J . Phys. Chem. 1985,89, 2979-2982
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Effects of Molecular Organization on Photophysical Behavior: Lifetime and Steady-State Fluorescence of Chlorophyll a Singlets in Monolayers of Dioleoylphosphatldylcholine at the Nitrogen-Water Interface M. L. Agrawal, J.-P. Chauvet, and L. K. Patterson* Radiation Laboratory and Department of Chemistry, University of Notre Dame,' Notre Dame, Indiana 46556 (Received: February 25, 1985)
The fluorescence lifetimes of the chlorophyll a singlet have been measured in spread monolayers of dioleoylphosphatidylcholine at the nitrogen-water interface. Determinations were made as functions of both chlorophyll concentration and compression. A single exponential decay with a lifetime of 5.5 f 0.3 ns was observed at 0.0084 mole fraction chlorophyll; this falls to 1.0 f 0.1 ns at 0.0800 mole fraction. Such findings indicate a limiting quantum yield approaching that found in isotropic polar media as well as vesicles; it is about 90% of that found in nonpolar media. Fluorescence and excitation spectra have also been obtained. An unstructured fluorescence band is observed with a maximum at 678.5 h 0.5 nm, indicating that the monomer is the predominant fluorescent form of chlorophyll a. Fluorescence intensity data in the same concentration range are presented for comparison. Plots of both intensity and lifetime, normalized to values obtained at infinite dilution, as functions of molecules/cm2, coincide quite closely. The agreement between these two types of data-while indicating that statistical chlorophyll a pairs are involved in the quenching mechanism-suggests that both fluorescence and energy transfer from such pairs compete with the process of quenching.
Introduction The organization of various constituents of the photosynthetic apparatus, especially the chlorophylls, within the thylakoid membrane is known to be crucial to the mechanism of light harvesting by the chloroplast. Any system selected as a model for investigating the dependence of chlorophyll photophysical behavior on the structure of its environment should provide both the means for controlling the organization of the chromophore within its surroundings as well as monitoring it. It has long been recognized that, by these criteria, the spread monolayer at the nitrogen-water interface provides an excellent system for such study. Components of the reaction environment may be confined to decreasing areas in controlled way, forcing accommodation to the space available by rearrangement. Any changes in interactions within the layer may be monitored by the macroscopic parameter of surface pressure and area per molecule while photophysical behavior may be monitored by some parameter such as fluorescence lifetime or yield. Due to self-quenching, early work with pure chlorophyll failed to give measureable fluoresence.2 Later studies which incorporated chlorophyll into monolayers of various lipids and related host molecules clearly demonstrated the dependence of fluorescence on concentration, separation of chromophores, and the nature of the host m a t r i ~ . ~These - ~ systems exhibited significant self-quenching, but the quantum yields were not explicitly measured nor were lifetime data obtained. Several studies have been carried out to investigate the interaction of chlorophyll with various hosts (phospholipids, phytol, etc.) by means of their force-area behavior. These have shown that such systems exhibit ideal mixing or closely approach Recently, measurements of fluorescence intensity have been carried out for chlorophyll a (Chl a ) (1) The research described herein was supported by the Office of Basic Energy Sciences of the US.Department of Energy. This is Document No. NDRL-2670 from the Notre Dame Radiation Laboratory. (2) Ke, B. In 'The Chlorophylls", Vernon, L.P., Seely, G. R., Eds.; Academic Press: New York, 1966; pp 253-79. (3) Tweet, A. G.; Gaines, G. L.; Bellamy, W. D. J . Chem. Phys. 1964, 40, 2596-2600. (4) Trosper, T.; Park, R. B.; Sauer, K. Photochem. Photobiol. 1968, 7 , 451-69. (5) Costa, S. M. De B.; Froines, J. R. Harris, J. M.; LeBlanc, R. M.; Orger, B. H.; Porter, G. Proc. R . SOC.London, Ser. A . 1972. (6) Gonen, 0.;Levanon, H.; Patterson, L. K. Isr. J . Chem. 1981, 21, 271-6. (7) Heithier, H.; Mohwald, H. Z . Naturforsch. 1983, 38c, 1003-10.
0022-3654/85/2089-2979$01.50/0
in such a lipid system, dimyri~toyllecithin.~ Interpretation of kinetic behavior from fluorecence data, however, ultimately depends critically on the values of fluorescence lifetime assumed from the system under study; but, to date, real time measurements of fluorescence decay have not been reported for chlorophyll pigments in spread monolayers at the gas-water interface. The present study provides singlet lifetime measurements for Chl a in a monolayer of dioleoylphosphatidylcholine (DOL) as a function of chromophore concentration and compression. Along with steady-state measurements of fluorescence spectra and intensities, these data demonstrate that in such a system the quantum yield of fluorescence approaches that found in homogeneous solution and that the concentration quenching observed is, indeed, related to dynamic processes at Chl a concentrations below 0.1 mole fraction.
Experimental Section A rectangular teflon Langmuir trough was used for these studies. Surface pressure measurements were carried out by means of a Cahn 2000 electrobalance fitted with a Wilhelmy plate. The fluorescence intensity measurements from the monolayer were taken with a fiber optic system connected to a Spex-Fluorolog single-photon-counting fluorometer. The detailed description of the fluorescence setup can be found elsewhere." All emission measurements were taken in ratio mode recording which corrects the data for long-term fluctuations in excitation light intensity. Lifetime measurements on the fluorescent layers were made by means of single-photon-counting equipment in our laboratory which is also described in detail e l ~ e w h e r e . ' ~ -To ' ~ illuminate the monolayer with pulses of exciting light, the optics of a Nitromite nitrogen laser (PRA, London, Ontario) are focused on the water surface at an angle sufficient to assure that incident light is not scattered downward toward the bottom of the trough. A fiber optic bundle is positioned to gather light from the portion of the (8) Almog, R.; Berns, D. S. J . Colloid Inrerace Sci. 1981, 81, 332-40. (9) Aghion, J.; Dupont, I.; LeBlanc, R. M. J . Colloid Inrerace Sci. 1981, 82, 569-71. (10) (a) Tancrede, P.; Munger, G.; LeBlanc, R. M. Biochim. Biophys. Acta 1982, 689, 45-54. (b) Tancrede, P.; Parent, L.; Paquin, P.; LeBlanc, R. M. J . Coll. Interace Sei. 1981, 83, 606-14. ( 1 1) Loughran, T.; Hatlee, M. D.; Patterson, L. K.; Kozak, J. J. J . Chem. Phys. 1980, 72, 5791-7. (12) Federici, J.; Helman, W. P.; Hug, G. L.; Kane, C . ; Patterson, L. K. Comput. Chem., in press. (13) Subramanian, R.; Patterson, L. K. J . Phys. Chem. 1985.89, 1202-5.
0 1985 American Chemical Society
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The Journal of Physical Chemistry, Vol. 89, No. 13, 1985
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Figure 1. Excitation (Exc) and emission (Em) profiles for Chl a in DOL monolayers at the nitrogen-water interface. The excitation profile (monitored at 679 nm) (-) was taken at 26 dyn/cm and 5.5 X loi2Chi a molecules/cm2, The excitation spectrum of Chl a in benzene taken with the same apparatus is included (---); the emission profile (excited at 430 nm) was taken at 4 dyn/cm and 6.4 X 10l2Chl a molecules/cm2. IO
surface illuminated by the laser and to transport it to the emission monochromator. Compression of the layer proceeded at rates not exceeding 1 .O A*/(molecule min). Both fluorescence spectra and lifetimes were taken at various stages of compression. In all cases, the background signal from a clean, aqueous surface was subtracted from the emission of the layer to obtain the emission spectrum of Chl a in the layer. For all measurements a sealed chamber containing the trough was thoroughly flushed with nitrogen prior to introduction of the surface active materials. The aqueous subphase was also deaerated. A nitrogen flow was maintained throughout the experiment to avoid backdiffusion of oxygen into the chamber. A relative humidity of -90% and a temperature of 23.0 0.5 OC were maintained throughout the measurements. The Chl a was kindly furnished by F. Villain (Laboratoire de Physico-Chimie des Pigments Vegetaux, ENS, Saint-Cloud, France). The lipid was furnished by PL Biochemicals.
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Results Both the emission and excitation profiles for this system are given in Figure 1. For purposes of calibration, an excitation spectrum of 2 X 10” M Chl a in benzene solution was taken with the monolayer spectrometer and was found to compare within a few percent to the absorption spectrum of the same solution. The excitation spectrum in the Soret band from the layer resembles closely the absorption spectrum published for Chl a monolayers by Bellamy et aLi4and gives no evidence that emission arises from aggregated chlorophyll. The emission spectrum, with its maximum at 678-679 nm coincides closely with that found in micellar solutions of triton X-100 and magnesium lauryl s ~ 1 f a t e . I ~It is, however, shifted from the values found for polar solvents such as the alcohols which lie around 672-674 nm.I6 Measurements of Chl a fluorescence maxima in benzene and methanol were made using our optical arrangement. The values obtained were 672 and 6 7 4 nm each within 0.5 nm of literature values.I6 With slits of 2-nm bandpass, a band as wide as that given in Figure 1, and the signal-to-noise ratio shown, this uncertainty o f f 0.5 nm is reasonable. As expected, the positions of both excitation and emission maxima reflect an interaction of the chromophore with a highly polar microenvironment. No satellite peaks were observed which would indicate fluorescent aggregates of the type suggested by observations in other host m a t r i ~ e s . ~ J ’ , ~ ~ Emission-decay profiles taken at Chl a mole fractions of 0.0084 (14) Bellamy, W. D.; Gaines, G. L.; Tweet, A. G. J . Chem. Phys. 1963, 39, 2528-38. ( 1 5) (a) Chauvet, J.-P. Ph.D. Thesis, University of Paris-Sud., 1983. (b) II’Ina, M. D.; Borisov, A. Yu. Biochim. Biophys. Acta 1981, 637, 540-5. (16) Szalay, L.; Tombacz, E.; Singhal, G. S. Acta Phys. Acad. Sci. Hung. 1970, 35, 29-36.
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Figure 2. Emission decay profiles for Chl a in spread DOL monolayers: (a) 4.7 X l o i 2Chl a molecules/cm*, 7 = 2.2 ns; (b) 0.79 X lo’* Chl a molecules/cm2, T = 5.5 ns. X2 values were 1.16 and 0.78. Excitation wavelength = 337 nm, emission wavelength = 679 nm.
and 0.048 (II = 2 dyn/cm) are given in Figure 2. The traces may be seen to follow single-exponential kinetic decays and yield lifetimes of 5.5 and 2.1 ns, respectively. While the number of counts per channel is not of the magnitude normally obtained in bulk solution with a low-intensity high-repetition-rate light source, repeated measurements with this system have shown a probable error of about *5-7%. Plots of first-order rate constant vs. Chl a concentration at surface pressures of 2, 10, and 30 dyn/cm are (17) Seely, G. R.; Senthilathipan, V. J . Phys. Chem. 1983, 87, 373-75. ( 1 8 j Subramanian, R.; Patterson, L. K.; Levanon, H. Photochem. Photobiol.,in press.
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The Journal of Physical Chemistry, Vol. 89, No. 13, 1985 2981 0.90-
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