J . Phys. Chem. 1987, 91, 6322-6326
6322
Thermodynamics and Klnetics of the Interaction of Merocyanine 540 with Hydrophobic Structures. 2. Binding of Merocyanine 540 to Soybean Phosphatidylcholine Oligolamellar Liposomes and to Mitochondria G. Dodin* Institut de Topologie et de Dynamique des Systemes de Wniversitd Paris 7 , associP au C.N.R.S., 75005 Paris Cedex, France
and J. Dupont Laboratoire de Biologie VZgZtale IV, CNRS-UA 1180, UniversitP P. et M . Curie, 75231 Paris Cedex, France (Received: March 24, 1987; In Final Form: July 7 , 1987)
Merocyanine 540 (MC 540) binds to soybean lecithin liposomes with an affinity constant K,F of 10 f 2.5 pM. It is estimated that each liposome, whose mean radius is 70 nm, can accommodate approximately 6000 dye molecules. MC 540 binds also to potato mitochondria, submitochondrial particles, and mitochondrion inner membrane with a similar affinity (Kamito= 5 f 1 pM). This study suggests that the number of a bound merocyanine molecules per molecule of surfactant or phospholipid is roughly constant in SDS micelles, phosphatidylcholine liposomes, and mitochondrial membranes. The T-jump kinetic investigation of MC 540 shows that the binding process to liposomes, submitochondrial particles, and mitochondrion inner membranes is identical with that previously described for addition to micelles (ref 8) though occurring in different time scales. Moreover our general kinetic model accounts satisfactorily for other kinetic observations which were rather assigned to equilibria taking place on or within the liposomes (ref 9).
The cyanine dye Merocyanine 540 ( M C 540) is widely used to probe membrane chemical and electrical states through changes of its optical properties (absorption and emission) induced by binding.1-5 The growing interest of M C 540 as a specific pho-
MC 540
tosensitizer of malignant ceW7calls for quantitative information on dye/membrane interaction which should include affinity constant determination and measurement of dye/membrane complex lifetimes which can be derived from the kinetics of the reversible addition of the dye. This latter parameter can be expected to be relevant in understanding the interference of the dye/phospholipid bilayers complexes with cell membranes or with cellular processes. The study of M C 540 addition to anionic and cationic surfactant micelles, which supposedly mimic membranes satisfactorily as they themselves are organized hydrophobic aggregates, has allowed the estimation of the binding affinity constant (4 pM) and of the kinetic rate constants of dye fixation ( k 2 = 3 X lo8 M-’ s-I) and complex dissociation ( k 2= 1200 s - ~ ) . ~ ( 1 ) Sims, P. J.; Waggoner, A. S.; Wang, C. H.; Hoffman, J. F. Biochemistry 1974, 13, 3315. (2) Cohen, L. B.; Salzberg, B. M.; Davila, H. V.; Ross, W. N.; Landowne, D.; Waggoner, A. S.; Wang, C. H. J . Membr. Bioi. 1974, 19, 1. (3) Valinsky, J. E.; Easton, T. G.; Reich, E. Cell 1978, 13, 487. (4) Masamoto, K.; Matsuura, K.; Itoh, S . ; Nishimura, M. Biochim. Biophys. Acta 1981, 638, 108. ( 5 ) Humphries, G. M. K.; Lovejoy, J. P. Biochem. Biophys. Res. Commun. 1983. 11 I . 768. (6) Sieber, F.; Spivak, J. L.; Sutcliffe, A. M. Proc. Narl. Acad. Sci. USA 1984, 81, 7584. (7) Atzpodien, J.; Gulati, S . C.; Clarkson, B. D. Cancer Res. 1986, 46, 4892. (8) Dodin, G.; Aubard, J.; Falque, D. J . Chem. Phys. 1987, 91, 1166. (9) Verkman, A. S . ; Frosch, M. P. Biochemistry 1985, 24, 7117. ~
~~
0022-3654/87/2091-6322$01.50/0
A recent kinetic investigation of the M C 540/phosphatidylcholine liposomes system at high liposome/dye ratio (when all dye is bound to the vesicle surface) has led to the conclusion that bound merocyanine exists as a mixture of exchanging monomers present together with a dimer.9 These studies prompted us to investigate the relevant thermodynamic and kinetic parameters of MC 540 binding to soybean phosphatidylcholine vesicles, mitochondria, mitochondrial inner membrane, and submitochondrial particles at low and medium phospholipid/dye ratio, in order to check (i) the adequacy of the kinetic model developed for the micelle/dye system8 and (ii) to gain insights into the binding step itself, which was not considered in Frosch’s and Verkmann’s workg and which would permit measuring complex lifetimes.
Experimental Section M C 540 samples, buffered at pH 7.4 (NBS primary buffer 0.0087 M KH2P04,0.034 M Na2HP04) with an ionic strength of 100 mM, used in the kinetic and thermodynamic studies, were prepared as described previously.8 Spectrophotometric and kinetic experimental setups were as described earlier (8). Oligolamellar Soybean Phosphatidylcholine Liposome Preparation. The liposomes were kindly provided by Mrs. Durieux (Pharmacie centrale des Hopitaux) and were prepared as follows: 500 mg of phospholipid was dissolved in chloroform/methanol mixture and then dried. Diethyl ether and 50 mL of buffer pH 7.4, I = 100 mM were used to resuspend the film. The suspension was then mixed with a mixer to yield an emulsion. The average diameter of the vesicles as measured with a nanosizer (Coultronics) is 140 nm. The mean number of bilayers per liposome, observed with the electron microscope, is 3.3. The concentrations of liposomes were expressed in terms of phosphatidylcholine (PC) molar concentration (the average molecular weight of soybean lecithin being 778). Liposomes were introduced in the spectrophotometric cuvettes and T-jump cell at the final concentrations of 1-15 pM. In this range the low turbidity of the suspension produces only a little light scattering. The electric field variation during the T-jump capacitor discharge did not alter the scattering. As a consequence, the single beam spectrophotometric detection of the T jump could 0 1987 American Chemical Society
Binding of M C 540 to Soybean Lecithin
be used confidently. In the measurements of the affinity constants, taking advantage of the double beam spectrophotometer, equal amounts of vesicles were introduced on both channels thus fully avoiding scattering perturbations. Mitochondria and Submitochondrial Particle Preparation. Potato mitochondria suspensions, in 10 m M phosphate (0.3 M mannitol) buffer, were prepared according to a described procedure. Mannitol, which ensures isosmotic conditions, was checked not to complex the dye. Mitochondrial inner membranes were discarded from outer membranes by centrifugation after osmotic shock of mitochondria in 25 m M phosphate buffer (without mannitol). Mitochondrial concentrations were expressed in terms of protein concentration. Though the medium did not contain calcium ions or bovin serum albumin (those constituents, usually present in mitochondrial suspensions, cause undesirable interactions with the dye), mitochondria fully retain their phosphorylating ability for at least 24 h. The same buffer was used in the spectrophotometric and kinetic experiments. It was observed that whole or sonicated mitochondria (suspensions with a low turbidity) have similar affinities to M C 540. As above, the organite samples (whole or sonicated) were introduced in both channels of the spectrophotometer. T-jump experiments proved not to be feasible when whole mitochondria were used because of a large shift of light scattering induced by the strong variations of the electrical field during the capacitor discharge. This effect was not disturbing with inner membranes at a concentration of 12 mg/mL or with sonicated mitochondria (submitochondrial particles) which thus were used in the kinetic investigation.
The Journal of Physical Chemistry, Vol. 91, No. 24, 1987 6323
0
0
i
700
Figure 1. Titration of 4 pM MC 540 by soybean lecithin liposomes (pH 7.4 I = 0.1 M) at room temperature. Liposome concentrations are
expressed in terms at phosphatidylcholine[PC] concentrations in moles. (1) [ E ] = 0, (2) 2.56 X lom5,(3) 5.12 X (4) 7.68 X lV5, ( 5 ) 14.08 (6) 20.48 X
X
(7) 26.88 X
n..... 6
Results Thermodynamics. The absorption spectrum of free aqueous merocyanine at concentration around 10 pM shows two maxima at 500 and 535 nm unambiguously assigned to dimeric and monomeric structures of the dye, respectively.8~10 When the concentration is further increased (>20 pM), an absorption develops around 600 nm as a result of increasing aggregation of the dye. When amounts of liposomes or of mitochondria are added to the aqueous dye a new absorption band shows around 515 nm with an isosbestic point at 530 nm (Figures 1 and 2). A similar behavior was observed when M C 540 is added to cationic and anionic micelles* and membranes" and must be definitely attributed to the reversible addition of the aqueous dye to the phospholipid bilayer. Further addition of PC vesicles or mitochondria does not bring about significant modification of the absorbance. The formation of PC liposomes and mitochondria/merocyanine complexes may be viewed as arising from binding of the dye to a set of identical and independent sites.* The overall binding equilibrium is thus characterized by an intrinsic constant
0;
aJ C
0
e VI
n
Q
01
[El[Dl K, = [CI
[c]
where [E], [D], and stand for the equilibrium concentrations of, respectively, phospholipids, dye (whatever its form, monomeric or dimeric) and complex. If there are n binding sites per P C molecule or per milligram of mitochondrial protein, the conservation of the overall dye yields: concentration [Do] [Dol = [Dl +
Figure 2. Titration of 2.5 pM MC 540 by potato mitochondria (whole organites) in pH 7, I = 0.01 M at room temperature. Mitochondrial concentrations are expressed in terms of protein concentrations in mg/ mL. (1) [mito] = 0, (2) 0.06, (3) 0.12, (4) 0.18, ( 5 ) 0.27, (6) 0.39.
The absorbance A a t X is Ax =
[ED[D]
+ tc[C]]l
then
Titration of M C 540 by varying quantities of vesicles allows the determination of the K / n ratio.
(10)
Dixit, N. S.; Mackay, R. A. J . Am. Chem. SOC.1983, 105, 2928.
(1 1) Smith, J. C.; Graves, J. M.; Williamson, M. Arch. Eiochem. Eiophys. 1984, 232, 430.
A. and A, being, respectively, the absorbance of free and fully where complexed dye. The plot of A - A,, versus A - Ao/ [E],
[ E ] has been taken as PC or protein overall concentrations to
Dodin and Dupont
6324 The Journal of Physical Chemistry, Vol. 91, No. 24, 1987
*
I
, \ ,
,
10-3IA-A,/
[PC], I
Figure 3. Plot according to eq 1 from the titration of MC 540 (4 pM) by liposomes at X = 565 nm. -KaPC/npc = loa M.
Figure 5. Scatchard plot (eq 2) obtained from titration of lecithin liposomes ([PC] = 6.4 X lo-' M) by MC 540 at X = 575 nm (c,,plcx,575 = 70000 M-I cm-I). See text.
0'
I
1
I
1
5
10
15
20
-
10 (A-Ao)/ [MITOIo
Figure 4. Plot according to eq 1 from the titration of MC 540 (2.5 pM) by potato mitochondria at X = 570 nm. -Kamit/nmlt= 0.16 mg of pro-
tein/ml. a first approximation, gives a reasonably good straight line from which K / n is estimated to loT4M for liposomes and 0.16 mg protein/mL for mitochondria (Figures 3 and 4). At high dye/phospholipid ratio, the site occupation parameter, u defined as
is close to the maximum binding site per PC mole, npc,or per milligram of protein, nmit (see spectra 2 on Figures 1 and 2). Computing the concentration of free dye from that of bound dye as estimated from the spectral absorption at 575 nm (where bound merocyanine is the only absorbing species with a molar coefficient of 70000 M-' cm-', see ref 8) leads to the conditions uF 3 0.1 and umit3 29 nmol of dye/mg of protein. It is worth noting that this latter value is close to that estimated for M C 540 binding to beef heart submitochondrial particles." Taking npc= 0.1 and nmit= 29 nmol of dye/mg of protein leads to K,F = 10 f 2.5 pM and Kbmit= (5 f . l ) pM, respectively. Alternatively, separate estimates of the affinity constants and maximum number of binding sites can be obtained from the titration of a fixed amount of phospholipids by increasing the quantities of dye, which should be well represented by a Langmuir isotherm. Thus the site occupation parameter is related to the affinity constant and n by the typical Scatchard equation
_a -- - 1[ n [DI
Ka
-
u]
where [D] is the concentration of free aqueous merocyanine estimated from the concentration of the bound dye as measured by the absorbance at 575 nm (where, as stated above, there is only a little optical density arising from other species) with c575 = 70000 M-' cm-l for bound merocyanine (Figure 5). The affinity constant of inner membranes as determined by this procedure is KarM = 3 f 1 pM. It must be stressed that PC liposomes and mitochondrial membranes have similar affinities toward merocyanine. These equilibrium constants must also be reconciled with those previously measured in the binding of MC 540 to SDS micelles6 and to beef heart mitochondrion membranes" (2.5 pM and 7.2 p M, respectively).
The subsequent kinetic study designed to determine the mechanism of dye addition and to estimate the residence time of the dye on or within the phospholipid bilayers will be understood on the basis of the preceding equilibrium discussion and data.
Kinetics Merocyanine 540/PC liposome or mitochondria solutions at a ionic strength of 0.1 and 0.01 M, respectively (as in the thermodynamic study) subjected to a fast temperature jump show a biphasic relaxation spectrum: (1) A fast phase, not time-resolved (
