MOLECULAR TOPOGRAPHY AND FUNCTION OF PERIDININ IN THE
PERIDININ-CHLOROPHYLL a-PROTEIN COMPLEX
by
SIVARAMAKRISHNA PRASAD KOKA, B.S., M.A.
A DISSERTATION
IN
CHEMISTRY
Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Approved _
Accepted
December, 1977 Ho, 7^
ACKNOWLEDGMENTS
I am extremely grateful and deeply indebted to my professor. Dr. Pill-Soon Song, for his constant guidance, patience and encouragement throughout my studies for the
Ph.D. degree. I am thankful to my fellow graduate stu dents for their help and cooperation. The efficient typing work of Mrs. Evelyn Gaffga is well acknowledged.
I continue to be grateful to my former professors,
Dr. Robert R. Kuntz (University of Missouri) and Dr. Richard
D. Doepker (University of Miami), without whose encourage ment I would not be where I am now.
Last but not the least, my indebtedness persists forever to my parents and family, without whose help I would not have been able to pursue higher studies.
XI TABLE OF CONTENTS
ACKNOWLEDGrffiNTS ii
LIST OF TABLES vi
LIST OF ILLUSTRATIONS vii
I. INTRODUCTION AND STATEI/IENT OF PROBLEM. ... 1
Introduction 1
Statement of Problem 2
II. LITERATURE REVIEW 3
Carotenoid-Chlorophyll Complexes . 3
Energy Transfer from Carotenoid
to Chlorophyll 9
Exciton Interactions 12
Role of Carotenoid As A Photoreceptor 13 Singlet Oxygen-Induced Photo- oxidation and Its Inhibition ... 14 Singlet-Triplet Transitions of Photosensitizer Dyes 21
III. MATERIALS AND METHODS 22
Materials 22
Methods 24
Theoretical Section 24
Critical Distance Calculation. . . 24
iii IV Intermolecular Distance Cal culation 25 Polarization by Photoselection. . 26 Experimental Section 28 Corrected Emission and Excita tion Measurements 28 Fluorescence Quenching 34 High Resolution Emission and Excitation Measurements 34 Low Temperature Measurements. . . 37 Circular Dichroism 38 Absorption Spectra 39 Fluorescence Lifetimes 39 Temperature Dependence of PCP and Chlorophyll a Fluorescence. . 40 Dynamic Depolarization 40 Photo-irradiation 44 Actinometry 45 IV. RESULTS 50 PCP (Glenodinium sp.) - Absorption and Polarization ... 50 PCP (Glenodinium sp.) - Fluor- escence Excitation 50 PCP (Glenodinium sp.) - Circular Dichroism 51 PCP (Glenodinium sp.) - Fluorescence Emission 60 PCP (G. polyedra) - Absorp tion and Polarization 71 PCP (G. polyedra) - Fluores cence Excitation 71 PCP (G. polyedra) - Circular Dichroism 78 PCP (A. rhyncocephaleum) - Absorption and Polarization. ... 78 PCP (A. rhyncocephaleum) - Fluorescence Excitation 79 PCP (A. rhyncocephaleum) - Circular Dichroism 84 PCP (A. carterae) - ..Absorp tion, Polarization and Circular Dichroism 84 PCP (A. carterae) - Fluor escence Excitation 89 PCP (A. carterae) - Fluor escence Emission and .Fluor escence Lifetimes 89 Dynamic Depolarization 98 Fluorescence Quenching 98 "Reconstitution" of PCP 105 Temperature Dependence of PCP Fluorescence 110 Photobleaching of Chlorophyll a^ . 113 Rose Bengal Sensitization and Singlet Oxygen Quenching 122 V. DISCUSSION 157 VI. CONCLUSIONS 185 LIST OF REFERENCES 189 LIST OF TABLES
Table Page 1. Intensity of the Bausch & Lomb monochromator Xenon lamp at 557 nm 47 2. The fluorescence maxima and lifetimes of PCPs. 96 3. The calculated values of In ^j~V.^^ with re ciprocal of absolute temperature^'^for PCP (Glenodinium sp.) 118 4. Photobleaching of chlorophyll a in solution and in PCP complex 123 5. Photobleaching of PCP in D20 125 6. Quantum yields of photooxidation of chloro phyll a in the absence of singlet oxygen quencher 129 7. Quantum yields of photooxidation of chloro phyll a in the presence of B-carotene. . . . 133 8. Quantum yields of photooxidation of chloro phyll a in the presence of benzoquinone (in ethanol-benzene) 137 9. Quantum yields of photooxidation of chloro phyll a in the presence of menadione 141 10. Quantum yields of photooxidation of chloro phyll a_ in the presence of a-tocopherol. . . 145 11. Quantiom yields of photooxidation of chloro phyll a^ in the presence of benzoquinone (in ethanol) 149 12. Quantum yields of photooxidation of chloro phyll a in the presence of DABCO 153 13. Photooxidation of chlorophyll a-singlet oxygen quenching rate constants. • 179
VI LIST OF ILLUSTRATIONS
Figure Page
1. Structure of peridinin 4
2. Isolation and purification of PCP complexes 7
3. The state of oxygen molecule and their electronic configurations 16
4. Optical layout of the Perkin-Elmer MPF-3 spectrophotometer. .... 30
5. Block diagram of Perkin-Elmer spectro photometer with Hitachi corrected spectra accessory 32
6. Block diagram with optical layout for high resolution spectrometer 35 7. Block diagram of the Phase-Modulation cross-correlation spectrofluorometer. ... 41
8. Absorption spectra of native and de natured PCP from Glenodinium sp 52
9. Fluorescence excitation polarization and absorption spectra of PCP (Glenodinium sp.) 54 10. Corrected fluorescence excitation spec trum of PCP (Glenodinium sp.) at room temperature recorded on Perkin-Elmer spectrof luorometer 56
11. Fluorescence excitation spectra of de natured and native PCP (Glenodinium sp.) recorded on high-resolution spectrofluor ometer 58
Vll Vlll Figure Page
12. Absorption and fluorescence excitation polarization spectra of chlorophyll a in ethanol 61
13. Circular dichroism spectrum of PCP (Glenodinium sp.) 63
14. Circular dichroism spectra of de natured PCP (Glenodinium sp.) and chloro phyll a 65
15. Polarized fluorescence excitation and absorption spectra of denatured PCP (Glenodinium sp.) 67
16. Fluorescence emission spectrum of PCP (Glenodinium sp .) 69
17. Absorption and polarized fluorescence ex citation spectra of PCP (G. polyedra) ... 72
18. Absorption and polarized fluorescence ex citation spectra of PCP (A. rhyncocephaleum) 74
19. Corrected fluorescence excitation spectra of (a) PCP from G. polyedra and (b) A. rhyncocephaleum. 76
20. Fluorescence excitation spectra of PCPs from Glenodinium sp., G. polyedra and A. rhyncocephaleum 80
21. Circular dichroism spectra of PCP from (a) G. polyedra and (b) A. rhyncocephaleum 82 22. Absorption spectra of PCPs from A. carterae and G. polyedra and fluorescence excitation polaFization spectrum of PCP from A. carterae 85 23. Absorption spectra of peridinin in ethanol at room temperature and 77K 87 IX Figure Page
24. Circular dichroism spectra of PCP from (a) A. carterae and (b) A. rhyncocephaleum. 90 25. Fluorescence excitation spectra of PCPs from A. carterae, A. rhyncocephaleum and G. polyedra 92
26. Fluorescence emission spectra of PCPs from A. carterae, A. rhyncocephaleum and Glenodinium sp 94
27. Fluorescence spectra of chlorophyll a_ as a function of potassium, iodide 99 28. Stern-Volmer plot of fluorescence quench ing of chlorophyll a—fluorescence inten sities vs. KI concentration 101
29. Stern-Volmer plot of fluorescence quench ing of chlorophyll a—fluorescence lifetimes vs. KI concentration 103
30. Absorption spectra of PCP as a function of acetone concentration 106
31. Effects of acetone on the CD spectrum of PCP 108 32. CD spectrum of PCP after "reconstitution" from 30 per cent acetone Ill 33. Fluorescence emission of PCP as a function of temperature 114
34. Plot of In A{1/TV against reciprocal temperature for PCP 116
35. CD spectra of PCP (Glenodinium sp.) as a function of temperature 120
36. Stern-Volmer plot of photooxidation of chloro phyll a in the absence of singlet oxygen quencher 131 X Figure Page
37. Stern-Volmer plot of inhibition o-f chlorophyll a^ photooxidation by B~ carotene 135
38. Stern-Volmer plot of inhibition of chlorophyll a photooxidation by benzoquinone (in ethanol-benzene) 139
39. Stern-Volmer plot of inhibition of chlorophyll a photooxidation by menadione . . 143
40. Stern-Volmer plot of inhibition of chlorophyll a^ photooxidation by a- tocopherol 147 41. Stern-Volmer plot of inhibitions of chlorophyll a photooxidation by benzoquine (in ethanol) 151
42. Stern-Volmer plot of inhibition of chlorophyll a photooxidation by DABCO .... 155
43. Absorption and fluorescence (hypothetical) spectra of peridinin in ethanol 161
44. Absorption spectrum of chlorophyll a nor malized with respect to the peridinin fluorescence spectrum 163
45. Probable molecular arrangement of PCP complex 167 46. Corrected fluorescence spectrum of PCP (Glenodinium sp.) protein 172 CHAPTER I
INTRODUCTION AND STATEMENT OF PROBLEM
Introduction
The isolation of tiie photosynthetic light-harvesting pigment complex, peridinin-chlorophyll a-protein (PCP) from marine dinoflagellate algae (1-5) opened the doors to under standing how the light-harvesting carotenoid, peridinin, transfers its energy to chlorophyll a in the complex. Up to now such an understanding has not been possible due to the non-fluorescent nature of the carotenoids and their extremely short excited state lifetimes (6). Carotenoids absorb light in the wavelength region where chlorophyll does not absorb, thus efficiently utilizing the light in the entire visible range for photosynthesis by the energy transfer. However in the absence of protein no energy is transferred from caro tenoid to chlorophyll such as in solution (6), and the PCP complex, unlike other carotenoid-chlorophyll-protein com plexes, containing a highly water-soluble protein, provides an excellent model to study the energy transfer mechanism.
The carotenoid, in addition to its role as light- harvesting pigment is thought to be the protector of chloro phyll a from singlet oxygen induced damage. The low-lying
1 triplet level of carotenoid quenches the singlet oxygen and prevents oxidation of chlorophyll a. An examination of the PCP complex is necessary to understand how the carotenoids act as donors.
Statement of Problem This dissertation deals with the determination of the molecular topography of the PCP complex that is required for the energy transfer from peridinin to chlorophyll a, the mechanism by which peridinin acts as donor and the role of peridinin as chlorophyll a protector from singlet oxygen in duced damage. The behavior of chlorophyll a_ in the PCP com plex is compared with its behavior in solution in the absence of apo-protein. The study of the role of carotenoid as chlorophyll a protector is ideally suited with PCP due to the presence of peridinin itself being bound to the apo-protein along with chlorophyll; thus, the PCP complex is a system that occurs just as in vivo in the light-harvesting algae in nature. There is no need to add the carotenoid as in in vitro studies to chlorophyll to study the chlorophyll protecting function of the carotenoid. CHAPTER II
LITERATURE REVIEW
Carotenoid-Chlorophyll Complexes The water-soluble PCP complexes have been isolated (1-5) from the marine dinoflagellates, Cachonina niei, Gleno dinium sp., Gonyaulax polyedra, Amphidinium rhynocephaleum and Amphidinium carterae (Plymouth 450). The total molecular weights of the PCPs range from 34,500 for Cachonina niei to 39,200 for A. carterae. The protein moiety of PCP (Gleno- diniimi sp.) has a molecular weight of 35,500 consisting of two polypeptides each with a molecular weight of about 15,500 while the PCP from G. polyedra is composed of single poly peptide of molecular weight, 34,500. The chromophore mole cular weight is 3,413 consisting of four peridinin and one chlorophyll a molecules bound to the apo-protein by non- covalent linkages. However, the chromoprotein from A. carterae is deterinined to have a molecular weight of 39,200 and is comprised of an alanine-rich apo-protein of molecular weight 31,800 associated with nine peridinin and two chlorophyll a molecules bound noncovalently (5). The brick red color of the PCP is due to the high concentration of peridinin. The 3 Fig. 1, Structure of Peridinin. o protein subunits of Glenodinium sp. have isoelectric points of pi 7.4 and 7.3. The PCP from G. polyedra has a pl value of 7.2 and the PCP from A. carterae has a pi of 7.5 (1-5). Fluorescence excitation spectra of purified PCP indicated efficient energy transfer from peridinin to chlorophyll a (3), lending support to the reported role of peridinin as an accessory pigment in photosynthetic oxygen evolution. The chromophores dissociated from the protein on treatment with sodium dodecyl sulfate (SDS).
Algae were grown in a sea-water medium enriched with nitrate, phosphate, soil extract, vitamins and minerals (7) and harvested by continuous flow centrifugation (8). The harvested cells were purified as described (1, 3, 5) in Figure 2. Column chromatography was used to separate protein samples for molecular weight determination (3) and the two major chromoproteins isolated and purified from Glenodinium sp.,were analyzed for amino acid composition. Pigments were extracted after the methods of Jeffrey (9) employing thin layer chromotography (TLC). Chlorophyll a_ concentrations were determined from absorption spectra of the acetone and ether extracts using the equations as de scribed (3). Peridinin was estimated by two-dimensional TLC (3) using the molar extinction coefficient of 8.44 x 10^ M"-*- cm"-'- at 466 nm (9) . Fig. 2. Isolation and purification of PCP complexes. Ciilture
growth (sea-water medium)
harvesting by continuous flow vy centrifugation
Harvested cells (frozen under nitrogen in the cold)
resuspension
\/ sonication
Crude Preparation
concentration by vacuum dialysis
recentrifugation Sephadex G 100 V 4°C
Chromoprotein
dialysis
Sephadex chromatography
\/ ion exchange
Purified PCP (Purity monitored by polyacrylamide gel electrophoresis, isoelectric focusing) The PCP from A. carterae was purified and character ized by a somewhat different procedure (5). At the present time, it is not clear whether the stoichiometry of all the
PCP complexes isolated from several dinoflagellates is the same.
Peridinin has a melting point of 107-109° C, is an orange-red pigment, readily separable from other carotenoids by chromatography. The str\icture of peridinin was established by Strain and others (10) .
Energy Transfer from Carotenoid to Chlorophyll
Energy transfer with efficiency of the order of 40-50 per cent, from carotenoids to chlorophyll a was detected in black lipid membranes (BLM), but only 10 per cent in pigment solutions, when the mean distance between pigment molecules was
23 8 in both systems (11). The fluorescence quantum yield of chlorophyll a^ in such solutions was only 2 per cent of that found in BLM. The large enhancement of energy transfer ef ficiency in BLM is due to the suppression of the collisional quenching of the excited singlet states by ground state mole cules. Deactivation of excited states by internal conversion may also be suppressed in BLM since this process is affected by rotational freedom of motion. According to present views, the molecular arrangement which results in efficient energy transfer in BLM may also be present in thylakoid membranes. 10 Energy migration was also detected in monolayers and thin films containing chlorophyll a and carotenoid ag gregates (12). Measurements of absorption, fluorescence and excitation spectra of monolayers and films of mixtures of chlorophyll a with carotenoids were made at different con centration ratios and densities. 3-Carotene does not affect the aggregation of chlorophyll a when the latter is in ex cess. Chlorophyll a. promotes the aggregation of 3-carotene molecules, but inhibits the formation of larger crystalline aggregates absent in mixed layers. The amount of 'aggregated' chlorophyll a drops considerably with an increase in the amount of 3-carotene. "Solubilization' of chlorophyll a_ by 3-carotene (if the latter is in excess) leads to an increase in the concentration of chlorophyll a^ in such mixed films. The properties of films containing fucoxanthin, a pigment differing from 3-carotene by the presence of polar groups and those of films containing zeaxanthin, are the same' as those of chlorophyll films with 3-carotene. Comparison of excitation and absorption spectra suggested that all chloro phyll a and 3-carotene forms are involved in excitation. The maximum energy transfer efficiency (about 50 per cent)
is attained when chlorophyll a is in excess (density = 0.30 2 nm /molecule), that practically all 3-carotene donors are surrounded by chlorophyll a acceptors. If the concentration of 3-carotene is increased until a ratio of 1:1 is attained, 11 the efficiency of energy transfer dropped to about 25 per
cent indicating that the efficiency of energy transfer be
tween carotene molecules preceding transfer to chlorophyll
a. is low. The energy transfer between different chlorophyll a forms was also studied (12). With increasing density of
the monolayer, an increasing number of different aggregates appears, and the efficiency of energy transfer from chloro phyll 669-72 nm to these forms reaches 100 per cent. The most efficient acceptors are most likely semicrystalline forms, with absorption peaks at 720-730 nm appearing in con siderable amounts only in films.
Song and Moore (6) attributed the bleaching of the main absorption band and appearance of a new band of exciton interactions between two stacked lycopene molecules in ethanol- water mixture. However, 3-carotene does not show much anam- alous bleaching under identical conditions. They observed no fluorescence from 3-carotene and lycopene at 77K using a high resolution spectrofluorometer equipped with a photon counter (13). The mean lifetime of the B state of 3-carotene — 1 4 — 5 was calculated to be 10 sec at a quantum yield at 10 .
Several conclusions were made some of which are stated as follows. The demonstrated matching of the action spectrum for phototropism and phototaxis with the absorption spectrum of a carotenoid by adding water to the ethanolic solution does not provide the necessary support for a carotenoid as 12
a photoreceptor for phototropism and phototaxis. A direct excitation of carotenoids _iri vitro does not populate the triplet state and it is unlikely that the intersystem cross ing i^ vivo is drastically enhanced. Due to lack of radi ative emission from the carotenoid polyenes involved in photo synthetic light-harvesting, it is unlikely that the mechanism of energy transfer to chlorophylls is via Forster dipole- dipole coupling. Due to the short excited singlet state life time, carotenoids are kinetically unlikely candidates for primary photoreceptors.
Exciton Interactions
An exciton state is defined as the collective excita tion of an assembly of molecules, in contrast to the local ized excitation of each individual member of the assembly.
The like molecules are resonantly coupled to one another so strongly that it is not possible to excite one molecule in dividually.
Kasha et^ aJ^. (14, 15) proposed the molecular exciton model for molecular aggregates and their relevance to excita tion energy transfer. They dealt in detail with different types of intermolecular electronic interactions; mathematical
(15) and non-mathematical (14) treatments are provided. Cri teria are proposed for strong-coupling and weak-coupling exciton cases (14). Possible exciton band structures and selection rules are indicated for strong coupling case. 13
Diagrams for exciton band structure in molecular dimers, linear molecular polymers and helical molecular polymers with various geometrical arrangements of transition dipoles are illustrated. In strong-coupling exciton cases, pro nounced spectral effects of molecular aggregates compared with individual molecules are predicted, with very fast resonance transfer rates (^lol^ sec~l) depending on the in verse ciibe of intermolecular distance. In weak-coupling ex citon cases, no significant spectral changes are predicted, but the transfer rates are still high ('^^10 to 10 sec ) and preserve the inverse-cube inteirmolecular distance depen dence .
Exciton interactions in bacteriochlorophyll-protein complexes, each subunit containing as many as five bacterio- chlorophyll a. molecules, were observed in absorption and circular dichroic (CD) spectra (16). Bacteriochlorophyll- reaction center complex absorption and CD spectra also could be resolved into several components due to exciton splitting (17). CD has proved to be a useful tool to detect interac tions between molecules that are closely spaced and in re solving these components.
Role of Carotenoid As A Photoreceptor Controversy continues to exist among the photobiolo- gists as to whether carotenoid or flavin is the photoreceptor for phototropism and phototaxis in plants. Evidence available 14
so far is more in favor of flavin than carotenoid for being the photoreceptor as reviewed by Song (18). It is concluded that carotenoid more likely plays the role of a secondary photoreceptor.
Singlet Oxygen-Induced Photooxidation and Its Inhibition
Many types of photosensitized oxidation which may be important in biological systems are available. Most of the photosensitized oxidations proceed by way of triplet sensitizer. Oxygen has two metastable singlets, with spectroscopic sym metry notations Z (37 kilocalories) and A (22 kilocalories), see Figure 3. The •'-A state is long-lived and, survives more collisions than Z^ under the same conditions (19). In the presence of suitable acceptors, oxygen and sensitizers, very efficient reactions may occur. Several dyes as well as chloro phyll are excellent sensitizers for all types of photooxida tion. In the presence of light these sensitizers provide singlet oxygen, A . Photosynthetic organisms are apparently y protected by carotenoids against the lethal effects of their own chlorophyll (prevention of "photodynamic action"). Mutants lacking carotenoids are rapidly killed in the presence of light and oxygen (20, 21). Singlet oxygen can also be pro duced by chemical oxygenation by hypochlorite-hydrogen per oxide (22, 2 3). Foote and Brenner (24) carried out photo- oxygenation of 3-methyl-l-(2,6,6-trimethyl cyclohexen-1-yl) 15
-1,3-butadiene, sensitized by Rose Bengal in methanol.
Numerous dye-photosensitized autooxidations as well as oxi dations by the reaction of sodium hypochlorite and hydrogen peroxide of olefins, and dienoid compounds were carried out
(19, 22-27). The active species in both the types of oxida tions is molecular oxygen in an excited singlet state. Ex cited singlet molecular oxygen produced by reaction of H„0 and sodium hypochlorite (22, 23, 25-27) or by radio-frequency discharge (28) gives products which are very similar to those of the photooxidations. Two alternative mechanisms have been proposed for photosensitized oxidations as follows (22,
23, 26, 27).
Sens "^ ) Sens (1 ) "^Sens > -^Sens (2 ) T 3 •^Sens + O2 ) -Sens-O-O- (3a)
•Sens-0-0*+A ^ ^^2 + Sens (4a)
•^Sens + •^O ^ Sens + -'-O2 (3b)
•^02 + A > AO2 (4b)
(where sens = sensitizer, A = acceptor)
The two mechanisms differ only in steps 3 and 4. Evidence will be presented later that will show that it favors the intermediacy of singlet oxygen.
Mechanism proposed for the photooxidative degrada tion of polymers such as polystyrene film in benzene (29) and with quinones as sensitizers (30) involve reactions of Fig. 3. The states of oxygen molecules and their elec tronic configurations. 17
Occupancy of State highest orbitals Energy
h- /\ 37 kcal <3 \1
/\ 22 kcal g I' \ ground state g 18 singlet oxygen molecules with polystyrene. Singlet oxygen may be formed in the reaction between excited benzene ring in polystyrene and molecular oxygen (29). The reaction rates are strongly increased by quinones (30). In the presence of quinones, it has been suggested that this photodegradation of polystyrene occurs by energy transfer from the excited triplet states of quinones to molecular oxygen (30). 3-Carotene has been found to be an extremely effi cient quencher of singlet oxygen (19, 31-39). Certain tran sition metal chelates also quench singlet oxygen but with less efficiency as compared to 3-carotene (35, 37, 39). The most efficient chelates quenched 0^ at close to the diffu sion controlled rate (35, 39), as did 3-carotene (32, 34-36, 39). As already stated, chlorophyll is among the most effec tive sensitizers for dye-sensitized photooxygenations of or ganic substates (36). Photosynthetic organisms, however, are apparently protected from the lethal effects of their own chlorophyll by carotenoids. Since both carotene and oxygen quench triplet sensitizers at a diffusion-controlled rate (oxygen to give singlet oxygen), quenching of triplet chloro phyll by carotenes cannot be responsible for the protective effects unless the local concentration of carotene greatly exceeds that of oxygen (34). Foote et CLI. (32, 36) obtained a bimolecular diffusion-controlled rate of 3 x 10^ M~ sec" for the singlet oxygen quenching by 3-carotene. This rate 19 value and the rates obtained for the same 3-carotene quench ing by Farmilo and Wilkinson (35) of (1*3 - 0-2) x 10''-'^ M~ sec"-^ and by Merkel and Kearns (40) of 2 x lO"*-*^ M""^ sec" are in good agreement. The decay rate of singlet oxygen was estimated to be 3.9 ± 0.4 x lO'^ sec" by Farmilo and VJilkinson (35), 4.2 X lO"^ sec" by Merkel and Kearns (40) and 1.0 x 10^ sec by Foote et al. (36). It is important to note in these quenching experiments that 3-carotene is not appreciably con sumed; the kinetics and control experiments show that at least 1000 O2 molecules are quenched for each molecule of 3-caro tene consiimed (36). An attractive mechanism for singlet oxygen quenching by carotenoids would involve energy transfer from singlet oxygen to the carotene, in a process that is the reverse of the reaction by which singlet oxygen is produced by energy transfer from triplet sensitizer to oxygen (22, 41): ^02 + car > -^car + ^0 ' (5) Such a mechanism would be tenable only if the triplet energy of 3-carotene is below or near that of singlet oxygen (22.5 kcal) (22, 41). Also the large quenching constant of 1.3 x 10 M~ sec" suggests that the triplet 3-carotene has an energy lower than that of 0,(-'-A ), i.e., 8000 cm""'- (35). ^ g It is known that 3-carotene quenches chlorophyll a (triplet energy 29 kcal), apparently by an energy transfer mechanism (42, 43). If the quenching of chlorophyll a triplet is an 20 energy transfer process, then the triplet energy of 3-carotene must be lower (36). This interpretation is consistent with the sharp dependence of triplet energy on the length of the conjugated system; triplet energy would be expected to in crease with decreasing length of the conjugated system; in this interpretation, systems with seven double bonds would have triplets that lie above 22 kcal, whereas those with nine or more would have triplets below or near this value (32, 36). So the rate of quenching is a sensitive function of the length of the conjugated polyene chain and parallels the pro tective action of natural compounds (32). This mechanism would suggest that cis-trans isomerism might accompany quenching (36) . The isomerization of carotenoids sensitized by chlorophyll a_ appears to occur only in the di rection cis > trans (44-48). All-trans-3-carotene does not appear to be isomerized, but experiments with 15-15'-cis' 3-carotene show it is rapidly isomerized, at least partly to the all-trans isomer, sensitized by singlet oxygen (33, 36). The photostationary state probably consists mainly of trans-B ear otene. Variety of other compounds was investigated for sing let oxygen quenching ability. DABCO (1,4-diazabicyclo [2'2'2] octane), hydroquinone and diethylsulfide were found
1 • 3 to quench 0„ effectively, their rates being about 10 lower than that of 3-carotene (36). 21
Singlet-Triplet Transitions of Photosensitizer Dyes" Quantum yields of triplet state formation (49) or intersystem crossing probability (50, 51) of some xanthene and thiazine dyes, fluorescein, eosin, erythrosin, methylene blue, etc., have been determined. Bowers and Porter (49) obtained values of 0.05 - 0.02, 0.4 - 0.07, 0.71 - 0.10 and 1.07 - 0.13 for fluorescein (Fl) dibromofluorescein (FlBr_) eosin (FlBr ) and erythrosin (FH.) respectively. Halogena- tion increased the intersystem crossing efficiency (49). In all cases, the fraction of excited singlet molecules which does not undergo fluorescence or intersystem crossing must be quite small (49). Intersystem crossing probability deter mined by Nemoto, et al_ in ethanol solution produced values for fluorescein, eosin and erythrosin of 0.007, 0.43 - 0.04 and 1.1 - 0.06 respectively (50) and in aqueous solution, values of 0.021, 0.64 and 1.0 for florescein, eosin and erythrosin respectively (51). CHAPTER III
MATERIALS AND METHODS
Materials Samples of peridinin-chlorophyll a_-protein complexes from Glenodinium sp., G. polyedra and A. rhyncocephaleum were a gift from Professor Francis T. Haxo, Scripps Institution of Oceanography, San Diego. PCP, A. carterae was a gift from Dr. H. W. Siegelman, Brookhaven National Laboratory,' Nev; York, PCPs from Glenodinium sp., G. polyedra and A. rhyncocephaleum were dissolved in 2 nm Tris buffer, pH8.4 whereas PCP from A. carterae was dissolved in 2 nM Tris buffer, pH7.4. The Tris buffer was prepared from Trizma-HCl and Trizma base Cfrom Sigma Chemical Co.) in double distilled water. The PCP complexes from G. polyedra and A. rhyncocephalexam were kept in saturated sucrose solution and were dialyzed against Tris buffer at 4°C prior to use. Rose Bengal was purchased from J. T. Baker Chemical Co. Menadione, DABCO, benzoquinone, a-tocopherol and ubiqui none were obtained from Sigma Chemical Co. DABCO was kept in dessicator. Menadione and benzoquinone were recrystal- lized in benzene and ethanol respectively. 3-carotene and cholesterol were purchased from Hoffman-LaRoche and 22 23
Eastern Organic Chemicals respectively. Potassium iodide was recrystallized in hot water. Solvents petroleum ether, ethanol and spectroquality glycerol were bought from J. T.
Baker Chemical Co., Fisher Scientific Co. and Matheson
Coleman & Bell respectively. Reagent grade acetone was obtained from Fisher Scientific Co. Powdered sugar was purchased from the local Furr's supermarket stores.
Chlorophyll a was prepared from spinach according to the procedure of Strain and Sherma (52). Two grams of spinach leaves were blended in 60 ml cold acetone in a chilled high-speed blender for 2 min. The supernatant was collected by filtration into a separatory funnel. 4 0 ml of cold petro- leum ether and 100 ml of cold saturated NaCl solution were added to the supernatant shaker and the upper layer collected.
The solution containing chlorophyll a, and other components was then separated and purified by column chromatography by eluting from a sugar column. The blue-green chlorophyll a. containing layer was eluted and subjected to column chroma tography or thin-layer chromatography for further purifica tion. The purity was checked by the absorbance ratio of the
Q to the Soret band of chlorophyll a. 24 Methods Theoretical Section Critical Distance Calculation In the PCP complex, the distance between peridinin and chlorophyll a at which the energy transfer efficiency from peridinin to chlorophyll a_ is 50 per cent is defined as the critical distance. The critical distance has been calculated on the assumption that the fluorescence spectrum of peridinin is the mirror image of its absorption spectrum as carotenoids do not fluoresce. The fluorescence intensity in arbitrary units is plotted against the wavelength fre quency in wave numbers (cm~ ). The area under the hypothetical fluorescence spectrum is then determined by weighing the paper corresponding to the fluorescence spectrum. The normal ization constant, N, is given by N = l/(area of fluorescence). In order to obtain the normalized area of fluorescence, e x Ipj, X N (where e = extinction coefficient of the acceptor, chlorophyll a, I„^ = fluorescence intensity of the donor, — c u peridinin and N = normalization constant) is plotted against wavenumbers. A value of 2.42 x 10 has been obtained for N. The area of the normalized spectrum is the overlap integral, J (D = donor, peridinin; A = acceptor, chlorophyll a). The critical distance is given by the equation, (8.79 X 10"25) K^ (f)^ R ^ = -A—:; Jr.. (6) o J ^4 DA 25 where R^^ = critical distance, K = orientation factor, assumed to be approximately ^[2/3, (53), (J)^ = quantum yield of the donor, peridinin, in the absence of the acceptor assumed to be 10"^ (limitation of the instrument), n = 1.446 (refractive index of the solution of 80 per cent glycerol in water) - 4-1 and V = 1.3986 x 10 cm -^ (average frequency - donor maxi mum) , The value of K^ = 2/3 is calculated from the equa tion (53) for the interaction energy between two dipoles, K = cos (j)j-,^ - 3 cos (j)j3 cos (j)^ (7) where (ji^ and 4)^ are the angles between the individual os cillators and the line connecting them, i.e., the line con necting D and A (<{)£)= 45° and cj)^ = 90°) and (f)j-)^ is the angle between the directions of the two (donor and acceptor) os cillations {-Qj^ = 45°) . The overlap integral was estimated to be 1.18.
Intermolecular Distance Calculation By using a dipole-dipole interaction model, the approximate exciton splitting, e , is given by the equation (54),
E+ =^ ± ^ (8) R3 using the definition of oscillator strength, f = 4.70 X 10^^ V M^, ( 9)
e+ = ± f (10) 4.704 X 10^^ V R3 26
where M is the transition moment in e.s.u., R is the point- dipole-point-dipole distance, taken as the distance between centers of mass of two peridinin molecules and v is the fre quency of cm of the monomer (peridinin) spectrum. The oscillator strength is also given by
f = 4.15 X 10"^ / e(v) dv (11)
where e is the extinction coefficient. The oscillator strength, f can be calculated for peridinin from integration
of the visible absorption band by plotting e against v.
Polarization by Photoselection
Fluorescence excitation and emission polarization
are techniques used to assign the relative orientations of the electronic transition moments. When a set of randomly oriented molecules in a rigid glass solution are excited by a plane polarized light, only the molecules parallel to the electric vector of the incident polarized light absorb the radiation. The degree of polarization is given by
P = —L'-;.. __ (12) I„ + I.L where Ijj and I^ are the intensities of the components of the beam resolved in directions parallel and perpendicular to the direction of incident polarized light. Normally, vertically polarized light is employed as the incident source,
Aizumi and McGlynn (55) proposed the following formula for 27 computation of polarization degree:
•""EE " """EB ^•^BE'''^"'"BB^ P = "l 7~1 (I /I ) ^13) •EE EB ^ BEZ-'BB^ where subscripts E and B refer to the vertical and horizontal orientations of the Glan-Thompson prism polarizers, re spectively- The light emerging from the grating monochro mator was found to be partially polarized to a small extent (55-5 7). By taking the second set of parallel and perpen dicular elements (i.e., based on the horizontally oriented excitation) into consideration, a correction for the effect of the emission monochromator and instrumental scattering could be made (55). The polarization from the theoretical approach was derived by Levshin (58), Perrin (59, 60) and Jablonski (61). If the angle between the absorption and emission oscillators is 0, it is shown (58-61) that the principal polarization is given by p = (3 cos^0-l)/(cos^0 + 3) (14) for vertically polarized exciting light. When 9 = 90° (i.e., when the absorption and emission oscillators are mutually at right angles) the polarization is -1/3 and.when 9=0° (i.e., when the absorption and emission oscillators are parallel) the polarization is 1/2. The angle of approxi mately 55° corresponds to a polarization of zero. It is only in rigid media where rotation cannot occur that 9 is 28 clearly identified with the angle between absorption and emission oscillators as an intrinsic molecular property and not an amount of rotation undergone by the molecule during its excited state lifetime.
Experimental Section Corrected Emission and Excita- tion Measurements A Perkin-Elmer Model MPF-3 Fluorescence Spectrophoto meter with Hitachi corrected accessory was used. The excita tion and emission monochromators are constructed in Czerny- Tiirner type mounting. The dispersing element in both mono- chromators is 600 lines/mm gratings glazed at 300 nm, with a 1/4 meter focal length collinating mirrors. A continuously variable slit from 0.13 to 5.16 mm was given with correspond ing band pass from 1.0 to 40 nm. A R106 type photomultiplier tube was used. This type of PMT has a Cs-Sb phtosensitive surface, resulting in a S-13 response which is highly sensi tive in 200-600 nm region. Xenon 150 watt lamp was used as light source. In order to obtain a corrected excitation spectrum, the incident light beam is split by a beam splitter and made to strike the fluorescent material, Rhodamine B. The fluores cent light from Rhodamine B is passed through the reference detector and the signal is amplified. This serves as the reference signal for the recorder. The quantiom yield of highly 29 concentrated Rhodamine B is almost constant over the wave length range of 200 to 600 nm and its fluorescence intensity is proportional to the quanta of exciting light. The light passed through the beam splitter strikes the sample cuvette. The sample emission is directed through the emission mono chromator into the sample detector and amplifier. The sample amplifier output is proportionally related to the reference amplifier output in the ratio recorder. The excitation spec trum is thus corrected for the wavelength dependency of source output and monochromator efficiency. The MPF-3 is designed to provide emission or excita tion spectra in the direct or ratio mode. In the direct mode, the sample photomultiplier signal is ratioed to a constant voltage and recorded (i.e., a fixed voltage is imposed on the reference channel of the recorder), whereas in the ratio mode the sample photomultiplier signal is ratioed to the reference photomultiplier signal to achieve long-term stability of measurement. To achieve corrected emission spectra, a programmed potentiometer is interlinked with the emission monochromator and a series of correcting variable resistors. The system is designed to correct the instriomental characteristics of the monochromator and detector. This is done by changing the amplifier gain in the sample circuit having the programmed potentiometer. Fig. 4. Optical layout of t photometer.
1. Xenon light source 2. Plane mirror 3. Entrance slit 4. Beam splitter 5. Detector 6. Collimating mirror 7. Grating 8. Reference (Rhodamine B) 9. Reference detector 10. Sample 11. Exit slit 12. Sample detector 31 Fig. 5. Block diagram of Perkin-Elmer spectrophotometer with Hitachi corrected spectra accessory.
Light path J^
Electronic signal feed-in ^
Mechanical interconnection ------^
1. Xenon light soiurce 2. Pre-programmed potentiometer 3. Excitation monochromator 4. Detector 5. Beam splitter 6. Rhodamine B 7. Filter 8. Interconnection assembly 9. Interconnection assembly 10. Mirror 11. Sample 12. Reference detector 13. Pre-programmed potentionmeter 14. Emission monochromator 15. Sample detector 16. Sample amplifier 17. Reference amplifier 18. Ratio recorder 33 34 Fluorescence Quenching Fluorescence spectra were recorded as described in the above section. Chlorophyll a solution iOD.^n = 0.22) in ethanol-Tris buffer, 3:2 v/v was excited at 430 nm. The chlorophyll a solution contained 0, 0.08, 0.2, 0.36, 0.6, 1.0 and 1.8 M solution of potassium iodide. Potassium iodide solution was prepared in Tris buffer. Fluorescence spectra of PCP, Glenodini\3m sp. and A.' carterae solutions in the ab sence and presence of 1.0 and 1.8 M KI were also recorded. The PCP solutions were diluted to obtain the same fluores cence intensity as the chlorophyll a solution in the absence of potassium iodide.
High Resolution Emission and Excitation Measurements The instrument consists of two Jarrell-Ash 0.25 meter Ebert monochromators of 82-440 and 82.441 series in a syn chronous tandem. This arrangement reduces scattered light and therefore produces a monochromatic light of higher spectral purity. Light from a 150-watt Xenon arc lamp (Hanovia) is collected by an ellipsoidal mirror and focused onto the en trance slit of the synchronous tandem set. The sample emis sion is observed at right angles to the exciting light by the emission monchromator which is a 0.5 meter Ebert spectro- Q ° meter (Jarrell-Ash 82-000, linear dispersion of 16 A, 0.2A first-order resolution). The spectrometer has 118 0 grooves/ Fig. 6. Block diagram with optic layout for high resolu tion spectrometer.
1. Reflecting mirror collimator 2. Xenon light source 3. 45° plane mirror 4. Excitation monochromator collimating mirror 5. Excitation monochromator grating 6. Condensing lenses 7. Sample 8. Emission monochromator grating 9. Heated insulating window 10. Photomultiplier tube 11. Refrigerated PMT chamber 12. 1120 amplifier/discriminator 13. 1105 Data converter (photon counter) 14. 1106 power supply console 15. Insulaced cooling hoses 16. Constant temperature cooler (ethanol as coolant) 17. Honeywell strip chart recorder 18. Emission wavelength drive 19. 0.5 M scanning emission monochromator 20. 0.25 M Ebert excitation monochromator (2) 21. Excitation wavelength drive 22. Emission monochromator collimating mirror 23. Polacoat UV sensitive polarizers (emission and excitation) 36 37 mm grating with spectral range from 1900A to 9100A and is coupled to an EMI 9659 photomultiplier detector. The photo multiplier tube (S-20 response) is housed in a refrigerated photomultiplier tube chamber. Model TE-104 from Products and Research, Inc. The PMT is constantly cooled to maintain a temperature of -40° C by circulating ethanol through the cooling unit. The operation of PMT at -40° C enables one to operate the PMT at a higher voltage with minimum background noise. A 1100 series photon counting system including the model 1120 amplifier/discriminator and a model 1105 data con verter console (both from SSR Instrument Co., Santa Monica, Calif.) converts the photomultiplier tube output data into three forms: indication on a front panel meter, a direct digital counting and recording on a Honeywell strip chart recorder. Recorded spectra were not corrected for the re sponse characteristics of the instrument.
Two Glan-Thompson polarizers were mounted on a frame perpendicular to each other. There are two possible orienta tions for each polarizer, vertical and horizontal. The sample cuvette is placed in between the two polarizers. Photo selection method has been discussed earlier in the theoretical section.
Low Temperature Measurements Low temperature (200 K) excitation, emission and polar ization spectra were recorded on the high-resolution 38 spectrofluorometer. For the low temperature measurements, the PCP in Tris buffer (or in 1 per cent sodium dodecyl sul fate) and glycerol (1:4 v/v) was sealed in a selected Pyrex glass tubing of 6 mm diameter and about 45 mm in length so as to fit into the sample mount of a cryocooler optical chamber (Model 20, Cryogenic Technology, Waltham, Mass.). The sample was then allowed to equilibrate for 1 hour at 200 K before proceeding with the measurements. The closed-cycle cryocooler is based on the follow ing principle. Helium is compressed in a liquid state and in a closed-system, helium undergoes an expansion-compression cycle while circulating through the cylinder. The tempera ture gradient causes heat to flow from the load into the ex panded helium via the cylinder wall. A temperature controller is connected to the tip heater circuit. The thermocouple im mediately adjacent to the sample tip is linked to the temper ature controller on the control panel. When a desired temper ature has been selected, the controller will automatically switch to the heater or the cooler, alternately, to maintain the system at this particular temperature within + 1 K. The temperature control range is approximately 12 K to 300 K.
Circular Dichroism The circular dichroic spectra were recorded on a JASCO-20 CD-ORD spectropolarimater which was modified to enhance the signal-to-noise ratio by replacing the Pockel 39 cell and associated circuitry with a Morvue photoelastic modulator (PEM-3) and lock-in amplifier (PAR Model 121). Spectra were recorded at room temperature.
Absorption Spectra
Room temperature absorption spectra were recorded on a Cary 118 C absorption spectrophotometer.
Fluorescence Lifetimes Some of the problems faced in the pulse method for lifetime measurements are alleviated in the phase fluorometry. While in homogeneous emitting populations the analysis is simple, it is complex for heterogeneous emitting populations. The fluorescence lifetimes were measured at room temperature with an SLM-Model 480 phase-modulation spectro- fluorometer, utilizing continuous light that has been sinu- soidally modulated. A 1 kilowatt xenon arc lamp from Oriel Optics was used as the light source. Appropriate filters (such as 0.63 Um red filter) from Oriel Optics were used in the emission light path to insure homogeneous emission. Lifetimes were measured at light frequencies of 10 and 30 MHz. The holographic grating monochromator of the instru ment is corrected for aberrations and has a linear disper sion of 2 nm/mm. The modulator contains r.f. electronics as well as the Debye-Sears ultrasonic tank. The tank contains 40 an x-cut quartz crystal and a very delicate gold plated reflector. The output is controlled by a lens and a slit. The photomultiplier tubes, Amperex 56 DUVP and 56 TVP cover the spectral range from UV to 700 nm. The OP-480 optical module is provided with uv transmitting optics and Glan- Thompson polarizers. The lifetime instrument is interfaced to a Hewlett- Packard Model 9810-004 calculator with optional printer. This interfacing allows direct calculation and readout of lifetimes, polarization data and dynamic depolarization.
Temperature Dependence of PCP and Chlorophyll a Fluorescence Fluorescence spectra and lifetimes were measured as already described. Temperatures varying up to 75° C were maintained by circulating water from the Haake Instruments Type F 4 391 constant Temperature Circulator through the in let and outlet of the sample compartments of the Perkin- Elmer and SLM Model-480 instruments. The Haake Temperature Circulator was provided with a Thermoregulator for tempera ture setting. The actual operating temperatture is read on a control thermometer.
Dynamic Depolarization The dynamic depolarization measurements were carried out on the same instrument, SLI4 Model-480, used to measure fluorescence lifetimes. The rotational relaxation time p Fig. 7. Block diagram of the Phase-Modulation cross- correlation spectrofluorometer.
1. Xenon lamp power supply 2. Xenon lamp 3. Monochromator 4. Ultrasonic tank 5. Turret cuvette holder 6. Photomultiplier tubes 7. Sample compartment with filter 8. RF generator 9. Phase shifter 10. Hewlett-Packard calculator 42 « i 43 was determined as described by Weber and Mitchell (62) using the equation
2a a^ + (1- (jJT, ) + ll±!^oll = 0 (15) 1-P 2 tan A •1-^ P^ o2 ^ ^o with
a = m (16) m = (3-P^)/(1-PQ2) (17) In the above equations a)(= 2TT X modulation frequency f) is the circular frequency of the exciting light, TQ the fluor escence lifetime and P^ the limiting polarization. The tangent of the differential phase delay, tan A, is given by the following equation
tan A = 2TTfAT = OJAT (18) where the differential lifetime AT is the value measured directly as the difference between parallel and perpendicu lar decay components. The rotational relaxation was measured at two different frequencies, 10 imz and 30 MHz, to detect anisotropic rotations in the case of non-spherical molecules. Non-spherical molecules produce-different differential life time values which give rise to different p values. The attainment of the maximum value for tan A,
tan A. PnUn0^)0^T 0 (19) i"^^ 1 +[(1+(O^TO2)(1-PQ2)]^ is an indication of isotropic rotations. From this equation, the maximum Ax expected is -^-0.2 nsec. 44
Photo-irradiation The phtolysis of Rose Bengal-chlorophyll a solutions in ethanol or benzene in the presence or absence of singlet oxygen quenchers was carried out at 557 nm with a Bausch & Lamb Model 33-86-07 monochromator fitted with a 150 watt Xenon lamp (from Hanovia). The monochromator grating was constructed with 1200 grooves/mm. The photolysis solution of 3 ml was prepared by adding 1 ml of 0.3 mM Rose Bengal solution in ethanol, 1 ml of chlorophyll a. solution of opti cal density ^^^3.0 in ethanol and 1.0 ml of the inhibitor solution in ethanol or benzene. At the final concentration of Rose Bengal of 0.1 mM most of the incident light (100%) was absorbed and the final absorbance of chlorophyll a was '^l.O. Continuous flow of oxygen gas through the photolysis solution was provided. To prevent solvent evaporation, the oxygen gas was bubbled through the solvent, ethanol, so that the gas was saturated with the solvent, prior to passing through the photolysis solution. The photolysis solution was placed in a 10 mm path length Pyrocell cuvette provided with a rubber stopper having an inlet and an outlet for the flow of oxygen gas. The sample cuvette was placed in the cell compartment of the Hitachi Perkin Elmer Model 139 UV- Visible spectrophotometer. An opening was provided in front of the sample compartment for the photo-irradiation of the sample cuvette. Both the entrance and exit slits of the 45 monochromator were set at slit width of 6 ram corresponding to a bandpass of 38.4 nm (linear reciprocal dispersion = 6.4 my/mm). The monochromator exit slit was placed at a distance of 30 cm from the sample cuvette with its slits open to their maximum slit width. Light was collimated onto the cuvette by a lens at a distance of 20 cm from the cuvette, The photooxidation of chlorophyll a was followed by measur ing the absorbance reading at 660 nm with the entrance to the sample compartment closed to prevent light from falling on the sample while taking the OD readings.
Actinometry The procedure that was followed to determine the in tensity of the Bausch & Lomb monochromator 150 watt Xe lamp was essentially the same as described by Calvert and Pitts (63). Potassium ferrioxalate (City Chemical Corporation) was recrystallized several times from hot water and a 0.15 M solution was prepared. A standard calibration graph for the analysis of the Fe^"^ complex for use with the spectro photometer yielded a value for the extinction coefficient of 1.11 X 10^ liters/mole-cm. The number of ions of Fe^^ formed during the phtolysis (npg2+) was calculated from the equation,
2+ = •6-02 3 X 1020 VT V, logi n (In/I) (20) Fe 46
where v^ = the volume of the actinometer solution irradi
ated (3.0 ml),
V2 = the volume of the aliquot taken for analysis
(see Table 1),
v^ = the final volume to which the aliquot V2 was diluted (25 ml),
log]_0 (IQ/-'-^ ~ ^^^ measured optical density of the solu
tion at 5100 A° (see Table 1),
I = the path length of the spectrophotometer cell
used (1 cm), and e = the experimental value of the molar extinction
coefficient of the Fe2"'" complex as determined
from the slope of the calibration plot (1.11 x 4 10 liters/mole-cm).
The value of the quantum yield, '^•^^2+ i'^ccn) was calculated from the equation,
R I^® *557 = -^^ ^ *480 ^ -H^ (21) ^480 I557 where Rcry and R^pn ^-^^ ^^^ rates/min of the actinometer solution at 557 and 480 nm respectively, ^rrn and '\>AQQ are 2+ the quantum yields of Fe at 557 and 480 nm respectively, and I??-, and I^^,, are the Xenon lamp intensities at 557 and 557 4B0 480 nm respectively. R^^^ = 0.0029, R^g^ = 0.0528, <^^^^ =
0.068 (calculated), ^.^^ = 0.94 (from Calvert and Pitts (63), 4 8 0 PP- 784) , I^f^/I^® = 14.94/11.33 (from the Xe lamp spectrum 4 8 0 557 in the Bausch & Lomb monochromator manual). Table 1. Intensity of the Bausch s Lomb monochromator 150 watt Xenon Lamp at 557 nm.. 48
Absorbance Volimie of the Intensity of Xe lamp at 5100 S aliquot, V-,(ml) (quanta/sec.)
0.412 2.3 8.63 X 10-^^
0.507 3.0 7.64 X 10^^
0.091 2.5 8.76 X lO-*-^
0.084 2.5 8.11 x lO-"-^ 49
The light intensity incident just inside the front window of the phtolysis cell was calculated from the equa tion (see Table 1,
•i ^T^ 2+ I-^ = £^ quanta/sec (22) °
The value of e[A]£ = optical density of the 0.15 M actinometer solution at 557 nm was 0.06 3. t is the time of irradiation of the actiometer solution in seconds. CHAPTER IV
RESULTS
PCP (Glenodinium sp.) - Absorption and Polarization Figure 8 shows the absorption spectr\am of PCP (Gleno dinium sp.) in Tris-glycerol (1:4). The molar ratio of peri dinin to chlorophyll a in the PCP complex has been estimated to be 4 to 1 from the absorption spectr;am based on a peri dinin molar extinction coefficient of 8.44 x 10"^ M" cm" . The complex was found to be stable even after repeated freeze (200K) - thaw cycles. The denaturation caused by 1 per cent sodium dodecyl sulfate is to shift the chlorophyll Qy band and peridinin band to the blue and red, respectively. Figure 9 shows the fluorescence excitation polarization of the native PCP (Glenodinum sp.). A polarization value of about 0.1 at the PCP emission maximum of 675 nm has been obtained for the entire main absorption band of peridinin. The polarization increases sharply for the chlorophyll Q^ band and attains the maximim at the Qy band maximTom.
PCP (Glenodinium sp.) - Fluores cence Excitation Figure 9 shows the fluorescence excitation spectrum
50 51 of PCP (Glenodinium sp.), not corrected above 600 nm, re corded on the Perkin-Elmer spectrofluorometer. The high resolution fluorescence excitation spectrum of the same PCP is shown in Figure 11. The ratio of excitation at chloro phyll Qy band to peridinin B band is nearly identical ('V'4) with the absorption spectrum. Figure 8, when the excitation of the former (667 nm) was corrected. Figure 10, or in the completely uncorrected spectrum as seen in Figure 11. Since this excitation ratio remains nearly unchanged at the peri dinin maximum (475 nm) absorbance of 0.3 and at a lower ab sorbance of 0.16, the excitation spectra shown in Figures 10 and 11 indicate very efficient (100 per cent) energy trans fer from peridinin to chlorophyll a. Figiure 11 also shows the fluorescence excitation spectrum of the denatured PCP (Glenodinium sp.). Denatinration completely destroys the intra-complex energy transfer as seen by the lack of exci tation in the peridinin absorption region (Figure 11). The denatured PCP fluorescence excitation spectrum is es sentially identical to chlorophyll a absorption spectrum (Figure 12).
PCP (Glenodinium sp.) - Circular Dichroism Figure 13 shows the CD spectrum of PCP (Glenodinium sp.). The expanded CD of the chlorophyll Qy band is also shown. There is no indication of any intermolecular inter actions between chlorophylls or between PCPs as seen by the Fig. 8. Absorption spectra of native (. ) and denat\ared ( ) PCP from Glenodiniimi sp. 53
absorbance ( )
E
{—) aouDqjosqo Fig. 9. The spectrum of polarization of fluorescence excita tion (excitation band-pass 0.5 nm and emission at 675 nm, 2 nm bp) of PCP:(0) Glenodinium sp. in Tris-glycerol (1:4) at 200 K, recorded on a single-photon counting spectrofluorometer. Absorbance at the peridinin and chlorophyll peaks were kept at 0.3 and 0.07, respectively, in order to avoid self-depolarization due to chlorophyll-chlorophyll energy transfer. The absorption spectrum of the PCP is also included for reference. 55
tn ^ to CVJ _ QJ» O C) o' d o 1 1 1 1 1 1—
£ c
oouoqjosqo Fig. 10. The corrected fluorescence excitation spectrum of PCP (Glenodinium sp.) at room temperature. The spectrum above 600 nm was not automatically corrected by the MPF 3 spectrof luorometer. 57
--. E 1 S CL 0/ xi E E c c O cn d. CD .o' a E X o UJ a. /< Fig. 11. Fluorescence excitation spectra of (a) denatured and (b) native PCP (Glenodinium sp.) recorded on the single-photon counting spectrofluorometer. 59
^01X ,.s siunoo '^l (D) 60 lack of CD splitting of chlorophyll Q band. The most im portant and interesting feature of the CD spectrum is that the peridinin absorption band is split into two approximately equal bands with positive and negative elliplicities (463 and 538 nm) . Denaturation completely destroys the CD band splitting. Both chlorophyll a_ and peridinin remain boimd to the apoprotein as seen by the changes in CD minima shown in Figure 14 (686, 673 and 665 nm for native, denatured PCP of Glenodinium sp. and free chlorophyll a, respectively). The fluorescence polarization for chl a of denatured PCP (Glenodinium sp.) remains at near the maximum value. Scat tered light in the case of denatured PCP samples is a likely cause for the consistently higher polarization degree through out the spectrum (Figure 15). In addition to the denatured PCP CD spectra, the fluorescence polarization data of de natured PCP also indicate that some chlorophyll a molecules are still bound to the apoprotein.
PCP (Glenodinium sp.) - Fluores cence Emission Figure 16 shows the fluorescence spectrxom of PCP (Glenodinium sp_^) recorded on the high resolution instru ment. The fluorescence spectrum produced by the excitation of PCP at 475 nm is identical to chlorophyll a fluorescence emission spectrum with the emission maximum red shifted to 675 nm. The chlorophyll fluorescence is due to energy trans fer from peridinin, as already stated. The fluorescence r r
Fig. 12. Absorption spectrum of chlorophyll a^ in ethanol. The fluorescence excitation polarization was measured in ethanol at 77K on the high resolution monophoton counting spectrofluorometer. The visible band CD. of chlorophyll a^ v;as kept at 0.07 in order to minimize self-depolarization arising from the strong overlap between the Q absorption and emission bands. The Pariser-Parr-Pople SCF MO CI Calculated polarization axes of 0 and Q bands are also shown (insert) . 62
Q'O Fig. 13, Circular dichroism (CD) spectrum of PCP (Glenodinium sp.) absorbance '^0.3 in Tris-glycerol (1:4) at room temperature. The insert shows the CD spectrum for the visible band of chlorophyll a^ of the PCP complex at an expanded scale. 64
,01X VV Fig. 14. Circular dichroism spectra of denatured PCP (Glenodinium sp.) in Tris-glycerol (1:4) (top) and chlorophyll a_ in ether (bottom). 66
tOlx^VV Fig. 15. The polarized fluorescence excitation spectrum of denatured PCP (Glenodinium sp.) in Tris-glycerol (1:4) at 200 K. The absorption spectrimi is also shown. 68
83UDqjosqo Fig. 16. The fluorescence emission spectrum of PCP (Gleno dinium sp.) in Tris-glycerol (1:4) at 200 K, recorded on the single- photon counting spectrofluorometer. Fluorescence lifetime data from four different modes [phase (tan (b) and demodulation (1/2'n'f) ijm~ -1) ] of measurements are also included in the figure, where (J) is the phase shift, f is the modulation frequency, and m is the attenuation or demodulation ratio which equals cos S s g B B 1 E 0. 4 n m 5.48 4 5.72 0 CL iv «> Xi E (J >. c "& E »•< CM c CJ no GO d C fO in o ID 47 5 .a id c E vt O (\l /< LLI c E c 01 X vs^unoo '^I 71 spectrum of chlorophyll a overlaps strongly with its absorp tion spectrum. PCP (G. polyedra) - Absorption and Polarization Figure 17 shows the polarized excitation spectrum of PCP (G. polyedra) . The absorption spectriim is also included. The polarized excitation spectrum, was measured in order to determine the orientations of the two components of the ex citon transition moments with respect to the Qy transition moment of chlorophyll a. Fluorescence polarization with respect to the chlorophyll fluorescence was measured at 200 K with the PCPs in Tris-glycerol (1:4). The G. polyedra polarized excitation spectrxom shows a slightly smaller polar ization degree for the positive CD band as compared to Gleno dinium sp. (Figure 9). These results indicate that the ori entations of both components of the peridinin CD bands are 45-50° with respect to the Qy axis of chlorophyll a. De naturation causes substantial distortions of the transition moment orientations. The absorption spectrum also indicates the same peridinin to chlorophyll a molar ratio of 4 to 1 as for Glenodinium sp. (Figure 9) . PCP (G. polyedra) - Fluores cence Excitation The corrected fluorescence excitation spectrum of PCP from G^ polyedra at room temperature is shown in Figure 19. The uncorrected fluorescence excitation spectra of PCPs Fig. 17. Absorption and polarized fluorescence excitation spectra of PCP (G^. polyedra) in 2 nM Tris-glycerol 91:4) at 298K and 200K, respectively. The monitoring fluorescence wavelength was 675 nm. The band pass for emission and excitation polarization measure ments on the single photon coimting spectrofluorometer were 2 and 0.5 nm, respectively. The absorbance was kept low, 0.16 at peridinin absorption band and <0.05 at the chlorophyll visible band (Q ) to eliminate self-depolarization due to the overlapping Qy absorption and emission bands. 73 Fig. 18. The polarized fluorescence excitation spectrum (excitation band pass 0.4 nm) of PCP (A_. rhyncocephaleum) in Tris- glycerol (1:4) at 200K. The absorption spectrum is also included. 75 Fig. 19. The corrected fluorescence excitation spectra of (a) PCP from G. polyedra and (b) A. rhyncocephaleum in Tris-glycerol (1:4) at room temperature. Absorbance at the peridinin band was kept at <0.3 [0.I6 for (a) and 0.29 for (b)J, so that direct comparison with the corresponding absorption spectra (Figxrres 8, 17 and 18) is meaningful. 77 78 from Glenodinium sp. and G. polyedra recorded on the single- photon counting spectrofluorometer are shown in Figure 20. Similar to PCP from Glenodinium sp., PCP from G. polyedra also shows 100 per cent energy transfer within experimental error, as indicated from the fluorescence excitation spectra (Figure 20) . The 100 per cent energy transfer efficiency is estimated from the identical ratios of the penidinin to chlorophyll maxima in both the absorption and fluorescence excitation spectra. The ratio of peridinin to chlorophyll maxima is estimated to be 4:1 (Figures 17 and 20). PCP (G. polyedra) - Circular Dichroism Figure 21 shows the circular dichroism spectrum of PCP from G. polyedra. The CD spectrum of this PCP is almost identical with PCP (Glenodinium sp.) with positive and nega tive eliipticities of 464 and 541 nm compared with 463 and 538 nm for Glenodinium sp. PCP (A. rhyncocephaleum) - Absorption and Polarization Figure 18 shows the absorption spectrum of PCP from A. rhyncocephaleum. The molar, ratio-of peridinin (B band) to chlorophyll a (Qy band) is 4:1 just as for the other PCPs described. The absorption spectrum shows a shoulder at 510 nm to the 4 75 nm band of peridinin. This shoulder is most prominently observed in this PCP as compared to 79 other PCPs. Figure 18 also shows the fluorescence excita tion polarization spectrum of PCP from A. rhyncocephaleum. The polarization degree for the entire main absorption band of peridinin at 200K in Tris-glycerol is about 0.05 just as for PCP from G. polyedra and slightly less than for PCP from Glenodinium sp. The polarization degree increases sharply for chlorophyll Q^ band and attains a maximum just as for free chlorophyll a. The peridinin B band polarization of 0.05 indicates that both the CD components B and B_ are oriented at an angle of about 50° with respect to the chloro phyll Qy band. PCP (A. rhyncocephaleum) - Fluores cence Excitation Figure 19 also shows the corrected fluorescence ex citation spectrum of PCP from A. rhyncocephaleum taken at room temperature. The uncorrected fluorescence excitation .spectrum recorded on the single-photon counting spectrofluor ometer is shown in Figure 20 along with that of PCPs from G. polyedra and Glenodiniiom sp. Similar to the other PCPs, PCP from A. rhyncocepha 1 elom also exhibits 100'per cent energy transfer efficiency from peridinin to chlorophyll a. This is indicated from the identical ratios of 4:1 for peridinin to chlorophyll band maxima in both the absorption (Figure 18) and fluorescence excitation (Figure 20) spectra. ff Fig, 20. Fluorescence excitation spectra of PCPs ( ; Glenodinium sp., • ; A. rhyncocephaneum, : G. polyedra) in Tris-glycerol (1:4) at 298K, recorded on the single-photon counting spectrofluorometer with an excitation band pass of 0.4 nm. 81 CO in ^ "lo" CVJ 01 » ,_oas s;uno3 '^i Fig. 21. Circular dichroism spectra of PCP from (a) G^. polyedra (absorbance 0.16) and (b) A. rhyncocephale\mi (absorbance 0.29) in Tris-glycerol (1:4) at room temperature. The baseline for the CD cell and solvent is also indicated. 83 84 PCP (A. rhyncocephaleum) - Circular Dichroism The CD spectrum of PCP from A. rhyncocephaleum is compared with that of PCP from G. polyedra in Figure 21. The 475 nm peridinin absorption band of PCP (A. rhyncocepha- leum) is split into two CD bands with positive and negative eliipticities at 462 and 54 5 nm respectively. PCP (A. carterae) - Absorption, Polarization and Circular Dichroism Figure 22 compares the absorption spectrum of PCP isolated from A. carterae with that from G. polyedra. Figure 23 shows the absorption spectriom of peridinin. The ratio of the absorbance of peridinin band to that of chlorophyll a Q band is 4.0 and 3.9 for PCPs from A. carterae and G. polyedra respectively (Figure 22) . It is also significant that the room temperature spectrum of peridinin (A^j^^^^ 476 nm and 509 nm (Figure 23) . The peridinin absorption band in the PCP (Figure 22) shows a broad shoulder beginning at approximately 510 nm. Since the peak at 509 nm does not represent a separate electronic transition but due to different vibrational level belonging to the main absorption band, B ^ A transition of carotenoids (64), therefore the CD band with a negative el- liplicity cannot be assigned to a separate electronic transi tion, but is best resolvable in terms of a dimeric exciton Fig. 22. Absorption spectra of PCPs in Tris-glycerol (1:4) at room temperature. Solid line, A^. carterae; dotted line, G^. polyedra: The spectrimi of polarization of fluorescence excitation (excitation band pass 0.5 nm and 2 nm bp for emission at 675 nm) of the A. carterae (-0-) PCP in Tris-glycerol (1:4) at 200K, recorded on the high resolu tion monophoton counting spectrofluorometer. The optical densities at 475 nm for PCPs of A. carterae and G^. polyedra are 0.34 and 0.79, respectively. 86 /-: / \ / 0.5 1 "J 0.4 O o 0.3 0.2 0.1 do° o ° 0 •-0.I 300 400 500 600 700 Fig. 23. Absorption spectra of peridinin in ethanol at room temperaturperature (solid curve) and 7'7 K (dotted c\irve). The chemical structure of peridinin is also shown. 88 'Q'O 89 as already stated (Figure 24). The electric transition dipole moment orientation of both the positive and negative CD bands is the same (45'\^50°) with respect to the Q absorp tion band of chlorophyll a as can be seen from the fluores cence excitation polarization spectrum (Figure 22). For comparison, the CD spectrum of PCP from A. rhyncocephaleum is also shown in Figure 24. The PCP complexes from Gleno dinium sp. and G. polyedra also exhibit the same CD and polarization characteristics as A. carterae PCP. PCP (A. carterae) - Fluores- cence Excitation Figiire 25 shows the fluorescence excitation spectra of PCPs from A. carterae compared with those from A. rhyn cocephaleum and G, polyedra recorded on the single photon counting spectrofluorometer. They all show about the same ratio of the peridinin excitation maximiom to the chlorophyll excitation maximum indicating 100 per cent energy transfer from peridinin to chlorophyll a. This ratio is close to the corrected excitation ratio as well as the absorbance ratio. PCP (A. carterae) - Fluorescence Emission, and Fluorescence Lifetimes Figure 2 6 compares the fluorescence emission spec trum of PCP from A. carterae with that from Glenodinium sp. Table 2 siimmarizes the fluorescence emission and lifetime data for all the four PCPs. An interesting observation is Fig. 24. CD spectra of PCP from A^. carterae (A) in Tris (2 nm, pH 7.4) and A^. rhyncocephaleum (B) in Tris-glycerol (1:4). The dotted and broken lines represent the base line for these PCP spectra (OD^-yc ^^^ = 1.08 and 0.29, respectively), respectively. Glycerol had no apparent effects on the CD spectra of all PCP complexes examined. The rotational strength of the negative (cor responding to B^ in Figure 45) and positive (corresponding to BJ peridinin bands are -3 x 10~ and 2 x 10"-^^, respectively. 91 Fig. 25. Fluorescence excitation spectra of PCPs ( carterae; •••• •••, A. rhyncocephaleum; -----, G. polyedra) in Tris-glycerol (1:4) at 200 K, recorded on the monophoton counting spectrofluorometer with an excitation band pass of 0.4 nm, 690 nm. em 93 700 600 500 400 300nm Fig. 26. Fluorescence emission spectra (uncorrected) of PCPs ( , A^. carterae (top) and A_. rhyncocephaleum (lower) ; , Glenodinium sp.) in Tris buffer pH 7.4 (A. carterae) and 8.4, respectively. Band pass 2 nm for emission and 5 nm for ex citation A = 475 nm. 95 620 660 700 740 nm Table 2. The fluorescence maxima and lifetimes of PCPs in Tris-glycerol (1:4) at 200 and 298° K, respectively. The latter was measured by both the phase and demodulation methods at two modulation frequencies. 97 (0 n CM 00 u in o ID > o in tn ^ •H -.V M '^ 3 EH . > o ro in to r~ o a in •H 33 i "3" •^ a 0) g S3 c CO in 00 p ft •H o C ^.—' in 'J •H in .* -0 CN Q 0 • C o o (U ro r-l o Qi o ^ 0) GO CN CN U 0 ro 0 -P m IW ft in rf .a< -o tn c OJ 3 3 0 H IH (fl H > (U o CN CN M o C (U 0 s 'J' "a" •H +J OJ Q •sH +yJ M i >H H in , 1 0) e tftn a (fl j; CN (u m EH ^ M ft * s tfl OJ •H ^ o ft fi 0) 0 o •H >i c ft T! H >1 0 0 J= a ft M 6 <\ Dynamic Depolarization The rotational relaxation times of the PCP complexes have been obtained from the dynamic depolarization data as described in the experimental section. The PCP of A. car terae showed a single isotropic rotational relaxation time of 33 nsec in Tris buffer at room temperature. The exhibition of a single rotational relaxation time indicates that the molecule is spherical. Fluorescence Quenching Figure 27 shows the fluorescence quenching of chloro phyll a by KI. Different concentrations of KI up to 1.8M [limited by solubility in ethanol-water (3:2, v/v)] have" been used. Figures 28 and 29 show the Stern-Volmer plots of the fluorescence quenching of chlorophyll a_ and PCPs by KI in terms of fluorescence intensity and fluorescence lifetime measurements, respectively. The second order quenching rate constants of 1.35 x 10^ M~-^ sec""'' and 1.26 x 10^ M~ sec"-"" have been estimated from Figures 2 8 and 29 respectively. Fig. 27. Fluorescence spectra (uncorrected) of chloro- phyll a vw„._(CD, „ = 0.22) in ethanol-Tris buffer (3:2) as a — 430 nm function of potassium iodide (in M) . A^ex^ = 430 nm (chl soret band) 100 620 660 700 740 nm Fig. 28. The Stern-Volmer plot of fluorescence quenching (I^ and l2; fluorescence intensities with and without quencher, re spectively) of chlorophyll a^ (-8-) in ethanol:water (3:2 v/v) and PCP (Glenodinium sp. and A. carterae)(-0-) in Tris buffer at room temperature. 102 ^ Fig. 29. The Stern-Volmer plot of fluorescence quenching (TjT and T,; fluorescence lifetimes with and without quencher, re spectively) of chlorophyll a_ in ethanol-water (3:2, v/v) (-6-) and PCP (Glenodinium sp. and A^. carterae) (-0-) in Tris buffer at room temperature. 105 These two rate constants show a good agreement between the steady state (intensity) and dynamic (lifetime) measurements. On the other hand, the fluorescence of chlorophyll a in PCPs is not quenched at all by 1,0 and 1,8 M KI (Figures 28 and 29) . The addition of KI to PCPs did not alter their absorp tion or CD spectra, indicating that neither dissociation of chromophores nor denaturation. of the protein occurred even at a KI concentration of 1.8 M. The diffusion-controlled rate for bimolecular quenching (Figures 28 and 29) is re duced. It should be noted that the small quenching rate constant for chl a in Figures 28 and 29 is due to the fact that a steric factor of 0.1 arising from the large surface area of the chlorin ring (ca. 46 A ) and small quencher (I, 4.7 £ ). "Reconstitution" of PCP Attempts were made to reconstitute the PCP after separation of chromophores from the protein or from denatur ation by addition of acetone. Figure 30 shows the effect of acetone on the absorption spectrum of PCP from Glenodinium sp. Both chlorophyll and peridinin bands undergo signifi cant changes as the concentration of acetone is gradually increased to 50 per cent. At 40 per cent acetone, the chloro phyll Q band shifts to 7 35 nm. Chlorophyll a apparently precipitates out almost completely at 50 per cent acetone. The effect of acetone on CD spectra is shown in Figure 31. Fig. 30. Absorption spectra of PCP (Glenodinium sp.) in Tris buffer as a function of acetone concentration (in%) at room temperatiore. , 10% acetone; , 30%; 40%; and , 50%. 107 Fig. 31. Effects of acetone on the CD spectrum of PCP (Glenodinium sp.) in Tris buffer at room temperature OD.^j- = 0.163 for 10% acetone solution, 0.144 for 20% acetone and 0.192 for 30% acetone. 109 110 As the acetone concentration is increased, the character istic CD band of chl a and the splitting of peridinin band are lost. Addition of 40 per cent or more acetone to the PCP solution produced large CD signals in the wavelength region above 660 nm. Three distinct CD maxima and minima were observed at 785 nm (+ ellipticity), 767 nm (- elliptic- ity) and 736 nm (+ ellipticity), indicating aggregation of chlorophyll. PCP in 50 per cent and 30 per cent acetone were lyophilized separately to remove all traces of the sol vent, redissolved in Tris buffer and incubated at room tem perature overnight. No indication of any reconstitution was detected in the case of PCP treated with 50 per'cent acetone. However, a partial reconstitution was observed in the case of PCP treated by 30 per cent acetone. This partial recon stitution was detected by the peridinin CD band splitting but the chlorophyll CD band was not restored (Figure 32) . This may indicate that the peridinin molecules rebind to the protein, while chlorophyll a either remains unbound or binds at one or more sites on the protein forming aggregates. The addition of acetone also quenches the fluorescence of chlorophyll a of PCP (when excited at 475 nm) . This is also indicative of chlorophyll aggregation. Temperature Dependence of PCP Fluorescence Figure 33 shows the fluorescence intensity of PCP Fig. 32. CD spectrum of PCP after "reconstitution" from 30% acetone. 112 113 (Glenodinium sp,) as a function of temperature. The depen dence on temperature of the fluorescence of PCP has been measured between 25 and 75° C, In this range, the shapes of the emission spectra remained unaltered, but the quantum yield of emission decreased as the temperature was increased. The fluorescence quantum yields (l/cj)) of PCP at different temperatures (—) are given in Table 3, Figure 34 shows In ^~/Z ^ ^^ ^ function of ^, where (j) is the fluorescence quan tum yield and T is the temperature in degrees Kelvin, Figure 34 shows a biphasic behavior with a slope yielding an acti vation energy of 12 kcal/mole from 25° C to 55'^^60° C and with a much higher slope above 60° C with an activation energy of 31 kcal/mole. This behavior is also reflected in fluorescence intensity as a function of temperature. The fluorescence intensity of PCP decreased slowly with increas ing temperature up to 55'V'60° C. Above 60 C, the rate of fluorescence quenching by temperature was much greater. The CD band splitting in the CD spectra gradually disappeared as the temperature of the PCP solution was gradually in creased, with the sharpest decrease occurring between 55 and 65° C (Figure 35), Photobleaching of Chlorophyll a The photobleaching techniques have already been de scribed in the experimental section. Free chlorophyll a Fig. 33. Fluorescence emission of PCP (Glenodinium sp.) in Tris buffer (pH 8.4) as a function of temperatiore. X.em 475 nm. 115 640 680 720 760nm Fig. 34. Temperature dependence of PCP fluorescence. In —p.|. ,^?' have been obtained graphically by measuring the slopes of the negative tangents to the curve obtained by plotting the values of l/(y against 1/T. 117 Table 3. The calculated values of In cl[ 1/T) with reciprocal of absolute temperature for PCP (Glenodinium sp.) in Tris buffer (pH 8.4) . °I~7'/iyi| values have been obtained graphically by measuring the slopes of tne negative tangents to the curve obtained by plotting the values of 1/(|) against 1/T. 119 d(- T X 10-^ •'/*^ xlO-3 1^ d{-l/ 1.03 3.36 0.65 6.47 1.09 3.30 0.73 6.59 1.16 3.25 1.00 6.91 1.22 3.19 1.43 7.27 1.25 3.14 1.82 7.51 1.39 3.10 2.25 7.72 1.45 3.05 3.43 8.14 1.72 3.00 5.40 8.59 3.13 2.96 11.33 9.34 5.56 2.92 17.00 9.74 Fig. 35. CD spectra of PCP (Glenodinium sp.) as a fionction of temperature. (A) 55° C. (B) 65° c. 121 122 solution in ethanol-water (3:2 v/v) undergoes rapid photo- decomposition when irradiated with 660 nm light in the pres ence of oxygen. However, no photobleaching of chlorophyll a of PCP (Glenodinium sp,) in Tris buffer (saturated with oxygen) has been observed when irradiated with red light. Table 4 shows the rates of photobleaching of chlorophyll a in solution and in the photosynthetic light-harvesting pig ment (PCP) complex from marine dinoflagellates. The photo- irradiation has been carried out with free chlorophyll a and chl a of PCP in the HO as well as D2O solutions. The singlet oxygen mediated photooxidation did not occur for chlorophyll a of PCP (Glenodinium sp.) even in D^O solution in which the singlet oxygen lifetime is much longer, 20 ysec in 020* Table 5 shows the absorbance values of peridinin (475 nm) and chlorophyll a (665 nm) maxima before and after 12 hours of 660 nm light irradiation. Rose Bengal Sensitization and Singlet Oxygen Quenching Rose Bengal sensitized photooxidation of chlorophyll a has been carried out by irradiation at 557 nm in ethanol and ethanol-benzene solutions. The singlet oxygen-induced photooxidation of chlorophyll a is inhibited by singlet oxygen quenchers in the following order: 3-carotene, a-toco pherol, benzoquinone, DABCO, manadione, KI, cholesterol and ubiquinone. 6-carotene proved to be the most efficient of Table 4. Photobleaching of chlorophyll a^ in solution and in peridinin-chlorophyll a - protein (PCP) complex. 124 •H c o o •H o§ o ft o u ft ft u s ft c •H C •H c S -H O O ft°^ O O ft >— Q) o 4-1 H x; «(0 u tu O c C m in oo o o to CN tn +J 4-) >H OJ O o u E c C o 00 ro ro in ro CN ID o VD tn 4J (0 Table 5. Photobleaching of PCP in D^O. 126 Absorbance of PCP Absorbance of PCP Irradiation time in D„0 at in D-O at (hours) 475 nm 668 nm 0.0 0.837 p.205 12.0 0.780 0.198 127 all the singlet oxygen quenchers studied. The studies with cholesterol and KI quenchers are affected by solvent and sol ubility limitations. Table 6 shows the quantum yields for the photooxidation of chlorophyll a at several chlorophyll concentrations in ethanol in the absence of singlet oxygen quencher. A plot of reciprocal quantum yield against re ciprocal chlorophyll concentration produced a straight line. The intercept of this straight line yielded a quantum yield of 0.168 at infinite chlorophyll a concentration. Figure 36 is the plot of reciprocal quantum yield against reciprocal chlorophyll concentration. Tables 7, 8, 9, 10, 11 and 12 show the quantum yields of the photooxidation of chlorophyll a in the presence of the singlet oxygen quenchers, 3-carotene (in benzene), benzoquinone (in benzene), menadione (in ben zene) , a-tocopherol (in ethanol), benzoquinone (in ethanol) and DABCO (in ethanol), respectively. Figures 37, 38, 39, 40, 41 and 42 show the plots of reciprocal photooxidation quantum yields against the concentration of the singlet oxygen quenchers, 3-carotene, benzoquinone (in benzene), menadione, a-tocopherol, benzoquinone (in ethanol) and DABCO, respec tively. Of all the quenchers tested, only 3-carotene has the ability to quench singlet oxygen at diffusion-controlled rate. The singlet oxygen quenching efficiency of all other quenchers is much lower than that of 3-carotene. Concentra tions of the order of 1.0 yM 3-carotene effectively quench 128 the singlet oxygen thereby reducing the rate of chlorophyll a photooxidation compared to that without the quencher. Much higher concentrations (1.0 mM) of the quenchers other than 3-carotene are required to reduce the rate of chloro phyll a photooxidation. The singlet oxygen quenchers, KI (0.05 M) and cholesterol (0.1 M), have little effect on the rate of photooxidation even at such high concentrations. Higher concentrations of these quenchers cannot be made due to solubility restrictions, as already mentioned. Table 6. Quant\im yields of photooxidation of chlorophyll a in the absence of singlet oxygen quencher. 130 r r-l H H rH o O H rH O O O H o H H O H r-t r-l X rXH X Mi-e- >< X X X o O b CN m .0 0 in \D 03 CTi CN m '* in ID 4J •e- m — 3 r-, Cr< •a I rH ro CN IN CN CN rsj CM 0) E I I I 1 1 CN O O O O 1 1 •c^ ^ rH H o O tn r-l r-i r-l Ho 6 0) !«! X X 3 H X X X 4-1 3 O H O H CO in OD r^ S " r^ in ro CN 3 rH fN H Ol O s O t(nU 1 ro H rH rH e O <-l rH r-i r-i O o O o u H r-l H rH. o O \ H r-l 4-1 0) X X rH C(OU tHn X X X X X « 3 VD in VD o o rH 00 ID in O OJ ro r-l r-i (M H « H H r-l r-l rH (Mo l ro ro O o O ro ro ro O O O H lu CN in o o O CN ID 00 o r\i in H f^J u c "^ .5f o ^ I I I I I I I o O II £ o o o O o H 4-1 C H X o in O H D O o CN o ro o C 00 in o O MH ^ "sj" ro U 0 in Fig. 36. The Stern-Volmer plot of photooxidation of chloro phyll a_ in the absence of singlet oxygen quencher; reciprocal quantum yield (molecules/cm -quanta) against reciprocal concentration (M~ ) of chlorophyll a^ in ethanol (correlation coefficient = 1.00, inter cept = 5.95 and slope = 0.03, from least squares analysis). 132 CI/(chl)]xlO -3 Table 7. Quantiim yields of photooxidation of chlorophyll a_ in the presence of the singlet oxygen quencher, 3-carotene in ethanol- benzene (1:2 v/v). Chlorophyll a concentration 1.25 x 10~^ M. 13 o ro ro ro ro O O O o o r-l H H r-l H Hl-e- X X X X X o O ^D o r-^ .7 5 in VD VD (X) to ^ § — 3 _ 0^ •a I '3' rH ro I ^ 0) E I I o o o o 1 '>.X r-i tn r-l o 6 tu X X 3 H X C u o H (0 Q) 3 H Of 0 s o cn I ro E N CM rvj rsi r^i o -H r-i r-i H r-i O O o O o 44 in r-l rH H rH H X « rH X X X X 3 O ^ in H 'fl' u •^ CN 0) in r>) o H 0 S c -H OJ E o in ro r- ro 4J < M Z^ 2 S "^ Q ^ O fH H H O O <1 O O O O O c c 0 0) VD VD VD VD •H 4J I I I I I 4J 0 O O o ro !H ^ o o U (0 S H 4-1 0 — X C I X OJ CQ rM in c CN ro CTi in 0 IW U 0 VD' ro Fig. 37. The Stern Volmer plot of inhibition of chlorophyll a photooxidation by the singlet oxygen quencher, 3-carotene; recip rocal quantum yield (molecules/cm -qioanta) against concentration (M) of 3-carotene in ethanol-benzene (1:2, v/v) (correlation coefficient = 0.93, intercept = 5.1 x 10"^, slope = 7.86 x 10 , from least squares analysis). 136 •~t ^ - ro O - OJ X * O OD CD £-Olx(0/|) Table 8. Quantum yields of photooxidation of chlorophyll a^ in the presence of the singlet oxygen quencher, benzoquinone in ethanolbenzene (1:2 v/v). Chlorophyll a. concentration 1.25 X 10~5 M. ~ 138 n ro m o o ro ro r-i o o H|-e- X in o o t^ CO o 00 H a^ in VD CO o H to +J •e:;:^- (c0 w rj v I rHro I I I in Q) E 1 •H O o o o o NX r-i o to H r-l X X g o X 3 rH in cri 4J 3 '3' ro OJ o rrl (S0 '0^) 3 H cn 0( 0 s o to I CM CN og ro rH e O O r-i o H o Q) \ o 4-1 tn (fl 0) K rH 03 3 CO 00 CO U '3' o ro 03 Q) H o^ 0 s C CO CN r^ in ro H O 00 •p e r^ VD H H O o O i5 \ O O O o O « Q o < HH o 0 c c 0 o ro ro ro ro ro •H c I I I +J •H O 10 3 ^ o o O o r-i I-I 4-1 o -^ X X X X X c N C o o in o in f CN ro in u Fig. 38. The Stern-Volmer plot of inhibition of chlorophyll a^ photooxidation by the singlet oxygen quencher, benzoquinone; reciprocal quantimi yield (molecules/cm-^-quanta) against concentra tion (M) of benzoquinone in ethanol-benzene (1:2, v/v) (correlation coefficient = 0.997, intercept = 5.21 x lO"^, slope = 7.'^2 x 10^, from least squares analysis). 140 o CD ,CD c.O|X(0/l) Table 9. Quantum yields of photooxidation of chloro phyll a in the presence of the singlet oxygen quencher, menadione in ethanol-benzene (1:2 v/v). Chlorophyll a^ concentration 1.25 x 10"^ M. 142 ro ro ro ro ro O O O o o H H H H H X X X X X H -e- CO ro in o o in VD CO H VD I^ 00 (T\ 0 s in o CO o CN c o cy> t^ r» VD (U -rl H o o o O 4-1 e o o o o O CO \ * O < c 0 CN CN CN rv) Oi •rl I I ' ' u +J o (0J-i t3 (0 o o o o c c ^ H r^X r-lX r-lX r^^X Q) 0) S u E ^ X CM ro in c O IH U 0 Fig. 39. The Stern-Volmer plot of inhibition of chlorophyll a^ photooxidation by the singlet oxygen quencher, menadione; recripro- cal quantum yield (molecules/cm -quanta) against concentration (M) of menadione in ethanol-benzene_(1:2, v/v) (correlation coefficient 0.999, iintercepr t = 5.42 x 10"^, slope =1.13 x 10 , from least squares analysis) 144 00 CD £.OIx(0/|) Table 10. Quantum yields of photooxidation of chlorophyll a^ in the presence of the singlet oxygen quencher, a-tocopherol in ethanol. Chlorophyll a concentration 1.59 x 10~ . 146 ro ro ro ro ro o O O O O O H H H r-l H r-i X X X X X X rH -e- o o o in .9 2 .8 0 H in VD t^ CO CTi o (0 4J ^ C -e- CO — 3 in rHro I 1 I I I I O a) e o o H o •rl D tn H E OJ 3 H CTi X in (Tl VD CM +J 3 H Ol O in c o (0 Q) cn 3 H Oi O s u tn I CM CN rM CN ro H H r^i H H e O O O O O \o H H H o CD tn X 4-1 0) (0 H VD O) 00 00 O) 3 O) Ol O ro Ol u 0) (Tl 00 H O s c CD -H O) O r«- 00 cn CN 4J a cn CO p~ vD in in (0 \ o o o o o o O:; Q o o o o o o o < MH O 0 o •H Q) 4-1 X (0 a^ •SH 0 s +J u >— C o 0) 4J o I c 0 s u Fig. 40. The Stern-Volmer plot of inhibition of chlorophyll a photooxidation by the singlet oxygen quencher, a-tocopherol; re ciprocal quantym yield (molecules/cm -quanta) against concentra tion (M) of a-tocopherol in ethanol (correlation coefficient = 0.988, intercept = 5.71 x 10"^, slope = 3.92 x 10^, from least squares analysis). 148 o lO c-O|x(0/|) Table 11. Quantum yields of photooxidation of chlorophyll a^ in the presence of the singlet oxygen quencher, benzoquinone in ethanol. Chlorophyll a concentration 1.50 x 10" M. 150 1 ro ro ro ro ro o O O O H o O rH H H H H H|-e- X X X X X X in o O .1 5 Ol .8 0 CO .5 0 CN VD VD t~- 00 O) 10 . H rfl —• (0 ... =• r-i I Ol in in Olro I H 1 1 o I 1 •::? s H o o o o >i u r^i H ^ \ X X X E tn ro ro ro 3 cu VD ro o (n 4J H CO r-i C 3 (0 O cn CO 3 0) 0< H 0 s o 0) to O) I Ol Oi H H r-i H rH O H O O o O o H o \ rH 0) tn X 4-1 fO in in VD 3 CN o o 00 CU cn rH O s c ^ in r~ in VD I-- (U -H (3^ CO r- VD in "^i 4-1 E o o o o o o \ o o o o o o S Q O < UH 0 (U c c 0 0 ro ro ro ro ro ro •H c I I I I I I +J •H o o o o o O 3 ^ r-i (0 W S H r-l 0 -- X X X X 4-1 N X X in in in Ua (cU o in o fN ro in o Fig. 41. The Stern-Volmer plot of inhibition of chlorophyll a photooxidation by the singlet oxygen quencher, benzoquinone; re ciprocal quantum yield (molecules/cm -quanta) against concentration (M) of benzoquinone in ethanol (correlation coefficient = 1.00, intercept = 5,47 x 10 , slope = 1.35 x 10 , from least squares analysis). 152 Table 12. Quantum yields of photooxidation of chlorophyll a^ in the presence of the singlet oxygen quencher, DABCO in ethanol. Chlorophyll a concentration 1.25 x 10"^ M. 154 ro O "5r O O O rH r-l o H r-l H X X r-il-e- X X X o O ro o O .3 0 O VD (0 ^ 4-1 -e- c —' (0 in I I I in r-i I I r o o O o o euro r-i r-l H r-i "1:1 6 X >i o X X ro o in ^ \ (Tl o cn CN E w in cn 3 (U VD 4J H (C 0 5 «> 0( H 0 s o ^ CTl 00 CO 00 O CTl VD ro r-i O o o O O O o o O Rat e D/mi n • ^ o d d o • o c ro ro ro ro ro 0 1 1 1 •H o o 1 1 1 4-1 uo o O O (0 H H H H § X X X 4J Q X X C CU •H O 0. 5 0 1. 0 3. 0 6. 0 0. 0 0c o Fig. 42. The Stern-Volmer plot of inhibition of chlorophyll a photooxidation by the singlet oxygen quencher, DABCO; reciprocal quanttmi yield (molecules/cm -quanta) against concentration (M) of DABCO in ethanol (correlation coefficient = 1.00, intercept = 5.55 X 10 , slope = 1.48 x 10 , from least squares analysis). 156 MxlO' CHAPTER V DISCUSSION The extremely short excited state lifetimes ('vlO -14 sec) and the nonfluorescent nature of carotenoids (6) made it difficult to investigate how the light-harvesting caro tenoid pigment transfers its light energy to chlorophyll for photosynthesis. The isolation of the highly water- soluble PCP complex (1-5) from marine dinoflagellate algae provided an excellent model to study how this energy transfer occurs in conditions in vivo. The following discussion deals with the characterization of the PCP complex in order to understand how this energy transfer from the carotenoid, peridinin, to chlorophyll a occurs in the PCP complex and hence in plants in general in conditions in vivo. It is not very clear to conclude from the absorption spectrum (Figure 22) whether the peridinin to chlorophyll a ratio in PCP from A. carterae is 9:2 (or 4.5:1) (5) and if it is different from the other three PCPs (peridinin: chl a, 4:1) since the chlorophyll Q^: peridinin B ratio is essentially identical, i.e., 1:4.0 for A. carterae PCP and 1:3.9 for other PCPs. Figures 24-26 show that PCP from A. carterae possesses the same spectroscopic characteristics 157 158 as the other PCPs. The molecular orientation of peridinins with respect to the chlorophyll a Q axis (Figure 22, fluor escence polarization) and the CD splitting of the main ab sorption band (Figure 24) of peridinin is exactly similar to other PCPs. There is no indication of the presence of two chlorophyll a molecules in one PCP complex as shown by the lack of CD splitting of the chl Qy bands (chl-chl interac tions in the 650-700 nm region). Other considerations should also be taken into ac count to come to conclusions as to whether A. carterae PCP possesses 9 peridinins and 1 chlorophyll a molecules as was carefully investigated and concluded by Haxo, et a3^ (5) . The A. carterae PCP protein is relatively small in size as indicated by its molecular weight of 39,200. The question then arises as to how 11 chromophores, 9 peridinins and 2 chlorophylls can be accommodated within the protein molecule which is globular (dynamic depolarization and fluorescence quenching data). The fluorescence quenching results indicate that the chlorophyll molecule or molecules (two) are not exposed to the solvent but rather buried inside the protein crevices since the chl fluorescence cannot be quenched by the external quenchers. However, the possibility of two chloro phyll molecules in a crevice is already ruled out due to lack of chl-chl interactions in CD spectra (Figure 24). Appar ently, the entrance to the crevice is so narrow that the 159 hydrated iodide ion cannot penetrate into it to quench the fluorescence. Based on the results obtained, absorption, polarization, CD, fluorescence excitation, dynamic depolari zation and fluorescence quenching, it can be said that PCP from A. carterae is similar to other PCPs. The results obtained on PCP clearly show that tjhe energy transfer process from peridinin to chlorophyll a is very efficient. Moreover, in order for energy transfer to occur, a very specific molecular topography of the PCP com plexes is required since denaturation causes loss of energy transfer. Therefore there exists a relationship between the energy transfer efficiency and molecular arrangement of the PCP complexes. Peridinin showed no fluorescence in native or de natured PCPs even at 17 K on the single photon counting spec trofluorometer. This indicates that the fluorescence quantum yield of peridinin is less than 10-1-5 0 -4 (limitatio. . . n of the instrument). This means that the mean lifetime of the peridinin excited state (•'"B) is on the order of less than or equal to 10-1 4 - 10-1 3 sec (les,, s tha^ n 1,„-10 4 sec for values of fluorescence quantum yield less than 10 , radiative life- , -9 • • time of the -^B state of peridinin being 10 sec) . This is too short a lifetime to transfer excitation energy from peri dinin Ifi state to chlorophyll Q^ state. Critical distances corresponding to these quantiom yields have been calculated 160 to be 5.8'V'8.6 S, assuming that a hypothetical fluorescence spectrum of peridinin is roughly a mirror image of its ab sorption spectriim (Figure 43) (65,66). The Forster vertical distance calculation is already explained in the methods section. Figure 44 shows the absorption spectrum of chl a normalized with respect to peridinin hypothetical fluores cence. Since the critical distance for 50 per cent energy transfer is 5.8'\^8.6 A, the distance between peridinin and chlorophyll a for 100 per cent efficient energy transfer in the PCPs must be substantially less than the 5.8'^8.6 A cal culated. The rate-limiting step in the energy transfer process from peridinin to chlorophyll is the chlorophyll emission. Thus the rate constant for the transfer of energy from peridinin to chlorophyll a needs to be only greater than 2 x 10^ sec""*" (fluorescence emission lifetime of chloro phyll a in PCP-^-S x 10"^ sec) . On the other hand the calcu lated critical distance of 5.8'^'8.6 2 is based on a peridinin excited state lifetime of lO"^"* -lo""^^ sec. These arguments based on lifetime and critical distance between monomeric peridinins and chlorophyll a are contradictory to the effic ient energy transfer in PCPs. It should also be noted that the critical distance calculation based on Forster energy transfer is valid only if the rate of energy transfer from peridinin to chlorophyll a is much less than that of the Fig. 43. Absorption and fluorescence (hypothetical) spectra of peridinin in ethanol. The hypothetical fluoresence spectrum was drawn on the basis of a characteristic mirror image relation ship found for smaller polyeres. 162 I g X £ Fig. 44. The absorption spectrum of chlorophyll a^ normalized with respect to the peridinin fluorescence spectrum. 164 1-43 1-51 I-59 1-67 cm-'xIO'* 165 vibrational relaxations of peridinin. The very short life time of peridinin and its lack of fluoresence must be effec tively overcome in PCPs. The absorption spectra of PCPs (Figures 9, 17, 18, 22) exhibit a shoulder at approximately 510 nm which is most prominent in PCP from A. rhyncocephaleum. This is an indication of the presence of two components in the main absorption band (B <= ^A) of peridinin, one blue- shifted maximum and the other, red-shifted shoulder. This has been confirmed by the CD spectra (Figiires 13, 21 and-24) which show the main absorption band of peridinin being split into two distinct approximately equal ellipticity bands of opposite signs. The rotational strengths of both the bands are almost equal. This indicates exciton interactions be tween two peridinin molecules. The four peridinins present in one PCP complex form two sets of dimeric excitons. Denaturation completely destroys CD band splitting though peridinins are not dissociated from the apoprotein as indicated by induced CD of peridinin (Figure 14). This clearly suggests that the unique structural feature of four peridinins arranged to form two sets of dimeric excitons is essential for energy transfer in native PCPs. Fluorescence polarization data shown in Figures 9, 17, 18 and 22 suggest that the orientation of both the ex citon components of the peridinin band corresponding to the CD maximum and the CD minimum are approximately the same. 166 i.e., 45-50 with respect to the fluorescence emission of chlorophyll (Q^ polarization axis). Figure 45 shows the possible molecular arrangements of the PCP complexes, satisfactorily accommodating all the available data. As already discussed, the PCP protein is spherical and provides a crevice to accommodate the chromophores. This indicates that the proposed molecular topography (Fig ure 45) is not coplanar though the exciton transition moment vectors are maintained at 45-50° to the Q axis. On the y basis of only the polarization data of fluorescence excita tion shown in Figures 9, 17, 18 and 22, one can propose a molecular arrangement with each peridinin making contact at four corners of the chlorophyll chlorin ring, but this arrangement is inconsistent with the CD data discussed pre viously. The maximum and minimum of CD bands in Figures 13, 21 and 24 yield an exciton split of 3000 cm" which corres ponds to a distance of about 12 A between the centers of mass of two peridinin transition moment vectors in each dimeric exciton. The peridinin transition moment vector lies along the long axis of the molecule. The 12 A intermolecular distance between two peri dinin molecules in each dimeric exciton is calculated from the equation 10 as described in the theoretical section. The sufficiently large CD exciton split of 3000 cm" indi cates that the dimer model is preferred over the tetrameric Fig. 45. A probable molecular arrangement of chlorophyll a^ and peridinins based on relative orientations of transition moments (double arrows) of Q (fluorescence) and B^, B_ (exciton) transitions. 168 (a) .^"N^" 169 array and that there be no interaction between the dimeric excitons. Smaller exciton bandwidth is expected for the tetramer with larger intermolecular distance(s). Another alternative geometry for four closely coupled oscillators that result in two allowed transitions in absorp tion and CD is also possible (14, 15). A rectangular or tetrameric array can be formed by bringing the peridinin dimer pairs in Figure 45 closer together and placing the four peri dinins on the perimeter of a sphere above the chlorophyll plane. This results in two allowed and two forbidden transi tions (14). This tetrameric array is also consistent with the excitation fluorescence polarization observed. The forbidden components are assumed to be spherical. The two dimer pair model is favored over the tetramer on the basis of the observed data including the magnitude of the exciton split and small perturbation of the peridinin ab sorption spectriom. The asymmetric nature of the chlorophyll n-electron cloud and apoprotein makes it unlikely for two forbidden exciton states of the tetramer to be so rigidly forbidden that the absorption and CD intensities are prac tically zero. The other possible tetrameric arrangements such as the stacked, linear and alternate translational models are inconsistent with the fluorescence polarization and CD data. The stacked and linear dimer arrangements should produce 170 hypsochromic (blue) and bathochromic (red) shifts respec tively (14), but such shifts are not noticeable in the ab sorption spectra (Figures 9, 17, 18 and 22) . It is likely that the exciton interaction signifi cantly lengthens the lifetime of the peridinin excited state for energy transfer to occur. It is also probable that non- or near-degenerate exciton coupling between the peridinin and chlorophyll excited states contribute to the efficient energy transfer. Since hydrophobic fluorescence probes such as ANS (1- anilino-8-naphthalene sulfonate) do not bind to the PCPs, the protein surface is highly hydrophilic. The lack of ANS bind ing is judged by its fluorescence and polarization degree. The addition of ANS to equi-molar concentration of the PCP protein did not produce any enhancement of ANS fluorescence. The ANS fluorescence is increased significantly as well as blue-shifted upon binding to a protein hydrophobic surface. In the presence of PCP, the ANS fluorescence is reabsorbed by peridinin in the peridinin absorption region. The polar ization degree of ANS remained at or near zero both in the absence and presence of PCP. Significant increase in polar ization degree should be observed, if indeed ANS binds to the PCP protein. Fluorescence lifetimes of ANS showed an increase but varied considerably between measurements in the presence of PCP, indicating heterogeneous interactions between PCP and ANS. The increase in lifetimes may indicate 171 weak interactions between ANS and PCP. No strong non- covalent binding can be predicted due to the lack of en hancement of ANS fluorescence and polarization degree. On the other hand, the chromophore binding environment is hydro phobic. The trp or tyr fluorescence is enhanced readily upon PCP denaturation, suggesting that these residues are in the hydrophobic binding environment of the chromophore. The trp or tyr fluorescence is being quenched by energy transfer to peridinin and/or chl a (Figure 46). Attempts to reconstitute PCP from acetone denatura tion were only partly successful (Figures 31 and 32) . Peri dinin molecules re-bind more readily at the original bind ing site, but chlorophyll a CD band is not restored. The CD maxima and minima of reconstituted PCPs are nearly identical to those of native PCPs. Since re-binding of peridinin does not require chl a binding, peridinin and chlorophyll a are bound on opposite side walls in the crevice or there is no substantial molecular overlap between the chl a and peri dinin planes. The temperature dependence of PCP fluoresence can be described in terms of two rate constants, one temperature dependent and the other temperature independent. chl ^^^-^ ^chl (23) k- Ichl ^ > chl + hVf (24) Ichl ^^ > chl + A (25) lehl !ll—^ chl + A (26) Fig. 45. The corrected fluorescence spectriom of PCP (Glenodinium sp.) at room temperature in Tris buffer before (A) and after (C) denaturation with 1% SDS. (B) Fluorescence of 1% SDS solution. 173 280 320 360 400 nm 174 If k is the temperature independent rate constant and k^, the temperature dependent rate constant, the quantum yield of PCP chlorophyll a fluorescence is related to the tempera ture by the equation 1 1 - ^2 , ^3 ^-E/RT * "^ " k7 k^ ^^^^ Differentiating with respect to temperature and taking the logarithm. The emission rate constant, k^, is temperature independent and is equal to 6.6 7 x lo"^ sec"""- [radiative lifetime of chl a 15 nsec (67)]. If k^ = k^e-EAt ^here k^ is the frequency factor calculated to be 7.5 x 10^^ sec"^ and k^, the temper- 2 -1 ature dependent rate constant is equal to 2.78 x 10 sec at 20° C. The temperature independent rate constant, k2 is 7 -1 equal to 1.4 x 10 sec . The activation energy of 12 kcal/mole for 25 to 55° C may correspond to breaking of hydrogen bonds and hydro phobic associations in the PCP protein. Above 55° C, the activation energy of 31 kcal/mole may correspond to the de naturation of PCP. Since no photodecomposition of chlorophyll a of PCP occurs when irradiated with 660 nm actinic light, chlorophyll a is effectively protected by peridinin from photodynamic 175 damage (Tables 4 and 5). Oxygen apparently enters the chromo phore binding crevice and becomes singlet oxygen upon energy transfer from triplet chl a. The singlet oxygen so produced is immediately quenched by the adjacent peridinin with its low-lying triplet prior to the oxidation of chl a by singlet oxygen. On the other hand, free chlorophyll a in the absence of carotenoid or other singlet oxygen quencher, is not pro tected against photodynamic damage induced by singlet oxygen (Table 4). The singlet oxygen generated by energy transfer from triplet chlorophyll a attacks the ground state chl a molecule causing its photodecomposition. The rate of photo- decompensation of chlorohyll a by singlet oxygen is enhanced by the presence of a photosensitizer such as Rose Bengal. The following scheme is proposed for the Rose Bengal sensitized phtooxidation of chl a: RB |a__^ V (29) IRB ° > RB + hVp (30) IRB i-5» RB + A (31) IRE ^2_^ 3RB (32) 3j^ 3_-, RB + A (33) 3RB + 3o2 ^i-^ ^02 + RB (34) lo^ ^5 .^ ^''^ ehl + lo, -J^^ ^chlO^ (36) 2 In ^7 , 3Q + 3o (37) Q + ^02 176 Where RB is Rose Rf^nrrai ID-D • T, O ^ Kose Bengal, -^RB IS Rose Bengal singlet, ^m is Rose Bengal trinlo^ 3^, A ^ A. • -, ^ T^ripiet, O2 IS triplet (ground state) oxygen, O2 is singlet oxygen, chl02 ^^ photooxidation products of chlorophyll a, Q is the singlet oxygen quencher and \ is the triplet quencher. The rate of chl photodecomposition is given by, „ _ -d(chl) , , 1 ^ ir~= ^6 (°hl) (S^ (38) d(^RB) _ I ^ ,1 , 1 -^ a - ko (^RB) - k;^ (IRB) - k„ (-^RB) = 0 (39) (-^RB) = ^a k^ + k^ + k2 (40) d(^05) 3 ^ - -^ = k^ (3RB) (30^) - k^ i\) - kg (chl) (I02) - k^ (Q) (I02) = 0 (41) flo \ - ^4 (3RB) (30?) ( O2) - —- (42) ^5 + kg (chl) + ky (Q) o ^i-^^ = k_ (IRB) - k_ (3RB) - k, (3RB) dt '^ 3 4 (^02) = 0 (43) ,3 V ^2 ("^RB) (3RB) = —^ i— (44) ^3 + k4 (3o2) Substituting the value of (•'•RB) from equation (40) into equation (44), 3 ^2 ^a CRB) = ^ (45) (VV^2) C k3tk4 (3o2)] Substituting equation (45) in equation (42), 177 (IQ ) _ V2^a(\) 2' (kQ+k;L+k2) Ck3+k4(3o2)l 1 [k5+k6(chl) + k7(Q)] ^^^^ From equations (38) and (46), we get kg(chl) k4k2la (^02) R ^W^2^ [k3+k4( 30^)3 1 (47) [k5+kg(chl) + k7(Q)] la = i = [ko+ki+k2) [k3+k4(3o2)] R k2k4(3o2) Kg (chl) Ck5+k6(chl) + k7(Q)] (48) I = [1 + kp+^l ] [1 + ^3 ,,_-j * k2 k4(3o2) k5 + ky (Q) [1 + ^ ] (49) ^6 (chl) In the absence of quencher, when (Q) = 0, , ^ k- + KT ^3 ^5 [1 + . ] (50) ^6 (chl) 1 1 ^o •'• ^1 For the plot of -x vs _±— , the intercept is [1 + j^ ^ (chl) J<2 [1 + —T- 3 ]. The concentration of oxygen is taken to ^4 (•'O2) be 10" M and the singlet oxygen decay rate constant, kg, is taken to be 1 x 10 5 sec —- ^1 (36) . 178 In the absence of Q, the singlet oxygen quencher, k -t-k-i }j the intercept [l + ^2 3 tl + k (JQ )1 is equal to 5.95 and the slope is equal to 3.00 x 10-2 (Figure 36). Therefore, k^/kg = 5.04 x 10"3 and kg = 1.98 X lo"^ M"-'^ sec"-"- (51) In the presence of Q, the intercept k +k k k ^^ "• ~k7^'^ ^1 •" k4 (3^^) ] [1+ kg (chl) ] is equal to 5.23 X 103 (Figures 37-39) from which k^/k^ = 1.1 x 10-2 D 6 and kg = 0.91 x 10"^ M"^ sec"^ [(chl) = 1.25 x 10"^ M]. (52) Thus there is good agreement of the biomolecular rate constant (for the oxidation of chl by singlet oxygen), kg, in the ab sence and presence of the singlet oxygen quencher. The bi molecular collisional rate constant, k_,, for the quenching of singlet oxygen by g-carotene has been estimated from the slope in Figure 37. 7.86 X 10^ (53) ky = 1.65 X 10^0 M"^ sec"-*- (54) Rate constant, k_, is calculated for other quenchers in a similar manner from Figures 38-42. Table 13 shows the rate constants, ky, measured for the quenching process of the singlet oxygen by the quenchers, 3-carotene, benzoquinone (in ethanol-benzene), menadione, a-tocopherol, benzoquinone (in ethanol) and DABCO. Quenching of singlet oxygen by 6-carotene Table 13. Photooxidation of chlorophyll a - singlet oxygen quenching rate constants, ~ 180 Biomolecular quenching rate constant Singlet oxygen quencher k_ (M"-'- sec" ) 3-carotene 1.55 x 10 Benzoquinone (in ^ ethanol-benzene) 1.62 x 10 Menadione 2.37 x 10 g a-tocopherol 1.05 x 10 Benzoquinone (in _ ethanol) 3.40 x 10 7 DABCO 3.11 x 10 181 takes place by a collisional mechanism which is diffusion- controlled, in good agreement with values obtained by others (35, 36, 40). Since the bimolecular quenching rate con stants for other singlet oxygen quenchers are several fold less than the diffusion-controlled reaction, their quenching mechanism(s) are different from collisional quenching. In the reaction scheme proposed for the Rose Bengal sensitization of chlorophyll a photooxidation, the reaction step, 3RB + chl 3. ^chl + RB i^ which triplet state Rose Bengal transfers its energy to chl to produce triplet chl and other triplet chl reactions have been omitted. Whether neglect of this energy transfer from Rose Bengal triplet to chl is justified or not has been verified by the resulting rate constants, ky for the singlet oxygen quenching. The resulting of a k^ value of 1.65 x 10^ M" sec" for singlet oxygen quenching by 3-carotene suggests that it is appropriate to neglect the Rose Bengal-chl energy transfer sequence. Though the intersystem crossing probabilities are available for certain xanthene and thiazine dyes (49-51), they are not available for Rose Bengal. Since xanthene dyes other than fluorescein, i.e., dibromofluorescein, eosin (tetrabromofluorescein) and erythrosin (tetraiodofluorescein) 182 have singlet-triplet transition probability greater than 0.4, (erythrosin being 1.0), it can be assumed that Rose Bengal (tetrachlorotetraidodo fluorescein) also has a singlet-triplet transition probability greater than 0.5 and possibly approaching that of erythrosin, since halogenation increases singlet-triplet transition probability. This means that the Rose Bengal sensitization of chlorophyll a photo oxidation occurs via Rose Bengal triplet as proposed earlier in the reaction scheme. Since the first proposal by Foote et aJ.. (19, 21, 32, 36, 68) about the possible role of 3-carotene as the pro tector of chlorophyll from photodynamic damage by its own sensitization, there has been speculation and interest as to how this occurs in, vivo. In vitro studies clearly showed 3- carotene as an efficient singlet oxygen quencher, quenching singlet oxygen at diffusion-controlled rate (35, 36, 40) . 3-Carotene and other carotenoids are present in large quan tities in plants complexed with chlorophyll-proteins. Though such complexes have been isolated in the past, their lack of solubility in aqueous solution made it difficult to study the photodecomposition of chlorophyll and its prevention by caro tenoids. A study of the prevention of singlet oxygen-induced photodecomposition of chlorophyll in a carotenoid-chlorophyll- protein complex involves conditions close to conditions in vivo. It is thus interesting and significant to study a 183 system that compares closely to the system iri vivo. PCP is such a system which is well-defined with the complex being highly water-soluble. The in vitro studies clearly show that chlorophyll a is photodecomposed by singlet oxygen when irradiated with red light, the photodecomposition rate being enhanced in D2O due to the existence of singlet oxygen for ten times longer length of time than in HO. The in vitro studies have been performed to show that chlorophyll is indeed photo- oxidized by singlet oxygen. They also indicate that the addition of singlet oxygen quenchers do inhibit the photo oxidation of chlorophyll by collisional or some other mech anism depending upon the quencher. Thus the finding that 3-carotene and other singlet oxygen quenchers, some of which being present in plants, inhibit photooxidation of chloro phyll raises the question as to how this happens in plants. Studies on a system comparable to iri vivo system can answer this question. In the PCP system, the photooxidation of chlorophyll a is fully prevented by the carotenoid, peridinin, as al ready mentioned. The photodecomposition of peridinin when chlorophyll is irradiated indicates that oxygen indeed enters the hydrophobic crevice of the PCP and becomes singlet oxygen by energy transfer from chlorophyll a, the singlet oxygen 184 then being quenched by the low-lying triplet of the adjacent peridinin. This confirms the results obtained in vitro in the absence of protein and throws light on how peridinin protects chlorophyll from singlet oxygen-induced damage in vivo. CHAPTER VI CONCLUSIONS All the fo.Mr PCP complexes from Glenodinium sp., G. polyedra, A. rhyncocephale\am and A. carterae contain one molecule of chlorophyll a and four molecules of peridinin per-mole protein, from the absorption spectra (Figures 8, 17, 18, 22). Even the PCP from A. carterae which is thought to contain two chlorophyll a and nine peridinin molecules shows the same molar ratio as the other three PCPs, judged by the absorption spectrum (Figure 22). Peridinin has an absorption maximum of 475 nm whereas chlorophyll a maximum is at 665 nm. While the absorption spectra (Figures 8, 17, 18, 22) indicate a 4:1 ratio for peridinin to chlorophyll a in PCP, the fluorescence excitation spectra (Figures 20, 25) also indicate the same ratio of 4:1 for the fluorescence excitation intensities of peridinin to chlorophyll a maxima. From this, it can be concluded that the energy transfer ef ficiency from peridinin to chlorophyll a is 100 per cent. Circular dichroism spectra (Figures 13, 21, 24) of all the four PCPs indicate that the main absorption band of 185 186 peridinin is split into two CD bands with positive and nega tive eliipticities at 463 and 538 nm for Glenodinium sp., 464 and 541 nm for G. polyedra, 462 and 545 nm for A. rhyn cocephaleum and 464 and 540 nm for A. carterae. The chloro phyll minim;am of PCP (Glenodinium sp.) at 686 nm (Figure 13) is blue-shifted to 673 nm upon denaturation (Figure 14) in 1 per cent SDS suggesting that chlorophyll remains bound to the apoprotein. Peridinin also remains bound to the apopro tein as indicated by the induced CD minimum in the peridinin absorption region (Figure 14). No chl-chl interactions are indicated in the CD spectra. The fluorescence excitation polarization spectra (Figures 9, 17, 18, 22) of all the four PCPs indicate that the polarization degree for the entire main absorption band (i.e., both CD bands) of peridinin is 0.05'\^0.1 with respect to the chlorophyll Q^ emission. This means that each of the positive and negative components of the CD spectrum is at an angle of 45-^50° with respect to the Qy band of chlorophyll a. Based on the CD and polarization data, a probable mole cular arrangement for the four peridinin and one chlorophyll a molecules has been proposed (Figure 45). Fluorescence quenching experiments (Figures 27-29) reveal that the chromophores are buried inside a crevice. Lack of ANS binding on the protein surface indicates that the surface is highly hydrophilic and so the crevice (interior) 187 is highly hydrophobic in nature. Dynamic depolarization data (e.g., rotational relaxation time of A. carterae PCP 33 nsec) suggest that the shape of the PCP protein is spher ical. Therefore, the molecular arrangement (Figure 45) is not coplanar. Only partial "reconstitution" of PCP is possible from 30 per cent acetone (Figure 32). While peridinin re- binds to the PCP protein, chlorophyll a remains unbound sug gesting peridinins and chlorophyll bind on opposite sidewalls of the protein. The temperature dependence of PCP fluorescence (Fig ures 33, 34) shows a biphasic behavior with an activation energy of 12 kcal/mole from 25 to 55° C and 31 kcal/mole from 55 to 75° C. While the former corresponds to the break ing of hydrogen bonds and hydrophobic associations, the latter corresponds to protein denaturation. Chlorophyll a is not photodecomposed in H2O or D2O when irradiated with red light (Tables 4, 5). However, per idinin absorbance is decreased with this irradiation. This indicates that oxygen indeed enters the hydrophobic crevice of PCP to become singlet oxygen by energy transfer from chlorophyll. The singlet oxygen so generated is immediately quenched by the low-lying peridinin triplet. The protection of chlorophyll a from singlet oxygen- induced photooxidation in the PCP complex which is similar to 188 in vivo conditions is compared with in vitro studies where the singlet oxygen quencher is added to chlorophyll. Chloro phyll is not protected against singlet oxygen-induced damage in the absence of quencher. However, the addition of quench ers, 3-carotene, a-tocopherol, benzoquinone, DABCO and mena dione inhibit the photooxidation of chlorophyll a in that order. The photosensitizer. Rose Bengal, enhances the rate of chlorophyll photodecomposition. The mechanism of Rose Bengal sensitized photooxida tion of chlorophyll a involves nine steps as already shown. 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