Langmuir 1990,6, 1739-1742
1739
Effects of Molecular Organization on Photophysical and Photochemical Behavior. Interactions of Oxygen and Pyrene-Bearing Probes across the Air-Lipid Boundary in Spread Monolayers' Maria Bohorquez and L. K. Patterson* Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556 Received December 27, 1989.I n Final Form: April 29, 1990 Oxygen quenching of excited singlet pyrene probes in spread monolayers of dioleoylphosphatidylcholine (DOL), dipalmitylphosphatidylcholine(DPL), and dielaidylphosphatidylcholine(DEL), has been measured by time-resolved fluorescence. The probes used were a pyrene-bearing phosphatidylcholine (2PyPC) and 1-pyrenedodecanoicacid (Py-22). In all casea, oxygen quenching was diminished by compression of the monolayer system. Plots of k,, the pseudo-first-order rate constant for quenching of Py-22* by oxygen, vs area per lipid molecule in the layer approached linearity more nearly than those involving 2-PyPC. This behavior may arise from the greater freedom of movement associated with Py-22. In all cases involving DPL, the phase transition behavior of the lipid introduced irregularities into the plot of area/molecule vs quenching rate constant.
Introduction The influence that lipid mono- and bilayer structures exert on kinetic processes for which such structures may act as matrices has been a focus of study for membrane biochemists and, incresingly, for those who view lipid molecular organization as a readily adjustable parameter for mechanistic control in such diverse areas as solar energy storage, microelectronics, and molecular recognition. Among their characteristics, these structures have the unique ability to provide a physical barrier to species crossing the boundary between aqueous and lipid regions or between the gas and lipid phases. The possibility for manipulating the efficiency of passage across this barrier has implications for a whole range of applications to the areas mentioned above. A model which lends itself readily to the study of such barrier properties is the spread monolayer at the airwater interface. In addition to controlling the packing of the lipid components and any substrate in the layer while measuring external penetration, one may monitor the state of organization through surface pressure-molecular area data2 Recently, the penetration of iodide from the aqueous subphase into monolayers of dioleoylphosphatidylcholine (DOL) and dipalmitylphosphatidylcholine (DPL) was studied through time-resolved fluorescence quenching of pyrene-bearing lipids incorporated into the layer^.^ I t was shown that for both lipids the rate of diffusion to the pyrene was linearly related to the area per molecule of lipid or more specifically to the space between lipid molecules. The most striking aspect of this finding was that the phase transition which occurs during compression of DPL had little effect on the linearity of the relationship. At 50 Az molecule, where there exists a tight-packed organization of the lipid, quenching could no longer be measured in a DPL layer. For the DOL system, however, there was a
*
Author to whom correspondence should be addressed.
(1) The research described herein was supported by the Oftice of Basic Energy Sciences of the Department of Energy. This is Document No. NDRL-3062 from the Notre Dame Radiation Laboratory. (2) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience: New York, 1966. (3) Bohorquez, M.;Patterson, L. K. Thin Solid F i l m 1988,159,133137.
residual "leak" of iodide a t areas below 85 A2 probably associated with the presence of two cis double bonds in the lipid. In the present study, we have addressed questions concerning interaction of oxygen from the gaseous phase with components of spread monolayers and the manner in which organization of the layer may affect those interactions. Here we monitor monomer excited-state quenching of a pyrene-bearing lipid by oxygen in monolayers of DOL, DPL, and dielaidylphosphatidylcholine (DEL). The physical characteristics of the latter fall somewhat between those of DOL and DPL. For comparison with the pyrene-bearing lipid, quenching has been measured for a long-chain fatty acid probe in DOL and DPL. I t may be seen that the rates of pyrene quenching and, hence, penetration of oxygen are markedly affected by the organization of the layer, but that quenching follows different relationships to lipid organization than did iodide.
Experimental Section The single photon counting apparatus and associated Langmuir trough equipment used in these experiments have previously been de~cribed.'~~ However, for these studies the excitation source employed was a mode-locked, Q-switched Quantronix NdYag system which provides an 80-ps pulse with a frequency of 5 kHz at the third harmonic, 354 nm. Integrated power at this frequency is about 10 mW. Dioleoylphosphatidylcholine (DOL), dipalmitylphosphatidylcholine(DPL),and dielaidylphosphatidylcholine (DEL) were purchased from Avanti Biochemicals. Concentrations of DOL and DEL were determined by standard microphosphate analysis techniques. The probe 2-(6-pyrenylhexanoyl)-cu-palmitoyl-sn-glycerophosphorylcholine (2-PyPC)was obtained from KSV Chemicals, Helsinki, Finland. 1-Pyrenedodecanoicacid (Py-22) was purchased from Molecular Probes. Premixed N2-02 was supplied by Mittler. Results Force Area Data. The surface pressure-area isotherm of DEL was measured over a pure aqueous phase on the Langmuir trough. Isotherms of DOL and DPL have (4)Federici, J.; Helman, W.P.; Hug, G. L.; Kane, C.; Patterson, L. K. Comput. Chem. 1985,9,171-177. (5) Subramanian, R.; Patterson, L. K. J. Phys. Chem. 1985,89,12021205.
0743-7463f 90f 2406-1739$02.50f 0
0 1990 American Chemical Society
1740 Langmuir, Vol. 6, No. 12, 1990
E
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Bohorquez and Patterson 50
1
- 0.95
-
-0.85 ;r a -0.75 x -0.65
-0.55
- 0.45
'
u;J ?
-
0.35 I20
2-1
a r e a /molecule [ Figure 1. Plot of k , the pseudo-first-order rate constant for quenching of 2-PyP8* by oxygen in a DOL monolayer (E), as a function of lipid area per molecule. The force-area curve for the lipid is also given (-). Measurements were carried out under a 1 '; 02 atmosphere.
area /molecule
[ A21
Figure 2. Plots of k , the pseudo-first-order rate constant for quenching of 2-PyP&*by oxygen in a DEL monolayer, as a function of lipid area per molecule. The force-area curve for the lipid is also given (-). Measurements were carried out under a 1%02 atmosphere (a) and a 2% atmosphere (m).
previously been measured in our laboratory and are given in figures below for comparison with the associated lifetime data. Lifetime Measurements. Time-resolved fluorescence mesurements were made with 0.015 mf of 2-PyPC or Py22 in monolayers of lipid under a nitrogen atmosphere and subsequently under one containing either 1% or 2 % oxygen. Lifetime data in all cases were analyzed in terms of the equation
G ( t ) = (yle-tl/'l + (yZe-'2/'2
(1)
where 71 denotes the decay of the pyrene monomer fluorescenceand 72 would generally be governed principally by excimer growth. Here 7 2 reflects some trough background signal due to the very low concentrations of probe present whose small fluorescent signals were further diminished by quenching. Data presented here were taken from 71 a t 380 nm, the wavelength of monomer fluorescence. Pyrene excimer behavior has previously been explored in detail for these probes in DOL,8v7 but for the purposes of the present study in quite dilute pyrene systems, quenching of the very small contribution from excimer has not been treated separately. Rather, the pseudo-first-order quenching of fluorescence by oxygen is taken from 71 according to the relation kq
= l/?02 = 1/70bs- l l 7 N 2
(2)
where 1/7,,bs is the observed rate of monomer decay, k, is the pseudo-first-order rate constant for quenching with oxygen a t the oxygen concentration stated, and 1/w2is the rate of monomer decay at 380 nm under nitrogen. This simple treatment is sufficient to illustrate the dependence of oxygen quenching on organization of the layer. 2-PyPCIDOL. The quenching of 2-PyPC by oxygen in a DOL monolayer is given in Figure 1as a function of area/molecule lipid. Also included is the surface pressurearea isotherm for this system so that one may relate quenching to organization of the layer both in terms of lipid molecular area and surface pressure. It may be seen that between 110 and 90 A2/molecule the decrease in k, is somewhat limited; from 90 to 80 A2 k, falls by approximately a factor of 2 and then a plateau occurs during further compression. Comparable plateaus have been observed for I- quenching. (6) Subramanian, R.; Patterson, L. K. J. Am. Chem. SOC.1985, 107, 5820-5821.
(7) Bohorquez, M.; Patterson, L. K. J. Phys. Chem. 1988, 92, 4218-
22.
area/molecule [ A 2 1
Figure 3. Plot of k,, the pseudo-first-order rate constant for quenching of 2-PyPC* by oxygen in a DPL monolayer (El),as a function of lipid area per molecule. The force-area curve for the lipid is also given (-). Measurements were carried out under a 1% 02 atmosphere.
2-PyPCIDEL. Oxygen quenching in DEL layers as shown in Figure 2 differs somewhat from that observed for DOL although general features of the surface pressurearea curve are quite similar to that of DOL. While the initial k, and the value of k, around 80 A2 are similar to those in DOL, curvature of the k, plot is markedly less than that for the analogous DOL measurements. A t higher surface pressures (e.g., 30 mN/M), DEL can be slightly more compressed, and a correspondingly lower value of k, may be seen under these conditions compared to the DOL system. In this system, both 1% and 2 % oxygen atmospheres were used, and the 2 % data are normalized on the plot. The agreement in kinetic data for two 0 2 concentrations suggests dependence of quenching on oxygen pressure. 2-PyPC/DPL. By contrast, the quenching rate of 2-PyPC in DPL behaves in a manner parallel to the other two systems only over the lipid expanded region, i.e., until the commencement of the phase transition at around 85 A2/molecule. The data are given in Figure 3. During the subsequent liquid-expanded to condensed phase transition, there is some slight recovery; this is followed by a sharp drop in quenching as the layer is compressed beyond the inflection region at the end of this transition. Py-22/DOL and Py-22/DPL. Py-22 quenching was examined in DOL and DPL, and the relevant data are presented in Figure 4. This probe contains 22 carbons between headgroup and probe; in principle, the pyrene chromophore could extend beyond the hydrocarbon groups of the host lipids. In DOL under expanded conditions, the initial quenching rate is marginally higher than for
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Langmuir, Vol. 6, No. 12, 1990 1741
Lipid Organization and Quenching E
40
coefficient of the probe (taken to be small compared to
30
Do), Po is the mole fraction of 02 in the gas phase (0.01 here), and Na is the total number of gas molecules per cubic
\
z 2 3 g
centimeter of gas phase. If one assumes that the diffusion coefficient Do will approach in magnitude that found in a monolayer or bilayer, then eq 3 in a monolayer may be written
20
?CL!
8
IO 0
0
= 4?rr(Do(Ybl)((Yml/abi)PdVa
(4) (4’)
100
80
60
area/molecule
120
[A21
Figure 4. Plot of k,, the pseudo-first-orderrate constant for quenching of Py-22* by oxygen in a DOL monolayer (n),as a function of lipid area per molecule. Measurements were carried out under a 1% 02 atmosphere.
,E
50
11
-jo.ep_
/f J d -
40.6
X
\
a
where r is taken to be 5.5 A.9 Such comparison with the monolayer also requires one to make some relative estimate of (Y for the vesicle and for the monolayer. If abilayer (ab11 and amonolayer (ami) are taken to be equal, then = (1.85 x 1013)(2x 10~)(10-~) 4 x io5 In the limit, however, one might suggest that a t the water surface amonolayer approximates more nearly an organic phase. Subczynski and Hyde measured the partiton coefficient between DML bilayer vesicles and water to be ~ 3 . One l ~ may then make the estimate that (Ybilayer = 30wakr or about 0.09 a t 25 O C . l l With the assumption that the monolayer approximates an organic solvent, one may reasonably take (Ymonolayer to lie between O.23amehmol and 0 . 2 6 ( ~ h e x a n e , labout l~ 2.5-3.0 times (Ybilayera One may then obtain an estimate for the diffusion-controlled collision rate in the separate monolayer “phase” as
v)
40
60
80
100
0
I20
area/molecule [A21 Figure 5. Plot of k,, the pseudo-first-orderrate constant for quenching of Py-22* by oxygen in a DPL monolayer @), as a function of lipid area per molecule. The force area curve for the lipid is also given (-). Measurements were carried out under a 2 % 02 atmosphere. Values of k , are normalized to 1% 02.
2-PyPC under analogous condition. However, the k, values fall rather linearly with area per molecule, and a t 75 A2/ molecule the quenching rates for the two probes in DOL are comparable. In DPL, where the experiments were carried out with 2 T oxygen, there is also a marked contrast with the 2-PyPC measurement (see Figure 5). The drop in k, up to and through the liquid-expanded/condensed phase transition is less marked (see Figure 3). However, the rate constant approaches 0 at the end of the transition.
Discussion To discuss the role played by lipid organization in limiting Py* quenching, it is necessary to consider the possible mechanisms by which 0 2 may interact with the probe. Two possible pathways for diffusion-controlled encounters suggest themselves. First, one may consider the monolayer as a separate phase and apply diffusion criteria analogous t o those found in treatments of membranes and vesicles. For example, Windrem and Plachy measured the product (Doa) in dimyristoyl-L-aphosphatidylcholine (DML) and DPL bilayer vesicles where Do is the oxygen diffusion coefficient (cm2s-l) and cy is the Bunsen coefficient of solubility.8 An upper value for the function is 2 X 10-6 cm-2 s-l. As applied to the Smoluchowski equation, one obtains for the frequency of collision between probe and 02 w = 4ar(Do + D,)crPdVa (3) where r is an interaction distance, D, is the diffusion
(8)Windrem, D. A.; Plachy, W. Z. Biochim. Biophys. Acta 1980,600, 655-665.
w
-
1 x lo6 s-l for 1%0,
which represents a rate about 10% of the value observed in the expanded monolayer. By contrast to the separate phase approach, one may assume the monolayer film to be a simple surface and consider collisions on the surface to originate from the gas phase. Here we use simple kinetic gas theory to calculate collisions of 02 molecules on a surface target of about 30 A2. The dimensions of pyrene are 3 X 8 X 11 A, and one assumes an area roughly equivalent to that occupied by a hydrocarbon chainsg With an average speed of 02 a t 4.4 X lo4cm/s and 1% 02,one arrives a t a collision frequency of 1 X lo7 s-l in close agreement with experiment a t low surface pressure. Of course, the exact collision cross section is rather difficult to estimate as the effects of lipid orientation on the random orientation of the probe have not been established. The results of these two approaches would, however, suggest that interactions of pyrene with gas-phase 02 moving across a lipid surface boundary, if not the dominant mechanism for quenching, certainly must contribute to the behavior observed here. The results given above indicate that the character of the lipid hydrocarbon chains and geometry of the probe play significant roles in determining interaction of probe chromophore with a gasphase quencher. This is in contrast to quenching in these same systems (save DEL) by iodide from the ~ u b p h a s e . ~ In those measurements, all systems exhibited somewhat comparable dependence of quenching rate on area/ molecule. The differences between k, for 2-PyPC in DOL and DEL must relate to the cis double bond a t the CS position in DOL compared to its trans analogue in DEL. This characteristic tends to make DEL somewhat more compressible than DOL. The region of the DOL layer around the Cg position on the hydrocarbon chain is that in which both cis double bonds and the pyrene chro(9)Sackmann, E.Z.Phys. Chem. (Munich) 1976,102,391-416. (10)Subczynski, W. K.;Hyde, J. S. Biophys. J . 1983,41,283-286. (11) (a) Wilhelm, E.; Battino, R.; Wilcock, R. J. Chem. Rev. 1977,77, 219-262. (b) Wilhelm, E.;Battino, R. Chem. Rev. 1973, 73,l-9.
1742 Langmuir, Vol. 6, No. 12, 1990
mophore will reside. One might speculate, then, that in DOL the region around the inflection in the S-shaped k, curve corresponds to Py filling into the cavities created by the cis groups while the portion of the chains furthest from the water surface order themselves into a more efficient barrier due to dispersion forces. By this scheme, DEL without such cavities (though one expects some perturbation of packing here)12 would pack around the chromophores in a less discontinuous fashion. Nevertheless, DEL can pack more tightly at the higher surface pressure, and this is reflected in a lower k, a t the compression limit. That k, for 2-PyPC in DPL shows a marked dependence on phase transition is not wholly unexpected. Indeed, interaction of spin probes with 0 2 in vesicles has been shown to diminish sharply a t transitions to less fluid phases.lO The small rise in k, throughout the long phase transition, however, is difficult to explain. The chromophores will certainly affect local ordering of the lipid chains under conditions associated with phase transition so that microscopic phase behavior which occurs in the vicinity of the pyrene chromophore may differ markedly from the macroscopic behavior reflected in the surface pressure-area curves. In the range from 80 to 55 A2/ molecule, there will be microdomains of both liquidexpanded and -condensed phases.13 The formation of condensed-phase domains might conceivably relieve local “pressure” around the chromophore and provide a slightly less well ordered barrier to 02 penetration during the early part of the phase transition in DPL. The Py-22 probe differs from 2-PyPC in two important respects: (a) there is a single chain in the molecule; (b) the long hydrocarbon chain bearing the pyrene provides the chromophore with considerably more freedom of movement than experienced by pyrene in 2-PyPC. Data for both DOL and DPL reflect this flexibility, which leads to a less discontinuous dependence of k, on compression than was found with the more constrained 2-PyPC. In fact, for the Py-22/DOL system the linear relationship observed suggests an evolution of chromophore position in the layer. Somewhat parallel behavior may be seen in the Py-22/ DPL system. An approximately linear decrease in k, (12) Feher, A. I.; Collins, F. D.; Healy, T. W. Aust. J.Chem. 1977,30, 511-519. (13) Miller, A,; Mohwald, H. J. J. Chem. Phys. 1987,86, 4258.
Bohorquez and Patterson
occurs in the liquid-expandedregion, and the behavior over the phrase transition gives a rather small decrease in k,, more as one would expect than was evidenced with 2-PyPC/DPL. The drop to nearly 0 for k, above the LCSC phase transition indicates that the lipid is able to coalesce into a very effective barrier to oxygen penetration. The behavior in both Py-22 systems indicates further that the probe is not squeezed upward above the monolayer as one might intuitively expect but rather, especially in Py-22/DPL, can be deeply buried in the monolayer or perhaps even sequestered into the region near the phosphatidylcholine headgroups.
Conclusion If one may roughly expect efficiency of barrier organization against 02 penetration to reflect ordering of the host lipid around a probe, then one may see that such organization is strongly dependent on both the host barrierforming lipid and the constraints that the probe molecule places on the target chromophore. It may be supposed, for example, that the long chain associated with Py-22 allows positioning of the pyrene to be much more readily accommodated to the packing requirements of the lipid than is the case for the short-chain moiety on 2-PyPC. The subtlety of these requirements and their effects on barrier formation are nicely demonstrated in the differences between the 2-PyPCIDOL and 2-PyPCIDEL systems in which the sole difference between the two systems is the change of one cis to one trans double bond in each 18carbon lipid chain. It is clear that the chains, and not the headgroups, play the dominant role in barrier organization. This statement of the rather obvious may not be extended to penetration of quenchers from the liquid phase (e.g., I-) where less distinct differences in quenching behavior with changing lipid and chromophore were ~ b s e r v e d . ~ These systems, which are so sensitive to organization of the layer, appear to provide a diagnostic approach to elucidation of lipid packing around substrates. Further, they may provide valuable insights to be utilized as one progresses toward the design of molecular barriers for control of reaction rates.
Acknowledgment. We acknowledge the assistance given by Mr. Louis Sehl of Professor Frank Castellino’s laboratory in the microphosphate analysis.
