AN INVESTIGATION OF THE PHYSICAL CHEMICAL CHARACTERISTICS OF LIGHT - INDLICED EFFECTS IN PIGMENTED BILAYER LIPID MEMBRANES Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY PAUL SHIEH ‘ 1975 W7 g1. .. 1.1535125: RY , ’Micbggm Stare This is to certify that the thesis entitled AN INVESTIGATION OF THE PHYSICAL CHEMICAL CHARACTERISTICS OF LIGHT_INDUCED EFFECT IN PIGMENTED BILfiH§R LIPID MEMBRANES. presene y Paul Shieh has been accepted towards fulfillment of the requirements for Doctor degree in Biophys i c s “fine ‘ M - or professor 0-7639 FEW It ABSTRACT AN INVESTIGATION OF THE PHYSICAL CHEMICAL CHARACTERISTICS OF LIGHT-INDUCED EFFECTS IN PIGMENTED BILAYER LIPID MEMBRANES By Paul Shieh Since their discovery by Rudin and his associates, bilayer lipid membranes have been used to investigate the mechanism of photoeffects. Explanations of the mechanism of photoeffects in BLM (Bilayer Lipid Membrane) may be classified into several models. Among the models currently proposed, the redox electrode model has recently gained more support, mainly because of its ability to explain the way charges are generated by light as well as the way these charges are transported across the membrane. Nevertheless, the developments of more definitive experi- ments are required to support this model more fully. According to the redox electrode model, pigmented BLM behaves similarly to a metallic surface or an inert redox electrode. That is, the pigmented ELM is capable of facilitating electron transfer in inducing the redox reaction. Three experiments were carried out to study the electronic conducting characteristics of pigmented BLM: including comparative measurements of the potential of BLM and platinum cup; the electrostenolysis in BLM; and the measurement of redox reaction in carotene or xanthophyll incorporated BLM in the dark. These systems were also illuminated to detect any light-induced electronic processes. Paul Shieh The results from BLM electrostenolysis measurement indicated that the electron was driven across the membrane from one side to the other side of biface and a metallic mirror was formed from the discharge of the metallic ion on the biface. A linear relationship between the redox potential difference of two redox couples and the observed membrane potential was found. It implied that BLM containing carotene or xantho- phyll functioned like an inert redox electrode, capable of accepting electrons on one side of biface and releasing electrons on the other side. Based on the biophysical aspect of the primary process of photo- synthesis currently understood, three fundamental questions can be asked: 1) how can light energy be utilized more efficiently; 2) by what mechanisms are light-induced carriers transported; and 3) how can proper conditions be established for separating generated redox products? One of the major aims of this investigation was to construct a BLM to look for the possible mechanisms and provide the best answers for the above questions. The detailed mechanism of the primary process in photosyn- thesis is still not quite known. However, it is speculated that the primary process could involve the mechanism of the energy conversion process. This process requires that the electron reverse directions in the redox potential gradient when light is applied. A theoretical treatment to correlate the Chl-BLM (Chloroplast Bilayer Lipid Membrane) photoresponse and the redox potential differ- ence across the membrane was made. Experimental evidence in support of this correlation was given. In summary, this study has accomplished the following: 1) distin: guishing clearly between electronic and ionic conduction across the BLM; 2) finding conditions in which the BLM behaved, in the dark, exactly Paul Shieh like an inert redox electrode; and 3) making biological applications of the BLM, such as studying the possible mechanism of the primary process of photosynthesis. AN INVESTIGATION OF THE PHYSICAL CHEMICAL CHARACTERISTICS OF LIGHT-INDUCED EFFECTS IN PIGMENTED BILAYER.LIPID MEMBRANES By P \L' J Paul Shieh A THESIS Submitted to MiChigan State University in partial fulfillment of the requirements for the degree of DOCTOR.0F PHILOSOPHY Department of Biophysics 1975 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. H. Ti Tien for the guidance and encouragement, as well as friendship, which he generously extended throughout the course of this investigation. Especially, it is due to Professor Tien's deep insight into the nature of bimolecular lipid membrane that the successful formation and characterization of the membrane system used in this work have been acheived. Thanks are also due to Dr. Peter T. Kissinger, Dr. Gabor Kemeny, and Dr. Estelle McGroarty for the invaluable discussions rendered during the course of this work and for critical reading of the thesis. A lasting sense of gratitude and appreciation is extended to the author's wife, Diana and daughter, Kate and parents for their patience, understanding and encouragement which they expressed throughout this study. Financial supports were obtained from the National Institutes of Health Grant GMr1497l and from College of OsteOpathic Medicine, Michigan State University. ii TABLE OF CONTENTS Page ACWOWIIEDGMNTS OOOOOOOOOOOOOOOOOOOOOO0.00...OOOOOOOOOOOOOOOOOOO ii LIST OFTMLES O...0.0.0.0000...OOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOO Vi LIST OF FIGUES 00......0.0.0.0....OOOOOO'OOOOOOOOOOOOOOOOOOOOOOO Viii GI‘OSSAM OOOOOOOOIOOOOOO0.000000000000000000.0.00...OOOOOOOOOOOO x Chapter I O ‘ INTRODUCTION 0 O O O O O I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 O O O 1 II 0' LITERATURE REVIEW 0 O O O O O I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 8 Redox Reaction in Artificial Membrane..................... 8 Various Models for BLM Photoeffects....................... lO Photosensitizing Process.................................. 16 Energy Conversion System.................................. 21 Energy Conversion Process Performed in Photosynthesis..... 24 III. EnERImNTAIJOOOOOOOOOOO0.0...OOOOOOOOOOOOOOOOOOOOOO0...... 25 1. Material Used and Solution Preparation................ 25 a. Chl-BLM Forming Solution Extraction............... 25 Oxidized Cholesterol Membrane Forming Solution Preparation.............................. 26 b. Chemical Solution Preparation..................... 27 2. Procedure............................................. 27 a. Photoresponse Meqsurement......................... 27 b. Membrane Resistance Measurement................... 28 c. Chlorophyll Concentration in Spinach Chloroplast Measurement........................... 28 d. The Standard Redox Potential Difference Between Two Redox Couples Measurement............. 34 e. Photosensitizing Measurement in BLM............... 34 Page IV. RESULTSOOOOOOOOO ...... 00.0...0.0.0.0..OOOOOOOOOOOOOOOOOOOO. 38 1. The Determination of Electronic and Ionic Conduction in Bilayer Lipid Membrane................... 38 a. A Comparison Study Between the Platinum Cup and the BLM............................................ 39 l. The Iodide Ion Diffusion Measurement........... 39 2. The Electronic Conduction Via Redox Reaction Measurement.................................... 40 3. Studies of the Factors Governing the Electronic Conduction Via Redox Characteristics in BLM and Platinum Cup............................... 41 b. Electrostenolysis in the Bilayer Lipid Membrane.... 43 c. The Redox Reaction in Oxidized Cholesterol BLM Incorporated carotene, in the Dark................. 48 2. Light Energy Utilization and PhotOproduct Separation in the Bilayer Lipid Membrane.......................... 48 a. The Asymmetrical Location of Photoactive Pigment in BLM.......HHH.................................. 48 b. The Nature of Photoactive Pigments................. 57 c. The Effect of Charge Carrier and Electric Fields Upon the Ox-ChO-BLM Photoresponse.................. 9O 3. The Photosensitizing Process and Model Studies of the Electron TranSport in Photosynthesis............... 92 a. The Photoreduction of M; Viologen Via Electron ‘ Transport Frmm D (Electron Donor) to M. Viologen.. 93 b. The Photooxidation of Ascorbate and DCPIPH Via Electron TranSport From Ascorbate To A1 (Electron Acceptor From Photosystem 1)....................... 94 c. The Photosensitizing Process in Ox-ChO-BLM and Chlorophyllin..................................... 100 d. The Photoreduction of A , the Electron Acceptor From Photosystem-Z, in 1-BLM.................... 101 e. The Resumption of Electron Transport Activity Prohibited by Inhibitor, in the Presence of Proper Electron Donor....................»........ 103 f. Operational Separation of Two Photosystems in Chl-BLM by Using Light Wavelength of 700 mu....... 105 v. DISWSSIONOOO0.0.0...OOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOOI...107 1. The Electronic and Ionic Conduction in Bilayer Lipid Membranes............................................. 107 2. Theory of Bilayer Lipid Membrane Photoresponse........ 120 a. Generation Consideration.......................... 120 b. Comparison Between the Observed Chl-BLM.photoemf and Redox Potential Difference of Redox Couples... 124 c. Factors Affect Standard Redox Potential as Well as Chl-BLM Photoemf............................... 124 Page d. Further Experiments in the Verification of the Theory.......................................... 131 3. The Energy Conversion Process and Nonenergy Conversion Process in Bilayer Lipid Membrane................... 142 BIBLIOGRAPHY ................................................. 151 Table 10. 11. 12. LIST OF TABLES Page The concentration of chlorophyll in spinach Chloroplasts extracts.0.00.00.00.000000000000IOOOOOOOOO... 33 The measurement of the potential change generated from the iOdide ion diffmion.OOOOOOOOOOOIOOOOOOOO0.0.0.0000... 40 The measurement of electronic conduction via redox reaction in BLM and pt cup................................ 41 The similarity between BLM and platinum cup in electron releasing—accepting and redox characteristics............. 42 The comparison of the redox potential difference and the observed membrane potential produced from redox reaction in OX‘ChO-‘BLM and in Chl-Bmoocoo...00000000000000.0000... 49 The Ox-ChO-BLM photoelectric effect in the presence Of nitm pigments.OOOOOOOOOOOOOOOOOOOOO0.0.0.0....00...... 58 The Ox-ChO-BLM photoelectric effect in the presence Of nitroso pigments...0....OIOOOOOOOOOOOOIOOOOOOOO00...... 59 The Ox-ChO—BLM photoelectric effect in the presence Of 820 dyeSOIOOOOOOOOI00......0.0.0....OOOOOOOOOOOOOOOOOOO 60 The Ox-ChO-BLM photoelectric effect in the presence Of aZine dyeSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.000...O 64 The Ox-ChO-BLM photoelectric effect in the presence Of ac1dicdyeSIOOOOOOOOOO0.000000000COOOOOOOOOOOOO0.00.... 65 The Ox-ChO-BLM photoelectric effect in the presence of xanthene dyes........ ..... ...................... ....... 69 The Ox-ChO-BLM photoelectric effect in the presence of anthraquinone dyes..................................... 74 vi Table Page 13. The Ox—ChOeBLM photoelectric effect in the presence ’ of phthalocyarline dyeSOOOOOOOOIOOOOOOOOO00......0.0.0.0... 78 14. The Ox-ChO-BLM photoelectric effect in the presence of triphenylmethane dyes.................................. 82 15. The Ox-ChO-BLM photoresponse in the presence of basic, acidic and neutral dyes at various bathing solution pH's.. 86 16. The enhancement of Ox-ChO-BLM.photoresponse in the presence of KI and tetraphenylborate...................... 91 17. The photoreduction of m. viologen by Chl-BLM photoresponse measurement................................. 95 18. The Chl-BLM photoresponse measurement of ascorbate and DCPIPH photOOXidation.000......OOOOOOOOOOOOOOOO0..0.. ..... 96 19. The Ox-ChO-BLM photoresponse in the presence of chlorophyllin and electron aCceptors...................... 101 20. The Ox-ChO-BLM photoresponse at the optimum pH for redox compomd SOIUbj-lity.OOOOOOOOOOOOOOOOOOOOOOOO0..0.0.0.0.... 102 21. The photoreduction of A , the electron acceptor from photosystem-Z, in Chl-B OOOOOOOOOOOOOOOOOOOOOOI0...00.... 103 22. The resumption of inhibited electron transport in Chl-BLM in the presence of proper electron donor.......... 104 23. The Chl-BLM photoresponse in the presence of m. viologen and ascorbate by light with different wavelengths......... 105 24. The comparison of Chl-BLM4photo-emf3and gtandard redox potential difference of X /X and Fe /Fe ................ 125 25. The comparison between the Chl-BLM photo-emf in the presence of two redox couples and the Chl-BLM photo-emf summed from that of presenting each individually.......... 132 vii LIST OF FIGURES Figure Page 1. Set up for BLM photoresponse measurement....... ..... ....... 30 2. Electric circuit for BLM photoresponse..................... 32 3. Cell arrangement for the redox potential measurement....... 36 4. The Ox-ChO—BLM photorssponse versus the electric field, in the presence of Fe and chlorophyllin.................. 54 5. The time course of Ox-ChO-BLM,photo-em§+at various membrane electric fields, in the presence of Fe and chlorophyllin. 56 6. The time course of Ox-ChO-BLM photoresponse in the presence Of 820 dyes...OOIOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 63 ‘7. The time courSe of OerhO-BLM photoresponse in the presence of azine dyes........................... ..... ..... 67 8. The time course of Ox-ChO-BLM photoresponse in the presence of xanthene dyes........................... ....... 73 9. The time course of Ox-ChO-BLM photoresponse in the presence of anthraquinone dyes............................. 77 10. The time course of Ox-ChO-BLM photoresponse in the presence of phthalocyanine dyes............................ 81 11. The Ox-ChO-BLM photoresponse in the presence of basic, acidic, and neutral dyes at various bathing solution pH's.. 85 12. The C3 -BLM time course photoresponse in the presence Of Fe salts and ascorbateOOOOOOCOOOOOOOIOOOOOOOOO00...... 99 13. The linear relationship between the redox potential difference of redox couple with respect to reference couple and the Ox—ChO-BLM/carotene membrane potential produced from redox reabtion across the membrane.... ....... 112 viii Figure Page 14. Time course Ox-ChO-BLM photoresponse with and without caroteneOOOOOO0.0.0.0.0000...0.00.0.0...OIOOOOOOOOOOOOOOOO. 116 15. Time course of photoresponse in Chl-BLM/FeCl3 with and Without T¢BOCOOOOOOOOOOOOOOOOCOOOOOOCOOCOCOO0.000.000.0000. 1'18 16. The plot of redox potential of organic compound versus pH.. 128 17. The BLM photoresponse versus light intensity............... 133 18. The light intensity dependent Chl-BLM photoemf............. 136 19. The light intensity dependent Chl-BLM photoemf............. 138 20. The Chl-BLM photoemf versus logarithm of Fe(CN)g3/Fe(CN)24. 141 21. Mechanisms of light-elicited phenomena in pigmented BLM.... 144 ix Ox-Cho-BLM or Cho. BLM T¢B Chl-BLM Photo-emf TMPD DCIB+,DCPIP+ TCIP+ NADP+ GLOSSARY Oxidized Cholesterol Bilayer Lipid Membrane Tetraphenylborate Anion Chloroplast Bilayer Lipid Membrane Light induced - electro motive force or net potential change (VL-VD) platinum cup membrane dark potential membrane light potential sodium buffered acetate potassium iodide phenazine Methosulfate Falvin Monoucleotide N,N,N',N'-Tetramethyl-o-phenylene-diamine 2,6-Dichlorophenol-indophenol Trichlorophenol-indophenol Nicotinamide adenine dinucleotide Electron acceptor from photosystem 1 Electron acceptor from photosystem 2 Electron donor from photosystem l open-circuit photoresponse closed-circuit photoresponse CHAPTER I INTRODUCTION Since the discovery by Rudin and his associates, bilayer lipid membranes (or BLM) have been used to investigate the mechanism of ion permeability, photovoltaic effect and photoconductivity. Hitherto, several hypothetical models to explain the mechaism of photoelectric effects in BLM that have been suggested by Tien (1968, 1974). These are: (1) solid state, (2) charge injection, (3) redox electrode, (4) dipole or electric double layer, (5) ionic diffusion, and (6) conformational change. A brief description of each mechanism and the supporting data are given in Chapter II. In view of the preliminary and qualitative nature of the data reported by many authors, only speculative explanations for these mechanisms have been given, and without more quantitative data, those explanations may be applicable only to Specific experimental conditions. For instance, the BLM of the type Kuhn and Ullrich (1972) and Hesketh (1969) used are impermeable to ions, such as Na+ or Cl-ions. But the electric double layer model they adopted to explain the BLM photoeffect would imply that the slow decay portion of the photoresponse was the result of the diffusion of the above ions. Therefore, this model seems to explain improperly the slow decay of the photoresponse for the type of 1 membrane used. Furthermore, their explanation of the fast rising portion of the photoresponse is based on the binding between the electron and the cation on one interface and between the positive chlor0phyll molecule and Cl—ions on the other interface. In fact, an electron captured by the electron acceptor could cause the reduction of this acceptor, resulting in a negative of photopotential on the electron acceptor side of the interface relative to the other interface. We have observed an opencircuit photoemf of about 140 mv in chlorOplast BLM in the presence of iodide ions and Fe+3 (Shieh and Tien, 1974). The observed photoresponse was positive on the iodide side, indicating that the iodide has been oxidized and Fe+3 reduced. Since this photo- response was a result of the light-induced charges separating in the absence of an electric field, it contradicts the finding of Hesketh, who observed no net charge separation in the absence of an electric field. In view of the BLM photoeffect, any proposed model to explain these basic phenomena must include a description of the way charges are generated by light as well as the way these charges are transported across the membrane. Among the models currently prOposed, the redox electrode model has recently gained more and more support, mainly because of its ability to explain the above phenomena. Nevertheless, the deveIOpments of more definitive experiments are required to support this model more fully. According to the redox electrode model, pigmented BLM behaves similarly to a metallic surface or an inert redox electrode. That is, a pigmented BLM is capable of facilitating electron transfer in inducing redox reaction. Therefore it must be able to provide satisfactory explanations to following observations: 1) the same photovoltaic phenomenon was observed in BLM as in metallic surface, 2) the electro- stenolysis -- the process of observed metallic mirror formation by the discharge of metallic ions -- was observed in BLM as on a metallic surface, 3) for carotene-incorporated BLM, a linear relationship between the redox potential difference of two redox couples and the observed membrane potential was found. An experiment confirming the first observation above -- the photo- voltaic effect in BLM -- was conducted by Tien in 1968, in the form of a photoemf measurement. Since the BLM photoemf was generated by the separation of charges (electrons and holes) resulting from the transport of these charges across the membrane, therefore the electronic conducting characteristics of BLM must play an important role in the above phenomenon. Hence an experiment to demonstrate the electronic conduction in BLM must be done in order to provide the complete explan- ation of the first observation above. A comparative experiment between the BLM and a platinum cup to look into these electronic conducting characteristics was conducted in this thesis. The similarity between these two systems was discussed. Electrostenolysis phenomenon was first observed by Becquerel in 1867. It was found that the electron was driven across the wall of a glass tube to discharge a Cu2+ ion. A metallic mirror was formed on the wall of this glass tube by the discharge of this Cu2+ ion. In line with this finding, if BLM can indeed facilitate the electronic conduction, the process of electrostenolysis should be able to be observed in this system. Thisshould be a most straightforward experflment illustrating the electronic conduction across the BLM and providing the explanation for the second observation. Because of the high dark resistivity of the BLM, the process of electronic conduction was not easy to observe in the system. However, it was suggested that the process could be made efficient by incorporating high conducting species, such as carotenes into the membrane. Hence, this carotene-incorporated BLM would function like an inert redox electrode, capable of accepting electrons on one side of the biface and releasing electrons on the other side. An experiment was carried out in this thesis to support the above hypothesis. The potentials of various redox couples were determined across oxidized cholesterol BLM‘With and without carotene. The relationship between the redox potential difference was discussed. The explanation for the third observation above was given. It will be shown that all experimental data tend to support electronic conduction and the redox electrode model. However, it is important to note, that any ionic contribution to BLM photoeffect can be checked in comparison with the photoresponse measurement of the platinum cup with and without the agar bridge, since the platinum wall alows only the electron transfer, while the agar bridge allows ionic diffusion. The biophysical aspect ot the primary process of photosynthesis as currently understood has been proposed by many authors (Clayton, 1965), Duysens, 1961), (Kok and Hoch, 1961), (Witt and Muller, 1961), (Hill and Bendall, 1960), (Kautsky and Appel, 1960), (Losada and Whatley, 1961). They suggested a formulation for the c00peration of two photochemical processes in a series linked by a chain of electron carriers (cytochromes, quinones dyes). One photochemical system can produce a strong oxidant which is capable of functioning as the precursor of 0 The weak 2. reductant produced from this system can be connected with the weak oxidant generated from another photochemical system through a series of electron carrier processes. The strong reductant produced from the latter systen is capable of reducing NADP which in turn reduces C02. Based on the above hypothesis, three fundamental questions can be asked: 1) how can light energy be utilized more efficiently; 2) by what mechanisms are light-induced carriers transported; and 3) how can proper conditions be established for separating generated redox-products?. Although the answers for the above questions are still not quite available at the present time, one other major aim.of this thesis attempts to construct a BLM to look for the possible mechanisms and providing the best answers for the above questions. In chloroplast BLM the pigmentS' were incorporated in the membrane. On the basis of energetics at the water-oil-water biface, the molecules in the BLM are oriented in such a way that the phytoylgroups extend inward and can be held by van der Waals forces, while the polar groups are located at the aqueous solution/membrane interface. The measurement of the bifacial tension of the membrane inplies that those chlorophyll molecules situated at the biface in the membrane are tightly compressed together (Ting, HuemOeller, Lalitha, Diana, and Tien, 1968). It seems that the porphyrin plates of the molecules would be oriented more or less perpendicularly to the biface. Only a few millivolts of photoemf were found at symmetrical conditions which indicated that the same number of porphyrin groups at two interfaces possessed about the same electron driving strength, though in opposite directions. In order to drive the electron flow undirectionally, there must be an asymmetrical distribution of porphyrin groups at two interfaces. The more porphyrin groups on one side of the biface, the larger the amount of electron flow toward this side, and therefore the greater is.the photoemf. In this thesis, 3 system has been developed to achieve this aim. That is, the photo- active pigments were introduced to one side of BLM aqueous solution. When the photoactive pigments were moved gradually toward the membrane surface and attached to this side of the membrane biface, the increased porphyrin groups on this side of the biface were expected. Besides the chlorophyll pigment, a study involving all dye elements has also been conducted. In addition, some factors must be considered from the above measurements, such as the charge carrier, the pH of the bathing solution and the electric field. Thermodynamically, the redox potential difference determines the direction of the transfer of electron. However, sometimes, in the presence of an external energy source, the light energy, the direction of this electron flow perhaps can be reversed, i.e., the electron flow can be driven agianst the redox potential difference. The above process is called "the energy conversion" because the light energy has been converted into the chemical energy and stored in the system. In photo— synthesis each photosystem was speculated to require light to drive the electron against the preeexisting redox potential difference. However, many details of the mechanism are still unknown. In this thesis, we attempt to use the BLM to investigate the mechanisms of the energy conversion process as well as the non-energy conversion process. An analogous electron transport mechanism of photosynthesis currently understood was studied in chloroplast BLM. Gyundel and Boguslavskii observed that the photo-potential of vitamin A-incorporated BLM, or carotene-incorporated BLM was linearly related to the logarithm of Fe3+/Fe2+ concentration, and the latter was linearly related to the observed redox potential of Fe3+/Fe2+. Therefore, a relationship must exist between the redox potential and the BLM photo- potential. A theoretical treatment to correlate the Chl-Blm photoemf and the redox potential difference across the membrane was made, and experimental evidence in support of this correlation was given in this thesis. Furthermore, factors affecting the redox potential value as well as the Chl-BLm photoemf were also discussed. In summing up, this study achieved three major tasks: 1) distinguishing clearly between electronic and/or ionic conduction across the BLM; 2) finding conditions in which the BLM behaved, in the dark, exactly like an inert redox electrode; and 3) making biological application of the BLM, such as studying the possible mechanism of the primary process of photosynthesis. CHAPTER II LITERATURE REVIEW REDOX REACTION IN ARTIFICIAL MEMBRANE Artificial membranes, either a thin solid structure such as a porous glass or a liquid membrane such as a BLMg'were found to perform electron conduction. "There are two fundamental questions one would like to ask: 1) is there any experimental evidence to support the idea of electron conduction in a thin membrane immersed in an aqueous environment? and 2) if so, what is the source of electron in the membrane?" As has been reviewed by Tien (1972), Braun (Braun, 1891, 1891) employed a glass tube with fine cracks filled with a dilute solution of chloroplatinic acid and immersed in a beaker containing the same solution, shining mirror ‘was observed on one side cf the glass barrier after passing a direct current of sufficient voltage. The electrodes used were platinum electrodes placed in the solutions across the glass barrier. The side 'where the metallic mirroeras deposited was facing the anode and on the other side the evolution of a gaseous product was frequently noted. Later Coehn, (Coehn, 1898) has investigated this phenomenon and gave a possible explanation. It simply means that a reduction occurs at the side of the ‘barrier where the positive electrode is situated, and an oxidation reaction 8 flwfl- takes place on the other side of the barrier. This explanation implies the movement of electron through the barrier. Becquerel (Becquerel, 1867, 1877), for more than a decade beginning in 1867, some twenty years prior to Braun's experiments, observed that when a solution of copper nitrate in a test tube with cracks was placed in a solution of sodium sulfide, metallic crystals were formed on the inner side, whereas a dark yellowish layer of liquid diffused from the outer surface of the tube to solution. It is evident that copper ions were discharged at one side, and the other side the dark yellowish liquid containing polysulfide was a result of the oxidation of sulfide ions. These two findings are termed "electro- stenolysis", meaning electrolysis in a narrow passage. Up to 1930, Fetcher et al found that, using a cellulose acetate membrane, ferrous and ferric ions can be oxidized and reduced respectively (Fetcher, Lillie, and Harkins, 1937). Many years earlier, Bethe and Torpoff used a membrane of collodin and observed an alkaline reaction on the side of the membrane facing the anode, and an acid reaction on the other side of the membrane. Another finding was that Ag+ can be deposited on a collodin membrane using a technique of Braun (Bethe and Torpoff, 1914, 1915). There were still very few studies in electrostenolysis before 1960. Kallmann et a1 (Kallmann and Pope, 1961) used a thin crystal layer of anthracene as the barrier and observed a number of redox reactions as well as photoconductivity. Photocurrent of the system could be as large as dark current in the presence of oxidizing agents such as Ce4+ and 12. More recently, the work of deposition of metals on the surface of various membranes has been described (Januseviviene and Kaikaris, 1965). The most recent artificial system to be mentioned is bilayer lipid membrane which has been intensively studied for the past decade as models 10 of biological membrane. Experimental evidence for electron conduction in BLM.has been provided by many investigators (Tien, 1968, 1971; Rosenberg and Jendrasik, 1968; Rosenberg and Pant, 1970; Jain, Strickholm and White, 1970; Tien and Verma, 1970; Hesketh, 1969; Mauzerall and Finkelstein, 1969; Alamuti and Lauger, 1970; Cherry, Hsu and Chapman, 1971; Rosenberg and Pant, 1971; Tien, 1972). Tien (1972) has extensively studied the electronic conduction process in Chl-BLM and has found that one side of Chl-BLM is oxidized and the other side is reduced when illuminated by light. The presence of ferric chloride on the reduced side and that of dye on the oxidized side will enhance Chl-BLM light induced photo-emf. The result has been confirmed by Boguslavsky et a1 (1972), where photopotential at BLM in the presence of Fe salts and thionin dye Can be explained as the basis of redox reaction taking place in aqueous and lipid phases. Recently, the enhancement of the photoresponse has been found by removing this photoactive pigment from the membrane and placed into the aqueous solu- tion (Shieh and Tien, 1974) a VARIOUS MODELS FOR BLM.PHOTOEFFECTS Bilayer lipid membrane (or BLM) has been used successfully for several years to investigate the mechanisms of ion permeability, photovoltaic effect and photoconductivity. Hitherto, the explanations that have been proposed can be classified as follows: 1) solid state, 2) charge injection, 3) redox electrode, 4) dipole or electric double layer, 5) ionic diffusion and 6) confromational change model. A brief description of each model and some experimental data to support this model are given. The photosynthetic pigmented BLM are pictured as simi- lar to liquid crystals. When this pigment is excited by light, some charge ll carriers (electrons and holes) were produced in BLM. In the presence of electric field, the electrons moved toward the positive electric field and holes toward the negative field. Tien (1968) first reported a 1.5 mv Chl-BLM photo-emf was generated and this photoemf was depen- dent on both light intensity and the duration of illumination. Using flash excitation, Huebner and Tien (1973) studied the photoresponse of Chl-BLM under the condition of FeCl3 chemical gradient, pH gradient and an electric potential gradient (Huebner and Tien, 1972). They inter- pretated this photoresponse as the result of the movement of photo- generated electrons and protons. For tocopherol and sphingomyelin BLM, in the presence of K1, an observed photoresponse of about 45 to 120 mv was observed by Kay and Bean (Kay and Bean, 1970). They suggested that electrons were generated by either excited tochpherol or iodide. The fast increase of this photoresponse was due to this electronic charge separation. The slow increase is due to the chemical change in the membrane. The charge injection scheme suggested by Kallmann and Pope was that the molecule may exist in the form of an exciton as a result of the absorption of photoactive radiation. This exciton is capable of dissociating into an eleCtron and a positive hole. In the presence of a membrane, this electron and hole can be injected into itself. If is assumed that electron and hole have different mobilities and lifetimes, a separation of charges in the membrane occurs, hence the observed photovoltaic effect. (Kallmann and Pope, 1960). Tien reported a 5mv photo-emf was obtained when yellow light (500-800 mu) was used to illuminate a thin lipid membrane prepared from spinach chloroplast pigment. A negative photoresponse in the illuminated side of biface 12 indicated that a large concentration of holes must be present at the dark side (Tien, 1968). In the redox electrode model the photoactive BLM is depicted as similar to a silicon cell comprising a photovoltaic generator in parallel with a membrane resistance and a capacitance. The excited photoactive species must be separated into charge carriers (electron and hole) either by direct electron emission or exciton dissociation. Actually this is a combination model of a solid state mechanism and charge injection mechanism. The dissociation of an exciton can be resulted by the external field, electron donors and acceptors. The photocurrent is produced when the positive holes move from one side of biface to the other side, or when there is a change in the rate of accumulation of Charge on the menbrane capacitance. The photogenerated Species (electrons) are attracted by the electron traps situated at the solution/BLM biface, with the density of the traps being greater on the side containing the added electron acceptor. The difference in concentration of photoelectrons across the BLM can give rise to the observed photo-emf. Tien and Verma (1970) reported their observation of Chl-BLM photoemf in the presence of some redox compounds in the aqueous solutions. They proposed that the electrons were ejected from the donor molecu1e=(excited Chlorophyll) to the electron acceptors, thereby effecting a reduction reaction. In the meantime, a large number of holes generated near the barrier drift toward the other side of the biface where electron donors are situated. In the absence of electron donor, water could serve as an effective donor, being oxidized. Verma (1971) also observed a photoresponse generation in Ox-Chl-BLM in the presence of methylene blue, or rhodamine and KMnOA. A negative photoresponse was observed in KMnO4 containing side. He 13 interpreted that dye Could solubilize or adsorb in the membrane similar to that of Chl-BLM. Light exCites the pigment to produce an electron, which can then be captured'by‘KMEnO4 to cuase its reduction and the hole can be captured by electron donor on the other side. The addition of phycocyanin to the same side of the cell with Fe3+/Fe2+ increasing the photoresponse in Chl-BLM and decreasing the fluorescence emission as reported by Ilani and Berns (1971). It was suggested that when Fe3+-phycocyanin complex was formed, the activation energy becomes lower than that necessary for electron transfer across the membrane and caused the reduction of Fe3+ ion. Pant and Rosenberg (1971) reported the generation of photovoltage with 12,NaI on one side and Hg(N03) on the other side of an oxidized cholesterol membrane. They also found that under the application of 50 mv across the membrane, a reflection metallic film was produced on the membrane surface. They suggested a couple redox reaction occurs across the membrane and is mediated by electron transport through the'membrane.’ Ilani and Berna (1972), experimentally, found that the photovoltage is dependent on the differenCe in redox potential of the solutions. They suggested that the conduction pathway is formed in the BLM as a result of illumination which causes the redox reaction. Gyundel and Boguslavskii (1972) found that a linear plot of BLM.photo-potential versus log of Fe3+/Fe2+ ratio can be Observed for a BLM unmodified and BLM.containing vitamin A, in the presence of Fe3+lFe2+ in the aqueous solution. Since they found that this linearity was also in the plot of the redox potential of Fe3+/Fe2+ versus Fe3+/Fe2+ concentration, it is suggested that the ratio of Fe3+/Fe2+ determines the membrane potential. In their interpretation, lipid molecules in the membrane will give up the electron to Fe3+ to be oxidized and this electron 14 giving up process will be eliminated if Fe3+ is replaced by Fe2+. Karvaly and Pant (1972) reported that a monotonic photoresponse was observed in 2 and FeCl2 in Ox-Chl-BLM when illuminated at 254 nm UV light and the sign of this photoresponse was coincided to that of a the presence of I redox reaction occurring near membrane interface. When FeCl2 was replaced by FeCl3, a biphasic photoresponse was found. It was interpreted that, besides the redox reaction event, a strong diffusable unknown component must be accompanied. Systematic studies of the effect of organic, inorganic redox compounds on the Chl-BLM photoresponse were systematically investi- gated by Shieh and Tien (1974). An enhancement of this Chl-BLM photo-emf of about 188 my was obtained in the presence of Fe3+ and ascorbic acid. In the dipole or electric double layer model, the BLM photoeffect was the result of double charge layers being built up near membrane/ solution interfaces (Hesketh, 1969). A light induced electron was bound with electron acceptor in one interface to form the positive charge layer and the positive chloriphyll molecule attracts Cl- ions at the other interface to form the negative charge layer. According to this hypothesis, the presence of a negative external field will tend to maintain this electrostatic field between the two chargezlayers and will reduce the net ionic (C1-) flux. Presumably, the ionic conduction is the Only conduction in membrane. A positive field increases the Cl- migration against the field and causes electrons to bind to the positive chlorophyll charge. In the absence of an external field, there will be no net charge separation. Experimentally, Kuhn and Ullrich (1972) reported that in a BLM, in the presence of a cyanine dye in the aqueous solution, its photovoltage rises rapidly and then decays gradually and when light is off, the voltage follows a sequence which is the reverse of the observed in light, but with negative polarity, They assumed that the excited pigment attracts electron from 15 donor P, forming a P+—D- dipole which immediately attracts ions from + the electrolyte solution, Cl-P -D“Na+. When the light is turned off electron returns from D- to donor (P). The change of photopotential from their maxima is explained on the basis of ionic charge diffusion (Na+ and C1-) across the BLM. In ionic diffusion model, it is assumed that the large size and 3 the low dielectric constant hydrophobic portion of membrane. Under the delocalized charge, such as I which can have sufficient solubility in applied field, this ion will move toward positive field and leave the membrane resulting in increasing the membrane resistance. In the absence of an electric field, no ionic diffusion can be observed (Mauzerall, 1969). This ionic diffusion model is first prOposed by Mauzerall to explain the result of his BLM (lipid, cholesterol, tocopherol) photoconductivity measurement in the presence of iodide‘and iodine. Trissel and Laeuger (1972) reported that their BLM photocurrent generation is due to the result of a hydrophobic ion (an oxidized form of the electron donor) jumping over the energy barrier to the other side of the BLM. The BLM.photoeffect.has also been proposed to produce from the membrane conformational change in light. It is this conformation change that results in the increase in membrane conductivity which in turn facilitates the charge carriers diffusion across the membrane. Tien and Kobamoto (1969) reported the BLM.photoeffect in the presence of carotene and retinals. The lack of photoresponse in carotene/BLM has been interpreted to be due to the completely free orientation of carotene within the hydrophobic interior of the membrane which results in no charge separation and no photoeffect. However, retinal has hydro— philic groups that can fix their orientation in the hydrophilic portion 16 of the membrane resulting in some charge separation. The cis-trans conformation change of retinal in the membrane induced by the absorption of a photon can induce in a change in the membrane, thus increasing its conductivity. PHOTOSENSITIZING PROCESS Since Engelmann (18884), there were many experiments intended to discover a simple system which could convert the electromagnetic energy into chemical energy with chlorophyll and related materials. All of these observations can most easily be assigned in two class- ifications. The first is a photosensitizing oxidation reaction with molecular oxygen in which the Chl is the photosensitizer, that is, the Chl absorbs the light and causes, in some way, the oxidation of some other substrate by molecular oxygen. The Chl itself is relatively stable and acts as a photosensitizing dyestuff which will cause oxidation of a good many substrates. One of the very early and more quantitative studies was that of Gaffron, in which he used Chl as a photosensitizer for the oxidation of allylthiOurea and, in fact, studied it so thoroughly that it could be used as an actinometer; that is,as a means of measuring actual light intensity in a beam, particularly in the red region of the. spectrum (Graffron, 1933). The number of allylthiourea molecules oxidized per quantum absorbed by chlorophyll, is approximately one. The other type of photochemical reaction in solution which Chl is known to sensitize is a hydrogen transfer from some reducing agent to some oxidized substance. One of the classic examples is the hydrogen transfer from materials such as ascorbic acid and hydrazine to dyestuff acceptors such as azo dye (methyl red). These reactions have been known for some time, and have 'been.studied extensively'in’the'Soviet Union._ One such reaction is 17 called the Krasnovskii reaction after the man who Spent a great deal of time Studying it (Krasnovskii, 1948; Krasnovskii and Brin, 1953; Krasnovskii, Gavriolova, 1951; Krasnovskii and Voinoskaya, 1949; Krasnovskii, Brin and Voinovskaya, 1949). Krasnovskii used ChlorOphyll and porphyrin model substances as sensitizers to transfer hydrogen from a variety of donors (ascorbic acid) to methyl red and other azo dyes. He did it under such conditions that he was able to show two steps as separate events, that is, the transfer of hydrogen from the hydro- gen donor to Chl to give some intermediate followed by the transfer of hydrogen from this intermeadiate to the hydrogen acceptor, giving back again the initial Chl. By cooling the reaCtion mixture, and performing the experiment in a basic solvent such as pyridine, Krasnovskii was able to show that Chl plus ascorbic acid, without the addition of a hydrogen acceptor, would go from a green color to a pink color. This pink color was presumed to be some intermediate, not necessarily bacteriochlorophyll, since the spectrum did not correspond. The reaction reverSes in the dark, and the pink intermediate is not a radical (Linschitz and HeiSSmen; 1957). It was suggested that Chl absorbs the photon first.‘ The excited Chl removes either a hydrogen atom, or an electron from the donor to give ' what Krasnovskii believes to be a radical which then dissociates to give a proton. This free radical can then go ahead and reduce another Chl, and the free radical ion of Chl can give up the hydrogen atom, or electron (or both) to the dyestuff (Dy) to give back the Starting Chl material and the partly reduced semiquinone of the dye. This, then finishes its reduction, either by combination.with a radical or by taking a proton directly off the hydrogen donor itself to give the colorless dyestuff and the dehydroascorbic acid or other dehydro-compound. This is a general l8 scheme which appears to apply for a whole variety of hydrogen donors, hydrogen acceptors, and sensitizing dyes. Koizumi et al reported in 1964, the kinetic studies of the photo— reduction of thiazine, oxazine dye and indophenol dye and phenazine dye by visible light (Koizumi, Obata and Hayashi, 1964). They found that thiazine dye, the only kind of dyes studied above that showed a first order kinetics in borate buffer and second order in phOSphate buffer. The rate of this photoreduction was found to be linear to the light intensity. The activation energy calculated for this system.was 4.2 Kcal. It is suggested that the light excited thiazine dye in its triplet state can interact with Other thiazine dye resulting in one positive charge in one dye and one negative charge in the Other. When these two charged dyes interact with OH, it will produce H202, CH3 OH, Dyez- and neutral dye. The reaction is particularly prominent when demethylation occurs simultaneously. In conclusion, Chl sensitized reaction can best be summarized into the following categOries as given by Cox and Kemp (1972): i) photo-oxidation of Chl itself, ii) photo-reduction of Chl by some compounds, such as vitamin-C, H28, phenylhydrazine, iii) Chl functions as an intermediate in the two stage electron transfer reactions. Process ii and iii may be important in photosyntheSis. In process iii, the transfer of an H-atom from the reductant to the oxidant does not readily occur by itself. The provision of an intermediate system will often facilitate it. The intermediate oxidant takes the H-atom or electron' from the reductant and gives to up to the oxidant. It thus serves as a go-between in an otherwise difficult chemical transaction. A g l9 photocatalyst may act similarly, except that it requires excitation by light to play its role. If the end result of a reaction is increase in free energy, this light excitation of the catalyst is indispensable to make the reaction possible. This is what photosynthesis is all about. Certain BLM, such as those formed from oxidized cholesterol or phospholipids which are not photoactive, can be sensitized by certain inorganic ions and organic dyes dissolved in the aqueous solution. For example, in the presence of I and I_ Mauzeral et.al have observed 2 light induced change in the conductivity of BLM of brain lipids, a-to- copherol, and cholesterol. The polyiodide ion is the species responsible for the obserVed photoeffect which serves as both the charge carrier across the membrane and the light absorber (Mauzerall and Finkelstein, 1969). The use of ultraviolet light to elicit photoresponses from BLM in an aqueous solution containing I-(Iz) has also been investigated by Kay and Chan (1969). In the visible light range no photoresponse.was found. But at wavelengths around 240 nm and 290 nm, very large photoresponses were found. They proposed that the photoresponse at 240 nm is due to the absorption of iodine, since no response was obtained in the presence of added 1-. The maximal effect produced around 290 nm was due to that of a-tocopherol. Pant and Rosenberg (1971) reported that a photopotential could be produced by UV excitation in the presence of photoactive inorganic ions. Recently, Karvaly and Pant (1972) have observed a biphasic photo- response in BLM with ferric chloride and iodine in the opposite sides of the aqueous solution at 365 nm and 254 nm wavelength illumination. In the same year, Gyundel et al have investigated the BLM photoeffect. 2+ in the presence of Fe3+/Fe . As a result of coupled redox reactions on opposite sides of the BLM, they suggested that there is an electronic 20 component in the conduction of charge across the BLM (Gyundel and Boguslavskii, 1972). Petkau (1971) repOrted that an iOnization phenomenon could be observed by light radiation of BLM formed from brain lipid dissolved in n-tetradecane and a transmembrane potential was generated during the irradiation. He suggested that this membrane potential was created as the result of a pH gradient across the membrane by the unequal radiation. During the irradiation, a non-equilibrium distribution of ions was created. Several photosensitizations of BLM by organic dyes were reported. Methylene blue, an azo dye, has been studied in the connection with chlorophyll by Mylinikov, (1968). He found that the spectrum of the photoconductivity was similar to the absorption spectrum of the solution. Tien reported a Change of the action spectrum of Chl-BLM in the presence of this dye (Tien, 1972). A lO-fold drop in membrane resistance in the presence of methylene blue was found by Verma (1971). He suggested that this membrane resistance drop was due to the solubilization of this dye into the interior of BLM and the formation of a complex“with the mem- brane. The efficienCy of methyl red in inducing photopotential has been studied by Evstigneev (1968). He observed that the dye, coated on the platinum electrode surface, was effective as an electron acceptor only in acid media (pH 5), and the effect increased with the concentra- tion of the dye.’ A noticeably enhanced absorption in the red region in the presence of methyl red in Chl-BLM‘was reported by Tien (1972). Methyl viologen, an electron acceptor extensively used in photosyntheSis studies, has been studied by Krasnovskii. He found that chlorophyll could be used to sensitize the reduction of methyl viologen by cysteine in the red region of light (Krasnovskii, 1969). A year before, this dye was reported 21 to be reduced by ascorbic acid in the presence of light-excited chloro- phyll (Evstigneev, 1968). In chloroplasts, methyl viologen appears to delay the rate of proton uptake, as Dilley has reported, (Dilley, 1969). In the Chl-BLM system containing ferric ions, the addition of methyl viologen to the Opposite side of bathing solution causing a tenfold reduction in the photoresponse according to Tien (1972). Phoeonap- thoxamine iodide dye was studied by Ullrich and Kuhn (1972). The membrane dark potential with the dye containing side positively charged, was ‘ created after the addition of this dye. The photo-emf was prOportional to the intensity of light. Kearns and Calvin and Tollin have reported the photoelectric effects in sandwitch cells of a phthalocyanine layer coated with either chloronil or TMD. The observed action spectrum did not follow the absorption spectrum of phthalocyanine (1958, 1960). Tien (1972) repOrted the photoresponse measurement of Chl-BLM in the presence of thionin dye. Two dramatic effects were obserVed. First, a new peak at about 580 nm in the opposite direction was induced, corresponding closely to its absorption maximum (600 nm). The second effect was an enhancement of absorption in the red region. ENERGY CONVERSION SYSTEM An energy conversion system is a thermodynamically unfavorable chemical reaction, an external energy source, such as light, must be input to initiate the reaction. When the light is turned off the back reaction quite often follows. L Weiss in 1934 found that Chl (in methanol) can be reduced by ferrous ion.in the presence of light (WeiSs, 1935). In the dark, a back reaction *would occur. This back reaciton is thermodynamically favorable and the 22 driving force of this back reaction is provided from the reduced form of chlorophyll. Krasnovsky in 1949, the Russian physical chemist, found that Chl dissolved in pyridine, can be reversibly reduced in light by ascorbic acid. The basic nature of the solvent (pyridine) seems to be essential for the display of the oxidizing prOperties of Chl (Krasnovsky, 1949). In the dark, the Krasnovsky reaCtion goes backward, showing that it is associated with the storage Of free energy (Rabinowitch and Govindjee, 1969). He went on with further experiments and found this reduced Chl can be re-oxi- dized again by a variety of oxidizing compounds, such as riboflavin, safranine and nile blue. (Krasnovsky and Erin, 1949, 1950) The net result is photocatalyzed reduction of theSe compounds by ascorbic acid. In many cases, this reaction leads to a net storage of chemical energy. For example, the well known biological catalyst, riboflavin (EO=0.2 volt) is reduced by ascorbic acid (EO=O.0 volt) in an illuminated Chl solution, overcoming an adverse potential gradient of 0.2 volt. Krasnovskii's experiment showed the reduction by ascorbic acid of compounds with redox potential down to -0.2 volt, thus bridging one_siXth of the 1.2 gap.fihat has to be bridged in photosynthesis. In 1962, Mathai and Rabinowitch reported the Chl sensitized photo- reduction of thionin by ascorbic acid, in an aqueous solution of pyridine. They found that the quantum yield (the rate of number of moles of dye reduced to the number of einsteins absorbed) is a function of the concen- tration of thionin, and independent of light intensity and Chl concentration. This finding is similar to what Livingston and Pariser (1956) reported on the Chl sensitized photoreduction of methyl red by phenylhydrazine. The difficulty of their experiment is that the primary oxidation and reduction 23 products were not separated. However, they suggested that, in photosyn- thesis, the energy rich products may be stabilized by distribution between two phases - a hydrophilic and a hydrophobic layer adjoining the pigment monolayer. Other dyes, sueh as methyl viologen, which have low redox potential (Eo=-O.5 volt) also have been used as an electron acceptor in the Chl sensitizing photo—oxidation of cysteine in solution (Krasnovskii, 1969) and in the Chl sensitizing photo-oxidation of ascorbic acid in solution (Evsrugneev, 1968). Many dyes are easily reduced to colorless leuodyes and reoxidized back to colored dyes. A well known example is methyl blue (Eo=0.0 volt), which can be reduced to colorless leuco-methyl blue by visible light (Usui, Obata, and Koisumi, 1961) without: the presence of electron donor. Methyl blue related dye "thinnin" was first studied by Weiss in 1935 to be reduced by light in the presence of ferrous ion (Weiss, 1935). The most extensive studies for this system came out more than one decade after WeiSs (Ainsworth and Rabinowitch,.1960;.Bowen, 1946; Rabinowitch, 1940; Cox and Kemp, 1972). The bleached system had a higher free energy that the colored one. It about the best initation we know of the postu- lated primary photochemical process of photosynthesis. Light is utilized in this experiment, as in photosynthesis, for an uphill oxidation- reduction reaction against the gradient of electrochemical potential. In contrast to photosynthesis, however, no enzymatic agents are present to stablize the energy-rich products, and the stored light energy is rapidly dissipated by a back reaction between ferric ions and leuco— thionin. In Rabinowitch's laboratory, the reaction was carried out in a way to preserve this energy. Here the reaction was carried out in an 24 emulsion of ether in water. The leucodye, formed in light, was extracted into ether, while the ferric ions stayed in water. the two solutions, the aqueous and the ether, could be separated in a separatory funnel. The products were thus prevented from reacting back. When the two solutions were stirred together again in the presence of alcohol, which makes them mutually soluble, the delayed back reaction took place and the color returned. Zolotovitskii et al used the method of pulse and steady photOpolar- ography to investigate the photoreduction of quinoid compounds, ribofla- vin and the sulphonates of 9,10-anthraquinone. They found that light excites this quinone, which then interacts with water to produce semi- quinone and OH- radicals. In the dark, the back reaction can proceed (Zolotovitskii and Korchunov, Elichis, Benderskii, 1970). ENERGY CONVERSION PROCESS PERFORMED IN PHOTOSYNTHESIS Photosynthesis probably involves two sets of primary photochemical reactions. One of them may involve photoreduction of an organic sub- strate by one pigment system; and the Other photo-oxidation of water by. another pigment system. If Chl is the photocatalyst, it will be oxidized in the first reaction and reduced in the second. A reaction between the oxidized form of Chl produced in one reaction and the reduced form produced in the second reaction is needed to close the sequence and restore the photocatalystic system to its original state. One is tempted to recall in this connection the Chl reversible photoreduction in pyridine solution. A remote, but plausible, analogy may exist between these two reactions in vitro and the two reactions of Chl (probably located in different molecular environments) in photosynthesis. This is only a speculation, but it does invite systematic experiments. CHAPTER III EXPERIMENTAL 1. Materials Used and Solution Preparation a. Chl-BLM Forming Solution Extraction The chlorophyll pigments used in this investigation were prepared from a bag of fresh spinach leaves obtained from a super market. A standard procedure for preparing Chl-BLM extracts was described by Tien et a1 (1968): 1. Remove the ribs and stalks from Spinach leaves, then wash and dry the leaves. Add.leaves slowly to the blender which contains 30Qm1 of (0.5 M sucrose + 0.05 M KHC03)-buffer solution at pH 7.5. First run them in the blender at low speed, then at high speed for 30 seconds after all leaves have been added. Filter the mixture through 4 layers of cheesecloth. Centrifuge the filtrate in 40 ml quantities (8 tubes) at low speed (variac at 40 volts) for 5 minutes. Discard the supernatant; Distribute another 100 ml of buffer solution to these eight tubes and transfer all of them into 4 tubes. Centrifuge at 40 volts for 5 minutes, then discard the supernatant. Wash the residue with 50 m1 H20 totally and let stand for 5 minutes 25 26 before another centrifugation. Now centrifuge them at high Speed (variac at 45 volts) for 10 minutes, then discard the supernatant. Extract the residue from above with 90 m1 of 2:1 petroleum ether and methanol solution in the blender at medium speed for 1 minute. This is the procedure to separate pigments and lipids from water and protein. Add above mixture to 2 tubes and centrifuge at low speed (variac at 30 volts) for 5 minutes. Pipet off the tOp layer from the tube into a round flask and evaporate to dryness at 40°C. Add 5 ml of 1:1 n-butanol and dodecane to the residue. This is the Chl-BLM extract. Oxidized-Cholesterol Membrane Forming Solution Preparation Freshly recrystallized cholesterol from commercial sources is oxidized in n-octane by bubbling molecular oxygen through the solution at its boiling point. The resulting colorless solution may be used directly for BLM formation procedure. A standard procedure was described by Tien and Howard (1969): 1. 2. Clean all glasswares thoroughly. Bubble nitrogen through 300 m1 of absolute ethanol for 30 minutes (via gas-dispersing tube). Add 25 g of cholesterol to 250 ml of nitrogenated ethanol. Warm the mixture to about 70°C until a clear solution results. Cool to 5°C and let stand for 1 hour (in a refrigerator). Filter the above solution with suction. Repeat steps 2-6, except use 181 ml of nitrogenated ethanol in step 3. 27 8. Lyophilize the combined filtered mass overnight. The residue should be white and fluffy. 9. Add 12 g of the material from step 8 to 300 ml of n-octane in a l-liter flask. 10. Heat the mixture to boiling (125°C) and bubble 02 at a rate of 100 to 125 cm3/min for 5.5 to 6.0 hours. 11. Cool to room temperature. The solution should be colorless with a white precipitate. 12. Pipet off the supernatant for BLM formation. b. Chemical Solution Preparation All compounds used either for bathing solution or other purposes were obtained from chemical companies and prepared without any further purification. Two concentrations of materials were selected. For completely H20 soluble compounds, the concentration was 10”1 M. For H20 insoluble dyes, the solutions were saturated. 2. Procedure a. Photoresponse Measurement The ChléBLM-emf or Ox-Chl-BLM-emf was studied in a setup as illus— trated in Figure 1. The cell arrangement is represented as follows: saturated aqueous aqueous saturated calomel solution BLM solution calomel electrode electrode The membrane potential was measured with an electrometer (Keithley, Model 610 B) through the connection of a pair of calomel electrodes via saturated KCl salt bridge. The output of the electrometer was fed into a chart recorder (Servo-Recorder, Model EUW-ZOA). An electric circuit for this meaSurement is illustrated in Figure 2. A BLM cell can best 28. be discribed as a parallel connection of a resistance and capacitance. Vp and Rp were BLM photobattery and photo-generated resistance. With the light on, a switch S1 R.p were connected parallel to Rm and Cm as suggested by Ilani et al was connected to the position 2, and Vp and (1972). When ChléBLM photoresponse was measured in the absence of external voltage sources, the switch S was opened. HOwever, the switch 2 could be closed when external voltage sources were applied. When an external current was passed to the BLM by an external battery (EE), through an external resistor (RE) which was in series with the BLM, the switch S3 was closed at position X. If only the external battery (EE) was applied, the switch S was closed at position Y. 3 b. Membrane Resistance‘Measurementi A dc membrane resistance was obtained by applying external voltage and resistor in series with the BLM. The polarization of the applied voltage could be controlled through the switch of the voltage divide. This input external resistance could be varied from 105 to 109 ohms. The membrane resistance was calculated according to Ohmls law for the circuit shown in Figure 2. It was RméRE. _ Vm Et'vu where REwas the input resistance, E was the applied voltage and Vm was E the membrane potential. For the best result, RE was adjusted so that V'm/EE could lie between 0.1 and 0.8. c. Chlorophyll Concentration in Spinach Chloroplast Measurement The chloroplast forming solution concentration can be measured through spectrOPhotometric technique. According to Beer's equation, 29 Figure l Set-up for BLM photo-response measurement RS light source, projector lamp shutter heat absorbing filter focus lens BLM inner chamber BLM outer chamber‘ magnetic stirring stirring motor calomel electrodes connection box electrometer variable voltage source resistance substitution box recorder 31 Figure 2 Electric circuit for BLM photoresponse membrane capacitance electrometer external battery voltage divide and switch for applying polarizing ‘potentials memb rane res is tance light generated resistance input resistance ' internal resistance of applying voltage source switches 1,2, and 3 BLM.photo-battery 33 the optical density (which is the logarthim of the ratio of incident light intensity to transmitted light intensity) is a linear relation to the concentration of absorbing substance, and the thickness of absorbing cell (d) O.D.=log I0 = A x(C)xD T 5 AS is the molar extension coefficient, which is the specific extinction (e) coefficient divided by the molecular weight of absorbing substance. The relative specific extension coefficient for Chl at various wavelengths from several authors has been collected by Rabinowitch (1951): TABLE observers e at red peak e at blue peak Zscheite and Comar (1941) 9.1xlO4 12x104 Zscheite, Wmar and Mack- 9.0x104 11.7xlO4 inney (1942) Mackinney (1940) 7.65x104 9.75x104, According to Beer's equation, the concentration of chlorophyll can be obtained by measuring the O.D with Cary-15 Spectrophotometer. Two 1 m1 spinach extracts were diluted to 500 ml and 2500 ml respectively. From the optical measurement, the concentrations of these two dilutions were obtained. TABLE-l The concentration of Chl in 500 ml dilution Wavelength O.D e (C) in (C) in 500 m1 dilution extract 430 0.19 12x104 1.6xlO-6 (M/L) 4x10'3(M/L) 660 0.15 9.1x104 1.6x10’6 (M/L) 4.13.2:10"3 (M/L) 34 TABLE-1 continued The concentration of Chl in 2500 m1 dilution Wavelength O.D e (C) in 2500 m1 (C) in dilution - extract 4 -4 -3 430 0.94 12x10 0.08x10 (M/l) 4x10 (M/l) 660 0.74 9.1x104 0.08x10-4CM/1) 4.05xlO-3(M/l) The spinach chloroplast extracts concentration obtained from the above measurments with different dilutions have ended up with similar results. This is 4 x 10'3 1M, which is consistant with that obtained by Lauger (1971) through fluorescence measurement. d. The Standard Rede Potential Difference Between Two Redox Couples Measurement The redox potential of redox couple is determined by using the set- up in Figure 3. A platinum electrode was immersed in the aqueous solution and its other terminal was connected to the active side of the electrometer. A calomel electrode was used as a reference electrode and was connected to the ground side of the electrometer. The aqueous solution was buffered acetate (10.1 M) pH 5 and the chemicals added to the aqueous solution has a final concentration of 10-3 M. There was 1:1 ratio of oxidized and reduced form in the redox couple. The redox potential difference between a redox couple and the reference couple (Fe3+7Fez+) could be obtained by subtracting the redox potential of this redox couple with referred to the reference electrode from that of reference couple with referred to the reference electrode. e. Photosensitizing Measurement in BLM Experimentally, BLM is formed in the usual manner on a hole in the side of a teflon cup separating two aqueous solutions. After the membrane 35 Figure 3 Cell designed for the redox potential measurement E electrometer C calomel electrode Pt platinum electrode S stirrer M motor 37 has reached the black stage, a known volume of the bathing solution on one side of the BLM is withdrawn, and an equal volume of solution contain- ing the dye is added. In most experiments the BLM is excited by a 150 watt projection lamp for open-circuit photo-emf studies. Undesirable effects, due to direct thermal heating of the BLM, are minimized by the use of heat-absorbing filters, and/or an 8-cm cell containing a 5% copper sulfate solution interposed between the light and the cell assembly. CHAPTER IV RESULTS 1. THE DETERMINATION OF ELECTRONIC AND IONIC CONDUCTION IN BILAYER.LIPID MEMBRANE The possession of the electronic or the ionic characteristics, or both in BLM is still not completely understood by investigators in studying the BLM photoeffect. What was lacking, in particular, was a clean-cut experiment to study the detailed mechanism of how these two characteristics work in BLM. If assuming a pigmented BLM is capable of facilitating electron transfer in inducing the rede reaction, then from a functional point of view it behaves similarily to a metallic surface or an inert redox electrode. According to the above assumption, some general phenomena which correspond to the Character of the metallic surface would be expected to be observed in pigmented BLM, such as the photovoltaic effect, the redox reaction in the dark or in the light, and the electro- stenolysis. Bearing this in mind, three experiments were carried out for this study. They include a comparative study between the BLM and a platinum cup, electrostenolysis in BLMS, and the study of redox reaction in BLMs containing carotene.’ The possibility of ionic conduction in BLM was also examined. 38 39 a. A Comparative Study Between a Platinum Cup and the BLM (Bilayer Lipid Membrane) The detailed mechanisms of electronic and/or ionic conduction in BLM have been studied by through the comparative measurements between the BLM and a platinum cup with an agar bridge. It is assumed that the BLM functioned similarly to that of a metallic surface with an agar-bridge. Since the platinum cup portion can allow only electronic conduction while the agar-bridge portion will only allow ionic conduction, hence from these comparative experiments one should be able to observe how elec- tronic and/or ionic components function in BLM. In order to have the complete experiment, measurements were also conducted with either a platinum cup or an agar-bridge alone. The aqueous solutions both in the platinum cup system and in BLM are buffered acetate (10'3 M) and pH at 4. The chemicals used have concentrations of 10"3 M, except tetraphenylborate (10.4 M) and thionin (lO-AIM). The potential changes across either the BLM or a platinum is measured by the electrometer. 1. The Iodide Ion Diffusion Measurement The diffusions of iodide ions across the BLM biface and across an agar-bridge were studied. Table-2 showed the results of these measurements. The positive sign of the potential Shown in Table-2 was referred to iodide ions contained side positive polarity. Since the agar-bridge allows only the ionic diffusion, hence the potential change of about 105 mv from agar-bridge measurement was produced by iodide ion diffusion across an agar-bridge. No potential change was observed across the platinum cup due to the BLM cup not allowing ionic diffusion. The potential change across a platinum cup with agar-bridge has been found to be exactly equaled to that observed in an agar-bridge system alone. 'This implies that iodide ions 4O diffuse through an agar-bridge placed_over a platinum cup. Since in the presence of iodide ions the oxidized cholesterol BLM.potential change has essentially the same value as that with an agar-bridge, it is suggested that BLM may possess ionic conduction like an agar-bridge does. TABLE-2 The measurement of the potential generated from the iodide ion diffusion (10"3 M) potential potential net potential system befOre I after I change by I added (mv) added (mv) difqui n (mv) NaAc/Ox-Chl-BLM/I-lNaAc o ‘ 100. 100, pH 4 NaAc/pt cup/I-lNaAc (pH 4) 0 O 0 NaAc/agar-bridge/I-lNaAc o 105 105 NaAc/pt cup + agar:bridge/ 0 110 110 I INaAc 2. The Electronic Conduction Via Redox Reaction Measurement The electronic transfer in coupling with the redox reaction in either dark or light was measured by the potential change in dark or the photoresponse in light. The photoresponse was measured after thionin pigment (10_4 M) was introduced to the side containing tetraphenylborate (10“ M). As are shown in Table-3, all systems except the agar-bridge have the positive photoresponse on T¢B and thionin containing side relative to the other.side. Since there has no electronic conduction through an agar-bridge, no photoresponse can be observed in this system. 41 TABLE-3 The measurement of electronic conduction via redox reaction in BLM and platinum cup potential change potential change photo- system by_addinng¢B, thionin in the light response a Ain.the dark VD (mv) . Ehvgvb:YD NaAc/Ox-Chl-BLM/Th,T4>B/NaAc 70 160 70 pH 4 H NaAc/pt cup/Th,T¢B/NaAc 140 230 90 NaAc/pt cup + agar-bridge/ 100 185 85 Th,T¢B/NaAc NaAc/agar-bridge/Th,T¢B/NaAc ' 16 16. 0 The photoresponse of about 85 mv in the pt cup and agar-bridge system is the result of an electron flow through the pt cup portion since a similar magnitude of photoresponse was observed with only pt cup. The same magnitude of photoresponse between the BLM and a pt cup can further suggest the electronic conduction via redox reaction in BLM as well. 3 Studies of the Factors Governing the Electronic Conduction Via Redox Characteristics in BLM and Platinum Cup The electronic conduction in BLM was studied in comparison with that in a platinum cup. This electronic conduction can be measured by applying several different techniques, such as by reading the potential change in the dark, or in the light; by observing the color change from the redox reac— tion in the light; and by electrostenolysis studies. In the potential change measurement, the factors which govern this electronic conduction via redox reaction, are the electron donor, the electron acceptor, the 42 existence of an electric field. These factors are investigated and the results are shown in Table-4. TABLE-4 The similarity between BLM and Platinum cup in electronic conduction and redox characteristics. The strength of these characters has been measured in terms of the magnitude of photo-response. Mechanism in ESV§UP in 2:5). Remarks -EhV Ehv ‘ a The electron accepting 10 35 D=T¢B from donor compound in light (D) b The enhancement of electron 15 25 AF Eosin accepting in light by the'~ 15 50 1M. blue presence of electron acceptor, 85 85 thiOnin in addition to electron donor 13 ' 11 safranine c The electron accepting in light-85 -85 VD=-100 mv was affected by the electric -55 -50 0 mv field, in addition to electron -40 -35 +50 mv donor (ToB) and acceptor (thionin) 43 The T¢B oxidation process occurred when lights were illuminated on both the Ox-Cho-BLM and a platinum cup surface.a The magnitude Of this T¢B oxidation, in terms of the photo-response, is 10 mv in platinum cup system and 35 mv in BLM system. This light-induced electron releasing process from tetraphenylborate to either the membrane surface or a platinum cup surface was enhanced in the presence of an electron acceptor. The orders of these enhancements both in the BLM and in a platinum cup were: thionin+ eosin, methylene blue+safranine. Besides the electron acceptor and the electron donor, the electronic conduction in the BLM and a platinum cup also seen to be affected by the electric field. The higher the applied electric field, with thetetraphenylborate Containing side being negative, the greater is the negative photo-response. b. Electrostenolysis in the Bilayer Lipid Membrane The BLM set-up for this electrostenolysis measurement is as usual: the membrane was formed on the hole of the teflon cup which separated two aqueous solutions. The ends of a pair of calomel electrodes immersed in the aqueous solutions were used to detect the membrane potential and the other ends were connected to an electrometer and external voltage source. The membrane forming solution is oxidized cholesterol dissolved in n-octane. The reaction mechanism on the membrane surface can be calomel [buffer buffer [calomel electrode acetate -1 (10 A)lx/ox'Cho-BLM/Y/acetate electrode. described as : Where X and Y are the chemicals used to produce the metallic mirror and which can be the same or different compounds. In electrostenolysis, the barrier requires a very low resistance for electronic conduction. Since T¢B was compound found to lower the membrane resistance by two or three orders of magnitude, it was added either to the aqueous solution or to the membrane. 44. Three experiments were carried out for this electrostenolysis study. Experiment I: the cell arrangements were H PtC16(two pieces/20 cc)200 A, 2 T¢B/Cho-BLM/H2PtCl6,T¢B and H PtCl6/Cho-BLM/T¢B. For the first cell 2 arrangement, an applied electric field of about 70 my was applied and the membrane surface began to form a dim mirror in 25 minutes. Meanwhile bright suSpensions appeared in the aqueous solution. These bright suspensions were produced by the direct interaction between HZPtCl6 and T¢B in the presence of light. They however, completely disappeared overnight. A direct contact between HZPtCl6 and T¢B could be prevented by using the second cell arrangement above. The mirror was formed when a 70 mv external electric field was applied. However, it wasn't clear whether the mirrors formed in both measurements could be made to disappear by reversing the electric field direction. Experiment II: the cell arrangement was Hg2(N03)2(5g/10cc)lOOA/Cho-BLM, T¢B/Hg2(NO3)2. The mirror was formed in half an hour under the applied electric field (at R1=100hm and VD=30mv). The mirror disappeared in half an hour when reversing the electric field. Hewever, the mirror could be formed again by applying a positive electric field. Since the membrane is transparent, there is no way to know on which side the Hg2(_N03)2 has been reduced to deposit Hg on the membrane. However, it was found that the mirror was unable to form in the absence of an applied current. Experiment III: the cell arrangement for this measurement was: Cu(N03)2/ Cho-BLM/Donor, where the donor could be NaZS, cystine, NaI. Cu(NO3) can also be used to substitute for Cu(NO3)2. The metallic mirrors were observed in all of the above situations. The brightness of the mirror was found to depend upon several factors, such as the concentration of Cu(NO3) membrane resistance, the aqueous solution pH, the charge carrier and 29 the external electric field magnitude. 45 Cu(NO concentration effect: 3)2 Varying concentrations of Cu(N03)2 solution were added to BLM outer chamber while NaZS (saturated, 5 x lq-zcc) was added to the inner chamber. The bathing solution was NaAc at pH 4. The brightness and the forming Speed of this Cu mirror were dependent on Cu(N03)2 concentration. A dull bronze color on membrane surface begins to be seen in very dilute copper nitrate concentration 4 x 10-4 M. A medium shine metallic 3 mirror can be observed in the presence of 5 x 10- M Cu(N0 A bright 3)2' metallic mirror is observed in the presence of Cu(N03)2 up to 2 x 10.2 M. The mirror surface formed at this concentration could be skimmed off with a syringe and a new mirror was formed continuously. The Speed of the mirror formation also depends on the concentration of Cu(N03)2; it forms 600 seconds, 200 seconds, 100 seconds after the addition of 4 x lO-4M/l, 5 x 10.3M/1, and 2 x lO-ZM/l Cu(N0 respectively. The 3)2 membrane potential change with respect to the formation of the metallic mirror also has been measured. Theimembrane potential of about 0 to 5 mv is observed in the presence of NaZS. However, the time of membrane potential increase depends on the concentration of Cu(N03)2; for example, the membrane potential begins to increase 200 second after the addition of 5 x lO-BM/l Cu(NO The membrane surface becomes cloudy white from 3)2' its black stage. The membrane potential rises to 50 my, when the metallic mirror forms on some portions of the membrane surface. The whole membrane surface will be covered by this metallic mirror as the potential rises to 200 mv. The higher the menbrane potential generated, the brighter the metallic mirror. However, the membrane potential will level off around 300 to 400 mN. The brightness of the metallic and the membrane potential were found to increase by increasing the concentration of Cu(N03)2. v46 Membrane resistance effect: Experimentally, the oxidized cholesterOl membrane resistance decreased as the concentration of Cu(N03)2 increased on one side of BLM aqueous solution. Since the brightness of the metallic mirror has been found to increase as the concentration Cu(NO3)2 increaSes, hence the brightness of the mirror is proportional to the decrease in membrane resistance. The membrane resitance can be as low as 107ohm when Cu(N03)2 concentration is up to 2 x lO-zM/l. It is assumed that the increase in Cu(N03)2 concentration will increase the rate of metal deposition. pH effect: The effect of the pH of BLM bathing solution (basic, neutral and acidic) on BLM electrostenolysis has been measured. With basic or neutral buffer acetate as the bathing solution, the addition of Cu(N03)2 results the generation of the membrane potential up to 150 mv. Instead of a mirror, only several bright spots can be seen on membrane surface. With acidic buffer as the bathing solution, the metallic mirror is formed and the membrane potential can be generated up to 300 mv. The mirror produced at the low pH of BLM bathing solution can be due to the high solubility of Cu(N0 and Na 8 in this low pH. 3)2 2 Charge carrier effect: Since, experimentally, the brightness of the mirror on membrane surface was found to increase as the membrane conductance increases, hence it is expected that the introduction of a charge carrier to the system would enhance the conduction as well as the brightness of the mirror. Some lipid soluble charge carriers, such as hydroquinone, Valinomycin, phenylhydrazine, ferroscene, tetramethylbenzidine, tocopherol (Eirotene and T¢B have been added either to the membrane or aqueous 47 solution. Among them, tocopherol carotene and T¢B show a positive effect on the mirror brightness. When T¢B 2 x 10.4 M*was added to both sides of the aqueous solution, the resistance drOpped by three orders of magnitude. The bright mirror begins to form.within 30 seconds after the addition of Cu(N0 Meanwhile the membrane potential starts to rise quickly. 3)2' The greater the potentialis generated, with the Na S side positive the 2 brighter is the mirror. The brightest mirror occurred near +300 mv and a net membrane potential change of about 380 mv was observed. Sometimes, this mirror was still stable under an applied voltage of 4 volts. This metallic deposit can either slide off by itself of can be skimmed off by a syringe. However, in both cases, the bright metallic mirrors were formed continuously. External electric field effect: The spontanous process between Cu(N03)2 and Na 8 is evident from 2 the formation of metallic mirror in the absence of the electrodes. The 2 3)2' current would affect the mirror formation. Experimentally, with Na electron is driven from Na S to Cu(N0 However, the applied external 23. (sat, S x lO—ch) and an applied field up to -200 mv, the mirror formation is delayed by almost 1000 seconds after the addition of (5 x lO-SM/l) Cu(N03)2. 0n the contrary, with an applied voltage of +100 to +200 mv, the mirror forms much faster than in the absence of an applied voltage. The effect of the electric field on the mirror formation can also be observed in Cu(N0 /BLM,T¢B/cystine. Under an applied voltage of +100 3)2 mv, with the Cu+ containing side positive, the whole membrane area begins to form a mirror in 10 min. However, under an applied negative electric field of -100 mN, the bright area disappears completely. 48 c. The Redox Reaction in Oxidized Cholesterol BLM Incorporating Carotene or.Xanthophyll in the Dark In the dark it was diffucult for the redox reaction to take place across the Ox-ChO-BLM due to its low electronic conductivity. However, the reaction was able to take place when carotene or xantho— phyll was incorporated into the membrane. The membrane conductance was increased two orders in magnitude with the addition of carotene (3mg carotene dissolved in 2 cc oxidized cholesterol solution). The cell arrangement used to study the redox reaction is as follows: NaAc/Fe3+/ Fe2+(lO-3M)/Ox-Ch0-BLM, carotene/XI/X/NaAc(10-1M) pH 5. X+ was the oxidized form and X the reduced form of redox couple added to the Opposite side 3+/Fe2+. The redox potential of redox couple was measured by using of Fe procedure (d) in Chapter III. Table-5 showed the redox potential difference of redox couple and the observed membrane potential produced from the redox reaction across carotene or xanthOphyll incorporated Ox-ChO-BLM. In addition, the membrane potential generated from the redox reaction across Chl-BLM was also listed in this table. 2. LIGHT ENERGY UTILIZATION AND PHOTOPRODUCT SEPARATION IN THE BILAYER LIPID MEMBRANE Light energy utilized in BLM, in terms of its photoreSponse, can be highly enhanced under the prOper conditions, such as: 1) the asymmetrical location of photoactive pigment in BLM, 2) the nature of photoactive pigment, 3) the presence of a charge carrier, and 4) the presence of an electric field. a. The Asymmetrical Location of Photoactive Pigment in BLM In order to use the light energy efficiently, BLM can be constructed in such a manner that photoactive species are added into one side of the 49 m.o+ o o om+ u om+ mu mm: om+ mu mm: No+ I ow+ NI omnllumnl om: man ooflnuuoqan n a so >| >u m mcmuoumo + zqmnaso zgmuano sqmnonoaxo no a a m > > 24mlonUIxo Q CHI +N > cm: can own oml OHHI quI 00H: A: noav o m m +m m owlomaom< mocmummwfip Hmfiucmuoe xovmu msu omNI owml ohm! owml ONMI omml owml ooql omcl omql ONNI omNI oaml A>av *AOMV Hmfiuamuoa woman man m2 Q- mloav Azovmm\m A: IOHV mHmua : o AZUme : A: uoav N Azmuoav oz\moz = A: loss Az,-oav Assuoflv+~cm\+qcm : A: -oHV A: uoav AzHIOHVw<\+w< = E ice A: uoav Asmuoav+~me\+mme mvwmca mvwmuso zumuseu as was zumuoaouxo as aosuomou nosey Scum vmoswoum Hmfiuamuoa memunama vm>ummno mSu was moamummmaw Hmfiucmuom xowmu mnu mo somfiumeaoo och mlmgm om+ oq+ ow+ 00+ moa+ NN+ msmuoumo + Zamlonolxo as .mwouuomam Hmaono mos mwouuomam muamummmu och .m me am om+ omH+ o~+ oma+ mm+ om+ mH+ oa+ oos+ mm+ m+ ~N+ -+ sN+ SAmIOSUINO n> $5.2 vmsafiuaoo mlmqm<fi oqal cos- cos- cad- osmu CNN- mmm- wow. wow! cowl A25 0 m x. Azmuoav aa>mamonfim A: nod emonOfie .z A: no: Az_uoav AssuoavafiaOHee Azmuoavmsan .2 Afuoduime As nod A: nod Azfinoevom\om Azmnofiv+mme\+mmm mwfiwefi mofimuso 51 BLM aqueous solution. A chemical asymmetry on both sides of the membrane/solution interfaces can be resulted from this arrangement. The photoactive species in the aqueous phase of this membrane system have a better chance than Chl-BLM interacting with the membrane surface, catching the light photon, and interacting with some other chemical compounds if they exist in the aqueous phase. The amount of light energy utilized by BLM was measured in terms of the magnitude of photoresponse produced. The photoactive species added to the aqueous solution is a chlorophyll related pigment, chlorophyllin. It is commercially available and is soluble in water. The BLM bathing solution used in the following measurement is KCl(lO-2M) and the pH at 5 to start on both sides of BLM. After Ox-ChO-BLM was formed at the black stage, a certain amount of aqueous solution was withdrawn from the BLM inner chamber and an equal amount of chlorophyllin (pH 8) was added. A new pH, is I established in the side chlorophyllin is introduced. Hence a pH difference across the BLM is created by this pH in chlorophyllin side and the pH in the outside. Experimentally, a positive membrane dark potential, from 10 to 20 mv, in chlorophyllin side relative to the other side was observed. The membrane resistance was logohm for this system. A two-step rising in photoresponse occurred. It took a 60 second light period to reach its meximmm.photoresponse. A positive photoresponse in chlorophyllin containing side relative to the other side implied that the oxidation of chlorophyllin and the reduction of the compound on the other side. The H+ions on the low pH side, opposite to chlorophyllin side, were suggested to be this compound to function as the electron acceptors in receiving electrons. 52 In another experiment, the electron accepting compound, such as Fe3+ ion, was added to the opposite side of chlorophyllin. A new pH, lower than 4 was established in the outside where Fe3+ ions (pH 2) were added. Hence a pH difference across the BLM was created between this new pH (pH % 3.2 in Fe3+ side and the pH (pH % 7.2) in chlorophyllin side. Experimentally, a positive membrane dark potential, from 200 to 250 mv, in chlorophyllin containing side relative to the other side was observed. The value of this membrane dark potential seems to correspond to this pH difference. A positive photoresponse, from 145 to 150 mv, in chlorophyllin containing side was observed. When the light was switched off, the membrane potential dropped back to the starting base line immediately and the photoresponse was repeatable. Besides the electron acceptor, the presence of the electric field also seems affect the magnitude of the photoresponse from the above system. It was found that the photoresponse could be higher than 360 mv under the applied voltage source. A plot of this photoresponse versus the membrane dark potential was shown in Figure-4. It was found that the Ox-ChO-BLM photoresponse was linearly related to the applied electric field. The greater the applied voltage, negative on the chlorophyllin containing side, the higher was the photoresponse. As shown in Figure-5, the time course of'Open-circuit photoemf has continuous flat portion after this system has arrived to the maximum (case-a). However, the photoresponse begins to drOp from this flat portion (case-b), when the membrane dark potential is reduced to about +140 mv under the applied voltage. The photoresponse decays immediately after passing its maximum, when the membrane dark potential is set between -100 my and -l7O mv (case-c and case-d). This decay portion 53 Figure 4 The Ox-ChO-BLM photo-emf versus the membrane electric field, in the presence of Fe3+ and chlorophyllin. The BLM aqueous solution was KC1(10-2M) pH 5. The photo- response is linearly related to the electric field, at least, within the range of 1'300 mv electric field. 9:: ct. NI 0 NI] Pq 55 Figure 5 The time-course Ox-ChO-BLM photo-emf at various membrane electric fields. Fe3+ and chlorophyllin were added to the opposite sides of BLM aqueous solution KCl(lO“2M) pH 5. 23 A3 2: CO CO ONTI m3 Noe / to/ O t l. mops as c to in 0.3 3v ._.. Toma om . .4. co >Eom>em 1.00m LP 0mm to 57 in photoresponse becomes significant as the negative electric field increases. All above phenomena could be explained in terms of light— induced proton diffusion across the membrane. In case-a and case-b, the concentration of light induced protons in chlorophyllin containing side is not high enough as compared with the large H+ ions in the outer chamber so that the diffusion of this light-induced proton is slow. In case-c and case-d, the concentration of these light—induced protons is large enough compared with the H+ ions in the outer chamber so that the diffusion of this light-induced proton across the membrane is faster. b. The Nature of Photoactive Pigments Besides the location of the pigment, the redox nature of the pigment, suchas its electron accepting strength also might play an important factor in determining the magnitude of the photoresponse. The stronger the electron accepting of the pigment is, the larger is the photoresponse. The experiments were designed in this section to survey the various pigment effect on the photoresponse. A complete classification of pigments can be found in Pratt, 1947 and Venkataraman, 1952. Where pigments were classified into nine groups based on their structures. Some commercially available pigments from each group were studied in this section. 1 General electric properties and photoresponse measurements of dye-containing Ox-ChO-BLM Since most dyes studied are commercial indicators which are sensitive to pH, our measurements were studied in buffered and non-buffered systems. In a buffered system, the characteristics of the dye was measured at pH 4. 58 In a non-buffered system, the characteristics of the dye was measured at a pH which is close to the dye solution pH. A systematic study of the dye from each group and its role in BLM photoelectric properties were‘made. Group-1 pigments: Dye elements from this class are the derivatives of naphthalene with an NO2 substitute group on the ring. Naphthol yellow is the representative dye from this class which was studied. Table-6 gives the experimental data from ChO—BLM photoresponse measurement in the presence of dyes from this group. TABLE-6 The ChO-BLM photoelectric effect in the presence of nitro pigments. The bathing solution was NaAc(10-1M) pH 4. system VD Ehv Rm remark ChO-BLM/Napthol yellow -8 -l 109 Napthol Y (lg/10 m1) 8 0.3 cc ChO-BLM/Napthol Y,KI 3 60 10 In a non-buffered system, a positive membrane dark potential on the naphthol yellow containing side was generated after this dye (pH 9.7) has been added to BLM inner side (pH 5). This membrane dark potential could be generated by this pH gradient established across the membrane. The extra H+ on the opposite side of the dye functions as an electron acceptor in receiving the electrons released from light-excited naphthol yellow. This results in a positive photoresponse on the naphthol yellow containing side. In the buffered system, naphthol yellow dye functions in absorbing light and accepting electrons. This electron accepting 59 property of the dye becomes significant when an electron donating compound is also present in the system. Only 15 seconds of light duration is required for this ChO-BLM, in the presence of naphthol yellow, to reach its maximum photoresponse. Meanwhile, a fast drop in membrane potential occurs immediately after the light is switched off. Group-II pigments: The dyes in this group contain nitriso group in general. Naphthol green B, a dye which belongs to this class, was studied. This dye solution was made by dissolving l g of it into 10 m1 H20. This is a high pH dye solution. Table-7 gives the characteristic effects of this dye on the Ox-ChO-BLM photoelectric properties. In either a buffered or a non-buffered system, there is always a membrane dark potential with positive polarity on naphthol green B containing side resulting from a pH difference across the membrane. In KCl, the positive sign of ChO-BLM.photoresponse indicates that the + H ions, in the outer chamber function as an electron acceptor in receiving electrons released from light excited dye molecule. TABLE-7 system VD Ehv .Rm remarks KCl(lO-2M)/Ch0-BLM/Dye*/KC1 3 6 109 N.G(lg/lO m1) pH 5 -1M 9 NaAc(lO )IChO-BLM/Dye/NaAc 10 -9 10 NaAc(10-1M)/Ch0-Blm/Dye,KI/NaAc 8 _3 40 50 10 KI(3 x 10 M/l) * Dye = naphthol green B 60 In the buffered system, the negative sign of Ox-ChO-BLM photo- response in naphthol green B containing side implied this dye was reduced. The electron accepting characteristics of this dye become significant in the presence of an electron donor. An enhancement of the photoresponse of about 50 mv was obtained in the presence of KI on the same side as the dye, where both do not interact in the dark. This system takes 60 seconds of light duration to reach its maximum photo- response. Group-III pigment: A11 dye elements in this group have a basic structure of -N=N- group on the center of the molecule. Two acidic and one basic dyes in this group were studied. Some experimental data, showing the effects of dyes from this group on BLM photoelectric properties are given in Table-8. TABLE-8 system VD Ehv Rm remarks -2M 3 KCl(lO )IChO-BLM/Azo dye/KCl -28 -8 10 A20 dye(lg/10m1) pH 5 0.2cc NaAc(lO-1M)/Ch0-BLM/Azo,Ki/NaAc 50 33 108 KI(3 x 10-3M/1) pH 4 KC1(10-2M)/ChO-BLM/Congo red/KCl l8 9 108 Congo(1g/10ml) pH 4 _ 8 red 0.3cc NaAc(10 1M)/Ch0-BLM/Congo red,KI/NaAc o 10 10 pH 4 _ 9 NaAc(lO lM)/Ch0-BLM/M. red,KI/NaAc 40 30 10 ph 4 NaAc(1O-1M)/ChO-BLM/M. orange,KI/NaAc 80 35 109 pH 4 NaAc(lO_1M)/Ch0-BLM/Bismark,KI/NaAc -3 15 108 brown 61 The efficiency of the dye elements in this group, in terms of ChO-BLM photoresponse, has the following order: M. orange + Azo dye +’M. red +-Bismarck brown + Congo red The lower efficiency of Congo red in the ChO-BLM photoreSponse may be due to the lower solubility of this dye at such low pH. Figure 6 shows the time courses of ChO-BLM photoresponses in the presence of dye elements from this group. The time of reaching maximum photoresponses are: M. orange, Azo dye +'M. red + Congo red, Bismarck brown t = 20 sec 30 sec 40 sec Group-IV pigment: Dyes in this group contain substituted amino group; -NH -NHCH 2’ 3’ N(CH3)2----. This group of dyes can be divided into three categories, such as azine, thazine, and oxazine. Dye elements used in our measurement were: azine group --— safranine thiazine group --- methylene blue, thionin, azure oxazine group --- nile blue other group --- aniline green The experimental data from.the measurement of ChO-BLM photoelectric effect in the presence of dye elements in this group are given in Table-9. In the buffered system, every dye element studied showed its electron accepting characteristics. This electron accepting prOperty of the dyestuff becomes more significant when an electron donating compound, such as K1, is added. The ChO-BLM has a positive polarity of photoreSponse with respect to the dye and KI containing side. When KI is added to the 62 Figure 6 -- The time cours Ox-ChO-BLM photoreSponse in the presence of azo dyes. 64 other side, the photoresponse becomes negative on the dye containing side. All these polarity changes are consistent with our hypothesis, that is; dye elements in this group function as electron acceptors in the buffered system. The addition of KI to the opposite side of the dye only decreases the contact between the dye and XI. TABLE-9 The ChO-BLM photoelectric effect in the presence of Group IV pigments. The bathing solution was NaAc(10-;M) pH 4 in every case. system VD Ehv Rm remark ChO—BLM/safranine -24 -4 109 safranine(lg/10 ml) 0.5 cc ChO-BLM/safranine,KI -5 78 108 ChO-BLM/M. blue -49 -4 109 M. blue(lg/1O ml) 0.5 cc ChO-BLM/M. blue, KI -37 100 108 ChO-BLM/thionin -40 -10 108 thionin(10-4M) ChO-BLM/thionin,KI -5 120 108 ChO-BLM/nile blue 0 -2 .109 nile blue(1g/1O ml) 0.4 cc ChO-BLM/nile blue,KI 22 10 108 ChO-BLM/azure-a -28 —4 109 azure-a(lg/10 ml) 0.05 cc ChO-BLM/azure-a,KI -15 40 108 ChO-BLM/aniline green 12 -l 109 aniline green(1g/10 ml) 0.05 cc 9 ChO-BLM/aniline green, KI 21 24 10 and thus decreases the magnitude of photo-response. A typical example is given in the case of thionin-KI system. 65 The order of electron accepting power of the dyes in this group is as follows: Thionin + Methylene blue + Safranine + Azure-a + Aniline G + Ehv? 120 my 100 mv 78 mv 40 mv 24 mv Nine blue Ehv? 10 my The time to initiate the light absorption by the dyes in this group and to induce the redox reaction also vary among the dyes. Figure 7 gives the time cours ChO-BLM photoresponse for each dye element studied. For dyes, such as thionin, methylene blue and nile blue, 60 second light duration is required to reach the maximum value. Most of the dyes in this group cause the membrane potential to drop immediately after the light is switched off. Group-V pigment: Dyes in this class contain a strong acidic group in the base (~803=) of their structure. Indigo carmine is a representative dye element of this class which was studied. Table-10 gives the experimental data from the measurement of ChO-BLM photoresponse in the presence of indigo carmine. TABLE-10 The ChO-BLM photoelectric effect in the presence of acidic pigment. The bathing solution was NaAc(lO-1M) pH 4 system VD Ehv R.m remark ChO-BLM/Indigo carmine .12 -l 109 Indigo carmine(lg/10 ml) 0.1 cc 8 ChO-BLM/Indigo carmine,KI 70 15 10 66 Figure 7 -- The time course of Ox—ChO-BLM photoresponse in the presence of azine dyes. t Thionin vi / ‘ i ‘ AZUIeA E 50 * N' hi mvj IleB M Blue "t 60 sec Safranine 68 The ChO-BLM membrane dark potential, in the presence of indigo carmine, is very smell which is due to the pH of the dye solution similarly to that of the BLM bathing solution. However, the electron accepting ability of this dye can be observed from the photoresponse measurement. The negative polarity of photoresponse on the dye containing side indicates the reduction of this dye. The presence of KI in the same side of the dye results in'a positive photoresponse on the dye containing side. This implies the oxidation of iodide and the reduction of indigo carmine. When Fe3+ ions were present in the opposite side of the indigo carmine (in the K01 aqueous solution), a large positive membrane dark potential was generated. This is due tova new Ph difference across the membrane having been established. Since Fe3+ ion is a strong electron acceptor, it is expected that Fe3+ will accept an electron from the light excited dye. A.positive photoresponse on the dye containing side was observed which is consistent with our hypothesis. GroupeVI pigment: The xanthene'pigments are characterized by the structure: c . 06" Dye elements belonging 0 this group in our studies are; Basic dye --- Fluorescein disodium salt Neutral dyes --- Anthracene, Eosin, Phenolphthalein Acidic dyes --- Rhodamine-b, Tetraiodofluorescein, Tetrabromophenolsulfonephthalein The experimental data on ChO-BLM photoelectric effect in the presence of dyes from this group is given in Table—ll. 69 TABLE-11 The ChO-BLM photoelectric effect in the presence of dye elements from xanthene pigments. The BLM bathing solution was NaAc(lO-1M) pH 4. * F. D. S. = Fluorescein Disodium Salt * T. I. F. 8 Tetraiodofluorescein ** 0.5 cc system VD Ehv R.In remark ChO-BLM/F. D. s.* 20 -5 109 F. n. S.(lg/lO ml) 0.3 cc ChO-BLM/F. D. S.,KI 30 70 108 ChO-BLM/anthracene 5 -1 109 anthracene(lg/10 ml)0.7 cc ChO-BLM/anthracene,KI 48 7O 108 ChO-BLM/phenolphthalein 7 -1 109 phenolphalein(lg/10 ml) ChO-BLM/phenolphthalein,KI 8 51 32 ' 10 ChO-BLM/rhodamine-b 4 0 109 rhodamine-b(lg/10 ml)0.5 cc -Ch0-BLM/rhodamine-b,KI —5 30 108 ChO-BLM/T. I. F.** -17 -1 109 T. I. F.(lg/10 ml)0.l cc ChO-BLM/T. I. F.,KI 25 30 108 ChO-BLM/T. B. P. P.*** -28 -1 109 T. B. P. P.(lg/10 ml)0.1 cc ChO-BLM/T. B. P. F.,KI 35 45 108 ChO-BLM/Eosin l7 -4 109 Eosin(lg/10 ml)0.02 cc ChO-BLM/Eosin,KI 15 165 109 s T. B. P. P. = Tetrabromophenolsulfonephthalein 70 The Ox-ChO-BLM stays at high resistance (Rmelogohm) in the presence of dyes from this group. However, the membrane resistance drops to 1080hm with the addition of KI to ChO-BLM dye. The sign of membrane dark potential depends solely upon the acidity or basicity of the dye. When an acidic dye is added to the inner chamber. H+ ions begin to diffuse across the membrane toward the outer chamber and results in a negative membrane dark potential in the dye containing side. When a basic dye is added to the inner chamber, H+ ions will diffuse across the membrane from the outer chamber toward the inner chamber and results in a positive membrane dark potential in the dye containing side. When the aqueous solution (NaAc) is replaced by KCl, the sign characteristics of membrane dark potential become more significant. In KCl aqueous solution system, if the dye solution has a pH greater than the pH of KCl, the observed membrane dark potential is positive on the dye containing side. If the dye pH is lower than that of KCl, the membrane dark potential is negative on the dye containing side. In buffer acetate, the electron accepting characteristic of the dye elements has been observed from photoreSponse studies. It was found that the dye was reduced in the light. This electron accepting characteristic of the dye elements becomes more significant in the presence of an electron donor, such as KI, in the BLM system. This electron accepting power of dye elements in this class decreases in the following order Eosin +~F. D. S. +- Tetrabromo- +' Phenolphalein + Anthracene phemolphalein Rhodamine B, Tetraiodofluorescene 71 Figure 8 shows the time course of photoresponse in the presence of dye elements from this class. It was found that the decay of 0x-Ch0-BLM photoresponse after the light was switched off, in the presence of dyes from this class, was very fast in comparison with BLM in the presence of other classes of dye. Two interesting phenomena were found in the measurement of ChO-BLM photoresponse in the presence of dye elements from this class. When rhodamine dye was introduced into the inner chamber of ChO-BLM, a colorless membrane, immediately a clear diffusion of this dye across the membrane toward the outer chamber could be observed. There was a continuous stream of this colored dye across the membrane. Since the stream of this dye was only across a certain area of membrane surface, it is suggested that there exist some pores in the membrane for this diffusion. Another phenomenon was ChO-BLM in the presence of fluorescein disodium salt and K1. A bright mirror was formed after the membrane was illumin- ated. The degree of brightness of this increased as the amount of light illumination increased. It is suggested that this bright mirror was the accumulation of some products in the membrane after light illumination. Group-VII pigments: The dye element in this class contains a basic structure: x I I \ / Alizarin is the most representative dye in this class, which has four additional hydroxyl ions attached to the side chain. Alizarin's derivatives are obtained when one of the hydroxyl ions is replaced by another ion or radical. The redox nature of dyes in this class can be measured by BLM photoresponse studies. The experimental data obtained from this measurement is given in Table-12. 72 Figure 8 -- The time course Ox-ChO-BLM photoresponse in the presence of xanthene dyes. Rhodam . 113., Eosin Anth racene Phenolphthalein 74 TABLE-12 The ChO-BLM photoelectric effect in the presence of dye from anthraquinone pigments. The bathing solution was NaAc(lO-1M) pH 4. system V E R remark 9 Cho-BLM/Alizarin 0 O 10 Alizarin(lg/10 ml)0.4 cc ChO-BLM/Alizarin,KI 5 75 109 ChO-BLM/Alizarin red 32 -3 109 Alizarin red(lg/10 m1) 9 0.1 cc ChO-BLM/Alizarin red, KI 80 63 10 ChO-BLM/Alizarin orange 13 0 109 Alizarin orange(lg/10 ml) 0.1 cc ChO-BLM/Alizarin orange,KI 4 20 109 ChO-BLM/Aminoanthraquinone -12 -l 109 Aminoanthraquinone(1g/10 ml) ‘ 0.1 cc 9 ChO-BLM/Aminoanthraquinone,KI 11 36 10 The experimental results indicate that every anthraquinone dye functions as an electron acceptor in the ChO-BLM. This electron accepting characteristic of dyes can be enhanced when an electron donating compound is also present. In general, this electron accepting property of anthraquinone dyes in ChO—BLM is determined by two factors: the chemical nature of the free radical attached to its side chain, and the solubility of the dye. Since in the nucleophilic substituting reaction of anthraquinone, the presence of an electron releasing group (like-0H,-SO3) deactivates the reaction and an electron withdrawing group (like -N0 -NH2) will activate the reaction, one would expect 2, that the magnitude of ChO-BLM photoresponse in the presence of alizarin orange (with ~N02), aminoanthraquinone (with ~NH2) should be greater than that in the presence of alizarin (with -0H), alizarin 75 red (with -SO3). However, the observed ChO-BLM photoresponse did not follow this hypothesis. Therefore, this electron accepting property of anthraquinone dye seems to be determined by the secOnd factor. Since the solubility of anthraquinone dyes decreases in the following order: alizarin + alizarin red + aminoanthraquinone + alizarin orange, one would ‘expect more molecules of alizarin, alizarin red to be available in the aqueous solution for absorbing the light and interacting with the electron donor. The magnitude of observed photoresponse was consistent with this hypothesis. Figure 9 gives the time course of ChO-BLM photo- response in the presence of anthraquinone dyes. It was found that the increase of ChO-BLM photoresponse was quite different in the presence of different anthraquinone dye elements. There was an immediate increase in photoresponse in the presence of alizarin red, but not in the case of alizarin orange. However, the membrane potential drOps immediately back to the base line in each element presenting case after light is switched off. Group-VIII pigment: Phthalocyanine appearsuto be based structurally on porphin: C“"CH C This porphin consists of fou pyrrol nuclei: HC II CH I l =.z,c- 76 Figure 9 -- The time course of Ox-ChO-BLM photoresponse in the presence of anthraquinone dyes. off 4r Alizarin V \Alizarinfl T . 40 mv Alizarin. : i Mi hv k_60 8604 78 Three representative dye elements from this class were studied: Phthaocyanine --- a basic dye without metal ion Mg-phthalocyanine -- an acidic dye with Mg++ ion Chlorophyllin -- a basic dye with Mg++ ion The experimental data from the measurement of ChO-BLM photoelectric effect in the presence of dye elements from this class are given in Table—13. TABLE-l3 The GhO-BLM photoelectric effect in the presence of dye elements from phthalocyanine pigments. The bathing solution was NaAc(lO-1M) pH 4. system VD Ehv 3R.m _ remark, ChO-BLM/phthalocyanine ' 4 0 109 phthalocyanine(lg/10 m1) ChO-BlM/phthalocyanine,KI 38 52 109 0.4 cc ChO-BLMJMg-phthalocyanine -1 0 108 Mg-phthlocyanine(lg/10 ml) ChO-BLM/Mg-phthalocyanine,KI ll 67 108 0.5 CC ChO-BLM/chlorophyllin 8 -8 109 chlorophyllin 0.1 cc from commercial The membrane potential of ChO-BLM is positive in the basic dye containing side and negative on the acidic dye containing side. The polarity of ChO-BLM dark potential is consistent with that of H+ ions diffusion which goes from low pH to high pH. Since the magnitude of ChO-BLM photoresponse is greater in the presence of Mg-phthalocyanine than in the presence of phthalocyanine, it seems that the electron accepting power of dyes in this class in increased when a metal ion is present in the porphin base. Different metal ions 79 would alter the electron accepting characteristics of dye element. In most dye systems studied, the enhancement of photoresponse can be obtained when some electron donor, such as K1 is also presented to the system. There is one exception though. In chlorophyllin dye system the addition of K1 to the system did not enhance the magnitude of ChO-BLM photoresponse. This is due to the interaction between chlorophyllin and KI in the dark. In a test tube, a color change can be observed immediately after mixing these two compounds. The time course of CHO-BLM photoresponse is given in Figure 10. Only 15 seconds of light duration is required for ChO-BLM with phthalocyanine to reach its maximum photoresponse, but 80 seconds with Mg-phthalocyanine system. Group-IX pigments: The dyes from this group have a general characteristic structure I Ph The substituted group in the side chain may contain methane radicals. Dye elements from this group studied were: Malachite green (pH 2) Crystal violet (pH 5) Pararosaniline (pH 8) Among them, malachite green and crystal violet are acidic dyes, pararosaniline is a basic dye. Table-l4 gives the experimental data of ChO-BLM photoresponse measurement in the presence of dyes from this group. Two special phenomena observed in ChO-BLM.with triphenylmethane 80 Figure 10 -- The time course of Ox-ChO-BLM photoreSponse in the presence of phthalocyanine dyes. * Mg-Phthalocyanine Phthalocyan e 82 pigments are: l) in a buffered system, a very large negative membrane dark potential was generated in the presence of any dye element from this group, 2) in a buffered system, the addition of the dye element to ChO-BLM caused the membrane resistance to drop from.1090hm to 108ohm. When KI was added to ChO-BLM, a further drop in membrane resistance occurred. When triphenylmethane dye was added to the BLM chamber, an interaction be- tween this dyeand the membrane surface occurred. This interaction may cause a membrane conformational change. The membrane resistance drop and the large negative dark potential generation were the result of this conformational change. TABLE-l4_ The ChO-BLM photoelectric effect in the presence of dyes from triphenylmethane pigments. The bathing solution was NaAc(10—1M) pH 4. system VD Ehv Rm remark ChO-BLMJMalachite green -45 -13 108 malachite green(1g/10 ml) 0.1 cc ChO-BLM/Malachite green,KI -17 5 107 ChO-BLM/Crystal violet -55 -5 108 crystal violet(lg/10 ml) 0.1 cc ChO-BLM/Crystal violet,KI -38 35 106 ChO-BLM/Pararosnailine 217 -1 109 paraosaniline(lg/10 ml) 8 0.1 cc ChO-BLM/Pararosaniline,KI 7 8 10 .Because of this low membrane resitance, the observed photoresponse in the presence of dyes from this group is small compared to the photoresponse in other dye groups. 83 2 Redox Nature of Dyes The character of aqueous solution, its pH, the dye solubility and the dye constituent could affect the redox nature of dye. Experiments to test the effect of these factors upon dye's nature have been done. 2H Effect When low buffer capacity salt solution was used as the BLM bathing solution, a pH difference across the membrane would appear in the presence of dye if the pH of dye solution wasn't the same as that of the BLM aqueous solution. Since the hydrogen ions in low pH side might have sufficient power to accept electron delivered from light excited dye, dye was apparently oxidized. However, the rate of electron transfer in this redox reaction might also depend on the concentration of the dye. Consequently, the solubility of the dye in the aqueous solution would affect the efficiency of electron transfer. Therefore, the magnitude and polarity of BLM photoresponse in the low buffer capacity BLM bathing solution will depend on which one of above factors dominates over the other. Several basic, neutral and acidic dyes were investigated. The membrane surface reaction was KC1(lO-2M) pH = x/Ox-ChO-BLM/Dye/KCl (10-2M)pH = x. Where x were varied from 3 to 9. Table-15 gave the observed Ox-ChO-BLM photoemf at different.pH. A.plot'of-0x-Ch0-BLM photoemf versus the pH of the bathing solution was given in Figure 11. For basic dye system, the photoresponse increases when the bathing solution pH is increasing. There is always a positive polarity of photoresponse in the basic dye containing side. The maximum photores- ponse seems to occur at pH which is close to the dye solution pH. For acidic dye system, the Ox-ChO-BLM photoresponse increases as the 84 Figure 11 The ChO-BLM photoresponse in the presence of basic, neutral, and acidic dyes at various KCl bathing solution pH's. The membrane surface reaction is: KCl(10-2M) pH = x/ChO-BLM/Dye/KCl(lO-ZM)pH = x, where x can be 3, 5 and 9. The polarity of BLM photoresponse is positive on the basic dye containing side throughout the whole pH range, negative on the acidic dye containing side. For neutral dye cases, there is a polarity that is positive for the photoresponse in low pH and a negative polarity in high pH. Basic dyes a a chlorophyllin b - congo red c methyl orange d phthalocyanine Neutral dyes e - alizarin f a thionin Acidic dyes g = azo dye , -H- h = alizarin red,Mg -phthalocyanine i = methyl blue 86 bathing solution pH increases. There is always a negative polarity of photoreSponse in this acidic dye containing side. The maximum photoresponse occurs at high pH of bathing solution. TABLE—15 The ChO-BLM photoresponse in the presence of basic, neutral, or acidic dyes at various bathing solution pH's. Dye ' BLM photorespbnse (mv) at: pH=3 pH=5 pH=9 Acidic dye Azo dye -l -4 -5 Methyl blue -4 -10 -20 Alizarin red -3 -5 -10 Mg-phthalocyanine -4 -6 -8 Basic dye Congo red O 9 15 Methyl orange 1 6 ll Phthalocyanine 2 3 4 Chlorophyllin 12 28 40 Neutral dye Thionin 3 -1 -20 Alizarin 3 -2 -6 The effect of two factors (the bathing solution pH and dye solubility) on Ox-ChO-BLM photoresponse differs for basic dye system and for acidic dye system. FOr basic dye system, the solubility of dye has more influence on the photoresponse than that of the pH of the 87 bathing solution. Therefore, the maximum photoresponse occurs near high pH of dye solubility region. For acidic dye system, the Ox-ChO- BLM photoresponse is determined mainly by the pH of the bathing solution and the pH difference across the membrane/solution interface. For neutral dye system, the Ox-ChO-BLM photoresponse will depend upon the dye itself near neutral pH. For example, if the dye is an electron acceptor, there will be a negative photoemf in dye containing side. However, when this neutral dye was added to the bathing solution of low pH, excess H+ ions appeared in the Opposite side of dye could function as the electron acceptor to receive electrons from the light illuminated dye. Therefore, the photoresponse becomes a positive polarity in dye containing side. When this neutral dye is added to the bathing solution of high pH, the excess H+ ions in dye side may serve as the electron acceptors to receive an electron from light illuminated dye. Consequently, the photoresponse has a negative polarity in dye containing side. The Effect of Dye Constituent The Ox-ChO-BLM photoresponse was reported to be enhanced when the dye in the aqueous solution could enter into the membrane. However, the details of how this occurs is still not quite known. From the structural point of view, a dye must possess a hydrophobic group in its constituent in order to be able to enter the hydrophobic portion of thefmembrane. From the functional point of view, a dye must possess a hydrophilic (or polar) group in order that it can sit at membrane/ solution interface ready for the electron releasing or accepting. Therefore, at constant bathing solution pH and high buffer capacity, 88 the redox nature of dye will be solely depended on its structure. Diagram A showed the relative electron accepting-releasing power of dyes. In this diagram, eosin dye from xanthene group is the strongest electron acceptor, chlorophyllin from phthalocyanine is the strongest electron donor, and malachite green, paraosaniline from triphenylmethane group are the weakest electron acceptors. Since almost every dye from dye group I to group IX contains the aromatic structure, it is not difficult for them to enter the hydrOphobic portion of the membrane. Since both the Ox-ChO-BLM photoresponse and the solubility of dye in the membrane seem to increase in the same order: benzene derivative + Napthracene derivative-+ anthracene derivative, the Ox-ChO-BLM photoresponse strictly depends on the solubility of dye in the membrane. Dyes belonging to anthracene derivative are methyl blue, thionin, alizarin-a, safranine, aniline-g from group IV and fluorescein disodium salt, phenolphthalein, tetrabronophenol-sulfon— ephthalein, eosin, anthracene from group VI and alizarin, alizarin-red, alizarin orange, aminothraquinone from group VII. The Ox-ChO-BLM has relatively large photoresponse in the prescence of the above dyes. The Ox-ChO-BLM has medium photoresponse in the presence of naphthracene derivative dyes, such as naphthol yellow, naphthol green from group 1, and small photoresponse in the presence of benzene derivative dyes, such as malachite green, crystal violet, and pararosaniline. Another dye constituent which affects the 0x-Ch0-BLM.photoresponse is the polar group of a dye. This polar group in a dye makes part of this dye ready to stay at the hydrophilic portion of the membrane. This character of dye is similar to the hydrophobic group of a dye which 175*- 150-4 125 - 100- Ehv (mv) 50- 25— Eosin (165 mv) * "Chlorophyllin (150 mv) Thionin (120 mv) —Methyl blue (100 mv) Safranine (78 mv) '—Alizarin (75 mv) F. D. S. (70 mv), Anthracene (7O mv) Mg—phthalocyanine (67 mv) Alizarin red (63 mv) Naphthol Y (60 mv) Phthalocyanine (52 mv) -Naphthol G (50 mv) Tetrabromophenolsulfonephthalein (45 mv) Azure A (40 mv) Aminoanthraquinone (36 mv) Methyl orange (35 mv), Crystal V. (35 mv) Azo dye (33 mv), Phenolphthalein (32 mv) Methyl R (30 mv), Rhodamine (30 mv) Tzniiine G (24 mv) Alizarin orange (20 mv) Bismarck B (15 mv), Indigo Carmine (15 mv) Congo red (10 mv), Nile Blue (10 mv) Pararosaniline (8 mv) Malachite G (5 mv) o .J Diagram A L The electron accepting power of dyes in the BLM system. * ChlorOphyllin functions as an electron donor. 90 makes a dye easily to go into the hydrophobic portion of the membrane. A typical example given here is dye from dye group VII. The basic structure of this group is where group X can be OH (alizarin), SO Na (alizarin red), NH2 (amino- 3 alizarin) and N02 (alizarin orange). Since the relative strengths of their polarities are N02 +-NH2‘+ SO3Na‘+ OH, the probabiity of these groups sitting at membrane/solution biface also follows this order. The magnitude of Ox-ChO-BLM photoresponse will be expected to increase in the same.order. The observed Ox-ChO-BLM photoresponse indeed follows this expectation and Eop is 75 my for alizarin system, 63 my for alizarin red, 36 my for alizarin (amino) and 20 my for alizarin orange. Another dye constituent affecting the Ox-ChO-BLM photoresponse is the electron deficiency in dye structure. A relative strong electron .accepting dye must contain a high electron deficiency in its structure. A typical example is dye in group IV, such as methylene blue, alizarin a, thionin, safranine, where each dye contains a 5N= group. This demands an electron to fill its electron deficiency if the redox process is taking place. Therefore, almost every dye in this group, except nile blue, is a strong electron acceptor in BLM system. Correspondingly, a large Ox-ChO-BLM photoresponse is observed. C. The Effect of Charge Carriers and Electric Field Upon the Ox-ChO—BLM Photoresponse The effect of charge carrier on BLM photoresponse has been shown in the BLM energy conversion system (thionin/ferrous). The Ox-ChO-BLM 91 has been found to increase the photoresponse by four times in the presence of tetraphenylborate charge carrier. The membrane conductivity also increases by 2 to 3 order in magnitude. The effects of charge carrier and electric field on the BLM photoresponse were observed. The system studied was T¢B/Ox-Ch0-BLM/Eosin dye/KI. Our previous experiment indicated that Ox-ChO-BLM/Eosin photoresponse was enhanced in the presence of KI (an electron donor) and the polarity of this photoresponse on the dye containing side was positively charged. It is expected that above response will be enhanced if an electric field with negative polarity in dye containing side presented prior to light illumination. Since it was found the presence of tetraphenylborate would generate a positive membrane dark potential in T¢B containing side, hence the presence of T¢B to the opposite side of eosin dye could be expected to end up in an electric field with negative polarity in dye side. The BLM.ce11 with this electric field arrangement would facilitate more light-induced electron .flow from KI to eosin. Table-16 gives the experimental data resulting from.this measurement. TABLE-16 The enhancement of ChO-BLM/Eosin photoresponse in the presence of K1 and Tetraphenylborate. (NaAc(10-1M) pH 4 as the aqueous solutions) T¢B conc (M/l) VD(my) VL Ehv)op(my) 1 x 10‘5 -45 140 185 5 x 10-5 -65 155-150 220-215 1 x 10'4 -95 150-165 245-260 3 1 x 10' ~90 135-145 225-235 92 An open-circuit photoemf of 245 to 260 my has been obtained. This photoemf magnitude is larger than the one obtained from,Fe3+/Ch1-BLM/ ascorbate system (Eop = 188 my). 3 THE PHOTOSENSITIZING PROCESS AND MODEL STUDIES OF THE ELECTRON TRANSPORT IN PHOTOSYSTEMS The green pigment chlorophyll is a common constituent of all photosynthesizing plant cells. It could be a physical agent in photosynthesis, collecting light energy and making it available for photosyntheéis, or it could serve as a photocatalyst, which acts as a light-activated chemical catalyst that takes an active, albeit reversible, part in the photosynthetic reaction (Rabinowitch and Govindjee, 1969). The question of its role in photosynthesis encourages studies of the photochemical prOperties of chlorophyll outside the plant cell. We are interested in knowing whether chlorophyll in vitro can serve as an oxidation-reduction photocatalyst and whether it can mediate the oxidation-reduction reaction involving the storage of light energy in BLM system. It has been suggested that the mechanism of electron transport in photosynthetic units can be studied separately by placing artificial electron acceptors, donors and the inhibitors in the proper positions in the electron transport chain. The following are some examples: 1) The presence of electron acceptors (A1) from photosystemrl, such as benzoquinone, mehtylene blue, indigo carmine, PMS, FMN, vitamin-K, Fe(CN)23, and electron donors (D1), such as DCPIPH, reduced PMS, reduced 93 TMPD, reduced diaminodurol and sodium ascorbate, the likely operation of electron transport in photosystem-l can be observed. 2) The presence of electron acceptors (A2) from photosystem-2, such as the indophenol dyes (DCIP+ and TCIP+), Fe(CN)g3, methylene blue, thionin, theonine, toluylene blue and FeCl3, the likely electron transport and 02 evolution in photosystem-2 can be observed. 3) In the presence of an inhibitor of 02 evolution such as NHZOH, the photoreduction of NADP+ may be effected to some extent; however, this reduction can be resumed in the presence of electron donor (D1) from photosystemrl. 4) An Operational separation of the photosystems can also be achieved by using light wavelength about 700 mu which activates photosystemrl only. The above suggested methods for studying electron transport in photosynthesis will be examined in the BLM model system. Hopefully in these studies, new methods in studying the detailed mechanisms of electron transport in photosysnthesis would be further established. a. The Photoreduction of M. Viologen Via Electron Transport from D1 (electron donor) to M. Viologen The photoreduction of m. viologen via electron transport from D1 to m. viologen in BLM‘was detected by measuring BLMthotoresponse. The electron donors used were DCPIPH, ascorbate, ferrocyanite, ferrous chloride and cysteine. The membrane surface reaction mechanism in the measurement of photoreduction via electron transport in m. viologen and D1 can be expressed as: NaAc(lO-1M) /M. Viologen (3 x 10-3M/1)/Chl-BLM/D1/NaAc(10-1M), pH 4. Table-17 lists experimental results from this measurement. Since 94 the magnitude of the membrane dark potential is small, the interaction between m. viologen and each electron donor does not seem to occur in the dark. The negative photoresponse in m. viologen is being reduced and ascorbate, DCPIPH, Fe(CN);4 , ferrous chloride or cysteine is oxidized. Here the chlorophyll pigment in the membrane dunctions as a photocatalyst; that is, the light has been absorbed by the chlorophyll to facilitate the electron transfer from D1 to m. viologen. When the light is switched off, all of the active chlorophylls go back to their ground state, which is observed from the drOpping of the membrane potential to the base line. Ascorbate is among the electron donors which when added to the Opposite side of m. viologen generates a 200 my photoresponse under an applied field. The action of the light seems to drive the electrons against the preexisting redox potential difference between the electron donor and m. viologen. This observed electron flow in Chl-BLM is similar to the suggested electron flow in photosystem-l in the presence of artificial electron donor (D1). b. The Photooxidation of Ascorbate and DCPIPH Via Electron Transport from Ascorbate and DCPIPH to A1 The membrane surface reaction for this measurement was; NaAc(lO-1M) pH 4,5/Ascorbate/Chl-BIM/AllNaAcflO-J‘M),pH 4, 5 or DCPIPH where Al, the electron acceptor from photosystemrl, which could be benzoquinone, K3Fe(CN)6, ferric oxalate and ferric chloride, FMN, vitamin-K. Table-18 gives the experimental data of the Chl-BLM photoresponse measurement in the presence of ascorbate, DCPIPH and A1. The Chl-BLM membrane resistance was decreased to 108 ohm when Fe salt was added to the opposite side of ascorbate. Since the membrane 95 TABLE-17 The photoreduction of Methyl Viologen by Chl-BLM photoresponse measurement. The bathing solution was NaAc(lO-1M) pH 4. system ** V (my) E E in out D hv)0p hv)close Ascorbats M. Violg en 10 97-100 170-220 (2 x 10 ‘M/l) (3 x 10 M/l) FeC12 _3 M. Vioig en 10 75-80 140-150 (4 x 10 M/l) (3 x 10 ‘M/l) cysteine3 M. Violo en 10 30-35 80-100 (3 x 10 W1) (3 x 10 M/l) ..Pc(cu)"_‘f3 M. Violg en —5 I 70-75 110-120 (3 x 18 W1) (3 x 10 11/1) * DCPIPH 3 M. Violo en 130 30-40 70-80 (1 x 10" M/l) (3 x 10' M/l) * - the bathing solution for this measurement is KC1(10 2M) pH 5. ** Ehv)op’ the Chl-BLM.photoresponse is positive in the inner side relative to the outer side in above table. dark potential is only several millvolts in magnitude for each system, it is presumable that there is no interaction between ascorbate and Fe salts in the dark. The Chl-BLM photoresponse seems to depend upon the ligand attached to the Fe octahedral basis. The observed photoresponse decreases in the following order: Fe salt = FeCl3 + K3Fe(CN)6 + Fe2(C204)3 Eh = 165-188 mv 64 my 40 my v)op 96 TABLE-18 The Chl-BLM photoreSponse measurement of ascorbate and DCPIPH photooxidation. The bathing solution was NaAc(lO-1M) pH 4. a. Ascorbate photooxidation system VD(mv) Ehv)op Ehv)close out in Ascorbate FeCl3 (2 x 10'3M/1) (5 x 10'3M/1) -2 -l68—-l88 -3oo K3Fe(CNZ9 -10 ~64 - (5 x 10 M/l) H _ _ _ Fe2(§204)3 10 4O (10 ‘M/l) " Benzoquinone 5 -25 -100 (10'3M/1) b. DCPIPH photooxidation (the bathing solution was KCl(lO-2M) pH 5. system VD(mV) Ehv)0p Ehv)close out in VD: -lOO 0 100 mv DCPIPH FeCl3 -170 -40 -45 -60 -70 (10'3M/1) " K3Fe(CN)6 -50 -35 -25 -55 -90 II Benz°qum°m355 -30 -2o -35 —80 " M. Viologen -l30 -30 -20 -50 -7O 97 the electron accepting power of an Fe salt depends upon the degree of its ionization in solution. FeCl3 is completely ionized in solution; therefore this compound has the greatest ability to receive electrons. K3Fe(CN)6 forms a complex in solution and the ligands (CN-) are bound tightly to Fe3+ ion so that an electron has to penetrate through the negative charge cloud in order to reach Fe3+ ion; hencefore K3Fe(CN)6 has a lower ability to accept electrons than FeCl Fe2(C 0 ) has a 3' 2 4 3 structural basis which looks like: 1 020 Re i czo4 7 204 C where the ligands (C204) are "bide tate" on the two positions of Fe3+ octahedral structure. It is much more diffucult for an electron to penetrate this negative charge cloud in order to reach Fe3+ ion itself. So, the Chl-BLM in the presence of Fe2(C204)3 has a smaller photoresponse than that with K Fe(CN)6. 3 Another interesting phenomenon is that the time course Chl-BLM photoresponses in the three Fe salt systems are different from each other. Figure 12 shows the Chl-BLM time course photoresponse in the presence of the three Fe salts on the opposite side of ascorbate. For the FeCl3, the photoresponse levels off after reaching the maxi- mum point as long as the light is still on. For the K3(CN)6, the photoresponse, after reaching the maximum point, stays at this point for a.while, then gradually drops to the base line before the light is 98 Figure 12 -- The Chl-BLM time course photore3ponses in the 4. presence of Fe3 salts and ascorbate. to 0m. sh. OBLIL :0 film- _oomo_._ >Eom >£m 100 switched off. For the Fe2(C202)3, the photoresponse, after reaching the maximum point, drops immediately to the base line before the light is switched off. If the slow component, as has been suggested, is due to the Hf ions (light-induced) diffusion, this implies that the membrane is more permeable for light-induced H+ ion in the Fe2(C2 case than oa’a in the K3Fe(CN)6 case. c The Photosensitizing Process in Ox-ChO-BLMrChlorophyllin The membrane surface reaction for this photosensitizing process in BLM can be expressed as: KCl(10-ZM)IX/ChO-BLM/chlorophyllin (0.1 cc)/KC1(10-2M) where X can be FeCl3, K3Fe(CN)6, Fe2(0204)3, or benzoquinone. The photosensitizing process studied here is the measurement of the light- induced redox reaction, or the electron transduction from chlorophyllin to the X compound. The main difference between this ChO-BLM system and Chl-BLM system is that the chlorophyllin pigment functions not only as the sensitizer, but as an electron donor to donate electrons to some electron accepting compound. After this reaction, chlorophyllin may not go back to its original ground state as the chlorophyll in Chl-BLM system does. This light-induced redox reaction between chlorophyllin and the X compounds can be measured in term of the ChO-BLM photoresponse. The polarity of photoresponse indicates the direction of this light- induced electron flow, or which compound has been reduced or oxidized. Table 19 gives the experimental data for this ChO-BLM photoresponse measurement in the presence of chlorophyllin and the other redox compound. In this ChO-BLM photosensitizing redox process, the electron accepting power of each X compound has been found to be pH.dependent. This could 101 TABLE-l9 The Ox-ChO-BLM photoreSponse in the presence of chlorophyllin and other redox compounds. The bathing solution was KCl(10-2M) at various pH's. pH of bathing Benzoquinone Fe2(C204)3 K3Fe(CN)6 FeCl3 solution VD Ehv)0p VD Ehv)0p VD Ehv)0p VD Ehv)0p 5 0 50 145 115 -7 5 210 150 7 2 65 185 45 -8 10 210 100 8 18 75 - - 38 28 165 80 be due to the fact that each X compound has its own maximum solubility at a particular optimum pH. When the compound is at this particular pH, many more molecules are available for electron accepting. Table- 20 shows the ChO-BLM photoresponse at the optimum pH for each X compound. The ChO-BLM has the maximum photoresponse in the presence of either FeCl3 or Fe2(C204)3 at pH 5 and in the presence of either benzoquinone or K3Fe(CN)6 at pH 8. The ChO-BLM.photoresponse of about 360 my obtained form chlorophyllin- FeCl3 system under the applied external voltage is the largest photores- ponse ever observed in the BLM system. d The Photoreduction of A2, the Electron Acceptor FromLPhotosystem- 2, in Chl-BLM As has been suggested, the electron transport in photosysteer can be studied by the incorporation of an artificial electron acceptor 102 TABLE-20 The ChO-BLM photoresponse at the optimum pH for X compound solubility. The bathing solution was KCl(lO-2M). X compound Ehv)op Ehv)close optimum pH FeCl3 150 360 5 Fe2(C204)3 115 185 5 K3Fe(CN)6 28 - 8 Benzoquinone 75 170 8 from photosysteer. This analogous electron transport mechanism.was examined in BLM model system. The artificial electron acceptors used were methylene blue, thionin, ferric cyanide, and ferric oxalate which were added to one side of the BLM aqueous solution. The bathing sol- utions were KCl and NaAc. The reduction of A2 is shown by the polarity of the Chl-BLM photoresponse. Table-21 lists the results from this measurement. The negative polarity of Chl-BLM photoresponse in each case indicated that all A2 compounds were reduced and the opposite side of the aqueous solution was oxidized in the light. In the absence of a proper electron donor, H 0 could serve as the available 2 electron donor to be oxidized (Tien, 1968). However, the resulting 02 evolution remains to be identified. The positive sign of the photoresponse indicates that Fe(CN);3 + side apparently works as an electron donor. This is due to the H ions on the opposite side of Fe(CN);3 having an electron accepting 103 power greater than Fe(CN)g3. TABLE-21 The photoreduction of A the electron acceptor from photo- 2’ system-2, in Chl-BLM. A compound bathing solution VDCmv) E E 2 op hv Pe013(1o'3M/1) NaAc(pH 4) -5 -3o- -60 -- KCl (pH 5) ~20 ~45 ~90 M. blue(lg/lO cc) NaAc(pH 4) ~49 -4 ~- 0.5 cc K01 (pH 5) ~65 ~20 ~80 Thionin(10-4M/l) NaAc(pH 4) ~40 ~10 KCl (pH 5) ~15 ~15 ~40 Fe(CN)g3(5 x 10-3M/l) NaAc(pH 4) 0 -8 ~37 pH 7 KCl (pH 5) -5 37 100 KCl (pH 7) -3 -2 ~10 Fe2(C204)3(5 x lO-3M/1) NaAc(pH 4) -10- -3o —20- -3o -- xc1 (pH 5) -110 -45 -- In order to observe the electron accepting character of K Fe(CN)6, 3 as an A2 from photosystem-2, a proper condition must be maintained (Boardman, 1963). Our observation suggests that this proper condition is the bathing solution buffered or at a pH similar to K3Fe(CN)6 solution (which has pH at 7.3). e The Resumption of Electron Transport Activity Prohibited by Inhibitor, in the Presence of Proper Electron Donor An electron acceptor, A1, such as m. viologen or NADP+ was added to one side of the BLM bathing solution and a photosystem-2 inhibitor (NHZOH) was added to the opposite side. The photoresponse was measured 104 to detect the reduction of m. viologen or NADP+. Later, an electron donor, such as ascorbate, KaFe(CN)6, was added to the same side as the inhibitor and the photoresponse was measured again. Table-22 lists some numerical photo-emf values from this measurement. The negative Chl-BLM photoresponse on the A1 containing side indicates the reduction of A1. However, the magnitude of this photoresponse is significantly decreased in the presence of inhibitor (NHZOH). Since this inhibitor blocks only the oxidation of n20 but not the other electron donors, presumably the electron transport activity _can be resumed by adding an external artificial electron donor. A large photoresponse regained in the presence of electron donor, ascorbate or K4(Fe(CN)6 indicated that the electron transport returned to normal. TABLE-22 The resumption of inhibited electron transport in Chl-BLM in the presence of proper electron donor. a. Ascorbate is the electron donor compound E hv)0p (mV) out in M. viologen ~18 NHZOH M. viologen -5 (inhibitor) Ascorbate M. viologen -80 (electron donor) 105 b. K4Fe(CN)6 is the electron donor. compound VDCmV) Eop hv out in Vb=100mv Omy ~100my + NADP -9 -4 ~80 -60 17 11112011 NADP+ -6 -1 -20 -1o 6 (inhibitor) K 4Fe (CN) 6 NADP+ -3 -2o -75 —4o 18 (donor) f 4 Operational Separation of Two_Photosystems in Chl-BLM by Using Light Wavelength of 700 mu. M. viologen (3 x lO-BM/l) and ascorbate (2 x 10_3M/l) were added to the opposite side of BLM bathing solution (NaAc, pH 4). A red light was used first, then a white light. The observed photoresponse changes are given in Table-23. TABLE-23 The Chl-BLM photoresponse in the presence of m. viologen and ascorbate by light with different wavelength. E E system op op by red light by white light Ascor1_)_§te/Ch1-BI..ML/M.V._.3 ~45 ~60 (2x10 M/l) (3x10 M/l) There are two possibilities for this photoresponse difference. One 106 is that the additional magnitude in photoreSponse by white light is contributed by photosystem-2 operation; the other possibility is that this additional photoresponse by white light is due to the light intensity difference between two lights. The latter possibility is not likely since the red filter did not cut much of light intensity from the light source. Therefore, this additional photoreSponse by white light must be due to the activity of photosystem-2 in Chl-BLM. This observation may be a direct evidence of the existence of the two photosystems in Chl-BLM. CHAPTER V DISCUSSION 1. THE ELECTRONIC AND IONIC CONDUCTION IN BILAYER LIPID MEMBRANES. Bilayer Lipid membranes, in general, exhibit the phenomenon of charge transport. It has been reported that charges could be trans- ported across a BLM devoid of photoactive pigments (Tien and Diana, 1968). The conducting species were thought to be ions in the membrane and in the BLM bathing solution. A BLM containing photoactive pigments was constructed with a lipid structure similar to that of non-photoactive pigments. It was then suggested that this membrane would be capable of ionic transport as well (Tien, 1968). However, even in the dark some electrons and holes were generated either thermally or by an external electric field; but the details of types of charges and how they move across the membrane are still not well known. It has been suggested by Tien that in the case of electronic conduction, the charges may be con- ducted in the membrane through the conjugated double bonds, such as carotene and xanthophylls which are abundantly present in the chlorOplast extracts. Nevertheless, a unique and clear-cut experiment is still needed in order to understand the mechanism of electronic and/or ionic conduction, in bilayer Lipid membranes. The pigmented BLM is capable of facilitating electron transfer in inducing the redox reaction. From a functional point of view it behaves 107 108 similarly to a metallic surface or an inert electrode. According to the above assumption, some general phenomena which correspond to the character of metallic surfaces would be expected to be seen in the pigmented BLM, such as the photovaltaic effect, the redox reaction in the dark, or in the light, and electrostenolysis. Bearing this in mind, three experiments were carried out to study these characteristics of BLM. They include com- parative measurements between a BLM and a platinum cup, electrostenolysis in BLMs, the measurement of redoc reaction in carotene or xanthophyll incorporated BLM. In the BLM-platinum cup comparative study, an oxidized cholesterol BLM containing a non-photoactive pigment was found to generate a large membrane potential of positive polarity in the presence of large ions (like I-), and a potential of negative polarity in the presence of protons (H+ion). A similar potential was observed when this BLM was replaced by an agar bridge. However, no potential was observed when this BLM was replaced by a platinum cup. Based on the above evidence it was suggested that the potential generated in the Ox-Cho-BLM was due to the diffusion of iodide ions crossing the membrane, which is similar to its diffusion across an agar bridge. The electronic conduction initiated by light in an oxidized cholesterol BLM or a platinum cup in the presence of photoactive pig— ments was investigated. The photoresponse was measured in detecting this light-induced electronic conduction across the Ox—Cho—BLM and a platinum cup. In a typical example, thionin dye was introduced into an Ox-Cho-BLM or a platinum cup. Light was absorbed by thionin, followed by the reduction of thionin and the oxidation of an electron donor, 109 such as TOB, KI. An agar bridge allowed no electronic conduction, so there was no photoresponse observed. The magnitude of photoresponses in the Ox-Cho-BLM or a platinum cut were found to depend upon the type of dye, the redox compounds and the external electric field. The most dramatic system was an Ox-Cho-BLM in the presence of eosin dye which contained K1 in one side and T¢B on the other side. An open-circuit photoemf of about 250mv was observed. In BLM electrostenolysis measurements, both spontaneous and non- spontaneous processes were investigated. In either process the electron must be driven across the membrane from one side of the biface to the other. A metallic mirror was then formed by the discharge of metallic ions on the biface. When the mirror was formed by a thermodynamic ally favoured redox reaction, such as Cu(N03)2/BLM/Na28, or cysteine, or K1, the process was a spontaneous electrostenolysis. In H PtCl6/BLM/H2Pt01 2 Hg2(NO3)2/BLM/Hg2(N03)2, or AgN03/BLM/AgN03, the electron was provided 69 via an external voltage source and was termed as a nonspontaneous electrostenolysis. Several factors affecting the degree of mirror brightness were studies such as the concentration of chemical reagents, the membrane resistance, the pH of the bathing solution, the charge carrier and the electric field. It was found that the metal deposition increased with an increasing chemical concentration. The metallic mirror was formed when the membrane conductance was high. In HZPtCl6/BLM/H2PtCl6, the mirror was especially difficult to observe when the membrane resistance was high (lanhm). The membrane conductance could become higher either by introducing highly concentrated chemical reagents or by adding charge carriers to the membrane. An example for the former case was Cu(NO3)2/BLM/Na23, where the bright mirror was formed when the Cu(NO3)2 llO concentration was high, up to lO-ZM. In an example of the latter case the same BLM was used, but, by incorporating carotene, the mirror could be formed even though Cu(N03)2 was diluted down to lO-aM. The brightness of the metallic mirror also depended on the pH of the bathing solution. In Cu(N03)2/BLM/Na28, the lower the pH of the bathing solution, the brighter the mirror became. The electric field, with positive polarity on the metal deposition side, assisted the mirror formation; but the electric field with negative polarity on the metal deposition side delayed the mirror formation. In the dark it was difficult for the redox reaction to take place» across oxidized cholestrol BLM due to its low electronic conductivity. However, the reaction was able to take place when carotene was incor- porated into the membrane. The membrane conductance was increased two orders in magnitude with the addition of carotene (3mg carotene dissolved in 2cc oxidized cholestrol solution). Figure 13 is a plot of the redox potential difference of two redox couples versus the observed Ox-Cho-BLM/ carotene potential. A straight line was obtained from the figure and the slope was approximately equal to one. This strongly indicated that Ox—Cho-BLM containing carotene functioned exactly as a redox electrode to detect the redox reaction between redox couples. In our additional experiment, the reference couple was replaced by other redox couple and it was found that the observed Ox-Cho-BLM/carotene potential was about the sum of individual membrane potentials in the presence of each redox couple to the opposite side of reference couple. A typical example was Ag+/Ag-M. Viologen couples. Here the redox potential difference calcu- lated from Table 5 was ~270mv and the observed Ox-Cho—BLM/carotene potential in the presence of them was also about ~270mv. 111 Figure 13 The linear relationship between the redox potential difference of redox couple with respect to reference couple and the Ox-Cho-BLM/carotenne membrane potential produced from redox reaction across the membrane. Fe3/Fe2+/(1O-3M) used as a reference couple, was added to one half cell containing NaAcpH.5. X+/X(lO-3M) was added to the other half cell. The active platinum electrode was immersed in this half cell. The redox potential difference across these two half cells was measured by the electrometer. The relative system with referred to each point in the plot is: 1. Reference couple (Fe3+/Fe2+)/Ag+/Ag(NO3) 2. " " /NO;/N0; 3. " " /Fe(CN)6-3/Fe(CN)6-4 4. ” " [2,6 DCPlP 5. " " /BQ/HQ 6. " " /M.blue 7. " " /Thionin 8. ascrobate/Ag+/Ag(NOS). 9. M. Viologen/Ag+/Ag(NOS). 10. Reference couple/Riboflavin ll. " " lM. Viologen 12. " " /Sn“/Sn2+ 113 The polarity and magnitude of the observed Chl-BLM dark potential in the presence of the reference couple and other redox couple indicated that Chl-BLM also contained small amounts of carotene in chloroplast extracts. This dark potential produced from the redox reaction was increased by increasing carotene content. In Fe3+/Fe2+-Ag+/Ag system, for instance the membrane potential reached the maximum value of about -l40mv in BLM where all chlorophylls were replaced by carotene. When the amount of carotene in the membrane was not sufficient to conduct the redox reaction completely, it was found that light could assist the completion of the redox reaction. For instance, in the case of Fe3+/Fe2+-Ag+/Ag, the membrane potential was only ~65mv when the membrane containted l=l ratio of chlorophyll and carotene. However, the potential could be increased to ~125mv when the membrane was illuminated by light. Furthermore, when an external field was applied to reduce this dark potential (VD=~65mv) to zero and then illuminated, the membrane potential still raised to ~125mv. The above evidence indicates that an Ox-Cho-BLM containing carotene functioned similarly to an inert redox electrode, and in the dark oxidation took place on one side of biface and reduction on the other side. The electron was moving across the membrane along the conjugated double bonds in carotene. Carotene not only assisted the redox reaction via electronic conduction in Ox-Cho—BLM in the dark, but in the light as well. No photoresponse was observed for a symmetrical Ox-Cho-BLM/carotene. However, a few photoemf could be observed in the presence of redox compounds. The photoresponse was 7 to lOmv with the presence of Fe3+/Fe2+and ascorbate. The photoresponse was enhanced by applying an external electric field in addition to the presence of the above redox compounds. The electron 114 conduction character of carotene in the membrane had two effects; speeding up the charges moving across the membrane and causing an increase in charge separations. These two effects could be measured in terms of the rate of photoresponse rise and the magnitude of photo- response. Figure 14 (a) demonstrates the time couse of the BLM photoresponse in the system KCl/FeCl3/BLM/chlorophyllin/KClpH=7.8. It was shown that the Ox-Cho-BLM incorporated with carotene (curve-a) needed only one-fourth of the total illumination time to reach the maximum photoresponse but the magnitude of photoresponse was double that of the carotene free Ox-Cho-BLM system (curve-b). Figure 14 (b) shows the time course of a BLM photoresponse for the system NaAc/FeClzl BLM/thionin/NaAc(pH.4). An Ox-Cho-BLM incorporated with carotene (curve-c) needed only one fourth of the total illumination time to reach the maximum photoresponse but it had doubled the magnitude of photoresponse when compared to the carotene free Ox-Cho-BLM system (curve—d). The electronic conduction mechanism of Ox-Cho-BLM may be extended so as to explain the electronic conduction of Chl-BLMS as well where charges (electrons and holes) produced by excited chlorophyll pigments in a membrane must travel along the conjugated double bonds, such as carotene or xanthophyll. Electrons are captured by an electron acceptor (like FeClB) on the biface and holes by an electron donor (may be H20) on the other biface. The protons produced from the oxidation of water, then would diffuse across the membrane to the other side. The time course photoresponse for this example given in Figure 15 (a) supported this postuate. The decay of photoresponse after it reached the maximum point was due to this proton diffusion. The details of how this proton was diffused across the membrane are still not clear. To most acceptable 115 Figure 14 Time course Ox-Cho-BLM photoresponse with and without carotent (a) is KCl/Fe3+(10-3M/1)/BLM/chlorophyllin/KC1(10-2M) pH 7.8 pH 7.8 (b) is NaAc/Fe2+(lO-3M/1)/BLM/Thionin(lO-4M/1)/NaAc(lO-1M) pH 4. Curve-a and curve—c are Ox-Cho-BLM photoresponse in the presence of carotene in the membrane and curve-b and curve-d are that of the 3+ 2+ absence of carotene. In both systems, Fe -chlorophyllin, and Fe - thionin, the Ox-Cho-BLM with carotene present have doubled in magnitude and been halved in time of illumination in reaching maximum photoresponse compared to the Ox-Cho-BLM without carotene present. 30 0" Ehv kmv) b 117 Figure 15 The time course of photoresponse in Chl-BLM/FeCl3 with and without TOB. (a) the decay follows after the reSponse reaching the maximum value, (b) the decay of the photoresponse completely disappears when T¢B has been incorporated in the chloroplast membrane or /Chl—BLM, T¢B/FeC13. 119 mechanisms were either that carrying protons by H20 which entered into the membrane or by breaking the balance of proton dissociation in lipid constituent in the membrane. Similar to the blocking of an electron from H O by NH 2 OH, this proton may also be blocked; this blocking could 2 result in no proton diffusion across the membrane. The chemicals, T¢B studied by many people, were found to be not only a self-difusable charge carrier across membrane (Liberman and Topaly, 1970), but blocked the dydrogen ion diffusion in light illuminated nigericin—treated chromatophore (Chance, 1968). Therefore in our studies, T¢B were added to the membrane and the mechanism of blocking proton diffusion was investigated. Figure 15 (b) shows the previous decay portion in photoresponse as having completely disappeared. This is evidence that proton diffusion was completely blocked. The charge carrier character of T¢B was evident from the magnitude of observed photoresponse. Here a double magnitude of photo- response was observed in the presence of T¢B as compared to that in the absence of TOB. 120 2 . THEORY OF BILAYER LIPID MEMBRANE PHOTO—RESPONSE a. General Consideration "A pigmented BLM is analogous to a photocell." The analogy of the two systems is that the photo—EMF can be a result of the electron concentration difference between two sides of the biface. The results obtained for the pigmented BLM can be explained by the model based on the Langmuir electron adsorption theory. A kinetic argument similar to the one given by Lange (Lange, 1938) has been described as follows: let Nout stand for the number of photoelectric active centers situated on the outer side of the biface, “Out expresses the number of active centers that photo-electrons have entered, and Nout-nout will be the number of empty active centers at the outer biface. Kinetically, the rate of photoelectrons entering the centers if given by a (N -n ) Ldt (1) o o o where L is the light intensity and a0 is a constant. The rate of emptying the active centers is described by the equation b n dt — (2) 00 at equilibrium, these rates must be equal; therefore a (N -n )Ldt=b n dt (3) o o o o o which can be written as Z (4) n =._g o C .2. 1+L where C = b /a . o o 121 As with photocells, the BLM has so great a light absorption that the intensity of illumination at the inner side of the biface is small. The concentration of free electrons on this side is then independent of the intensity of illumination and given solely by a constant m. If no¥m, there will be a difference in concentration of photoelectrons across the bifaces. This difference can give rise to the observed photo-emf. For the calculation of the EMF, the Nernst formula can be used for the diffusion potential. U -U n Eon” new“ Rifle. (5) h M In BLM, as in photocells, the mobility of the positive charges is small compared with the mobility of the electrons. Therefore, it follows that: E = 0.058 log no at T = 18°C op D1— =0.058 log EB. ~~~ (6) C (l + _2_)m L Equation (6) is therefore an expression relating the photo-emf and the intensity of illumination. At saturated light illumination, since C O L 1 + I2 1" therefore: E =0.058 log No --- — (7) 0p F 122 ie:, for a given photoactive BLM system under saturating light intensity, 3 maximum photo-emf will be obtained; it depends solely upon the ratio of photoactive centers across the bifaces. The BLM photoemf can also be derived from the redox reactions taking place across the biface. The reactions can be expressed as (Tien and Verma, 1970): Chl + hU + Chl*(excited) (8) dissociation - Chl* + Chl+ + e‘ A + e‘ + A- +)Ch1+ + D -> Chl + D+ Overall hu _ Reaction A,+ D + A + D+ The free energy change in terms of the activities of the reactants is O. 01 AG = AG° +RT 1n A"): (9) a (I A'D Since AG = nEF, divided by ~nF gives (1 0. AB = AE° - 3;; 1n GA“ - 13+ (10) nF . A ° 0‘1) Where AE is the measured cell emf and AE° is the standard redox potential difference between A/A' and D/D+ couples. Assuming the solutions studied are very dilute, it follows Henry's law that the activity can be replaced by the concentration. Equation (10) can be rewritten as: RT [A] [D+] AB = AE° ~.__ 1n (ll) nF [A'] [D] Since the BLM photoemf is the difference between its potential in light and the potential in dark (E = VL - VD), the BLM photoemf op can be expressed as 123 RT [A] [n+1 = (AE° - VD) - __ In (12) nF [A ] [D] The above equation indicates that Chl-BLM photo-emf is linear to the lagarithm of redox compound concentrations. In the case in which [A]/[A—] and [D+]/[D] are all unity, then Equation (12) can be written as: Eop = AE° - VD (13) Under the above circumstance, the Chl-BLM photoemf is linearly related to the standard redox potential difference between A/A‘ and D/D+ couples. The small deviation can result if the membrane dark potential is sifnificant. The Relation Between Chl-BLM Photoemf and Thermodynamic_guantities The relation between AG and AH is given by the Gibbs-Helmholtz Equation = ~AS = [ ] (14) Where AS is the variation of AG with temperature at constant pressure. The application of the Gibbs-Helmholtz Equation to the relation AG = ~nAEF allows us to calculate the AH and AS of the reaction from the temperature coefficient of the observed cell emf. Since the BLM photo~ emf is proportional to the cell emf in Equation (12), AH and AS of the reaction can be calculated from the observed BLM photoemf at various temperatures. Since the heat of reaction is the sum of hydration energy, ionization energy, electron affinity, etc., the direct association of Chl-BLM photoemf and hydration energy, ionization energy, electron affinity could be expected. 124 b. Comparison Between the Observed Chl-BLM Photoemf and the Redox Potential Difference of Redox Couples. Several redox couples are chosen for this measurement. The redox potential difference between these redox couples and Fe3+lFe2+ was measured by the method given in Chapter III procedure d, except for 2—OH-Napthaloquionone, ascorbate which were obtained from other authors. Table 24 indicates that most of the observed Chl-BLM photoemfs are closely related to the redox potential difference. However, the sign of Chl-BLM photoemf in many systems is opposite to that of rede potential difference. c. Factors That Affect Standard Redox Potential As Well As Chl-BLM Photoemf. Hydrogen ion concentration, insolubility of compounds and complex ion formation are considered to affect both the standard redox potential and Chl-BLM photoemf. Hydrogen ion concentratiog: The effect of hydrogen ion on the standard redox potential has been frequently discussed with reference to the MnOZ / Mn2+ system. Recently hydrogen ion also has been found to show great influence on the standard redox potential of organic compound. For example, in an organic redox BE 1n 2F reaction A? + A + 2c, the expression of redox potential is E = E°- .gAF . Where CA=’ CA are the concentrations of A? and A. However, A reactions such as ll..." AHZ AH“ + H" -------- (15) AH“ A: + 11+ (16) [Lo may occur after the redox reaction: A= Z A + 2e' where K1, K2 are the equilibrium constants for the above equations. Therefore, the total The comparison between Chl-BLM photoemf and standard redox potential 125 TABLE 24 difference of x+/x and Fe3+/Fe2+. Redox Couples in NaAc pH = 5 AE°(mv) Eop(mv) Remark outside inside Fey/FeZ't 0u2+/Cu1+ +60 +50”30 " Fe(CN)'g/Fe(CN)'g ~90 +70~90 opposite sign " CeZH/Ce3+ +30 ~23~ ~35 opposite sign it = = ~ . 8203/8406 ~95 +90 +98 opposite Sign " Sn4+/Sn2+ ~80 ~90 +80 ~+92 opposite Sign " cysteine/ ~90 +75 ~+90 opposite Sign cystine " Ascorbate +174(1) +165 ~+188 opposite sign " NOS/NOE ~80 +60.~+8O opposite sign " 2~OH~Napthaoquinone +130 (2) +140 (1). A E° = +174mv, this value is the subtraction of E° Fe3+Fe2+ from EoAsc- E° As/Asc+ (pH 4) = +166mv (after Burton, Ergeb Physiol (2). 43, 275 (1957). E° of Fe3+/Fe2+ (in acetate, pH 5) = +340 mv (after Clark, in oxidation-reduction potential of organic systems. Williams and Wilkans, Baltimore, Md. E0 2) lgapigg%pquionone (1960)). (at pH = 5) is 470 mv (after Clark, Chem Rev. 126 concentration of reduced species is not CA= but Cred which is the sum of CA=, CAM" and CAHZ' For convenience, CAH- and CAHZ can be expressed in terms of CA: by its relation with K1, K2 in Equations (15) and (16). Then, Cred can be simplified to be CA=(1+ [H+]2 + [H+] ). If one put CA= = Cred (1+ [H+]2 + [H "'l"1 into the E expression, E becomes 1(le K1 3T Bl Cred E = 13° + 2F 1n {1(le + [11+] + K2[H+]} - 2F lnczg— Eo-l 3:1: ~ 2F 1n KIKZ (17) Figure 16 gives a plot of E versus pH. Curve-3 gives a typical example of pH dependent E curve. The slope is 0.059 for pH<6 and becomes 0.029 at 610. Curve-1 is the curve of E for thionin/semithionin (Rabinowitch, 1940). The redox potential of thionin/semithionin decreases as pH increases. Its redox potential becomes +320my at pH 4. Since Fe3+/Fe2+ couple has the redox potential of about +340mv in acetate, the redox potential difference between these two couples is 20mv which is agreeable with the observed Chl-BLM photoemf of 19mv ~23mv in the presence of Fe3+/Fe2+ and thionin in buffered acetate (pH. 4). Insolubility of compound. In the case of Fe3+/Fe2+ system, if alkali is added to this system 3+ and Fe2+ will form Fe(on)3, Fe(OH)2 until [OH] = l g-ion/liter, Fe precipitates. The concentration of the two ions remaining dissolved in the alkaline medium, calculated from the solubility products, are very small and the redox potential becomes ~0.54 volt. This means the system has enormously increased its reducing power in such alkaline Figure 16. 127 The plot of redox potential of organic compound versus pH. curve ~1. curve -2. curve ~3. Thionin/Semithionin system (Rabinowitch, 1940) Methylene blue system (Clark, 1925) Theoretical curve of E-pH for organic redox compound. slope = 0.059 for pH<6 slope 0.029 for 610. 129 solution which will reduce Cu2+ to Cu metal or N03, N02 and NHZOH to NH3. Since BLM becomes very unstable at this high alkaline solution, the measurement becomes difficult. Complex ion formulation It has been found that some ligands (F’, H20) favour high oxidation state of metals in complex formation, whereas others (CN', R28) favour low oxidation state of metal in complex formation. However, as long as this complex forms, the redox ability of metal ion will change to some extent. Therefore, the value of redox potential should be changed from metal ion state at complex state. This standard redox potential of the complex can be obtained from the reaction kinetics. For a redox re- action type: (MLN)x+ + (x-y)e + (MLN)Y+ — (18) Where L is the ligand attached to metal ion (M) and N is the coordina- ting number. The redox reaction in Equation (18) can be expressed in a series of stepwise reactions, as: MX+ + (x— )e+ MY+ AG°= ~(x~ )E° F -- (19) y l y MX+/My+ K X+ MLN Mx+ + NL ( + ) (MLN)x+ AG§= ~RT anNX+ (20) K y+ MLN + M¥+ + NL ( + ) (MLN)y AG§= ~RT anNy+ — (21) +) overall X+ + reaction (MLN) + (x-y)e = (MLN)y AG° (22) . - X+ Where K(MLN)X+’ K(MLN)y+ are the overall Stablllty constant of (MLN) and (MLN)y+ complexes which are the measures of the stability of the complex in solution with reference to dissociation into metal ion and free radicals. Since the complex (MLN)x+ is formed step by step, its K value is then given by the product of these successive equilibrium 130 constants K = K1K2K3 ~-~~~~KN_1 KN. Since AG° is the sum of AGi, ~AG§ and A63, ~(x-y)E° X+ F = ~(x-y) E°x+ F+ RT 1n K _RT 1n K (MLN) /'+ (MLN)X+ (MLNir then at T=25°C E E ~ 0 059 (mom/(811,)” _ MX+/My+ ,'.-y (log K >---<23) (MLN)X+- log K(MLN>Y+ Equation (23) Shows that the standard redox potential of a metal ligand complex is not the same as the standard redox potential of metal ion. For the stable metal ion complex, the standard redox potential is less than that of a metal ion due to the contribution of the stability constant term in the above equation. In the case when one of the two couples presented in BLM forms the complex, then Equation (12) becomes: Eop+(E° - E° -vD)-§3_ 1n [MLNy+][D+] (24) (MLN)X+/(MLN)Y+ D/D+ (x-y) [MLNX+][D] Equation (24) indicates that Chl BLM photoemf is determined by the Standard redox potential of this metal complex which, in turn, is deter— mined by the Stability constant term. AG° value in Equation (22) depends on the strength of the metal— ligand bond, which includes how it donates electrons to metal ion in forming 6 bond, and how the overlap between ligand and metal ion orbital occurs in forming the H bond. The strength of this metal-ligand bond is usually expressed by the ligand field stabilization energy (AHL). In the case of an octahedral complex, this is the total energy difference between dB and dr orbitals in metal ion AHL = ~(0.4nE~ 0.6 nr)A —— (25) where n3, 111. are numbers of electrons occupying dB and dr orbitals. A is 131 the energy difference between dE and dr orbital of metal ion in ligand field. The value of this A is solely depended on the type of ligand attached. The effect of ligand on field strength has the order: I‘
Y+/Y(my) Ehv)obs (mv) 8n4+/Sn2+ Pe(CN)g'/Pe(CN)g' 10~ 20 20 25 8n4+/8n2+ Ce4+/Ce3+ -110~-103 ~90 - -100 cysteine/ 3_ 4 cystine Fe(CN)6/Fe(CN)6- 0~~5 my ~3 ~~5 133 2) Light intensity effect. Equation (6) expresses the dependence of the BLM photo-emf on the intensity of illumination for the whole light range. However, there are actually three sections that can be distinguished between the relation of BKM photo-emf and light intensity. Figure 17 is a schematic course of the BLM photo-emf dependent upon the intensity of illumination. Em ' (my) log (intensity of illumination) Figure 17. Ehv vs. log (intensity of illumination) In region I of small intensities of illumination, the ratio No/m in- creases only slowly and at very small intensities approaches the value 1. Therefore equation (6) can be written as: E =0.058lim No 1+C_o = 39 013 (1+ Co L L N -> o T O M + 0 = 0.058 1 CO = K.L L —— (30) where K is a constant. This suggests that in region I, the BLM photo— emf is merely proportional to the intensity of illumination. However, in region 11, at medium illumination, 134 C C 1+ 0 = ___ ._9 L L ) For this region, equation (6) becomes approximately: E = 0.058 log No 0? Co i:——1n = 0.058 log K'L — - (31) where K' is a constant. There appears a linear dependence of the photo~ emf on the logarithm of the illumination. For high light intensities, that is, in region III, it follows that: N N 1 N C limlfiW o = _2_1imi*n =._g .: 1+ _2 = 1 C m L C m L 1+ _9) m 1+ __9 L L Therefore, equation (6) becomes: E = 0.058 1 N - 7 op 08 E?- ( ) This is a similar expression to equation (7). That is, for a given photo~ active BLM system under saturating intensity of illumination, a maximum photo-emf will depend solely upon the ratio of photoactive centers across the biface, but is independent of the intensity of illumination. Figure 18 and Figure 19 are plots of BLM observed photo-emf versus light inten- sity in the presence of FeC13 and ascorbate. Medium light obtained by switching to the middle position on a Keystone movie projector was used in Figure 18 and the forward position on the Keystone movie projector (intense light) was used in Figure 19. It appears that ChliBLM photo~ emf is linearly increased with the intensity of illumination in the range of 0 to 100% middle position light intensity (indicated in Figure 18). A linear dependence of the ChliBLM photo-emf on the logarithm of the intensity of illumination was found in the range of 3 to 50% 135 Figure 18. Light intensity dependent Chl-BLM photo-emf response. This dim light source is obtained from a Keystone movie projector or which the switch is set at the middle position. The intensity is controlled by setting various degree gray filters between the light source and the membrane. It is found that Chl-BLM photo-emf increases linearly as the light intensity increases within the whole light intensity range. FeCl3(1O-3Mle) and ascorbate (lo-3M1e) were added to the aqueous solutions (NaAc pH. 5). I % (hanmylight) 137 Figure 19. The light intensity dependent Chl-BLM photo-emf response. The light source is provided from a Keystone movie projector with the switch at forward position. In region 11, the Chl-BLM photo-response is the exparential of light intensity. The plot of Chl-BLM photo-emf versus the logarithm of light intensity indicates the linearity between the Chl-BLM photo-emf and the logarithm of light intensity. In region 111, the Chl-BLM photo-response seems to level off at the saturated light point. Further increases in the light intensity did not affect the photo-response. FeCl3(lO-3Mle) and ascorbate (10'3M1e) were added to the aqueous solutions (NaAc pH.5). 109(1 % X102) l I I J [ I % (FonNard full light as I.) 139 forward position light intensity (indicated in Figure 19) region 11). The ChliBLM photo-emf seems to level off after the intensity of illumination has greater than 50% forward position light intensity. As has been shown in Figure 19, the curve in the plot of ChliBLM photo-emf versus logarithm of light intensity deviates from the straight line when the intensity has greater than 50% forward position light intensity. Region III in Figure 19 will be the area where the ChliBLM photo-emf is no longer dependent upon the light intensity, but on the conditions of redox compounds in the aqueous solution. 3) The evaluation of relative redox potential of redox couple by the ChliBLM photo-emf measurement. From equation (12), the expression of ChliBLM photo-emf is shown as: = 0+ - - RT (D) (A-) Ehv ( AE"x IX VD) 3p 1“ —-—-——(D+) (A) If (Af)/(A) is (Fe3+)/(Fe2+) and has the same ratio, then equation (22) can be simplified as; E = (AEO -V ) - 5?, (D) (32) hv X+/X D nF ln'TD;) The relative redox potential of couple X+/X can be evaluated from the plot of Ehv versus logarithm of (D)/(D+). Here ( Eg+/x. VD) term is the value of Chl-BLM photo-emf at a logarithm (D)/(D+) Of zero. Figure 20 gives such an example as Chl-BLM photo-emf versus the logarithm of (Fe(CN)-2/(Fe(CN)-2). The Chl-BLM photo-emf is linear to the logarithm of (Fe(CN)'2)/(Fe(CN)'g). The experimental value of Chl-BLM photo-emf is 75 mv when log (Fe(CN)-6)/(Fe(CN)-g) is zero. Since the membrane dark potential for most of Fey-[Fe2+ ~ Fe(CN)-2 measurements is around 15 my, the observed relative redox potential of Fe(CN)-g/Fe(CN)-g is about 90 my. This is about the redox potential value of Fe(CN)_g/Fe(CN) -2 obtained from the redox potential measurement. 140 Figure 20. The Chl-BLM photo-emf versus logarithm of [Fe(CN)-g]/[Fe(CN)'g]. According to the expression; Ehu= (AEO KW -vD> - 33 in ID] [A'] nF [D ' l [A] the value of (AE§+/x ~ VD) term is equal to Ehv when log [3]]Efig] = O. 142 3. The Energy Conversion and Non—energy Conversion Processes in Bilayeral Lipid Membrane a. Definition The observation of photoeffects in pigmented BLM affords an opportunity to Speculate on possible mechanisms of energy conversion as well as non-energy conversion. Figure 21 gives a scheme in repre- senting the energy relationships of electron donor and acceptor level of pigment as well as that of electron accepting and donating compounds. The higher the level on the vertical scale, the higher is the energy potential. The energy levels of the electron acceptor and donor must lie below the excited State and above the ground state of the pigment molecule, respectively. The energy of the absorbed photon is stored, therefore, in terms of the redox products. That is, after absorbing photon, the electron of the pigment in ground state will transit to a higher energy level. This electron finds itself in a higher energy level than the acceptor. At the same time, the donor becomes higher in the energy level than the vacancy state of the pigment molecule. As a result, the electron is able to transit from donor to acceptor and the pigment is back to its original state. In this case, the driving force of the reaction is the energy of the absorbed light quantum which raises the electron of the pigment from its ground State to a higher energy level. AS a result of this, the complete electron transition becomes possible. This type of process is named as "the energy conversion process". In the second case, shown in Figure 21 (b), a thermodynamically favoured reaction is able to proceed as a result of the lowering of activation energy. This type of a N process is named as the non-energy conversion process." 143 Figure 21 Mechanisms of light-elicited phenomena in pigmented BLM. (Tien and Sheih, 1974). The BLM is considered as an ultrathin layer of liquid cristalline phase separating two highly conducting aqueous solutions. (A) Energy conversion via energy transducing membrane where light is the driving force. The energy of the absorbed photon is stored in terms of redox products. (B) Photosensitized redox reactions where light mainly lowers the activation energy for thermodynamically favoured reactions. Aq Aqueous Aque _ ous sum solution I B SOIUtIOfl _..__""3“ $2 fié?‘ ACGGPtOi I I’ I FL) . I I loom, ET I Ii: I l I [,1 ‘ ’ I I : ’/"\e I I . v, Donor w’ h (""- __.... vw) _@__ Mr» -81- —70 .-I ”70A.- (A) (8) 145 Several redox couples have been employed in the measurement of the process. For convenience the redox couple in one side of BLM was fixed with Fe3+/Fe2+(lO-3M/l). The energy level of redox couple in the opposite side with reference to Fe3+/Fe2+ was determined by the redox potential difference measurement. ChlBLM photoemf technique was employed in the determination of the direction of light-induced electron flow. So far, the redox couple, such as Fe(CN)-2/Fe(CN)-2, 8203/8406’ Sn4+/Sn2+, cystine/cysteine, NOS/NOE, 2~OH~Napthaloquinone all have their energy levels lower than Fe3+/Fe2+. However, under the assistance of chlorophyll pigment in membrane and light quantum, the electron is able to be Transited from the low energy level to high energy level of Fe3+/Fe2+. The energy has been stored in terms of photoproducts in the aqueous solutions. They are energy conversion processes. The redox couples, like ascorbate, Cu2+/Cu1+ have their energy levels higher than Fe3+/Fez- (in acetate), and FeF6, Fe(NO3)6-3/Fe(N03)6-4 have their energy levels lower than Fe3+/Fe2+ (in acetate), light and chlorophyll are provided to assist the electron transition from high energy level to low energy level. This is a spontaneous process, since the system can proceed in the absence of external energy source, such as light. Light is provided to proceed the process much more efficiently. This kind of process is a non—energy conversion. Some other redox couples, like Ce4+/Ce3+, thionin/semithionin, have their energy levels higher than Fe3+/Fe2+ (in acetate, but the electron can be transited from the latter to the former under the assistance of light and chlorophyl pigment. Therefore, they are also classified as the energy conversion processes. b. Further research in using bilayer lipid membrane in looking into the process of energy conversion in photosynthesis. 146 The data obtained on the structure of chlorOplast lamellae with the use of electron microscopy have suggested that chlorOphyll is located in the lamellae in the form of a very thin bimolecular layer (Rabinowitch, 1951; 1959; Wolken, 1959; Giraud, 1962). It can be perceived that all or part of the chlorophyll molecules in these layers are arranged against each other, in pairs, at a short distance; that is, one of the molecules may be directly or indirectly linked with the enzyme of the cytochrome type capable of giving up an electron to chlorophyll, while the other molecule may be linked to an electron acceptor of the ferredoxin type in the chain of electron transfer to phosphopyridine nucleotides or in the phosphorylation chain. The former molecule is under conditions favourable to its photoreduction, while the latter is under those favouring photooxidation. The possibility that chlorophyll can participate in both reactions at different states has been examined. There exists a suggestion that in one of these reactions, chlorophyll undergoes photochemical oxidation while being reduced in the other (French and Fork, 1962; Duysens, 1962; Goedheer, 1962; Rabinowitch, 1962). Evstigneev suggested that, at a simultaneous absorption of light by chlorophyll molecules, their electrons pass from the excited state of the first chlorophyll molecule to the electron vacancy at the ground level of the second molecule. This direction of electron transfer can be due to the fact that electron transfer from the excited level of the second chlorophyll molecule to the ground level of the first chlorophyll molecule may be prevented under the action of electron donor properties of the enzyme bound with the first chlorophyll molecule, and under acceptor properties of the enzyme bound with the second chlorophyll molecule. As a result of this, a pair of chlorophyll molecules is formed with the 147 properties of ion-radicals, one of which is oxidized, while the second is reduced. The reaction of the oxidized radical with cytochrome, and that of the reduced radical with ferrodoxine, cause the chlorophyll pair to pass to the initial state, while the energy rich electron enters the chains of reduction reaction of photosynthesis. Therefore, from the energy point of view, the whole photosynthesis in green plant consists of two giant energy conversion processes (one in photosystem I and the other in photosystem 2) connected by electron transport chain. Since bilayer lipid membrane has been found to perform either energy conversion or non-energy conversion process, one would be able to use this type of bilayer lipid membrane in looking for the details of two giant energy conversion processes occuring in photosynthesis. Along this direction, some preliminary studies, in terms of model studies of electron transport in photosynthesis, have been done in this thesis. However, there still needs further research in order to have a complete picture of the detail mechanism in photosynthesis. CHAPTER VI SUMMARY A. The BLM is capable of facilitating electron transfer in inducing the redox reaction. Therefore, from the functional point of view, it behaves very similarly to a metallic surface or an inert redox electrode. Three experiments were carried out to study the elec— tronic conducting characteristics of pibmented BLM: including comparative measurements of the potential of BLM and platinum cup; the electro- stenolysis in BLM; and the measurement Of redox reaction in carotene incorporated BLM in the dark. 1. The photoresponse was measured to detect light induced electronic conduction across oxidized cholestrol BLM and a platinum cup. The photo- response in both BLM and platinum cup was found to depend upon the nature of pigment, the redox compound, and the electric field (Table 3, 4, p.41.. 42). 2. In BLM electrostenolysis measurement, both spontaneous and nonspontaneous processes were studied. The results indicated that the electron was driven across the membrane from one side to the other side of biface and a metallic mirror was formed from the discharge of the metallic ion on the BLM biface. The brightness of this metallic mirror depended upon the concentration of chemical, the membrane resistance, the 148 149 Charge carriers, the pH of the bathing solution and the electric field. 3. The potentials of various redox couples were determined across oxidized cholesterol BLM with and without carotene. Because of the high dark resistivity of oxidized cholesterol BLM, the measured potential across the membrane did not correlate with the experimental redox potential. When carotene was incorporated into the membrane, however, a linear relationship between the redox potential difference of two redox couples and the observed membrane potential was found (Figure 13, p.112). This implied that carotene—incorporated BLM functioned just like an inert redox electrode, capable of accepting electrons on one side of biface and releasing electrons on the other side. The evidence of this electron conduction in carotene-incorporated BLM strongly supported the hypothesis that light-induced electrons and holes must travel along the conjugated double bonds of carotene in a chloroplast membrane. The reasons were: (1) carotenes were abundantly present in chloroplast extracts, (2) chloro- phyll and carotene-incorporated BLM has its photoresponse increased as fast as that of chloroplast BLM. The rate of photoresponse rising was ' four times slower in BLM without carotene (Figure 14, p.116). B. The efficiency of light energy utilization in pigmented BLM, in terms of the photoresponse, was enhanced by four conditions: (1) the asymmetric location of pigment; (2) the use of pigments with strong electron-accepting tendancy (Diagram-A, p.89); (3) the introduction of charge carriers; and (4) the application of an electric field. The most dramatic system was T¢B/BLM/thionin,KI, where an open-circuit photoemf from 245 to 260mv was observed. 150 C. Two types of light-induced electron flows were observed in a chloroplast BLM. In the energy conversion process, the electron is driven against the redox potential difference across the membrane. But in the non-energy conversion process, the electron moves in the direction of the redox potential difference (Figure.21, Eh 141). Chlorophyll pigment in the membrane played a very important role in both the energy conversion process and the non-energy conversion process. D. 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