Macromolecular Assemblies in Polymeric Systems - American

Chapter 17

Reduction of Phospholipid Quinones in Bilayer Membranes

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Kinetics and Mechanism Charles R. Leidner, Dale H. Patterson, William M . Scheper, and Min D. Liu Department of Chemistry, Purdue University, West Lafayette, IN 47907-1393 1

Gel permeation chromatography, electron microscopy, H NMR spectroscopy, UV-Vis spectroscopy, and stoppped-flow kinetics have been employed to determine the structural and redox properties of quinone-functionalized phosphatidylcholine liposomes. These unilamellar liposomes (ca. 25-30 nm diameter from sonication or 100 nm diameter from extrusion) typically contain 2 - 20 mol% phosphatidyl-choline anthraquinone (DPPC-AQ) which can be reduced and reoxidized by solution reagents. The transmembrane distribution of DPPC-AQ is controllable (58 - 98 %outer) via phospholipid compositions and liposome preparation methods. The rate law for S O 2reduction of DPPC-AQ/DOPC, k = k k [S O 2-] / (k + k [S O 2-]) indicates the presence of two kinetically-distinct forms of DPPC-AQ. Comparison with the corresponding homogeneous rate constant suggests the identities of the two pathways. 2

obs

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-1

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The recent (1) c r y s t a l structure of the bacterial photosynthetic reaction center provides a striking vision of how nature positions, orients, and assembles redox molecules i n order to effect specific and efficient redox reactions (i.e.. charge separation). Conplimentary to rtotosynthetic energy transduction i s respiratory energy transduction within the inner mitochondrial membrane wherein charge i s transported across a phospholipid bilayer membrane, eventually leading to the reduction of oxygen and the formation of ATP (2). Figure 1 provides a representation of the Qcycle, a key sequence of redox reactions occuring at specific protein sites within the membrane. The performance of these photosynthetic and respiratory "reaction centers" depends c r i t i c a l l y en the position, orientation, and assembly of the redox molecules. Both energy transduction processes involve the transport of electrons through a phospholipid bilayer membrane by membrane-bound 0097-6156/92/0493-0202$06.00/0 © 1992 American Chemical Society

In Macromolecular Assemblies in Polymeric Systems; Stroeve, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


17. LEIDNER ET Al-

Reduction of Phospholipid Quinones

203

Figure 1. Representation of the Q-cycle i n respiratory energy transduction. (Reproduced with permission from ref. 2. Copyright 1986 Plenum.)



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MACROMOLECULAR ASSEMBLIES IN POLYMERIC SYSTEMS

quinones (2) · The motivation to prepare stxix±urally-defined redox assemblies, particularly containixig quinones within phospholipid membranes, i s obvious. To this end we initiated (3-8) a study of the stuctural, redox, and transport properties of quinonefunctionalized monolayers and bilayers as simple, chemical models for quinone-mediated energy transduction. DPPC-AQ i s a phosphatidylcholine anthraquinone that closely resembles simple phospholipids l i t e dipalndtcylphasphaticVlcholine (DPPC) and i t s dioleoyl (DOPC) and ethanolamine (DPPE) analogs (Scheme I). Unilamellar, quinone-f\incrtlcnalized liposomes, prepared by the sonication (£) or extrusion (â) of DPPC-AQ and the simple phospholipids, provide chemical assemblies with which to study the redox and transport properties of membrane-bound quinones. We present herein the use of gel permeation chromatography, electron microscopy, -^H NMR spectroscopy, UV-Vis spectroscopy, and stopppedflow kinetics to provide a description of the structural and redox properties of unilamellar liposomes containing DPPC-AQ. Experimental Section The simple phospholipids were purchased from Avanti Lipids (DOPC, DPPC, DPPE, and MPPC (mcnopalndtcylphosphati ); the anthraquinone (DPPC-AQ) and anthracene (DPPC-AN) analogs were prepared and purified as described previously (6). The purity (> 99%) of the phospholipids was verified by TLC or *H NMR. A l l lipids were stored i n a dessicator at -10°C. A l l other chemicals were reagent-grade or better and were used without further purification. TiipoficmR piêparation. Liposomes were prepared from a mixture of simple phospholipid (DPPC, DPPE, MPPC, or DOPC) and functionalized phospholipid (DPPC-AQ or DPPC-AN). Phospholipid mixtures were (20 mg) suspended i n 1 mL of 50 mM tricine (pH = 8.0), 0.2 M HC1, and 1 mM EDEA solution under N . The resulting suspension was either sonicated (20-40 minutes) or extruded (3) through 100 nm Nuclepore membranes under nitrogen. After sonication the liposomes were fractionated on a Sephadex G50 or Sepharose 4B columns. A l l manipulations were performed at 52°C for DPPC (T = 42°C) or room temperature for DOPC (T = -22°C). H NMR spectra were obtained an a Varian VXR500S spectrometer at 52°C using the standard Varian S2 pulse sequence. Electron iaicrographs were obtained with frozen, phosphotungstate-stained suspensions of liposomes. 2

c

X

c

Spectrophotometry and StoppedHPlow E>q?eriments. Details of the spectrcphotometric and kinetics experiments have been presented previously Q>). In short, the liposome eluate from the Sephadex column was diluted i n a cuvette i n a thermostatted cuvette holder. Reagents were syringe-injected into the liposome solution. The titration experiments were performed on a Hewlett Packard 7450 diode array spectrophotometer. The l i p i d œncentratation was typically 0.1 to 1 mM; the DPPC-AQ œncentration was typically 2 to 200 μΜ. The DPPC-AQ concentration was calculated from the extinction coefficient of the [MB3NŒ2AQ] (Br) analogue (e = 4750 K" ^" at 322 nm). Stoppped-flow spectrophotometry was performed on a High-Tech Stopped-Flow Spectrophotometer interfaced to a Zenith 151 computer by a MetraByte Dash 16 A/ card. M l solutions were thermostatted 1

1

D



17. LEIDNERETAL.

SCHEME I .


P h o s p h o l i p i d s employed t o prepare

liposomes.


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MACROMOLECULAR ASSEMBLIES INPOLYMERICSYSTEMS

at 25 + 0.1 °C. Excellent f i t to a single exponential was observed for most of the absorbance (of the product) vs. time traces. Some traces (especially at high [S 0 ~] ) deviated at short times and were f i t beyond the f i r s t h a l f - l i f e . Runge-Rutta simulations of [H Q] y§. time were performed on a Zenith 286 miarcoccputer using the Fortran program GEAR. 2

2

4

2


Results and Discussion The structural similarity between DPPC-AQ and simple phospholipids (Scheme I) i s evident. However, the presence of a bulky, anthraquinone head group affects amphiphile assembly; neat suspensions of the cone-shaped DPPC-AQ do not form liposomes. Mixtures with less than 25 mol% DPPC-AQ yield clear suspensions of liposomes. Liposomes containing 2-20 mol% DPPC-AQ are routinely prepared and are optically identical to those from DPPC, except for the quinone peak at 322 nm. As demonstrated by Figure 2, the elution volume and peak width of these functionalized liposomes from the size exclusion column (Sepharose 4B) matches that of unilamellar DPPC liposomes. Additionally, electron micrographs of frozen samples of DPPC and DPPC-AQ/DPPC liposomes are identical — a majority of the structures are 25 - 35 nm; a few larger objects are observed, likely due to fusion of the smaller liposomes during cooling. Thus, sonicated DPPC-AQ/DPPC liposomes are unilamellar with an average diameter of ça. 25-30 nm (9). Sonicated DPPCAQ/DPPC/DPPE, DPPC-AQ/DPPC/MPPC, and DPPC-AQ/DOPC liposomes likewise possess optical and size-exclusion chromatographic characteristics identical to those of the œrresponding ncn-functionalized liposomes. The mol% DPPC-AQ i n the DPPC-AQ/DPPC liposomes, calculated from the dry mass of the l i p i d mixture used to prepare the liposome, was verified using spectrophotometry and *H NMR spectroscopy (6). The dry mass mol% values are accurate to ± 15%. Wè have performed l i t t l e characterization of the extruded liposomes at this time, but we envision no reason for the extruded DPPC-AQ/DPPC or DPPC-AQ/DOPC liposomes to differ from the DPPC or DOPC analogs. Thus, we have unilamellar, 100 nm diameter liposomes containing 5-10 mol% DPPC-AQ. Spectrophotometry experiments. The liposome-bound quinones can be reduced by external, aqueous S 0 ~ or BH " and reoxidized by exposure to oxygen or addition of Fe(CN) ~. Figure 3 illustrates the UV-Vis spectra of a 8.7 mol% DPPC-AQ/DPPC liposome solution with sequential additions of external, aqueous S 0 ^ " . Note that the quinone peak ( A j ^ = 322 nm) decreases from 100% to 0% as the hydroquinone peak (Α^^χ = 384 nm) increases from 0% to 100%. Exposure of the hydroquinone solution to oxygen or addition of Fe(CN) ~ causes a fading of the yellow color and a concomitant regeneration of the quinone peak. Clearly, a l l of the DPPC-AQ amphiphiles within the liposome are redox-active. (Similar redox behavior i s observed with a l l of the liposome systems.) S 0 " penetrates the bilayer and therefore can reduce a l l quinones regardless of their location within the bilayer (10). In contrast, EH"~ does not penetrate into the bilayer (10). By measuring the fraction of DPPC-AQ remaining upon addition of excess BH"", we have 2

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6

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2

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4

17. LEIDNERETAL


ι ι ι ι ι I I I I I I I I I I 111 1I » I I I Ι I I I I » » » 1


o.a H

207

ι

ι ι ι

ι

ι

0 6.Β mol* DPPC-AQ/DPPC • DPPC

0.6 H

ο ο CO

ϋ

d «0.4 Ο 03

0.2

0.0 10

15

20

25

30

35

Eluted volume (ml) Figure 2. Size exclusion chromatograms for sonicated DPPC (•) and 6.6 mol% DPPC-AQ/DPPC (O) liposome solutions.

wavelength

(nm)

Figure 3. Spectrophotometry response of a 8.7 mol% DPPC-AQ / DPPC liposome solution to the sequential addition of external S 0 ". / 2

2

4



208


a means of measuring the fraction of the quinone residing within the outer monolayer of the liposome. Table I l i s t s the percent quinone reduced by E H 4 " i n both DPPC and DOPC liposomes. The E H 4 " typically reduces 90 ± 4% of DPPC-AQ amphiphiles i n DPPC liposomes cxxitaining 5-12% DPPC-AQ, the typical mol% DPPC-AQ used i n these studies. DPPC-AQ/DOPC liposomes possess a constant %outer (86 + 4) throughout the studied mol% range (3.7 to 24). Statistical considerations (a) alone considering a symmetric phospholipid distribution predict that 70% of the quinone amphiphiles should reside within the outer monolayer for small, unilamellar vesicles (SUVs) of 25 nm diameter. Our data show that the cone-shaped (large head group) DPPC-AQ i s incorporated preferentially (90% and 86% y§. 70%) into the outer, less hindered monolayer of the liposomes. This asymmetry can be manipulated through varying the phospholipid proportions and preparation methods. DPPC-AQ/DPPC liposomes with less than 5 mol% DPPC-AQ posssess a greater fraction of outer quinones (98% for 4 mol%) than those with 5-12 mol%. Apparently at low mol% the DPPC-AQ amphiphiles can be accommodated within the more favorable outer layer, but upon increasing mol% the inner monolayer i s populated. Irrarporation of the inverted cone-shaped (small head group) DPPE into DPPC liposomes pronounces the transmembrane asymmetry of DPPCAQ (Table I). This system i s an example of a completely asymmetric, functionalized liposome — one i n which a l l of the functionalized phospholipids reside on one side of the liposome. Incorporation of the cone-shaped MPPC into DPPC-AQ/EFFC liposomes has the opposite effect, only 72% (Table I) of the quinones are reduced by EH ". Using the 100 nm extruded liposomes relieves the geometric strain of the SUVs and leads to more statistical distributions. With extruded DPPC-AQ/DPPC liposomes, 58% (vs. 53% statistical) resides on the outer monolayer. This ability to manipulate the DPPC-AQ distribution and thereby engineer liposomes with variable and controllable structure should prove useful i n our attmepts to use quinone-fXinctionalized liposomes as simple, chemical models for biological processes. 4

nfR Studies. The 500 MHz *H NMR spectra of (sonicated) DPPCAQ/DPPC liposome solutions (Figure 4A) reveal the ^ resonances (11) of the majority DPPC amphiphiles and the diminutive peaks from the anthraquinone portion of DPPC-AQ at 7.8-9.0 ppm (1:4:2 ratio). Several of the DPPC resonances possess the double-peaked shape indicating different magnetic environments for amphiphiles residing on the outer and inner monolayers of the liposome (11). These resonances are partitioned 67:33 on the average, indicating that our quinone-fXinctiGnalized liposomes are structurally similar to the well-characterized DPPC liposomes — they are unilamellar and possess an average diameter of 25-30 nm. The observation of only one set of quinone resonances indicates that the quinone amphiphiles exist predominently i n one enviroment i n the liposomes. This i s i n agreement with the spectrophotometry results that ca. 90% of the incorporated quinones reside i n the outer layer. Observation of separate resonances for the remaining inner quinones i s impractical due to their low concentration and/or the substantial linewidths of the quinone resonances.




17. LEIDNER ET AL.


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Further insight into the structure of DPPC-A(yDFFC liposomes i s provided by nuclear Overhauser experiments. Figure 4B illustrates that irradiation of the 2-glycerol H at 5.6 ppra results i n substantial diminution of the 1-AQ H resonance at 8.9 ppm v i a a through-space coupling (nuclear Overhauser effect). Note the minimal effect on the other resonances i n the spectrum. À similar, although less dramatic, effect i s observed upon irradiating the NMe resonance; irradiating the resonances within the alkyl chains has l i t t l e effect. These simple experiments demonstrate that the 1AQ H i s located near the glycerol portion of the majority DPPC amphiphiles within the liposome. The quinone "head" group i s located at a position near the hydrophilic - hydrophobic interface, not extended out into solution. This description i s shown i n Figure 5. A subtlety of the NMR results i n Figure 4 i s the intensities of the quinone resonances with respect to those of DPPC. At low (< 4) mol% DPPC-AQ, the integrated intensities convert to the expected mol% DPPC-AQ; at higher mol% the integrations are too small (e.g., 4.3 mol% calculated vs. 6.0 mol% actual). This could indicate that a portion of the DPPC-AQ anphiphiles aggregate into a gel-like region and thus exhibits such severely-broadened resonances that they are effectively absent from the spectrum. The integrated intensities would reflect only the "fluid" DPPC-AQ aicphiphiles. Increasing temperature could cause some of these immobile DPPC-AQ asophiphiles to "melt" or become more mobile; the integrated intensities of the quinone resonances would increase. Such an effect, although slight, i s observed i n our experiments. Endogenous quinones are known (12) to aggregate within liposomes, so our chemical models may be mimicing even this aspect of the membranebound quinones. Despite this fortuitous similarity, a gel-fluid equilibrium would complicate any detailed analysis of the redox, transport, and structural properties of our system. We are investigating more closely the possibility of aggregation of DPPC-AQ amphiphiles. +


3

Kinetics. Reduction of (sonicated) DFPC-AQ/DOPC liposomes at room temperature proceeds at a rate readily measured using stopped-flow techniques. Figure 6 illustrates the reduction of DFPC-AQ/DOPC liposome solutions ([DPPC-AQ] = 2.3 μΜ) with S 0 ~. Satisfactory f i t to an exponential growth of H Q ( ) i s observed (see Experimental). The psuedo-first order rate constants (k^g) for the reduction of DPPC-AQ/D0PC by S 0 ~ were measured at various [DPPCAQ] and [ S 0 ~ ] . These data are presented i n Figure 7. Within the scatter i n these data, no discernible trend of k ^ g with [DPPC-AQ] i s noted. The leveling effect (saturation kinetics) exhibited i n Figure 7 i s accounted for by the rate law: 2

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4

2

klk [S 0 -] 2

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(1)

*obs = 2

K-l + k [ S 0 - ] 2

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4

with the best f i t parameters:



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17. LEIDNER ET AL.

Figure 4. 500 MHz -Ή NMR spectra of 6.7 mol% DPPC-AQ/DPPC liposome solutions at 52°C (A) ; same with irradiation at 5.6 pp (Β). (Reproduced with permission from ref. 6. Copyrioît 199] American Chemical Society.)



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Figure 5. Representation of liposomes rarfcaining DPPC-AQ. (Reproduced from reference 6. Copyright 1991 American Chemical Society.)



17. LEIDNER ET AL.

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time (s) 2

Figure 6. Time dependence of [H Q] following addition of S 0 ~ to DPPC-AQ/DOPC liposome solutions ([DPPC-AQ] = 2.3 μΜ); [S 0 ~] = 4140 μΜ (•), 513 μΜ (•), and 131 μΜ (X). Relative hydroqîinone concentrations were obtained from absorbanoe changes at 385 nm after correction for baseline changes. Solid lines are exponential f i t s for = 3.9, 2.9, and 1.5 s" . (Reproduced from reference 6. Copyright 1991 American Chemical Society.) 2

2

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4.0

3

QB

( )

*-l k

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2

QB + S 0 " 2

> H Q + 2 S02

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(4)

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2

S 0 " binding: 2

4

S

2

S

2°4 -AQ

2

2°4 "

(5)

L r P

*-l s

2

2°4 "

+ L r P

Q — >

H Q + 2 S02

(6)

2

where and QQ represent two different forms of DPPC-AQ (DPPC-AQ i n two different environments), represents liposome-bound, and represents aqueous. The mechanistic inplications are drastically different, but the functional form of the rate law i s identical i n both cases. The crucial aspect of the experimental data that permits dismissal of the S 0 " binding i s the observation of biphasic growth of H Q at high [ S 0 ~ ] , as illustrated i n Figure 8. Note that a single exponential f i t s beyond the f i r s t h a l f - l i f e (Figure 8B), but not the entire region (Figure 8A). The S 0 " binding mechanism i s inconsistent with an i n i t i a l fast phase, while the quinone equilibrium i s entirely consistent when i t i s recognized that the quinone equilibrium i s established at the outset of the reaction (addition of S ^ " ) . At high [S 0^ "] reaction (4) i s extremely rapid (k [S^^ "] » kn, k_^) yielding the f i r s t phase, while the remainder of the reaction proceeds from to give the rate law (1). At low [S 0 ~] the disparity i n the two phases i s minimal and a single exponential f i t s the data. Fitting the [H Q] vs. t data to the quinone equilibrium mechanism using Runge-Kutta T i T P

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17. LEIDNER ET Al-

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0.145

0.00

0.40

0.80

1.20

time (s) 0.145

0.12-

A

0.115 0.00

0.40

0.80

1.20

time (s) Figure 8. Experimental (A) and calculated ( ) [H Q] vs. time plots; solid lines were calulated with single exponentials for best f i t over entire time range (A) and over long time (B). 2

simulations was reasonably successful (cf. Figure 9). The biphasic behavior i s reproduced for the high [S 0 "] and the single exponential at low [ S 0 " ] . The best f i t to the data i s obtained for k - 4 - 5 s" , k_! ~ 5 - 6 s" , and k ~ 0.4 - 2.5 Χ 10 M" ^" 2

2

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1

x

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2

Estimating the values of the various rate constants i s straightforward, but identifying the nature of the quinone equilibrium i s more d i f f i c u l t . Fortunately, the NMR results



216


described above provide two possible scenarios identifying and QQ: gel + fluid and menfcrane-entoedded + solution-extended. In both cases, one form of the quinone (Qg) would react much more rapidly than the other form (Q^). Within the mentorane-erabedded + solutionextended scenario the Οβ form should resemble a solution species, so use of k for Me NCH A£r as an i n i t i a l estimate of k i s reasonable. k_i thus calculated i s 21.2 (+ 0.2) s , providing an estimate of kj/k.^ = 0.19 ± 0.01. This estimate i s reasonable for the membraneembedded + solution-extended scenario, but i s inconsistent with the curve-fitting described above (kj/k-i - 0.8). The other scenario i s consistent with k j / k ^ ~ 0.8, since this leads to [ Q & ] / [ Q B ] - 0.45. k thus estimated (-2 Χ 10 M" ^" ) i s significantly less than for the œrresponding aqueous reaction (1.1x10 tf"^" ). Such a difference i n rate constants could be due to the distance between the aqueous S 0 ~ and the lipid-embedded AQ, restricted access to the AQ (a steric conponent), or due to the change i n dielectric of the medium at the hydrophobic / hydrophilic interface. Although a more detailed data analysis w i l l be necessary to differentiate between the two scenarios, the gel-fluid scenario presently provides the best explanation of the kinetic data. 3

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Conclusions DPPC-AQ i s the f i r s t example Q) of a quinone-functionalized phospholipid. Quincaie-functionalized liposomes with varying phospholipid octrposition and transmembrane distribution can be prepared with DPPC-AQ. Incorporation of DPPC-AQ into DPPC and DOPC liposomes has no effect on liposome size, although i t does increase liposome permeability. NMR spectroscopy reveals that the AQ "head group" of DPPC-AQ resides near the hydrophobic / hydrophilic


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LEIDNER ET AL.


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interface of DPPC liposomes. The quinone-functionalized liposomes undergo facile redox reactions with solution reagents. The mechanism of S 0 " reduction involves electron transfer between solution S 0 ~ and liposcroe-bound DPPC-AQ. Our primary interest i n quijxne-functionalized arophiphilic assemblies stems from the desire to provide simple, chemical models for qpinone-mediated energy transduction. The present systems are a successful beginning; however, the biological quinones (2) reside deep within the bilayer membrane, possess considerable mobility, and interact strongly (if not necessarily) with meittorane-bound proteins. We must incorporate these concepts into our quinane-functionalized liposomes so that we can prepare well-defined, chemical models of the endogenous systems. 2

2

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2

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Acknowledgements, The authors thank the Purdue Research Foundation for financial support and Karie M. Horvath for preparing sairples of DPPC-AQ. Prof. Dale Margerum provided access to the High-Tech stopped flow spectrophotometer and useful comments on the kinetic data analysis. NMR experiments were performed on instruments funded by NIH Grant RR01077 and NSF/BBS-8714258. Literature Cited 1. Feher, G.; Allen, J. P.; Okamura, M. Y.; Rees, D. C. Nature 1989, 339, 111. 2. von Jagow, G.; Link, Τ. Α.; Ohnishi J. Bioenerg. Biomemb. 1986, 18, 157. 3. Leidner, C. R.; Liu, M. D. J. Am. Chem. Soc. 1989,111,6859. 4. Leidner, C. R.; Simpson, H. O'N.; Liu, M. D.; Horvath, Κ. M.; Howell, Β. E.; Dolina, S. J. Tetrahedron Lett., 1990, 31, 189. 5. Liu, M. D.; Leidner, C. R. J. Chem. Soc., Chem. Comm. 1990, 383. 6. Liu, M. D.; Patterson. D. H.; Jones, C. R.; Leidner, C. R. J. Phys. Chem., 1991, 95, 1858. 7. Liu, M. D.; Leidner, C. R.; Facci, J. S. J. Am. Chem. Soc., submitted. 8. Liu, M. D.; Duevel, R. V.; Corn, R. M.; Leidner, C. R. J. Phys. Chem., submitted. 9. Thomas, P.D.; Poznansky, M.J. Biochim. Biophys. Acta 1989, 978, 85. 10. Ulrich, Ε. L.; Gervin, M. E.; Cramer, W. Α.; Markley, J. L. Biochemistry 1985,24,2501. 11. Michaelis, L.; Moore, M. J. Biochim. Biophys. Acta. 1985, 821, 121. 12. Hinz, H.-J.; Korner, O.; Nicalou, C. Biochim. Biophys. Acta 1981, 339, 111. 13. Lambeth, D. O.; Palmer, G. J. Biol. Chem. 1973, 248, 6095 RECEIVED December 10, 1991


Macromolecular Assemblies in Polymeric Systems - American

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