ABSTRACT DE'IEMNATION OF PORPHYRIN RING ORIENTATION IN SPINACH CHLOFDPLAST EXTRACT CHIOFDPHYLL BLACK LIPID IVEMBRANES BY PHO'IUVOLTACE SPECTFDSGJPY By Herman G. Weller, Jr. Photovoltage spectroscopy with polarized light was used to investigate the structure of black lipid membranes formed from spinach chloroplast extracts. The photovoltage action spectrum of the chlorophyll black lipid membrane is similar to the absorption spectrum of the membrane—forming solution, with a red and principal blue peak. The maglitudes of these peaks were found to depend on the direction of polarization of the exciting light. 'Ihis is apparently a direct consequence of the dichroism of the mn‘brane. The polarized light photovoltage data were used to obtain information on the orientation of chloropmrll in the membrane. The chlorophyll principal blue transition mment was calculated to make an angle of“ 21 1 2° with the plane of the membrane; the red transition moment, an angle of 38 1 2°. From these angles, an angle (averaged over the chloroprwll a; and chlorophyll g in the membrane) of 1&5 .+. 5° was calculated for that Herman G. Weller, Jr. between the plane of the porphyrin ring and the plane of the membrane . DE'IERMATI ON OF PORPHYRIN RING ORIENTATION IN SPINACH CHIDROPLAST EXTRACT CHLOROPHYLL BLACK LIPID MENBRANES BY PHOI‘OVOLTAGE SPECTHDSCOPY By Herman G. WEller, Jr. A Thesis Submitted to Michigan State University V in partial fulfillment of the requirements for the degree of Master of Science Department of Biophysics 19714 'Do Yamba Wiiga, Bob Foester, Larry Morgan, and Abui Nbrgan ii ACIQ‘IOWIEDGMEN’IS The author wishes to express his gratitude to his major advisor, Professor H. Ti Tien, in whose laboratory this research was conducted. Interesting and informative discussion with the Master's Thesis Committee members, Professor A. Haug, Professor S. Izawa, and Dr. Tien, is also appreciated. The financial support of this work was provided by a National Institutes of Health Grant (GM—113971). 111 TABLE OF CONTENTS LIST‘OF TABLES . . . . . . . . . . . LIST'OF FIGURES . . . . . . . . . CHAPTER I. INTRODUCTION . . . . . . . . . II. ORIENTATION OF THE CHLOROPHYII.PORPHYRIN RDMG:DQP(HIH.NEWBRANE SYSTEMS AND IN BIOLOGICAL MEMBRANES: A.LITERATURE m 0 O O O O 0 0 O O O 0 Some Chlorophyll Chemistry . . . . Theoretical Work . . . . . . . .Model Systems . . . . . . Biological Membranes . . . . . . III. EXPERIMENTAL . . . . . . . . Apparatus . . . . . . . . Extraction Techniques . . . . . . Procedure . . . . . . . . . . IV. THEORETICAL CONSIDERATIONS . . . . F RESULES AND DISCUSSION . . . . . . VI. MISCELLANEOUS . . . . . . . . . Photoelectric Action Spectra with various Additional Components in.the Bathing Solutions . . . . . . iv Page V11 10 12 15 18 18 22 2h 29 35 A3 ”3 CHAPTER Page Preferential Polarized Light "Photo- Bleaching" of Chlorophyll Nblecules in the Membrane and the Attempt to Ehploy it for the Determination of a Rotational Diffusion Relaxation Time for the Membrane Chlorophyll Molecules . . . . . . . . . . . . . . 52 Effect of Voltage Applied to Spinach Chloroplast Extract Black lipid Membrane on the Dependence of the Photo-emf Action Spectrum upon the Direction of Polarization of the ExcitingLight......'....... 58 APPENDICES..................59 A. DIRECT PROPORTIONALITY OF THE PHOTO-EMF, Em, TO THE AMOUNT OF LIGHT ENERGY ABSORBED BY ms mam LEADS TO DIRECT PROPORTIONALITY OF ( ) /( ) TO'IHEDICHROICRA'I'IO . .Ej‘Yy.E1.7".Z . . . . . 59 B. IZERIVA'I'ION OF THE ANGIE, a, BETWEEN ms PLANE or THE PORPHYRIN RING AND ma NomAL,m,To'IHEMEMBRANE . . . . . . . . . . 62 BIBLIOGRAPHY.................67 LIST OF TABLES Table Page 1. List of the Equipment and Materials Necessary to perform the Chlorophyll Black Lipid Membrane Experiments Described in the Text . . . . l9 2. Orientations in the Spinach Chloroplast Extract Chlorophyll Black Lipid Membrane of the Transition Dipole Ivbments Responsible for the Chlorophyll Red and Principal Blue Absorption Bands, and Orientation of the Chlorophyll Porphyrin Ring . . . . . . . . . 39 3. Four Attempts at Fixation of the Membrane Chlorophyll Nblecules with Glutaraldehyde . . . . . 57 LIST OF FIGURES Figure Page 1. Schematic representation of the structure ofchlorophyllaorg . . . . . . . . . . . 8 2. Schematic diagram of the membrane chamber and the electronics for measuring the phOto-enf O O O O O O O I O I O 0 O O 0 21 3. Schematic diagram of optic set-up (top view) . . . . 25 A. (A) Schematic diagram illustrating the determination of the orientation of the membrane relative to the direction of propagation of the incident light, (topview)... .........26 (B) Schematic diagram illustrating the establishment of the direction of polarization of the incident light with the electric vector, E, vibrating parallel to the plane of incidence (top view) . . . . . 26 5. Schematic diagram of the transition dipole moment, M, of either the red or the principal blue chlorophyll absorption band (see text) . . . . 30 6. Absorption spectrum and photo-emf action spectra of the spinach chloroplast extract blacklipidmembrane . . . . . . . . . . . 36 7. Photo-emf action spectra of the spinach chloroplast extract black lipid membrane with 1 mM FeCl3 in the outer chamber and "water-soluble chlorophyll" in the imer chamber................ All 8. Photo-emf action spectra of the spinach chloroplast extract black lipid membrane with 1 mM FeCl in the outer chamber and o.1thhionin;intheimerchamber . . . . . . 1:6 vii Figure 9. Photo—emf action spectra of the spirech chloroplast extract black lipid membrane with 1 mM FeCl in the outer chamber and 0.3 m p—benz uirone in the inner chamber........... 10. Photo—emf action spectra of the spinach chloroplast extract black lipid membrane with 1 mM FeCl in the outer chamber and A mid L-asoorbi acid in the inner chamber........... 11. Shape of a typical plot of the photo-emf versustime.......... 12. Schematic diagram of the relation between the red transition moment vector, M , the principal blue transition moment vector, ”8’ and the normal to the membrare, IV . viii CHAPTERI INTRODUCTION In green algae and higher plants, photosynthesis is the process occurring in the chloroplasts in which (a) electromagnetic energy from incident visible light activates the reduction of nicotinamide adenine (NADP+) to NADPH and the oxidation of water, and (b) carbon dioxide is reduced to {CHZO}n. (Lehninger, 1970) The grana contain essentially all the photosynthetic pigments of the chloroplast as well as the enzymes required for the primary light—dependent reactions. The paired thylakoid membranes are the sites of the light-trapping systems in the chloroplast structure (Rabinowitch and Govindjee, 1969). The isolated chloro- plast lamellae when illuminated perform electron transport from water to ferredoxin, yielding oxygen gas and reduced ferredoxin. Phosphorylation of ADP to ATP accompanies this electron transport (Hill, 1937; Hill, 1965; Arnon _e_t_;a_l_., 19514). The correspondence hemeen photochemical action spectra and the light absorption spectra of various green algae and photo- synthetic higher plants have led to the conclusion that chloroprwll must be the primary light—trapping molecule in green cells (Clayton, (1971). Because of the complexity of the photosynthetic system detailed investigation of the primary physical processes of photo— synthesis, involving energy and electron transfer (Clayton, 1965), has been very difficult. A great deal has been inferred from the stucw of simpler systems. The propertiesof chloroplwll in soluticn (Goedheer, 1966), in the solid state (Ke, 1966; Cherry, 1968), and in monolayers (Ke, 1966) have been studied. The light reactions of photosynthesis and the associated electron transport reactions have been shown to occur within the internal membrane system of chloroplasts, while the (132 fixatim reactions occur within the stroma regions of the chloroplast (Trebst 93 _a_l_. , 1958; Park and Pon, 1961). The thylakoid membrane is com- posed of about 52% lipid and A81 protein by weight, with about 101 being chlorophyll (Park and Biggins, 196A). The lamellar structure of’the photosynthetic apparatus undoubtedly is important in its function. Thus, studies of chloro- phyll in solution or in crystalline form have provided a limited amount of infermation which is directly relevant to the role of’the membrane in photosynthesis. Artificial lipid.membranes have been employed to study various components of biological membranes in a bilayer environment. A great deal of evidence has been accumulated in recent years indicating that the gross structure of many biological mem- branes is that of a fluid lipid bilayer matrix in which are "dissolved" amphipathic intrinsic membrane proteins, lipoproteins, and glycoproteins. (See, for example, Singer and Nicolson, 1972; Bretscher, 1973. Many additional references are contained therein.) Electron paramagnetic resonance studies with phospholipid bilayer-s and rabbit sarooplasmic reticulum by McConnell and co-workers have indicated that lipids may be very mobile in the plane of the membrane (Kornberg and McConnell, 1971; Scandella _e_t_ gal. , 1972), but much less mobile in a direction perpendicular to the plane of the membrane (Kornberg and McConnell, 1971; McNamee and McConnell, 1973). Studies by Frye and Edidin on intrinsic membrane proteins complexed with fluorescent—labeled specific antibodies in the envelopes of human cells and mouse cells caused to fuse under the influence of Sendai virus, and by Nicolson and Singer on red blood cell intrireic membrane proteins complexed with specific ferritin- labeled antibodies, have shown that the proteins "dissolved" in the plasma membrane may also be quite mobile laterally. (Frye and Edidin, 1970; Nicolson and Singer, 1971a; Nicolson and Singer, 1971b; Nicolson and Singer, 1971c) The black lipid membrane has been introduced as a model system for the study of biologcal membrane components in a bilayer lipid matrix separating two aqueous soluticns by Mueller, Rudin, Tien, and Wescott. (Mueller, Rudin, Tien, and Wescott, 1962) The black lipid membrane exhibits many properties which are similar to those of biological membranes, e.g., thickness, resistance, capacitance, and interfacial tension (Tien, 1971). The chlorophyll black lipid membrane separating two aqueous phases has been proposed as a model system for the study of the primary processes of photosynthesis of green plants. Various properties of black lipid membranes in the dark have been measured, e.g. water permeability, bifacial tension, thickness, resistance, and dielectric breakdown (Ting gt _a_l_. , 1968). Recently, light— excitable properties such as fluorescence (Alamuti and Lauger, 1970) , absorbance (Steinemann gt__a_1_., 1971; Cherry _e_t_ 31;, 1971), and photovoltage effects (Tien, 1968) have been investigated. It has been found that with Fe3+ in one aqueous phase visible light incident on the spinach chloroplast extract chlorophyll-lipid bilayer induces a transmembrane voltage (Van and Tien, 1970) . This was to be expected since a "photovoltaic" effect in layers of chlorophyll a, t_)_, a+p_, and other pigrents applied to a metallic electrode lowered into an electrolyte had been observed and studied earlier by Yevstigneyev, Terenin, and co-workers (Yevstigneyev and Terenin, 1951; Yevstigneyev, 1962; Termin and Putseiko, 1961; Yevstigleyev and Savkina, 1963; Putseiko, 1963). More recently, Getov and J ordanova have found that in layers of chlorophyll a and :1 applied to a semi-transparent gold electrode illumination causes a "photo-emf," the gold electrode always being positive, and the spectral distribution of the photo— emf upon illumination on the electrode almost coincides with the optical absorption spectrum of chlorophyll (Getov and Jordanova, 1972). Tre chlorophyll black lipid membrane photo-emf may be comparable to the trans-thylakoid voltage calculated by Witt and co—workers from absorbence changes at 515 nm of chlorophyll p_ during electron transport and protOphosprorylation in spinach chloroplast preparations (Junge and Witt, 1968; Schliephake gt _a_l_., 1968 ; Witt, 1972). This calculation involved assumptims of concomitant trans—thylakoid proton transfer, thickness and dielectric constant of the membrane lipid layer, and the area of thylakoid covered by one electron transport chain. Witt gt _a_l_. arrived at values of about 50 mV for 1.5(10-5) sec of "saturating intensity" excitation at 630 - 680 nm, about 200 mV for the maximum voltage upon excitation of longer duration, and in permanent light a steachl-state value of about 100 mV. Tle light-induced emf of the artificial chlorophyll-lipid membrane has been found to depend on the wavelength of the illuminating light (Van and Tien, 1970). A "photo—emf action spectrum" can be obtained by scammg the visible wavelengths. The present work concerns tle finding that the magnitudes of the peaks of the proto-emf action spectrum depend on the direction of polarization of the exciting light. This appears to be a direct consequence of the absorption properties of the chlorophyll in the artificial membrane, and may be used to determine the orientation of the chlorophyll porphyrin ring in the black lipid membrane. (Weller and Tien, 1973) The principal blue transition moment was calculated to make an angle of 21 t 20 with the plane of the membrane; the red transition moment, an angle of 38 1 2°. From these angles, an angle of 145 r. 5° is calculated for that between the plane of the porphyrin ring and the plane of the membrane. These values are averaged over the chlorophyll _a and chlorophyll _b_ present in the membrane. There exists a possibility that the acidic bathing solution employed in these experiments converts some or all of the chlorophyll a and chlorophyll _b_ in the artificial membrane to pheophytin a and preophytin b, respectively, by the removal of the Mg atom from the center of each porphyrin ring. This pheOprytinization reaction would depend on the degree of exposure of the membrane chloropmll porphyrin rings to the aqueous phases. ' The porphyrin ring orientation angle determination in the experiments described herein does not supply erough information to , f ascertain the availability of the chelated chlorophyll Mg atom to N the acidic bathing solutions. Thus there exists the possibility that the porphyrin ring orientation angle obtained may be for pheophytin and chlorophyll. CHAPTER II ORIENTATION OF THE CHLOROPHYLL PORPIIYRIN RDIG IN MODEL MEMBRANE SYSTEMS AND IN BIOLOGICAL MMBRANFS: A LITERATURE REVIEW Some Chlorgihyll Chemistry Chlorophyll (Figure 1) is a molecule with an unusual combination of electron donor-acceptor properties (Katz, 1973). The ring V keto O=O group can function as donor, the central Mg atom as acceptor. In the absence of extraneous nucleophiles, donor-acceptor interactions form chlorophyll dimers, ((1112), and .oligomers, (Ch12)n. With mmoiunctional electron donors, mmomeric chlorophyll species form. BifUnctional donors may cross—link chloroplwlls through Mg atoms to form large polynuclear adducts of colloidal dimensions. Katz has examined the visible absorption spectra by com- puter deconvolution techniques and found considerable similarity between bulk or antenna chlorophyll in the plant and (0112)“. (Katz, 1973) Electron spin resonance studies have suggested that ESR photo—siglal I associated with the photosynthetic reaction center of photosynthetic organisms could arise in a special pair of chlorophyll molecules (Chl H20 Chl)+. Katz pointed out that these structures can be combined to give a structure that possesses both light—gathering properties and photoactivity, (Chlz)n(Ch1 H20 Chl). The Junction is readily Figure 1. Schematic representation of the structure of chlorophyll _a_ or _b_. In chlorophyll a, X is -CH in chlorophyll b, X is 3; -CH0 (after Lehninger, 1970). m m m m. m -CH /m\c/m2\ 2 m/ pmtyl chain m.14 . w m2\ /m2\ 2 m/m2\ 3 m . m /w2\ 2 m/ m2\ 3 a m/ mm3 10 effected by a keto 0-0—-Mg interaction between the terminal chlorophyll molecule of (Ch12)n and the chloropmll of the special pair that still l'es an Mg atom available for coordination. (Katz, 1973) This model accounts for both optical and ESR properties of plant chlorophyll. Such a structure can survive only if access of water to it is strictly limited, otherwise the entire structure will be converted to (Chl°H20)n. The electron-transport agents and the enzymes required for the subsequent chemical reacticns of photo- synthesis presumably would be in the membranes and hydrophilic regions of the chloroplast‘that surround the chloroptmrll. (Katz, 1973) Theoretical Work Theoretical work based on experimental observations has sugasted that chlorophyll molecules in photosynthetic systems are oriented, and that energy transfer would be much facilitated by suitable chromophore orientation. For example , the finding that triplet excitation is greatly enhanced, while the quantum yield of fluorescence is diminished mam-fold, in chlorophyll aggregates over monaleric chlorophyll suggested to Kasha that a suitable thylakoid concentration of chloroptwll might allow absorbed energy to be transferred in the chloroplast via excitation to a chlorophyll "exciton band," followed by triplet excitation. (Kasha, 1959) While discussirg energy reception and transfer in proto- synthesis , Calvin speculated about the orientation of chlorophyll in 11 . the chloroplast lamella and suggested that the porphyrin rings lie in a characteristic pattern, namely at an angle of about 145° to the stacking axis. (Calvin, 1958; Calvin, 1959) Seely calculated that emery transfer bemeen chlorophyll molecules by a "slow mechanism" (colpatible with Forster's inductive resonance theory) would be fastest when the chlorophyll transition moment vectors are in a collinear arrangement and very small when the vectors are parallel but an echelon by an angle of 60° (Seely, 1973a; Seely, 1973b). This suggested that an expeditious use of orientation would be to group as new chlorophylls as possible into collinear files, stagered 600 from each other, so that transfer would be rapid the length of the file but slow fiom one file to another. The files would lead to the reaction center with as few changes in orientation as possible. On the basis of x-ray crystallographic studies, Kreutz has postulated that the photosynthetic membrane is conposed of three layers: protein, porphyrin ring, and lipid. (Kreutz, 1970; Kreutz, 1972) He felt that the chlorophyll molecules are anchored in the protein layer by means of their phytol chains , and the contact between protein and lipid is established by the 90mm?Em ring which partially penetrate into the unsaturated fatty acid zones of the lipid layer. Based on the assumption that chloroplasts in the natural state exhibit a dichroic ratio of D 8 l for both the chloropl'yll red and principal blue absorption bands (with the exception of chlorophyll-695, for which D > 1) , Kreutz calculated that the porpmrin ring should make an angle of 5A.7° with the lamella plane. Model Systems Chlorophyll is very hygroscopic (Ballschmniter and Katz, 1969) and water is necessary to form mnicrocrystalline chlorophyll (Katz _e_t_ _a_l_. , 1968). Chlorophyll-water complexes have a similar electron paramagnetic resonance spectrum to photosynthesizing chloroplasts, wkereas anhydrone chlorophyll does not. (Katz gt _a_1_ . , 1968) An infrared absorption study of chlorophyll-water aggregates has indicated that the water is hydrogen-bonded both to the ring V ketone carbonyl and to the 0—2 ester carbonyl omen atoms of the adjacent molecule (Ballschmiter and Katz, 1959). From X—ray diffraction determination of the structure of MgTPP'Hz) (T‘imkovitch and Tulinsky, 1969), Mch-H20°205H5N (Fischer 3}; §_1_., 1971), and methyl pheophorbide a_ (Fischer 913 a._l_., 1972), Fischer and co—workers proposed a model of chlorophyll which has dimensions similar to methyl pheophorbide a with the Mg atom 0.50 3 out of the plane and a water molecule 2.02 (A) above the 1% atom (Fischer §_t_ _a_1_ . , 1972) . Hydrogen bonds comect the water molecule to the next chloromell. Repetition by simple translations would lead to molecular crowding, but repetition by a 2 screw axis would 1 permit a satisfactory fit. Hanson reported that chlorophyllide can form a morolayer consisting of close-packed porphyrin rings, and he assumed that the porphyrin planes are tilted at a 55° angle with the plane as in crystals. (Hanson, 1939) From fluorescence polarization study on chlorophyll glipid monolayers, at an air—water interface, Trosper and co—worlaers 13 concluded that in pure chloropryll _a monolayers the pigrent molecules are unordered, in chloroprwll _a_—monogalactolipid monolayers the chlorophyll molecules are randomly dispersed, and in chloropryll a_—"lipid" monolayers (with sulfolipid, oleyl alcolol, or castor oil as "lipid") the chlorophyll molecules are partially oriented (the porphyrin rings making an angle of from 0° to 50° with the interface plane depending on the surface pressure). (Trosper, 1968) V Brody investigated monolayers of chlorophyll g "complexed" with various electron donors and acceptors at an air-water interface (Brody, 1971). From the surface area/chloroptwll molecule in each case, he calculated the angle between the porphyrin plane and the water surface. He found, for example, angles of 39°, 37°, 189°, 116°, and 119° for chlorqnhyll _a_ "complexed" with phenazine methosulfate (P16), PMS + ascorbate, benzyl viologen (8V), ascorbate, and delvdroascorbic acid, respectively. Hoff incorporated chloropm'll _a_, chlorophyll b, and bacteriochlorophyll g in an oriented phospholipid multilayer and measured the orientation of the chlorophyll molecules by polarization absorbsnce spectroscopy (Hoff, 197A). The multilayer contained several Immndred monolayers , with one chloropmrll molecule per 200 phospholipid molecules. He found angles of 55A :t l.l°, 51.6 : 0.6°, and 51.7 t o.2° between the porphyrin rirgs and the plane of the multilayer for chlorophyll _a_, chloroplwll b, and bacteriochlorophyll _a_, respectively. From polarized absorption spectroscopy on an artificial ll} chloropnyll black lipid membrane with a chlorophyll concentration up to 2.5(1013) molecules/omz, Steiremann gt _a_l_. found values of 23 : 2°, 27 : 2°, 29 1 3°, and 29 a 2° for the angle between the principal blue transition morent and the membrane plane for chlorophyll a—phcsphaticwl etharolamine, chlorophyll a-dioleoyl— phosphatidyl choline, chlorophyll _a-phosphaticbrl serirne, and chlorophyll b—dioleoyl—prosphatidyl choline membranes, respectively (Steinemnann _e_:c_ _a_l_. , 1972). They found angles of 35 1 1°, 3& 1 1°, 36 1 2°, and 28 : 2° for the red transition mnorents in the same membranes (in the same order). From these angles, they calculated values of 1m 3 3°, “6 1 3°, 1&9 1 5°, and ‘42 .t 14° for the angle bemoan the porpryrin ring and the plane of the membrane for the above membranes (in the same order as above). From polarized absorption spectroscopy on six chlorophyll- egg lecithin bilayers in series separated by aqueous. phases, Cherry and co-workers obtained orientation angles of 26° and 29.50 for the chlorophyll a and chlorophyll t_> blue transition moments, respectively (Cherry gt a_l_., 1972). 'Ihey found angles of 36.5° for both the chlorophyll a and chloropmll 2 red transition mnorents. From these angles, they calculated values of “8° for chlorophyll _a_ and 51° for chloropryll _b_ as the angle between the porpmrrin ring and the plane of the membrane. Hoff criticized the work of Cherry _et _a_l_. on the grounds that (a) their technique is inherently much less semitive than his multilayer technique, and permits only measurement of the dichroic 15 ratio at one fixed angle, and (b) their values are calculated by assuming that only one dipole moment contributes to the blue absorption band. (Hoff,l9714) Biological membranes The first experimental results of Venice, Frey-Wyssling and Steinmann, and Ruch with polarized light microscopy on unicellular algae, W and Closterium, were interpreted by these researchers as an effect of the stacking of lamellae in the grena (i.e., textural dichroism) rather than as an orientation of pignents. (P'lenke, 1938; Menke, 1958; Frey-Wyssling and Steimnarm, 19148; Ruch, 1957) Goedheer mode absorption measurements in polarized moro- chrcmatic lignt on W and found a weak dichroism in light of 680 nm. (Goedheer, 1955) He concluded that there was a slight orientation of chlorophyll a molecules. Later, by means of linear dichroism and polarized fluorescence measurements on the unicellular algae Moth and Egglona, Olson and co-workers detected a form of chlorophyll with maximum dichroism at about 705 mm and maximum polarized emission near 716 nm. (Olson _e_t_:_ g. , 1961; Olson gt _a_l_., 1962; Olson _e_t_ g._1__. , 1961411; Olson _e_t_ 11., 196%) 'Iromas gt 9;. found a distinct dichroism at about 680 nm in spinach chloroplasts oriented at steel-water interfaces. They interpreted this to be due to 2% of the chlorophyll a—680 being oriented in the plane of the chloroplast lamellae. (Thomas 31; £1: , 1967) 16 Sauer and Calvin oriented spinach chloroplast f‘ragnents by electric field (Sauer and Calvin, 1962) or by velocity gradient (Sauer, 1965) and found a dichroic ratio significantly different from unity only at 695 mm. In the case of orientation in a hydrommamdc gradient, they snowed that the long wavelength absorption oscillator lies parallel "j to the streamlines of the sheer gradient, and assnmed this to be the direction in which the planes of the chloroplast larellae are oriented. They interpreted this dichroism at 695 mm as resulting from 51 of the chlorophyll _a_ which is strongly oriented. Morita and Miyazaki oriented the rod-shaped photosynthetic bacterium Rhodopeudononas palustris cells in a flow-gradient and lamellae in a thin film. (Morita and Miyazaki, 1971) They found small dichroism at 590 mm and large dichroism at 800 on and 870 nm. Geacintov gt _a_l. oriented inorella cells and spinach chloroplasts in aqueous suspension by means of a static magnetic field. (Geacintov _e_t_ a_}_. , 1971; Geacintov _e__t_ §_1__. , 1972; Van Nostrand gt _a_l_. , 1973) They found significant dichroism in the chlorophyll absorption band at 675 - 678 nm and polarized fluorescence at about 685 nm, the chlorophyll _a_ fluorescence band. They concluded that the bulk of the chlorophyll in _v_i_\_i_g is highly oriented with its red transition moment preferentially parallel to the plane of the lanellae. Breton and fellow researchers oriented spinach chloroplasts by application of a static mnagnetic field or by brushing then onto an optically polisred quartz plate. (Breton gt _a_l_. , 1973) 'Ihey oriented spinach chloroplast lamellae by brushing them onto a l7 polished quartz plate or by air—drying a drop of a suspension of isolated lamellae on the plate. They measured the linear dichroism spectrum of the oriented chloroplasts or lamellae with a spectro- polarimeter and calculated the orientations of the dichroic absorption bands' transition moments. 'Ihey found that the y-polarized transition moments of chloropryll _a_-680 and longer wavelength forms of chlorophyll _a_ lie at angles close to the lamellar plane (i.e. , at angles less than 25°-- 30° with the plane). Chlorophyll _a_-670 is less oriented or oriented at an angle slightly less than 35° with the plane. "Negative" dichroism in the Soret band of chlorophyll a implies that the directions of x—polarized transitions are at angles of about 142°. Chloropmll b—650 exhibited a low degree of order, making an angle less than 350 with tre lamellar plane. CHAPTER III EXPERIMENTAL aratus The equipment and parts required for the measurement of the spinach chloroplast extract chlorophyll black lipid mnenbrane photo-emf action spectrum and the chloropmll orientation in the membrane are listed in Table l. lhe experimental set-up is illustrated in Figure 2 (Fang, 1972; Van and Tien, 1970) and Figure 3. One requirement for the membrane chanber is that the directions of the light beam incident on the membrane and the lignt beam reflected from the mnenbrane be perpendicular to the plane of the glass through which each passes. ‘Ihe other requirements are that the mnenbrane chamber provide a stable support for the Teflon bealosr and be of sufficient heignt to allow the outer aqueous medium to extend above the hole in the beaker. The mnenbrane chanber must be isolated from the vibrations caused by the cooling fan for the arc lamp. 'Ihis was achieved by placing styrofoam pads under the lamp housing and under the stand supporting the membrane chalber. Good electrical irnsulation was obtained by the use of non-metallic supports and coaxial cable. The 2 mm diameter role for the membrane was bored in the side of lemon beaker below the level to which the aqueous medium, 18 19 Table 1. List of the equipment and materials necessary to perform the chloropmll black lipid membrare experiments described in the text. Item rbdel and Catalogue No. Manufacturer Electrometer, vibrating reed Electrodes (2) Light source, D.C. Xenon arc lam lamp rousing Lamp power supply Eyepiece Phonetic bars Optic lenses Cell assembly Glass cup , Teflon beaker Micrcpipette Shutter Mitochromator visible grating Synchrorous motor Cary Instruments 31 Fiber Junction calomel 39270 Hanovia, Type 976C (1000 W) Schoeffel LH-151N Selbeffel LPS 255 we 70,266 No. lno,u18 10ml Sampler Photographic Bausch and Iomb, Nbdel 5 Hurst, Model AR-DA (1/3 RPM) Applied Physics Corp. 272‘! S. Peck road Monrovia, California Beckman Instrument Co. 25511 Southfield Rd. Southfield, Michigan 118075 Ehgelhard Hanovia Inc. Newark, New Jersey Schoeffel Instr. Co. 2” Booker St. Westwood, New Jersey Ednund Scientific Co. Harrington, New Jersey 08007 Will Scientific Co. Box 63 Ann Arbor, Michigan 11810? Oxford Laboratories Obtaired locally Bausch and Iomb , Inc. Rochester, New York 111602 Hurst Co. Princeton , Indiana 20 Table l (cont'd). Item Model and Catalogue No . bonufacturer Coaxial cable anhenol-Borg Electronics Co. Broadview Chicago, Illirois Electrical No. 202 HH Obtained locally cornnectors Bakelite insulated phone tip .1 ack Syringe-dispereor )bdel PB-600-l (100 nil) Electrical noise Copper wire mesh shield Light polarizer Polaroid Hamilton Co. Whittier, Calif. Obtained locally Obtaired locally 21 Electrometer } Figure 2. Schematic diagram of the membrane chamber and the electronics for measuring the photo-emf. S, magnetic stirring bar. 8114, black lipid membrane. 22 0.1 M potassium acetate, was added. The membrane was formed by applying a small amount of the membrae-forming solution with a micro—syringe. 'Ib facilitate application, a 2 - in mm piece of 0.038" polyetmlere tubing was placed on the end of the micro- Miriam The positions of the lenses were adjusted until the exciting light was focused upon the menbrane. 'Iie visible wave- lengths were scamed by rotating the monochromator can with a smell motor (Hurst Nbdel AR DA) at approximately 2.5 rum/sec. The lignt- induced membrane emf was monitored with a calonel electrode in each aqueous prose and a Cary 31 electrometer. A convenient recorder speed was 2 in/min. Extraction Technicmes 'Ihe membrane-forming solution was prepared by isolating chloroplasts from commercial spinach and then extracting chlorophyll arnd lipoid materials . All glassware employed was rinsed with acetone and hot distilled water before use in order to avoid contamination by soaps. ‘Ihe specific steps in the extraction are given below (Tien and Howard, 1969) . l. 'Ihe ribs and stalks were removed from 10 oz fresh spinach. The leaves were washed thorongnly, then dried. 2. The leaves were added to a 300 mnl solution of 0.5 M 3 buffer (pH 7.5) in a Waring blender at low speed. When all the leaves were added, sucrose and 0.05 M KHCO high speed was used for 30 sec. 'li'e mixture was then 5. 23 filtered through for layers of cheesecloth; tre residue was discarded. I The filtrate was centrimged in approximately 110 mnl quantities at 8700 g for 5 minutes; the supernatant was discarded. The chloroplasts were re-suspended in the buffered sucrose solution (approximately 15 ml per test tube) using the Vortex mixer, then centrimged at 8700 g for 5 minutes , discarding the supernatant. The chloroplasts were broken by adding 25 m1 of glass- distilled water to each test tube, mixed with the Vortex mixer, tren allowed to stand for 5 mninutes. The mixture was then centrimged at 9700 g for 10 minutes; the supernatant was discarded. The residue was extracted with 90 mnl of 2:1 petroleum ether: methanol (by volune) in the Waring blender at medium speed for l mninute, then centrimged at 5100 g for 10 mninutes. The top layer was pipetted off into a my flask and evaporated to dryress. It was then re-suspended in 5 mnl of 1:1 n-butanol: dodecane (by volume), and stored inthedarkatO-lnoc. 21! Procedure The membrane was formed in 0.1 M acetate buffer, pH 5, across a circular aperture of 2 mm diareter in a Teflon beaker set inside a glass cup. After the menbrane had reached the black stage, Fe013 was added to the inner chanber to bring the 1=ne3+ ion concentration to 1 mM. 'ne open-circuit potential difference across the membrare was monitored by a Cary 31 electrometer via a calomnel electrode in tie aqueous phase on each side of tie membrane (Tien and Howard, 1969) . The black lipid membrane was excited with lig'nt from a 1000 w D.C. Xeon arc lamp (Hanovia, Type 976 C) which was passed througn a teat filter, a visible grating mornochronator (Bausch and Lomb, Model 5), a plano-convex lens, a shutter, a converging lens, a polarizer, and a collimator (Figure 3). During formation, the membrane was observed with dim green lignt(~525 hm). After application of the membrane-forming solution across the aperture, the membrane thinned first to a thickress of less than 1 um. At this stage, if the mnenbrare is observed at an angle with the normal equal to the angle of incidence of illuminating lignt, interference frings are seen (Figure 11a). In this way, the orientation of the mnenbrane relative to the direction of propagation of tie incident lignt was determined. What of the lignt reflected norm the membrane is polarized with the direction of vibration of the electric vector parallel to the plane of the membrane, i.e., perpendicular to the plane of incidence (Figure lb). The direction of polarization of the 25 I I g! ‘l . 3o? 98 nonpon Shoo mo ago? cause-Boom .m are .5338 oooonoaofiz 98 SEER. apogee n88 Figure ll. 26 (A) Schemnatic diagram illustrating the determination of the orientation of the membrane relative to the direction of propagation of the incident light, -'x (top view). 1, angle of incidence; r, angle of reflection. i = r = 4:. (B) Schematic diagram illustrating the establishment of the direction of polarization of the incident light with the electric vector, 15’, vibrating parallel to the plane of incidence (top view). 27 Incident beam W x6 (A) y Reflected beamn Observer Incident \ membrane beam Mr) Reflected beam Observer 28 illuminating light was varied, by rotating the polarizer about the direction of propagation, until the direction for which tie interference fringes were observed to have a minimum intensity. This established the direction of polarization.of the incidbnt light with the electric vector vibrating parallel to the plane of incidence. When the thickress of the membrane has fallen much below 1000 X, destructive interference gives rise to the optically "black" appearance (Tien and Howard, 1969). When the "black" menbrane is illuminated with exciting light, an electromotive force (open-circuit voltage) is generated across it, with the side in contact with Fe3+ icn.becoming:more negative than the other'sideu The magnitude of this "photo—emf" is dependent upon the wavelength of the exciting light, and a "photo-emf’action spectrum" can be obtained by scanning the visible wavelengtre (Van and Tien, 1970). CHAPTER IV 'IHEDRE‘I‘ICAL CCNSIIERA'IICNS Before presenting the results and discussing their significance, a consideration of sore aspects of theoretical back- ground upon which the present interpretation is based is in order. First, it is assumed that the chloropmrll black lipid membrane is a lyotropic liquid crystalline system with smectic structure. In a smectic structure tie molecules are arranged in layers, with their long axes parallel to each other and approximately normal to the plane of the layers. Tie molecules can move in two directions in the plane and can rotate about one axis (Brown, 1967). In the chlorcpmrll black lipid membrane, each transition moment is probably restricted to an direction which lies on a conical surface making an angle of o with the rormel, N, as depicted in Figure 5. The orientation of each transition mnonent, M, is given by the angle between M and the normal, N, to the membrane. The components of M along the two directions, y and z, of polarization of the incident light are 1% and M2, where Pk-Msinesindcosoi-Mcosesinc (l) Mz-Msinecosc (2) 29 Figure 5. 30 Schematic diagram of the transition dipole moment, M, of either the red or the principal blue chlorophyll absorption band (see text). -x, the direction of propagation of the exciting light. a? Black lipid xef 32 The dichroic ratio, D, which is defined as the ratio of the absorbence for morochromnatic light polarized in the y-direction (i.e. torizontally polarized) to that for the light polarized in the z-direction (i.e., vertically polarized) (Setlow and Pollard, 1962) , is given by D-fi— , (3) A2 If the absorption band for lignt polarized in the y-direction has the same shape as the absorption band for lignt polarized in the z-direction, then I Z - 1y (1) Z where I3, and 12 are the integrated intensities for the bands (Orchin and Jaffé, 1971). Since the integrated intensity of an absorption band is proportional to the square of the transition dipole moment, _‘fx. . a . i (5) A2 12 w: Dichroism of chloropl'yll black lipid membranes has been attributed to orientation of chloropmll molecules in the membrane (Crerry _e_t_a_1_., 1971; Qnerry gt gl_., 1972; Steinemenn 935931., 1972). The (kpendence of the magnitude of the blue and red peaks in the photo-emf action spectrum on the direction of polarization of the exciting lignt is apparently a direct consequence of the dichroism of the membrane. If so, then ratios of emf magnitudes of each peak 33 for the stated two directions of polarization of incident lignt should allow calculation of the orientations in the membrane of the transition dipole mmnents of the blue and red absorption bands. For each peak the ratio of photo-emfs, (Em )y/(Ehv)z varied less then 81 with a fourfold increase in light intensity. If the photo-emf, Em, is proportional to the amount of light energy absorbed by the mnenbrane, then the ratio of the emf magnitudes for horizontally polarized light to vertically polarized light for the red peak or the principal blue peak gives the ratio of the absorbances for these directions of polarization (see Appendix A), and WWW)" - "‘2' (6) membrane is given by the expression 2, 2 2 cosB cos 0R+coseB (9) (HAP'ERV E30138 AND DISCLBSICN A typical photo-emf action spectrum of a chloroplast extract black lipid membrane, obtained by the method outlined in the experimental section, is shown in Figure 6. . 'Ihe photo-emf action spectrum shows a slight dependence on the direction of wavelenan scan, with peaks shifting about 5 mm and each peak magnitude changing by a factoer of about 1.2. The peaks in the action spectrum are shifted to the red from the peaks for the bulk solution absorption spectrum. Otherwise, the photo-emf spectrum bears a strong resemblance to the absorption spectrum for chloroptmrll a, with peaks at 1130 arnd 660 on. Other researchers have observed a red shift in the red and blue peaks from the chlorophyll absorption spectrum in bulk to the absorption spectrum in chlorophyll black lipid membranes (Steinemam 9331;, 1971; Cherry 33gb, 1971). It is likely that this is responsible for the red shift in the pinto- emf action spectrum. The photo-emf blue peak is of greater magnitude for the incident light polarized perpendicular to the plane of incidence than for it polarized parallel to it. The situation is opposite for the red peak. From the photo-emf peak values for horizontally and vertically polarized light, the orientation 0 of each transition 35 Figure 6 . 36 Absorption spectrum and photo-emf action spectra of the spinach chloroplast extract black lipid membrane. .... , absorption spectrum of the spinach chloroplast extract membrane-ferming solution in bulk. -—-,‘photo— emf action spectrum of spinach chloroplast extract black lipid membrane, scan from 350 to 800 rmn. _, photo-emf action spectrum of spinach chloroplast extract black lipid membrane, scan from 800 to 350 nm. The action spectra have been corrected to show emf per unit light intensity. 37 Em (Photo-emf, mV) A (arbitrary unit) Wavelength (nm) 38 moment can be calculated with the aid of the equations deve10ped above. 'Ihe results are summarized in Table 2. ‘Ihe principal blue transition moment was calculated to make an angle of 21 i: 2° with the plane of the membrane; the red transition moment, an angle of 38 1 2°. From these angles, an angle (averaged over the chlorophyll a and chlorophyll b in the membrane) of 1&5 1 5° is calculated for that between the plane of the porphyrin ring and the plane of the membrane (Weller and Tien, 1973). These results can be compared with values for chlorophyll porphyrin ring orientation obtained from polarized absorption spectroscopy on artificial chloroprwll membranes by other researchers. For chlorophyll-egg lecithin black lipid membranes, Cherry gt _a;l_. found angles of 188° for chlorophyll a_ and 51° for chlorophyll 2 (Cherry _e_t_ a1. , 1972). Steinemann and co-workers found angles of M r 3°, 1:6 i: 3°, and 1&9 1 5° for chlorophyll a- phosphatich'l ethanolamirne , chlorophyll _a_-dioleoyl-phosphatich'l choline, and chlorophyll _a—phosphatidyl serine membranes, respectively. They found ”2 i '40 for chlorophyll b__-dioleoy1— phosphatidyl cholire membranes. 'Ihere exists the possibility that the acidic bathing solution employed in these experiments converts some or all of the chlorophyll a and chlorophyll t_n in the artificial membrane to pheoprwtin a and pheophytin _b_, respectively, by the renoval of the Mg atom from the center of each porphyrin ring. The central Mg atom of chlorophylls is readily displaced by strong and weak acids (Willstatter and Hocheder, 1907) . 39 an: 38 m H mm m H mm 84 sacs one as $3 m a m: m « Hm m u mm mod xwoo cram on Am I N\kv manage € 3 n ~55 no ocean one ovate—me. on» N more Egon .uo Q83 o5 been: “EB no ocean consume Savannah» Spruce in a zone 3&5 cowgoé: cause .3332? € 0 A :5 53382 6:895: can 5 p583 m 3539830 one w. HHEoeoEo one p26 engage one was? omens .mfio c2328 2.338% on» co schematic o5 . 85a :2quch 83 3305.3 can out #292030 on» non cabana-canon 3:85: 3036 53355 on» «0 05.55:. Enfi cacao nieces pompous 23526 fishes c5 5 8033:ch .m 3nt 1&0 In aqueous acetone, the rate of pheopmtinization is first order in acid concentration (Joslyn and Mackinney, 1938) and in chlorophyll concentration (Mackinney and Joslyn, 19110). The rate constant for Mg displacement in 20% aqueous acetone is S - 6 times larger for chlorophyll _a_ than for chloropl'yll _b (Schanderl _e_t_ _a_l_. , 1962). Activation energy for chlorophyll a was about 11 kcal. 'Ihe plneophytinization of chloropmn in the lipid bilayer would depend on the degree of exposure of the membrane ctnloroptwll porphyrin rings to the acid. Loss of m from chlorophyll has been found to be 13 times as fast in a chlorophyll monomolecular layer at an air-water interface (pH in) as in acetone (Rosoff and Aron, 1965). The rate in the monolayer is sensitive to pressure and the presence H ofO 0a , andrg". 2’ The rate constant for the mnonolayer pheophytinization mereased with increasing pressure. For example, the rate coretant 1 "-1 was 1.36(103) min' at an initial pressure of about 6 (hues/om (i.e. , a molecular area of 120 :2 per chlorqnmll molecule) and l.‘43(102) min-'1".-1 at 16 dynes/om. These results suggested that the onange in orientation of the chlorophyll molecules in the monomer is responsible for the availability of the porpmrrin ring m for reaction. However, the chloroplwll embedded in a lipid bilayer matrix may not be as exposed to the acidic bathing solution as chloroplwll in solution or in a monolayer. The porpm'rin ring orientation angle determdnation in the experiments described herein does not supply enougn intonation to ascertain the availability of ill the chelated chloropmrll It atom to the acid. Comparison of the photo—emf action spectrum and the absorption spectra of chloroptwll in solution and in a lipid bilayer with the absorption spectra of pheoprvtin in solution seems to indicate that a very small amount of chlorophyll in the chloroplast extract chlorophyll-lipid bilayer is pheopmrtinized. The absorption maxima for chlorophyll _a_ and chlorophyll _b in ether have been reported as 1630 nm, 662 rm and 1453 mm, 6142 nmn, respectively. The maxima for pheophytin a and pheoplwtin b in ether are 1408 mu, 667 mm and “314 mm, 655 nm, respectively (Goedheer, 1966). Pheophytinization shifts the principal blue peak about 20 mm to a lower wavelength and the red peak 5 - 13 nm to a higher wavelength. waever, Cherry gt; _a_l_. found maximna at 1039 nm, 672 nm and 1466 rm, 653 nm for chloropmll a and chlorophyll b_, respectively, in the chlorophyll-egg lecithin bilayer (Cherry _e_t_ a_1_. , 1972). ‘Ihese are red shifts from the bulk spectrum of 9 - 13 nm for the principal blue peaks and about 11 nm for the red peaks. Steirnemann and co-workers found maxima at 1&3? mm and 672 nm for chlorophyll a in the chlorophyll-dioleoyllecithin bilayer (Steinemam _e_t_ _a_l_. , 1971). ‘Ihese are red shifts of about 5 mm for the principal blue peak and 10 nm for the red peak from the bulk spectrum. ‘Ii'e photo-emf actiwn spectrum peaks (Figure 6) for the chloroplast extract chlorophyll black lipid membrane are red-shifted 20 - 25 nmn from the bulk absorption spectrum for the principal blue peak and about 3 nm for the red peak. Pheophytinization of chlorophyll, or even subsequent 112 oomplexing of a Fe atom in the center of the porptwrin ring, would change only slightly the directions of the red and principal blue transition mnonents relative to the symmetry axes of the porphyrin ring. For example, the direction of the chlorophyll red transition moment vector is shifted about llo by replacement of the chelated 1% atom by a Fe atom (Platt, 1956). lbs calculation of the angle of tilt, B, of the porphyrin ring is not unduly sensitive to the assurption of a right angle between the red and principal blue transition moments. For example, if this angle is perturbed up to 10° fromn the value of 90°, the resultant uncertainty in a is still only 2 5° (Cherry 9}; _a_l_. , 1972). The polarized light-induced emf in black chloropm'll-lipid mnenbranes is apparently a sensitive technique for investigating the orientation of chlorophyll in a bilayer lipid membrane. It requires only one membrane and a simple electronic set-up. Furthermore, the existence of the phenomenon of light-induced emf in artificial chlorophyll membranes suggests that a similar phenomenon may occur _ip_ :13 in the chloroplast thylakoid membrane during the process of transduction of lignt energy into chemical energy. OiAP'IERVI MISGZILANEOUS Photoelectric Action Spectra with Various Additional Conponents in the Bathing Solutions The dependence of the photo—emf action spectrum of the spinach chloroplast extract chlorophyll black lipid membrane upon the direction of polarization of the excitirg light was measured for the following four sets of bathing solution components (Figures 7 - 10). The exciting light was incident initially upon the solution shown at the left side of the membrane. 1. Fe3+ (lmfl) Black Lipid "Water-soluble" Membrane chloropmrll HAc (pH 5) HM (pH 5) 2. 3... .1; Fe (inn) Black Lipid Thionine (10 a) tambrane HAc (pH 5) HM (pH 5) 3. 3+ _u Fe (lmfl) Black Lipid p—Benzoquinone (3)(1o w) Membrane HAc (EH 5) HM: (PH 5) “3 Figure 7. Nil Photo—emf action spectra of the spinach chloroplast extract black lipid membrane with 1 mM FeCl3 in the outer chamber and "water-soluble chloropl'wll" in the inner chamber. 0.1 ml of "water-soluble chloroprwll" 4L- f‘rom KIRK Laboratories, Inc. , Plainview, New York (no concentration value was supplied by them) was added. _, action spectrum with unpolarized exciting light. ---, action spectrum with the exciting light polarized perpendicular to the plane of incidence. . , action spectrum with the exciting light polarized parallel to the plane of incidence. “5 E5 npwcoang 0mm 0mm. om: co: omm u a u- > . \'-' .. \\ SA . .hn I ... \ "‘floooooooo o . so I o. 00 UN 3m mwm m8 :3 .3 Figure 8. 1:6 Photo-emf action spectra of the spinach chloroplast extract black lipid membrane with l I!“ FeCl3 in the outer charter and 0.1 nM thionine in the imer charrber. _, action spectrum with unpolarized exciting light. ---, action spectrum with the exciting light polarized perpendicular to the plane of incidence. . .. . , action spectrum with the exciting light polarized parallel to the plane of incidence. "7 om» $5 533263 co.» omw om: o a m L J7 mo % ‘00. 00.40”." 00 I he cocooooJ/Oo oooowoo‘oo'ooko'oo on ’ \ooo /oooo coo. \ $- on. a $0 I coo. coo. \\ n... a. ’ .... x m , \... y mg x to ’ \ co ’ .000 a... \ LI u. /.n\ x \ m ... ms / x 09 1 'NH mmm ma so: om $5 Em Figure 9. 148 Photo-emf action spectra of the spinach chloroplast extract black lipid menbrane with 1 nfl FeCl3 in the outer chamber and 0.3 an p-benzoquinone in the inner chamber. ______, action spectrum with unpolarized exciting light. -—-, action spectrum with the exciting light polarized perpendicular to the plane of“ incidence. . . . . , action spectrum with the exciting light polarized parallel to the plane of incidence. Q9 Aenvafimwmqu>m3 om» co» 0mm cow ommn O In :1- O O :1- 8 m ...Zuln cocooooooto'ooo’oq’ 1 0 GP 0 u D o :7 \III 0 \ I oo 0N0 ooooooooo I I \oo‘o‘ooooo: oooooooooooo one... I ‘Il” \000 :00. I \oo mww coo-o. ooooooooy \ooo .000 000 o\o awn luo.: mm: mm: Es aim Figure 10. 50 Photo-our action spectra of the spinach chloroplast extract black lipid membrane with 1 mM FeC13 in the outer chamber and 14 HM L-ascorbic acid in the inner chanber. _, action spectrum with unpolarized exciting light. --—-, action spectrun with the exciting light polarized perpendicular to the plane of incidence. . . . . , action spectrum with the exciting light polarized parallel to the plane of incidence. 9.5 egg: o2. om» o8 omm . 3m om: 8: Rm 51 w . 4 if mm: om .v $5 Em 52 Fey-(11M) Black Lipid L-Ascorbic d Mm (h) (10" ) H mm (PH 5) HAG (DH 5) All of these spectra exhibit a dependence of the red and principal blue peaks upon the direction of polarization of the exciting light which is similar to that exhibited by the anthrane with only Finn3+ in one bathing solution. However, the addition of mother species of dnrounophore ("water-solnble" chloropmll or thionine) and/or the alteration or the photochenistry probably rembr incorrect an interpretation of charges in the ratio (Hwy/(Em), as reflecting only changes in the orientation of the menbrane chloropmrll ring. Preferential "Polarized Lijnt [Iihoto-Bleachirg" of Gnlorepm'll Molecules in the Meubrane arnd the Attenpg to Engloy it for the Determination of a Rotational Diffusion Relaxation Time for the Henbrane Chlorophyll Molecules Under continuous illunination of the spinach chloroplast extract chlorophyll black lipid nenbrane with light of either the red or the principal blue photo-emf peak wavelength, the photo-emf first (a) increases linearly (Figure 11), then (b) the rate or increase of F1” decreases until (c) Em remains constant or decreases. 'nne existence of this saturation of the capacity of the membrane to transduce light energy into energy stored in an electric field suggests a possible method for the determination of the 'difmsional \ A 53 an, (IN) A 3 .. 2 P I’M 1 .- a 4 a 1 1 o i 15 30 “5 60 >T1m (sec) Lignt on Figure 11. Shape of a typical plot of the photo-emf versus time. Excitation of the spirnach chloroplast extract chloropwll black lipid snonbrane is at either the principal blue peak or the red peak wavelength. The athirg solutions are 0.1 M acetic acid (pH 5) with 10' M F9013 in the inner chamber. See text for Further details. 514 rotation frequency of the chlorophyll mlecules in the humane. If the mennbrane chlorophyll molecules with their red (or principal blue) transition smarts more aligned in a certain direction —- e.g. , sore vertically oriented than horizontally —- could be preferentially "photo-bleached" with plane-polarized light, then there would be a snbsequent mart of time before rotational diffusion would again randonnlze the azimuthal orientations of the "bleached" chlorophyll nolecules. During this randomizing interval, excitirg lignt plane- polarized normal to the polarization direction of the previously ennployed "bleaching" lignt would give rise to a photo-emf of nearly "unbleached" nagnitude, whereas light polarized in the "bleaching" plane would result in a photo-emf of smaller mgnitude than that when "unbleached." I.e., the ratio (Ehv)y/(Fhv)z will have a value different than when the membrane is "unbleached." The time required to return to the "unbleached" value of (Ehv)y/(Ehv)z is then related to the rotational diffusion relaxation tire for the mennbrme chlorophyll molecules . In order for an experimental determination of the rotational diffusion frequency to be perfbrrned using preferential "bleaching" of nnenbrane chloropmll molecules with plane-polarized lignt, it would be desirable first to stow that the preferential "bleaching" can occur. I.e. , it must be shown that the unantnrane chloroplwll molecules can be "fixed" (e.g. , with glutaraldehyde, mo“, or coon) so that preferential "pl'otobleaching" can be observed over a period of tire of the order of a few seconm or sore. This technique for the determination of the rotational diffinsion relaxation tine for the chloropmrll molecules in the ‘ moraine is in some aspects similar to the techniques of Brown and Gone who established first that (a) dichroism of frog retinal rod outer segnant viewed end-on can be photo-induced by partially bleaching rhocbpsin with plane—polarized lignt following fixation with glutaraldebyde (Brown, 1972), and then that (b) the rapid decay of the dichroism induced by a flash of plane-polarized light provides a direct neasure of the relaxation time —- 20 usec --- of rhodopsin in the receptor membrane (Gone, 1972). Isolated rhodopsin is highly dichroic, absorbing light most strongly when the electric vector is parallel to the log conjugated chain of ll-cis retinal (Clayton, 1971). Rodopsin consists of the chronophore ll-cis retinal attached by a Schiff's base linkage to the lipoprotein opsin. Several chemical and structural properties of rhodopsin suggest that rotations of the chromonhore accurately reflect rotations of the entire molecule . Photoisonerization of retinal from ll-cis to all-trons initiates a bleaching process which consists of a series of configuration transitions in opsin, and leads eventually to the release of retinal: hv modopsin -—) Prelnml —-) Lani —)neta I (“98 nm) (5"3 nm) ('497 nm) (8 run) ~lv Retinal + main (—— Para L—weta II (387 m) (“65 mu) (330 m) 56 he absorption maxim are shown below the configurations . Retinal in rhodopsin and lunirhodopsin has an absorption maxinun at about 500 nm, whereas in prelnmirhodopsin the maxim shifts to 5113 nm. At the meta II stage, the specth shifts far to the blue, becoming similar to that of free retinal. Attemts were node to "fix" the chloronmu molecules in the spinach chloroplast chlorophyll black lipid nnenbrane with glutaralchmrde, and subsequently to induce a change in the ratio (Fifi/(Em): with plane-polarized light at the photo-emf red peak (Table 3). It was tbnnd that the presence of glutaraldemrde has no effect , or perhaps only a swell effect , on the morone photo-emf action spectrum. However, no change in the ratio (Shay/(E1102 upon excitation with plone-polari zed light was observed. Similar concentrations of mo“ also proved ineffective. Some coments m be in order on possible reasons for the ineffectiveness of glutar'aldelwde in "fixing" the nubrone chlorophyll molecules sufficiently to enable photo-induction of dichroism. Glutaralderwde has been used by researchers principally to stabilize proteins by cross-lirnldrg then. It may only react with lipids con- tainirg free annino MB (e.g. , phosphatidyl ethanolannnine) (Jornston and Roots, 1972). The reaction of an aldem'de with a primary amine is expected on the basis of classical organic reactions (i.e. , Schiff base formation). Althougn Brown observed very strong photoinduced dichroism with a 2 . 51 glutaraldemde concentration fixation of rl'noonpsin in the disk nenbranes (Brown, 1972) , there are inportent differences between A- 57 .852 a; ma o.~ a. om o; o; oSz o4 o4 a; m 3 m5 m5 802 o.m m4 o.m m S no fio ocoz o.m in new m 2 ll do 3383 hops” Locus hone—Eu non—BS ooh mm 6.25 $33 .8on 3:5 oases A55 Ea: 35 ago 5 mwcwno oooow poeéfi a e horseman peruse eroegeau .265 A ram—{”35 m5. 85 umm ao efioé . 3&3ng xmoa ow.» 5590on cofioom uemlouona on”; one: e: 8e consumed headaches, or? ..oonoeoap. he: acetate poo: xeeS 3390.320 poguxo 9830.830 sagas 05 .oogoggaw on mango wcazodnpm .ooEoogoiw 5? 89038 Sandpaper matafie as oo 8398 so 3953s .88 .m magma 58 his system and an artificial morbrane. 'lhe retinal rod outer segnnent is a tissue and rhodopsin is lat-31y protein. 'lhe black lipid unen- brane contaim only chloroptwll and lipids . It nust be noted that Vasquez and co-workers (vasquez _e_t a}, , 1971) ennployed a 23 glutaraldemrde concentration in fimtion of black lipid nenbranes for electron microscopy. The black lipid nervbrane types which they fixed were (a) lipidic -- total phospholipids of the cerebral cortex and cholesterol, (b) lipidic-proteolipidic --- with snail amounts of proteolipid from Electrophoms electrqnlax, and (c) protcolipidic -- from Electrophorus electroplax. Effect of Voltage _Applied to Spinach Wombat Extract Black Lipid Mennorone on the Dependence of the Photo-emf Action Spectrun upon the Direction of Polarization of the Exciting Lignt Externally applied voltages across the unenbrone up to 35 mV had no observable effect on the dependence of the photo-emf action spectrum upon the direction of polarization of the exciting lignt. APPENDICES APPENDIX A armor Pmpom'IonAm'rr or nus mono-am, rm, mmmorumammomabarm mama IEAIB TO DIRECT Paommovm or (Fifi/(31192. TO THE monorc RATIO The fraction of the light incident on the umbrene which is absorbed is (1A) where Io and IT are the intensities of the incident and the trans- mitted lignt, respectively. The fraction, 9, is related to the absorbance in the following mamer A - -log(1 - o) - -(l/2.303) ln(l - a) (2A) The red or principal blue peak absorbance of a chlorophyll- lipid bilayer is at best of the order of 0.010 (Cherry 9_t_ gl_. , 1971; Cherry g_t_ 9.1;: 1972; Steinenenn 2331;, 1971), which inplies a value of about 0.023 for 9. Expansion by Taylor series of the expression for the absorbance in term of the fraction, 9 , absorbed yields A - +(l/2.303) (p + 92/2 + 03/3 + 0V“ + "') (3A) 59 60 for (-1< o <+l). (Clayton, 1970) For the absorbance of the order of 0.010 (i.e. , for a value of p of about 0.023) the following approximation holds, with an error of less than :21, A - - p/2.303 (HA) Then, the ratio of the absorbances for nnonochronatic lignt polarized horizontally to that for the light polarized vertically is given by A}, cry {(10 - IT y)/(IG)} --— - —— - ’ (5A) A 9 (IO " Lr’z)(Io) where IT y and IT,z are the transmitted intensities for the incident ’ light polarized horizontally and vertically, respectively. Since the incident lignt intensity, 10, is the sane regardless of its polarization, (I - ) i- o ITJ (6A) Az (Io - IT,Z) If the assnmption is made that the photo-emf, Em, is directly proportional to the amount of light enery absorbed by the neubrane an . (Io ~LI.)tS <7A) wheretisthetimeofeiqnosureofthemenbronetotheligntandSis 61 the honoree area. Since the nnenbrane area, S, rerains constant and is exposed to either polarization of the incident lignt for the same time interval, t, (Eho)y (IO ' ITJ) .. ‘3' (8A) ‘2 (Ehv)z - (Io " I‘I',z) (Em)y @ an). ' n2 (9A) APPBWDIX B WATIWGF'IHEANGIE, 8, m MMOF'IHEPOWRJNGAND MNOML,N,'ID'IIEW 'No vectors of am magnitudes, one along the direction of the red trarsition noment, MB, and the other along fine direction of the principal blue transition monent , NR, porphyrin ring. ‘lhus, for ease of calculation, select both vectors, determine fine plane of the BC and 80, with magnitude m (Figure 12). ”B and MR are nutually perpendicular and make angles of GB and respectively, with the normal, 10. OR, Angle can - era (18) N is dram long enough that its projection, n cos 8, upon fine porprwrin plane has an endpoint, E, on fine diagonal E. Then, m-nsina (23) AngleAB-MgleAED-Angleflc-w/Z (38) E-E-mrér' (‘8) Since triangle so: is an isosceles rignt triarngle, 0 MgleBm-AngleBIE-w/H (SB) 62 53 —>~a Figure 12. Schenatic diagram of the relation between the red transition noment vector, , fie principal blue transition monent vector, , and the normal to the membrane, I. See text for mrther details. 614 CD— 8 m ’2'" (68) Applying the Pythagorean Theorem to triangle AED, KHz-n2 sinZB +E2 (7a) and to triagle AEC, Tile-n2 sinzs +fi‘? (m) Subtracting equation 88 fron equation 73 , Id? - Id?- - m?- - o2 (98) Applying fine Lav of Oosines to triangle ABD, Ive-nzermz-amoos 913 (nos) and b0 triangle ACE Ez-nzi-mz-ancoso R (118) Subtracting equation llB fron equation 1(8, fi-Ké-meoseR-cosoB) (res) Ehuating (1'52 - A?) in equation 958x! equation 128, mz—Ez-anwos eR-cos GB) (133) Substituting for E from equation MBinto equation 19. mz-(a/T-m2)-am(eoeeR-eoee3) (nus) El'é—Inn(cosoR-coseB)+m (158) 65 Squarirg both sides of equation 19, 2 -n(cosoR-cos03)+2nrn(cosoR-cos05)+m (163) Substituting for m2 fronn equation 168 into equation 73, 2 B52-n2sin284-n/2 (cos OR-cos GB)2 +nnrn(cos eR-coseB)+m2/2 (173) Tin—DQ-nZ-mz/Z-nz ooszsi-nZ/Z (008 93-008 93)2 +rm (cos OR" cos 88) (1%) Substituting for (m2 - n2) from equation lCB into equation 18B, 2 m2-2nncos OBI-Ima/Z-n2 cosza+n2/2 (cos oR-cos GB) + m (cos 0R - cos GB) (198) m2/2 -nrn (cos OB + cos OR) II n2/2 (cos 0R - cos 9B)2 . n2 0082 6 (2(8) Substituting the trigononetric identity (sinza - l - 00328) in equation 78, TAI_)2a-n2(1-cos28)i’fi-iz-nzanzcornea eta? (213) nacosZB - m2 I n2 - A152 (228) Applying the Law of (bsines to triangle BIB, nzcoszs - an2 4- E2 - 2m as) cos “5° (23) 66 nzcoszs - m2 - m2 - mm) f2— (2143) Equating (nzcoszs - E2) in equations 223 and 218, nZ-Zfiz-mz-Mm IT (258) flaming equation SB. nZ-Ez-amcoseB-mz (26s) Equating (n2 - H52) in equations 258 and 268, mz-m(fi) W-Zmncosea -m2 (273) an-E/E—IchoseB (288) Substituting for re I 2 from equation 158 into equation 2m, 2nn-{n(cosoRucosonB)-!-nnn}--32rncos(lB (2Q) In-n(coseB+cos OR) (3%) Substitutirg for m from equation 3m into equation 2m , 2 2 _ 2 2 n/2(coseB+coseR) n (cosaB+cosoR) 2 2 +n/2 (cos OR-cos OB) - nzcoszs (313) Siflplimns, cosB-ooszeRi-cosea (323) BIBLIOGRAPHY L Arnon, D. 1., Whatley, F. R., and Allen, M. 8. (1951!), _J_. Am. anem. 833. 76, 6321!. Ballschmiter, K. and Katz, J. J. (1969). g. _An_nn_. Chem. §_oc__. 91, 2661. Breton, J. , Michel-Villas, n., and Paillotin, c. (1973). Biochim. Bioptys. Acta 31", 112. Bretscher, M. 8. (1973), Science 181, 622. 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