon them by linear polymerization in the course of drying. Such asymmetry can be demonstrated directly with rhodopsin. A wet paste of rhodopsin-digitonin micelles, sheared between glass slides, becomes highly oriented, the rhodopsin chromophores lining up in the direction of shear, the retinal oxime produced by bleaching orienting more perpendicularly to the shear.