2788
J. Phys. Chem. 1993,97, 2188-2192
Fluorescence Lifetime Distributions of Various Phospholipids Labeled with Covalently Bound Diphenylhexatriene in the Erythrocyte Ghost Membrane E. Prenner,? A. Hermetter,?C. Landl,t H. Stiitz,?H. F. Kauffmann? and A. J. Kungl'J Institut fir Physikalische Chemie der Universitiit Wien, Wahringerstrasse 42, A - 1090 Wien, Austria, Institut fir Lebensmittelchemie und Biochemie, Technische Universitiit Graz, Petersgasse 12, A-801 0 Graz, Austria, and Institut fir Mathematik, Johannes-Kepler- Uniuersitiit, A-4040 Linz, Austria Received: December 10. I992
We present time-resolved fluorescence data from three different covalently diphenylhexatriene(DPH)-labeled lipids [ 1-palmitoyl-2-diphenylhexatrienpropionyl-glycerophosphocholine (DPH-PC), 1-palmitoy1-2-diphenyl-
hexatrienpropionyl-glycerophosphoethanolamine (DPH-PE), 1-palmitoyl-2-diphenylhexatrienpropionylsphingomyelin (DPH-SM)] in erythrocyteghost membranes. These biologically important lipids were analyzed regarding underlying fluorescence lifetime distributions and their location in different lipid domains. Multiexponential reconvolution yielded three component best-fits, with x2 that were somewhat greater than the ones obtained by exponential series distributional analysis. A bimodal lifetime distribution was found to be independent of the lipid under investigation. The centers and the widths of the distributions varied for the different lipids, indicating different organizations of the respective lipids in the red cell membrane. Sphingomyelin showed the narrowest distributional widths and the shortest lifetime (centered around 7.05 ns) compared with PE and PC. The erythrocyte membrane is proposed to generate a number of different micro-environments for the different DPH-labeled lipids, which is reflected in differences of lifetime distributional widths and centers. Synthetic time-resolved fluorescence data showed that the exponential series lifetime/amplitude spectrum recovered from a definite three-exponential decay (with lifetimes taken from the multiexponential reconvolution) is very similar to the according spectrum of the raw data. To decide whether multiexponential analysis is only a crude parametrization of what should be better described by a distribution of lifetimes, the criterion for deciding between "discrete" and "distributed" exponentials in high-quality data sets was suggested to be the better x2.
Introduction A variety of methods have proven to be useful for the investigation of the physical chemistry of lipids in synthetic and natural membranes. Very interesting results were obtained by fluorescence studies together with NMR and IR experiments. For this purpose, various fluorescence labels were introduced in the literature for studying the heterogeneity of natural membranes. Lipids of biological membranes are supposed to be distributed asymmetrically with respect to lateral and vertical membrane organization. The resulting formation of membrane lipid domains is of relevance for membrane function.' Non-covalently labeled membranes were used for most of the published results. However, since the localization and orientation of fluorescence labels are not accessible2J in general, the number of experiments using covalently labeled phospholipids is increasing. Time-resolved fluorescence spectroscopy is a very powerful method for the investigation of artificial and natural membranes since the transients reflect the micro-environment of the chromophore which is embedded in the lipid bilayere4J In our study we have used different lipids labeled with 1,6-diphenyl-l,3,5hexatriene (DPH).6 Especially in membranes, the fluorescence decay is affected by the molecular composition as well as the dielectric constant of the medium.',* The fluorescence decay of DPH has been shown todepend on thesolvent under consideration. Recently, Parasassi et al. proposed a two-state photophysical model for the fluorescence decay of DPH in solution and in artificial membranes8 The interconversion rate between the two excited states was shown to be sensitive to the micro-environment of the Author to whom correspondence should be addressed. Present address: Max-Planck-Institut fCr Biochemie Abteilung Strukturforschung, DW-8033 Martinsried, FRG. Technische Universitlt Graz. t Johanncs-Kepler-Universitlt. f Universitit Wen.
chromophore. The photophysics of DPH should, therefore, be a valuable tool for the investigation of natural membranes. Analysis of fluorescence patterns by distributions of lifetimes instead of discrete exponentials has been shown to be meaningful on a phenomenological basis particularly when complex decays, such as those resulting from the fluorescence of proteins and membranes, are considered. James and Ware have introduced the so-called exponential series method (ESM) for analyzing the complex fluorescencedecay of homotrypt~phan.~J~J' There, the number of possible sidechain conformations quenching the indole fluorescence made the description of the transients by a distribution of lifetimes more reliable than by the conventional sum of discrete exponentials.I2 This was judged by the x2-criterion as well as by a photophysical model which proposes a broader distribution of sidechain conformations for homotryptophan compared with tryptophan. Distributional analysis of phase and modulation data from proteins was recently reported by Alcala et a1.'3J4J5 The authors used an algorithm with Lorentzian shape for the lifetime distributions. Thus, the centers and the widths are the only parameters in their fitting routines. According to James and Ware, we have used a home-made ESM with no fixed shape of the distributions.16 Higher moments of the distributions can therefore be used to characterize the fluorescence pattern of the probe. In the following we will present one of the first studies on time-resolved fluorescence data from lipids covalently labeled with DPH in the erythrocyte ghost membrane. The aim of this work was to compare the fluorescence properties of three biologically important lipids and to see whether they are located in different lipid domains within the natural membrane.' Comparison of two different methods for data analysis has been drawn to show their limits regarding extractable and (bio)physically meaningful information. Distributional analysis of high quality data will be shown to yield better x2's than those
0022-3654/93/2091-2788104.00/0 , Q 1993 American Chemical Society ,
I
Phospholipids Labeled with Diphenylhexatriene
The Journal of Physical Chemistry, Vol. 97, No. 11, 1993 2789
bath (Haake, Karlsruhe, Germany). The donor membranes were separated from the ghosts by centrifugation (microcentrifuge, 5 min, 15000 rpm) (Hettich, Tuttlingen, Germany) followed by repeated washings of resuspended membranes with 3 ml Tris/ HC1 buffer each. Fluorescence Measurements. Time-resolved fluorescence was performed on a serial PRA 3000 transient configuration (Photochemical Research Association, London/Ontario, Canada) using a thyratron-gated hydrogen flash lamp. The full width at half maximum of the lamp instrumental function L ( t ) was typically 1.3 - 1.5 ns. Fluorescence photons were detected by a Hamamatsu R928 photomultiplier (Hamamatsu, Hamamatsu City, Japan) and time-correlated by single-photon timing.23Timeto-amplitude converted (TAC) output signals were collected in a Tracor Northern T N 1750 multichannel analyzer (Tracor Northern, Middleton, USA) and the fluorescence histogram then transferred to a MicroVAX I1 (Digital Equipment Corporation, Mainard, USA) for data analysis. No significant non-linearities in the TAC were detected. Instrumental response functions were collected bothat excitation wavelength and at emission wavelength C to rule out an energy-dependent delay (photomultiplier color effect) extractable as an artificial short-time exponential in numerical rise phase analysis. Scattering effects could be excluded by measuring blind samples containing no DPH, which gave no significant fluorescence response in the relevant spectral region. Typically, 2 X lo4- 4 X lo4counts in the peak channel maximum were collected. Transient Data Analysis. Two different methods for data Figure 1. Structures of DPH-PC,DPH-PE,and DPH-SM. A = analysis were used in this work. First, fluorescence raw data l-Palmitoyl-2-[ [2-[4-(phenyl-trans-1,3,5-hexatrienyl)-~hen~ll-eth~~lca~H(r)were analyzed by the usual nonlinear, least-squares reconbonyl]sn-glycero-3-phosphocholine(DPH-PC). B = l-Palmitoyl-2-[ [2volution technique24 [rl-(phenyl-tran Is ,3,5-hexatrienyl)-phenyl]-ethyl]carbonyl]sn-glycero3-phosphoethanolamine(DPH-PE).C = N - [[2-[4-(6-phenyl-trans-I ,3,5H ( t ) = L ( f )0 F(2) (1) hexatrien yl)-phenyl]ethyl]-carbonyl]-trans-4-sphingesine-1-phosphocholine (DPH-SM). where the molecular &pulse fluorescence F ( t ) is assumed to be a sum of discrete exponentials with both the amplitudes and the lifetimes floating in a free-fit procedure. obtained from multi-exponential reconvolution. Numerically simulated data and their ESM analysis will further aid to Second, for the evaluation of a potential distribution of fluorescence lifetimes from a (nonexponential) &pulse fluoresdiscriminate between a discrete three exponential fit and a bimodal cence response lifetime distribution.
Materials and Methods Lipid Syntheses. DPH-PC was prepared according to Kalb et 1). DPH-PE was generated following the procedure of Comfurius and Zwaal.” Sphingosylphosphocholine was prepared from bovine brain shingomyelin (Sigma, Deisenhofen, Germany) by acidic hydrolysis using a modified version (the reaction time was three times longer) of the procedure described by Kallerls and characterized according to Cohen et al.I9 Erythrocyte Ghost Preparation. Blood was drawn from healthy donors. Hemoglobin was removed from human erythrocytes according to Dodge et a1.20until the resulting ghost preparation was colourless. The phospholipid contents of membrane preparations were determined according to Broekhuyse.21 Cholesterol content was determined with a commercially available test kit from Boehringer (Mannheim, Germany). Preparation of Phospholipid Bilayers. Unilamellar vesicles (DPH-PC, DPH-SM) or multibilayers (DPH-PE) consisting of fluorescent phospholipids were prepared by the ethanol injection method.22 A solution of the lipid (60 nmol) in 25 pl ethanol was injected into 3 ml of Tris/HCl buffer (10 nmol, pH 7.4) at 37 OC under stirring. The preparation was stored over night at 4 OC in the dark before use. hbeling of Erythrocyte Ghosts with Fluorescent Phospholipids. The uptake of DPH-labeled lipid (60 nmol phospholipid) into erythrocyte ghosts (400 nmol phospholipid) at 37 OC was determined from the increase of fluorescence intensity at 430 nm (excitation 360 nm), using a RF-540 spectrofluorometer (Shimadzu, Kyoto, Japan) equipped with an external thermostatic
a1.6 (Figure
an exponential series method (ESM) developed in this laboratory16 was used in raw data analysis. In the ESM, F ( t ) in eq (2) is approximated by a coarse discretization. By using terms of a series of exponentials with fixed lifetimes T~ (usually equally, or logarithmically spaced over the time scale of fluorescence), the method allows the corresponding discrete set of amplitudes (a,) to be evaluated from the convolution integral (eq (1)) in a linear, least-squares free-parameter optimization. In order to obtain statistically significant results regarding centers and widths of the lifetime distributions, the data quality was kept as high as possible. As extensive experience in ESM analysis on known lifetime distributions has shown, reliable and reproducible results are obtained when the data quality reaches or exceeds 2 X lo4 counts in the peak channel maximum. Our ESM uses a hybrid-algorithm similar to that of AI-Baali and F l e t ~ h e r . ~In~general, . ~ ~ our ESM is robust and it works well on synthetic and high-accuracy real data. There is no cutoff required for deleting amplitudes of values close to zero during the reconvolution, and contrary to other exponential series techiques, our method is not restricted to positive amplitudes (puredecays). Also negativevalues for the amplitudes (rise phase) are possible. In all cases, a rectangular distribution of initial guesses was applied in the ESM probe function. Generally, 70 terms were set in the analysis. Details of the method are given elsewhere.I6 Synthetic fluorescence profiles have been generated according to eq (1) by convolving input lifetimes and amplitudes with an
2790 The Journal of Physical Chemistry, Vol. 97, No. 1 I, I993
Prenner et al.
TABLE I: MultiexpooenHal Reconvolution Parameters (Amplitudes ai and Lifetimes ~ i of) DPH-PC,-PE,and -SM Fluorescence = 430 nm Recorded at L = 360 nm. L,,,
0.04 DPH-PE DPH-SM
0.03 0.17
0.14 0.05 0.10
0.39 0.19 0.1 1 0.44
experimental (noisy) pulse. The thus received patterns have additionally been superimposed by Gaussian noise and the data quality was adjusted to 4 X IO4 counts in the peak channel maximum.
8.32 8.53 8.57 9.27 7.55
3.45 4.99 3.25 6.84
1.39 2.1 1 3.12
1.80 1.55 1.06 0.94 0.93
100000 I0000 1000
Results and Discussion The fluorescence quantum yield of the DPH-labeled erythrocyte ghosts was lower compared with protein-free artificial phospholipid vesicles (Prenner et al., manuscript in preparation). Due to efficient fluorescence quenching in the natural membrane we were not able to collect more than 40000 counts in the peak channel maximum, which is a quite high yield for biomembranes. However, as theX2criterionshows, the dataquality isgood enough to differentiate between distributions of lifetimes and the conventional sum of exponential terms. In Table I we present the multi-exponential reconvolution parameters obtained from the decay-analysis of DPH-PC, DPHPE, and DPH-SM fluorescence. Data sets from five different erythrocyte preparations of each lipid (including comparison of different donor erythrocytes) gave reproducible results within the statistical errors of the method. Moreover, reproducibility has been confirmed independently by phase modulation measurements (Prenner et al., manuscript in preparation). No effects of ageing could be detected since the samples were always freshly prepared before measurements. The results of two- and three-exponential parametrizations are given in Table I. For DPH-PC and DPH-PE, three exponential terms gave a better x2as well as better distributed residuals and autocorrelation compared with two exponential terms. In contrast, for DPH-SM no further improvement could be seen by incorporating a third lifetime component in the fit (data not shown). Regarding the best fit, the mean lifetime of DPH-SM is shorter than that of DPH-PC, which itself is shorter than that of DPH-PE. All decays have a lifetime (rl)with a low amplitude (al)which may reflect the interconversion rate between two excited states of DPH proposed by Parasassi et a1.8 Thus, this interconversion would be slowest for DPH-SM (7, = 3.12 ns) and fastest for DPH-PC (rl = 1.39 ns). Measurements devoted to the elucidation of the nature of the proposed excited state reaction should lead through a better understanding of the DPH photophysics to a deeper insight into the influence of its (natural) membrane surrounding. This will be the topic of future research in these laboratories. 73 is longest for DPH-PE and shortest for DPH-SM reflecting the different electrostatical interactions within the natural membrane (Figure 1): the polar headgroup of S M shifts the mean lifetime of DPH to smaller values compared with PC and PE. Moreover, the lipids could interact differently with other components like proteins of the erythrocyte membrane due to their different localization. Thus, intermolecular quenching should be more effective for DPH-SM, refering to chargeaccepting nearest neighbours during the fluorescence lifetime of DPH. In Figure 2 an overlay of the DPH-PC, DPH-PE, and DPHS M transient fluorescence is presented. For a better visual inspection only one lamp profile is shown. The decay pattern of DPH-SM clearly differs from both other lipids whereas the fluorescence profiles of DPH-PE and DPH-PC more closely resemble each other. Different kinds of interactions and thus
c.
100
i
10
0
20
40
60
80
100
nsec 2 0 -2
2 0 -2
2 0
-2
Figure 2. Fluorescence decays of DPH-PC (a), -PE (b), and -SM (c) recorded at A,, = 360 nm, bm = 430 nm with 0.4 ns/channel. The respective residuals are shown below.
micro-heterogeneity in the erythrocyte membrane should be responsible for these differences. The physical properties of such microdomains which affect fluorescence lifetimes are basically intermolecular quenching with identical molecules or with components of the matrix in a dynamical or a statical way (complexing with cholesterol,6 specific and nonspecific lipoprotein formation21), polarity effects, mobility effects, the ionic environment, dielectric effects,l vertical location of the chromophore in the membrane,S interfacial28 and transmembrane potential, etc. The significant difference in the decays of DPH-PE and DPH-PC, which differ only by three methyl groups at the terminal nitrogen (Figure l ) , may be due to the ability of DPH-PE to form hydrogen bonds leading to a longer lifetime for this label compared with DPH-PC which is incapable of forming hydrogen bonds. Analysis of the fluorescence decays of the three DPH-labeled lipids by an exponential series method to reveal underlying distributions yields the centers and full widths at half-maximum given in Table 11. A bimodal shape of the lifetime distribution with the centers cI and c2has been found for all three lipids under investigation. Phase modulation fluorescence measurements of the lipids incorporated in POPC ( 1-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine) vesicles have shown that they are indistinguishable with regards to their distributional widths in such a homogeneous environment (Prenner et al., manuscript submitted to Arch. Biochem. Biophys.). However, in the erythrocyte ghost
The Journal of Physical Chemistry, Vol. 97, No. 11, 1993 2791
Phospholipids Labeled with Diphenylhexatriene
TABLE II: Distributional Parameters (Fractions 4 , Lifetime Centers q ,and Full Widths at Half-Maximum wi) of DPH-PC, -PE, and -SM Time-Resolved Fluorescence substance DPH-PC DPH-PE DPH-SM
cl(ns) 7.85 8.01 7.05
fl
1.34 1.14 1.37
wl(ns) 2.8 2.9 2.5
fz
cdns) 1.63 1.47 1.79
0.48 0.46 0.64
whs) 0.76 0.78 0.78
XI
1.41
0.9 0.78
0.16
XI
0.12
U
0.08
aE
*E
t
0.04
0.00 0.0
2.0
4.0
6.0
8.0
10.0
12.0
0.0
2.0
4.0
nsec
6.0
8.0
10.0
12.0
nsec
I
"
'
I
u)
4
.-3 -
e a -
0.04
-
2 0.0
2.0
4.0
6.0
8.0
10.0
12.0 0.0
nsec
2.0
4.0
6.0
8.0
10.0
12.0
nsec
0.20
Figure 4. ESM analysis of fluorescence data numerically simulated according to the multiexponential reconvolution parameter sets of DPHPE (Table I): (a) shows the ESM lifetime/amplitudepattern of a definite two-exponential decay; (b) shows the lifetime/amplitude pattern of a definite three-exponential decay.
0.16 0.12 0.08
0.04 0.00 0.0
2.0
4.0
6.0
8.0
10.0
12.0
nrec Fipre3. ESM-analysisof DPH-PC (a), -PE(b), and -SM(c) fluorescence decays presented in Figure 2. 70 lifetimes were spaced linearly from I to 12 ns.
membrane DPH-SM is the shortest-lived substance, followed by DPH-PC and DPH-PE and the width (w,) of the lifetime distribution issmallest for DPH-SM (Table 11). Thus, the polarity shift of the DPH-SM lifetime center (Figure 3) is accompanied by a significant narrowing of the respective lifetime distribution. In terms of lifetime heterogeneity DPH-SM has, therefore, the most homogeneous micro-environment and could be less mobile within the erythrocyte membrane, resulting in a narrower distribution compared with the ESM of DPH-PE and DPH-PC. The small difference in the widths of DPH-PE and DPH-PC lifetime distributions is significant due to the high data quality collected which is necessary for a reliable ESM fit. Independently, this significant difference has been confirmed by phase modulation measurements (Prenner et al., manuscript in preparation). From Table I1 it becomes also clear that analysis by ESM yields the best x2 for the same data set compared with the twoand three-exponential reconvolution presented in Table I. Numerically, the best fit is therefore obtained by the distributional analysis. This is in agreement with the multitude of events occuring during the lifetime of a chromophore within a complex matrix (see above) which should render discrete values of decay parameters obsolete indeed. The lifetime distributions of the three DPH-labeled lipids are shown in Figure 3. From these graphs can also be seen the discreteness of the 'distribution" at c2 for all three lipids.
Although the x 2 is significantly improved when going from a two- to a three-exponential, and then further on to an exponential series analysis, one might argue that the x 2 obtained from a biexponential reconvolution is sufficiently low enough. We have numerically simulated fluorescence data on the basis of the twoand three-(mu1ti)exponential reconvolution parameter sets presented in Table I. We then ran ESM on these synthetical data sets and one representative example is presented in Figure 4 (the input parameters for the data generation were the parameters of DPH-PE, see Table I). The ESM-recovery of the synthetic twoexponential decay (Figure 4a) is in no way similar to the ESManalysis of the respective real data set (Figure 3b, DPH-PE). If the two exponentials found in the multiexponential analysis of the raw data should hold any significance, ESM would have found narrow distributions at the corresponding centers, too. The ESMrecovery of the synthetic three-exponential decay (Figure 4b) is, however, very similar to the ESM-analysis of the real data set (Figure 3b). Thus, ESM could be blamed for numerically hiding two discrete lifetimes 72 and 73 beneath one distribution. But since the x2 is significantly improved when a three component fit (Table I) is compared with an ESM fit (Table 11) of fluorescence real data, it becomes clear that the opposite should be true: multiexponential reconvolution is only a parametrization of the physically underlying lifetime distribution. Comparing Table I and Table 11, the distribution centered at cI is approximated by two exponentials in the decays of DPH-PC and DPH-PE. However, cI is similar to T3 for DPH-SM and hence no further lifetime ( 7 2 ) was necessary for the multiexponential reconvolution of this lipid, We thus conclude that the lifetime distribution of DPH-SM could be approximated by a discrete lifetime to a good extent and that hence the interactions of this lipid-as reflected in lifetime heterogeneity-within the natural membrane are very homogeneous.
2792
Prenner et al.
The Journal of Physical Chemistry, Vol. 97, No. 11, 1993
We have demonstrated that DPH-PC, DPH-PE and DPHSM show different fluorescence decay patterns when they are incorporated in erythrocyte ghost membranes. Organization into different lipiddomains within the natural membrane may account for thesedifferences. The multitude of possible interactions during the excited state lifetime ofthechromophore in such lipiddomains results in a multitude of deactivation channels of the excited state, leading to distributions of fluorescence lifetimes. In a recent paper by Toptygin et al.29theauthors haveshown that the radiative decay rate of DPH-like molecules is dependent on the orientation of the emission dipole when the chromophore is incorporated in an optically discontinuous and anisotropic environment like a membrane. A theory has been developed which takes into account the orientational dependence and which is sufficient to describe the complex fluorescence decay of DPH-like molecules in membranes. Thus, a nonexponential fluorescence decay is expected already from DPH alone when it is embedded in a discontinuous and anisotropic e n ~ i r o n m e n t .Differences ~~ in the characteristics of lifetime distributions of the three phospholipids as revealed by ESM distributional analysis may therefore refer to different orientational mobilities of the DPH-labeled phospholipids in the respective lipid domain.
Acknowledgment. Financial support by the Fonds zur F6rderung der wissenschaftlichen Forschung in Osterreich (Projects S 4615 and P7182) is gratefully acknowledged. List of Abbreviations DPH-PC DPH-PE DPH-SM ESM
l-palmitoyl-2-diphenylhexatrienpropionyl-g~y~rophosph~ choline l-palmitoyl-2-diphenylhexatrienpropionyl-gly~roph~ph~ ethanolamine 1-palmitoyl-2-diphenylhexatrienpropionyl-spbingomyelin exponential series method
References and Notes (1) Aloia, R. C.; Curtain, C. C.; Gordon, L. M. (Us. Lipid ) Domains and fheRelationship to Membranehnction; Advances in Membrane Fluidity
2, Alan (2) (3) (4)
R. Liss Inc.: New York, 1988.
Straume, M.; Litman, B. Biochemistry 1987, 26, 5113. Straume, M.;Litman, B. Biochemisfry 1987, 26, 5121. Williams, B. W.; Stubbs, C. D. Biochemistry 1988, 27, 7994. ( 5 ) Fiorini, R.; Valentino, M.; Wang, S.;Glaser, M.; Gratton, E. Biochemistry 1987, 26, 3864. ( 6 ) Kalb, E.; Paltauf, F.; Hermetter, A. Biophys. J . 1989, 56, 1245. (7) Zannoni, C.; Arcioni, A.; Cavatorta, P. Chem.Phys. Lipids 1983,32, 179. (8) Parasassi, T.; De Stasio, G.; Rusch, R. M.; Gratton, E. Biophys. J . 1991, 59, 466. (9) James, D. R.;Ware, W. R. Chem. Phys. Letters 1985, 120, 455. (10) James, D. R.;Ware, W. R. Chem. Phys. Letters 1986, 126, 7. (11) Wagner, B. D.; James, D. R.; Ware, W. R. Chem. Phys. Lefrers 1987, 138. 181. (12) Chang, M. C.; Petrich, J. W.; McDonald, D. B.; Fleming, G. R. J . Am. Chrm. SOC.1983,105, 3819. (13) Alcala, J. R.; Gratton, E.; Prendergast, F. G. Biophys. J. 1987,51, 587. (14) Alcala, J. R.; Gratton, E.; Prendergast, F. G. Biophys. J . 1987,51, 597. (15) Alcala, J. R.; Gratton, E.; Prendergast, F. G. Biophys. J . 1987, 51, 925. (16) Landl, G.; Langthaler, T.; Engl, H. W.; Kauffmann, H. F. J . Comp. Phys. 1991, 95, 1. (17) Comfurius, P.; Zwaal, R. F. A. Biochim. Biophys. Acta 1977, 488, 36. (18) Kaller, H. Biochem. 2.1961, 334, 451. (19) Cohen, R.;Barenholz, Y.;Gatt, S.; Dagan, A. Chem. Phys. Lipids 1984, 35, 371. (20) Dodge, J. T.; Mitchel, C.; Hanahan. D. J. Arch. Biochem. Biophys. 1963, 100, 119. (21) Broekhuyse, R. M. Biochim. Biophys. Acta 1968, 152,307. (22) Kremer, J. M. H.; v.d. Esker, M. W.; Patmamanoharan, J. C.; Wiersema, P. H. Biochemistry 1977, 16, 3932. (23) OConnor, D. V.; Phillips, D. 1984. Time-correlaredSinglePhoron Counting; Academic Press: London, 1984. (24) Grinvald, A.; Steinberg, I. Z . Anal. Biochem. 1974, 59, 583. (25) AI-Baali, M.; Fletcher, R. J . Operational Res. Soc. 1985, 36, 405. (26) Fletcher, R.;Xu,C. IMA J . Num. A n d . 1987, 7 , 371. (27) Williams, B. W.; Scotto, A. W.; Stubbs, C. D. Biochemistry 1990, 29, 3248. (28) Hermetter, A.; Kalb, E.; Loidl, J.; Paltauf, F. SPIE 1988,909, 155. (29) Toptygin, D.; Svobodova,J.; Konopasek, I.; Brand, L. J . Chem.Phys. 1992, 96, 7919.
