Photosensitized production of singlet oxygen by merocyanine 540 bound to liposomes

J. Photo&em.

Photobid.

B: Biol., 9 (1991)

281-294

281

Photosensitized production of singlet oxygen by merocyanine 540 bound to liposomes Maryse

Hoebeke,

Jacques

Laboratory of Experimental B-4000 Liege (Belgium) (Received

September

Keywords.

6, 1990;

Piette and Albert

Physics,

accepted

Institute

of Physics

December

van de Vorst B5, University

of Liege,

28, 1990)

Merocyanine 540, photosensitization,

liposomes, singlet oxygen.

Abstract The production of singlet oxygen by merocyanine 540 was studied in dimyristoyl-phosphatidylcholine liposomes using two singlet oxygen probes: 9,l O-anthracenedipropionic acid (water soluble) and 9,10-dimethylanthracene (liposoluble). Upper and lower limits of singlet oxygen quantum yield for bound merocyanine 540 were determined to be 0.055 and 0.015 respectively. The diffusion characteristics of singlet oxygen were examined using the isotropic enhancement effect of Da0 and the inhibitory effect of sodium azide. It was shown that ‘02 spent more than 87% of its lifetime in a vesicle environment. When the singlet-reacting substrate and the dye were both located in the bilayer, approximately 40% of the singlet oxygen remained in the liposomes where it was originally generated.

1. Introduction

Merocyanine 540 (MC540) is a fluorescent anionic dye which binds selectively to many types of electrically excitable cells, leukaemic and immature haematopoietic cells [ 11. This important biological property has been exploited in clinical diagnosis and in a procedure to eliminate leukaemic cells in a murine model of autologous bone marrow transplant [ 11. In addition, MC540 has frequently been used as a probe to study membrane potentials [2] and haematopoietic development [3]. Despite its importance in biology and medicine, little is known about the photochemistry of the dye and the molecular basis of its photosensitizing activity. Characterization of several photophysical properties of MC540, such as fluorescence lifetime, fluorescence and triplet quantum yields, has been made by Aramendia et al. [4] for free MC540 in ethanol solutions and MC540 bound to unilamellar liposome vesicles. A previous study from our laboratory has shown that, on visible light irradiation, free MC540 in solution undergoes an isomerization reaction, leading to the generation of singlet oxygen (‘0,) (51. The measured photoisomerization quantum yield (@is,,) is very high and dependent on viscosity unlike the singlet oxygen quantum yield (@lo,>

loll-1344/91/$3.50

0 Elsevier

Sequoia/Printed

in The Ncthcrlands

282

which is very low and remains unchanged whatever the solvent viscosity [ 61. photoinduced cis-trans isomerizations of organic molecules are important processes in biological systems [ 71, nothing is known about the biological consequences associated with the photoisomerization of MC540 located in cellular membranes. However, the production of ‘02 by MC540 bound to artiilcial membranes systems, such as dimyristoyl-phosphatidylcholine liposomes, has been demonstrated by the direct detection of ‘02 luminescence at 1268 nm [ 81. These workers also reported a dependence of ?O, production on oxygen concentration and an oxygen-dependent photobleaching of MC540 in the membrane bilayer. It has been shown that ‘02 possibly plays a major role in the cellular photokilling mediated by this dye Although

191.

Liposomes consist of two distinct regions: the intervesicle aqueous phase and the intravesicular part involving the bilayer of phospholipids and the aqueous phase entrapped inside the vesicle [lo]. Fluorescence polarization studies suggest that, in lipid bilayer membranes, MC540 can exist as a monomer in two exchangeable orientations: parallel and perpendicular to the plane of the membrane with the parallel molecules in equilibrium with the dimers [ 11, 121. When IO2 is generated by bound MC540 in the lipid bilayer of one vesicle, there are several pathways open to the molecule: diffusion through the vesicle where it has been generated, diffusion through the inter-vesicleaqueous phase and/or diffusion through the intravesicle phase. The aim of the present work is to examine the quantum yield of singlet oxygen production by bound MC540 and to determine the manner in which singlet oxygen diffuses through the lipid phase and the aqueous phase from the site where photosensitization takes place. Dimyristoyl-phosphatidylcholine unilamellar liposomes are used as a membrane model and two different kinds of ‘02 traps are employed: 9,10-anthracenedipropionic acid (water soluble) and 9,lOdimethylanthracene (liposoluble).

2. Materials

and methods

2.1. Chemicals MC540 and rose bengal (RB) were obtained from Eastman Kodak (U.S.A.). Dimyristoyl-L-a-phosphatidylcholine (DMPC) and 9,lOdimethylanthracene (DMA) were obtained from Sigma (U.S.A.). 9,10-Anthracenedipropionic acid (ADPA) was synthesized as described by Gandin et al. [ 131. Sodium azide (NaN,) and deuterium (DzO) were obtained from Merck (F.R.G.) and Sephadex G25 superfine was purchased from Pharmacia (Sweden). All other chemicals were of reagent grade. RB was purified by gel chromatography according to Gandin et al. [ 141. Commercial MC540 was used without purification. 2.2. Liposomes Solutions of DMPC (3.33 mg ml-’ dissolved in chloroform) were dried under vacuum in a rotary evaporator and resuspended in 3 ml of phosphate

buffer (pH7). The resulting multilamellar large vesicles (MLVs) were then transferred into an extruder (Lipex Biomembranes, Vancouver, Canada) where they were 6ltered through polycarbonate filters (0.1 pm pore size, Nucleopore Corp., Pleasanton, CA, U.S.A.) using pressures of up to 6900 Pa of nitrogen gas [ 151. This extrusion was carried out above the temperature of phase transition of the phospholipid (23 “C) and was repeated ten times. Previous studies have demonstrated that repeated extrusion of MLVs through polycarbonate filters (0.1 pm pore size) at moderate pressures produces a relatively homogeneous population of large unilamellar vesicles (LUVETS) [ 151. The stock of LUVETS was then diluted in phosphate buffer or DzO to obtain the desired concentration of phospholipids. MC540 and ADPA, if required, were added at this stage. Experimental conditions were selected to maximize the binding of MC540 to the vesicles, so that solution to membrane partitioning was minimal. Sikurova et al. [ 16, 171 have determined the dye concentration range in which all the dye in the suspension can be considered to be bound to the liposomes. According to their results, with a phospholipid to MC540 molar ratio of greater than 73, nearly all the MC540 molecules are bound to liposomes. In our experimental conditions, a ratio of greater than 97.5 was used and no absorption peak corresponding to MC540 molecules in water was observed. Furthermore, no destruction of ADPA molecules was detected with MC540 in phosphate buffer, which is in good agreement with the absence of ‘02 production observed by Feix et al. [8] for this dye in solution. Complete incorporation of MC540 into the phospholipid bilayer was checked by absorption spectroscopy (Perkin-Elmer 559 UV-visible spectrophotometer) and by gel filtration on Sephadex G25. The incorporation of DMA into the phospholipid bilayer was carried out by evaporating DMA solubilized in chloroform together with DMPC in a rotary evaporator. After hydration with the phosphate buffer, a freeze-thaw procedure using liquid Nitrogen (five cycles) was carried out before sample extrusion. The excess of DMA was removed by chromatography on Sephadex G25 equilibrated in phosphate buffer. No release of DMA was observed by dialysis during the time of the experiments. 2.3. Steady state irradiations The solutions were irradiated with filtered light from a slide projector (Pradovit RA150, Leitz, F.R.G.). A cut-off filter (Schott OG515, F.R.G.) was used to eliminate light under 500 nm. The relative number of photons absorbed by the dye was determined by the method described by Gandin and Lion (181. 2.4. Fhmescence

and absorption

measurem,t?nts

Absorption spectra were recorded on a Perkin-Elmer 559 spectrometer. The fluorescence measurements were carried out on an Aminco 500 spectrofluorimeter. Singlet oxygen monitoring was performed by following the bleaching of ADPA at 400 nm [ 191 or by following the decrease in DMA

284

fluorescence emission intensity at 410 nm. A thermostatically-controlled cell was used for all the determinations to monitor the temperature of the sample. Linear regression fitting of the experimental curves enabled the slopes of the ADPA or DMA decay to be determined. Previous studies have indicated that ADPA and DMA are suitable for IO2 detection in water and DMPC respectively [ 13, 19, 201. If the decay of ADPA or DMA is determined for MC540 in liposomes and for a dye, for which @Q~ is known, the ratio between the slopes (corrected for the absorption and deactivation constants of ‘02 via the solvent) gives the value of @Q for MC540 in liposomes [ 131.

3. Results 3.1. DAL4 photobleaching mediated by MC540-DMPC liposomes Figure 1 shows the photobleaching of DMA mediated by MC540 bound to DMPC liposomes in aerated or oxygen-saturated phosphate buffer and in DzO at 24.6 “C. This photosensitized DMA decay was monitored by the decrease in fluorescence intensity at 410 nm. The kinetics are clearly exponential. A small but significant enhancement of the rate of DMA decay is observed when D20 is used instead of Ha0 (the decay increases by a factor of 1.3). To verify the participation of ‘02 in the destruction of DMA, competition experiments were carried out using NS- as ‘02 quencher. When azide is present in the external aqueous phase, the rate of DMA bleaching is markedly reduced due to the quenching of ‘OZ. It should be noted that, even at high azide concentration (77 mM), the inhibition of the DMA fluorescence decay is not complete (Fig. 1) (between 40% and 50% of the DMA decays without azide). When NaN3 is added at various concentrations to solutions maintained at 26 “C, the ratio between the rate constants of DMA destruction in the absence and presence of NaN3 varies linearly with the inhibitor concentration. Analysis of the experimental data using the Stern-Volmer equation. K,/K= 1 +K,[N,-

]

(1)

where K. and K are the rate constants of substrate destruction in the absence and presence of azide respectively, gives K,= 2930 M-‘. This incomplete azide effect, together with the complete inhibiting action of nitrogen and the accelerating effect of oxygen-saturated conditions (Fig. l), indicates that part of the DMA photobleaching is due to ‘02 remaining in the bilayer. Moreover, the residual slope of DMA photobleaching in the presence of 77 mM NaN, is the same in Ha0 and DaO. The slope of the DMA decay corrected for light absorption [ 181 gives a relative measure of the ‘02 quantum yield (&,). Using the absolute value of @lo2 for MC540 in ethanol (0.007) (51, and taking into account the different rate constants of singlet oxygen deactivation by the solvent and by reaction with DMA together with their corresponding weighting factors (see discussion), a @loZvalue between 0.0 15 and 0.05 is obtained for MC540 bound to liposomes by comparison with MC540 under the same irradiation conditions.

285

0.6 I

E

0.4

02

t

t

I

I

I

2

L

6

I

6 time (min)

I

10

1

12

I

1L

I

16

Fig. 1. Bleaching of DMA fluorescence emission (&,,= 376 nm, A_== 410 nm) in the presence of MC540 bound to liposomes (temperature, 24.6 “C): A, in &-saturated aqueous solutions in the presence of 7.7 x lo-’ M NaNa; 0, in air-saturated aqueous solution; A, in DzO; n , in oxygen-saturated aqueous solution.

3.2. ADPA photo-oxidation mediated by MC540-DMPC liposms Figure 2 shows the loss of absorbance of ADPA measured at 400 nm and various temperatures during the photosensitization reaction mediated by MC540 bound to DMPC liposomes. In all experiments, the temperature was above the phase transition temperature (23 “C) of the DMPC liposomes. A two-phase decay is obtained and can be fitted to a double exponential function. The size of the discontinuity observed in these decays varies with the temperature of the liposome suspension. Two important characteristics can be deduced from these curves: (i) the discontinuiiy in the ADPA absorbance decay appears more rapidly when the temperature is increased from 24.2 to 30.1 “C and (ii) the slope of the slower mechanism increases very slightly

286 6_

-0

I_

‘=-q

-

2

4

6

8

10

12

14

16

18

20

22

2.4

time Imin)

Fig. 2. Photobleaching of ADPA (monitored at 400 nm) in the presence of MC540 bound to liposomes at 30.1 “C (m), 26.1 “C (Cl) and 24.2 “C (0). Inset: Stem-Volmer plot of the destruction of ADPA photosensitized by MC540 bound to Iiposomes in the presence of N,-. Ka and KS are the slower rates of substrate destruction in the absence and presence of N3-, respectively.

with temperature (except between 24.2 and 30 “C where it is not significant). At 38 “C, only one slope is detected which is 2.9 times faster than the slower slope measured at 24.6 “C (Pig. 3). When NaN3is added at various concentrations to the solutions containing 70 PM ADPA and 3.6 PM MC540 at 26 “C, the ratio between the slower rate constants in the absence and presence of NaN3 varies linearly with the inhibitor concentration (Pig. 2, inset). Analysis of the experimental data using the Stem-Volmer equation (eqn. (1)) gives K,=2297 M-‘. However, even 10 mM of aside does not inhibit the faster phase of the decay completely (Pig. 4). Whatever the NB- concentration used, the value obtained by subtracting the slope (KS) of the slower mechanism from that (Kf) of the faster mechanism remains constant and equivalent to the value of the residual slope observed when the reaction is carried out in the presence of 10 mM sodium azide. These results suggest that part of the ADPA bleaching occurs in the hydrophobic region of the lipid bilayer where ‘02 is not so easily accessible to quenching by aside. Moreover, no significant ADPA bleaching is recorded after saturation under nitrogen.

287

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-6

(J

-8

""----o

0 -10

-12

I

L

I

L

I

2

Z.

6

8

10

I

L

12 14 t i m e ( min )

L

L

I

I

L

16

18

20

22

24

Fig. 3. Bleaching of ADPA (monitored at 400 rim) in the presence of MC540 bound to liposomes: (3, 24.6 °C; I , 38 °C; A, 38 °C in D20.

Deuteration of the medium at 24.2 °C results in an increase in the slower decay rate by a factor of 3.4, with the residual slope (Kf-Ks) remaining constant (Fig. 4). At 38 °C, deuteration of the medium results in an increase in the single slope by a factor of 1.26 (Fig. 3) and 10 mM azide completely inhibits the bleaching. An increase in the two slopes at 24.2 °C is also recorded under oxygen-saturated conditions. The enhancing or inhibiting effects of the additives demonstrate that the two-phase ADPA destruction proceeds through a mechanism involving 102. The slower rate constant of ADPA decay, corrected for light absorption, allows an estimate to be made of the lower limit of singlet oxygen quantum yield (~1o2). Using the absolute value of ¢,o~ for RB in water (0.75) [21], and taking into account the different constants of 102 deactivation via the solvents together with their corresponding weighting factors, a ~,o~ value between 0.035 and 0.05 (at 24.2 °C) is deduced by comparison with RB under the same irradiation conditions. Furthermore, the correction factor due to the 102 independent destruction of ADPA b y the photosensitized reaction mediated by RB was also taken into account [ 13]. At 38 °C, ~,o~ increases by a factor of 1.3, (all determinations obtained by comparison with a standard at the same temperature). In each case, the extent of MC540 bleaching is less than 10% of the total MC540 absorbance measured prior to irradiation.

288

-6 t

-‘“,L 2

L

6

0

10

12

li

16

16

20

22

2.b

time [min) Fig. 4. Bleaching of ADPA at 24.2 “C (monitored at 400 nm) in the presence of MC540 bound to liposomes: 0, in nitrogen-saturatedaqueous solution; Cl, effect of NaN, (10 mM) in HzO; A, in HzO; A, in Hz0 bubbled with oxygen for 10 mh prior to irradiation; 0, in DzO.

4. Discussion The production of singlet oxygen by the photosensitizing action of MC540 bound to DMPC liposomes has been demonstrated by the direct detection of ‘02 luminescence at 1268 run [81. The quantum yield of singlet oxygen production (a&) in homogeneous conditions, e.g. in ethanol, is low and independent of viscosity [6]. However, in our experimental conditions, all MC540 is membrane bound. In lipid bilayer membranes, MC540 can exist as a monomer in two exchangeable orientations: parallel and perpendicular to the plane of the membrane with the parallel molecules in equilibrium with the dimers [ 11, 121. There is a fivefold excess of MC540 monomers oriented parallel to the phospholipid chain with their sulphonated group located near the membrane surface above the perpendicular monomers. The dimers are located deep within the membrane, perpendicular to the phospholipid chain. MC540 can also translocate to the opposite membrane surface. Previous studies [22] have shown that ‘OS may be formed in the lipid bilayer of one vesicle and may react with a quencher located in the lipid bilayer of another vesicle or in the water phase. The aims of the present study were to examine the quantum yield of singlet oxygen production when the dye is embedded in the hydrophobic bilayer (whatever its state or location) and to determine the manner in which ‘02 diffuses through the lipid phase and

289

the aqueous phase from the site where photosensitization takes place. In order to study the diiksion of ‘02 inside and outside the liposomes, two types of substrate were used: DMA (hydrophobic) which exhibits a strong affinity for the lipid bilayer [lo] and ADPA (water soluble) which is located in the water phase and is in contact with the membrane surface of the liposomes [ 191. When ‘Oa is generated by bound MC540, it can diEuse via two different pathways: into the intervesicle aqueous phase and/or through the intravesicle phase i.e. into the lipid bilayer or into the aqueous phase entrapped inside the liposomes. In the presence of DMA, ‘02 undergoes the following decay processes MC540 -

MC540* (light absorption)

MC540* -

3MC540 (intersystem crossing)

3MC540 + 302 ‘02 -

IO2+ MC540 (energy transfer)

3O2 (physical deactivation of ‘Oa)

IO2+ (DMA), 2 ‘02+N3-

-

(DMA-O&

(endoperoxide formation)

302 + N3- (quenching of ‘Oa by azide ion)

(2) (3) (4) (5) (6) (7)

Due to the heterogeneity of the environment, the rate of ‘02 deactivation is better represented by k,, = gwkdw+ g”k,”

(8)

where kdw and kdv are the partial constants for ‘Oa quenching in water and in the vesicle phase respectively and g” and g” are the corresponding weighting factors with g” +g” = 1. If R(‘02) is defined as the rate of ‘02 formation d[lO~]/dt=R(lO~)-(k,[DMA]+gwk~w+gvk,v)[’O~]

(9)

The rate of DMA destruction can be described by the following expression -d[DMA]/dt=

k,]DMAlR(‘O,) k,[ DMA] + k,“‘g” + kdvgv

(10)

With a DMA concentration of 1 PM and using the values 2.5 X lo5 s- ‘, 4x lo4 s-’ and 3.2 X lo8 dmT3 mol-’ s-’ for kdw, kdv and k, respectively [ 10, 231, k,[DMA] can be neglected with regard to kdwgw+ k,“g” and eqn. (10) can be simpiified to -d[DhJA]/dt=

kJDMWK’O,> k,“g” + k,“g”

(11)

Using eqn. (lo), the ratio between the rates of DMA destruction in Hz0 and DzO can be expressed as

290

gwkdDzo + gvkdv gwkdw+ g”k,”

(12)

This relationship allows g” to be determined as a function of 9”. The calculation of both weighting factors can be performed using g” +g’= 1. Values of g’=O.96 and g”=O.O4 are calculated with kdDZo=1.8x104 s-’ at 24.6 “C [23] and R(‘Oz) is constant. Taking into account the various errors in each measure in the g” calculation, we estimate a value in the range 0.87-1.00. The weak increase measured in Da0 strongly suggests that ‘Oa spends a considerable proportion of its excited state lifetime in the vesicle interior where no isotopic lifetime enhancement is observed. The g values indicate that ‘02 spends more than 87% of its excited state lifetime in a vesicle environment. When azide ion is present in the intervesicle aqueous phase, the ratio of the rates in the absence and presence of azide is given by

Ko - =1+ K

kNN- I g’“k,” + g”k,’

(13)

A kN value of 1.4X 10’ drne3 mol-’ s-’ was observed and is in agreement with literature values [ 241. It is important to emphasize that the highly hydrophobic DMA and MC540 molecules are both located within the bilayer membrane. These locations, together with the incomplete azide effect, lead us to suspect that an important part (between 40% and 50%) of the reaction between DMA and IO2 occurs within the same vesicle. In order to determine the quantum yield of DMA destruction in liposomes, we used the quantum yield of DMA destruction in ethanol as a reference [13]. The destruction of DMA by MC540 in ethanol is given by R’(‘O,)k,‘[DMA] - dIDMA dt = kd

(14)

where R’(‘0,) is the rate of IO2 production by MC540. Using 4.71 X lo7 dm3 mol-’ s-r [25] for the rate constant of DMA quenching by ‘02 in ethanol (k,‘) and lo5 s-’ [23] for the rate constant of ‘02 quenching in ethanol (kd’), a comparison between the slope in ethanol and the slope in liposomes, corrected for absorption, gives GloZ between 0.015 and 0.05 for bound MC540. The second type of probe used in this work was ADPA (more water soluble than DMA). The use of azide, which is located in the intervesicle phase during the photosensitized reaction mediated by MC540 bound to liposomes, gives a kN value of 2.5 X 10’ mol-’ s-‘. This value is in agreement with that published by Lindig and Rodgers [24] and therefore demonstrates that ADPA destruction is mediated by singlet oxygen generated by MC540 bound to liposomes. An important feature observed during the photosensitized destruction of ADPA is a strong dependence on temperature. Liposomes consist of two aqueous phases separated by a simple lipid bilayer with a defined composition and a large surface area. Thus, temperature-induced

291

changes in the bilayer organization influence the solute permeability and the permeation of water molecules across the bilayer [ZS]. Another parameter which is crucial in the experiments is the probe location. The experiments carried out with ADPA at 24.2 “C reveal the presence of a break point in the kinetics, an incomplete inhibition by sodium azide of the first part of the decay curve and a residual slope (&-KS) which is not influenced by D20. These results suggest that ADPA molecules are located in two different surroundings: some in the aqueous phase and some solubilized near or in the interfacial region of the liposomes. When ‘02 is produced by MC540 bound to liposomes, it can react directly with the latter molecules without leaving the bilayer, which explains the absence of Da0 isotopic and Naquenching effects. Using the same competitive reactions as for DMA, a comparison between the rate of ADPA photo-oxidation in water (slower slope) and DzO at 24.2 “C enables the apparent weighting factors to be calculated (gw=0.33 and g”=0.66). The g” value is overestimated because the ‘Oa which reacts directly with the ADPA molecules in contact with the liposomes is not detected by ADPA in the aqueous phase and so is not subjected to the Da0 isotopic effect. At 38 “C, there is only one slope, which is completely inhibited by 10 mM azide, leading to the determination of g”=O.955 and g”= 0.045. It should be noted that the values obtained for g” and g’ contain an error of 10%. The permeability of membranes to water depends on the degree of packing and the thermal mobility of the hydrocarbon chains. It has been shown [27] that the water permeability of the bilayer continues to increase when the temperature reaches the phase transition or above. When the temperatures are increase above 30 “C, the environment of the ADPA molecules becomes more and more aqueous. Using kinetic parameters, GloZ can be determined at 24.2 “C. The value is in the range 0.035-0.055. At 38 “C, @w,~increases by a factor of 1.3. The increase in ‘02 may have two origins: (i) all ADPA molecules at this temperature are in the aqueous phase (the molecules solubilized near or in the interfacial region of the liposomes could not be responsible for quenching the ‘02 produced by MC540 and then reducing the amount of ‘02 detected by ADPA in the aqueous phase); (ii) the change in temperature may affect the monomer-dimer equilibrium of MC540 in the liposomes, increasing the proportion of monomer and so ‘Oa production. Singh et al. [ 281 have recently shown that the ‘02 action spectrum closely follows the absorption spectrum of monomeric MC540 in these systems. From the results obtained with ADPA and DMA at the same temperature (24 “C), a Qlo2 value between 0.015 and 0.05 is obtained, which is at least twice as high as that measured using free MC540 in ethanol solution (0.007) [5]. 5. Conclusions The following conclusions can be drawn from the results of this study. (i) The QloZvalues obtained are at least twice as high as that measured

292

using free MC540 in ethanol solution. This may be due to the enhanced triplet quantum yield of bound MC540 as observed by Aramendia et al. (41, since the increase in &oz is of the same order as the increase in the triplet quantum yield. (ii) The temperature is an important factor in these experiments as it influences the stability of the liposomes. (iii) The two decay times observed for ADPA destruction indicate an inhomogeneous environment for the probe. (iv) The g values provide an indication of the average time spent by ‘02 in an aqueous or vesicle environment during its excited state lifetime. It was observed that ‘02 spent more than 87% of its excited state lifetime in a vesicle environment. (v) When the probe is located within the lipid bilayer, nearly 40% of the singlet oxygen molecules react directly with it without leaving the vesicle. This can be explained by the location of both MC540 and the probe (DMA) within the bilayer lipid membrane. (vi) All membrane cellular components are susceptible to modifications by singlet oxygen. The rates of reaction of singlet oxygen with fatty acids (4 x lo4 M-’ s-‘) and oxidizable amino acids (lo7 M-’ s-l) suggest that bound MC540 will promote the efficient photo-oxidation of membrane proteins probably at the level of histidine and tryptophan. However, it should be emphasized that the low rate of reaction between phospholipids and singlet oxygen does not necessarily mean that the oxidation products generated by this photosensitized reaction will not lead to important cellular modifications 1291.

Acknowledgments This work was supported by a grant from the Belgian National Fund for Scientific Research (contract number 1.5.178.90F, 1989-1990; Brussels). J. P. is a Senior Research Associate of the Belgian National Fund for Scientific Research.

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