Supporting Information. with Phospholipid Engineered Membranes

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1 Supporting Information Interaction of the Belousov-Zhabotinsky Reaction with Phospholipid Engineered Membranes Kristian Torbensen, a Federico Rossi, b Ottorino L. Pantani, c Sandra Ristori, d * and Ali Abou-Hassan a * a Sorbonne Universités, UPMC Univ Paris 06, UMR 8234, Laboratoire Physico-chimie des Electrolytes, Nanosystèmes InterfaciauX (PHENIX), 4 place Jussieu - case 51, Paris cedex 05 France b Department of Chemistry and Biology, University of Salerno, Via Giovanni Paolo II 132, Fisciano (SA), Italy c Department of Agrifood Production and Environmental Sciences, University of Florence, P.zle Cascine 28, Firenze, Italy d Department of Earth Sciences & CSGI, University of Florence, Via della Lastruccia 3, Sesto Fiorentino, Firenze, Italy

2 1. SAXS experiments and fitting Fitting of SAXS diagrams The GAP software models the SAXS intensity profiles of bilayer-based structures by the following equation: (1 Ndiff ) S( q) P( q) + Ndiff P( q) I ( q) = (1) 2 q where N diff is the fraction number of positionally uncorreraled bilayers (i.e. those forming non interacting vesicles), S(q) is the structure factor defining the spatial distribution of scatteres and describing the inter-particle interactions and P(q) is the absolute square of the bilayer form factor. In this work, a structure factor obtained by a modified Caillé theory 1,2 was chosen to model the bilayer interactions when quasi- Bragg peaks were evidenced in the intensity profiles. The electron density is modelled in the GAP software by a three-gaussians profile, 3,4 representing the polar head groups, placed at ± z H, and the hydrocarbon core. The standard deviation of these electron distributions are σ H and σ C, respectively. The terminal methyl group in the bilayer center corresponds to the minimum of the electron density profile, as sketched in scheme SI1. The amplitude (always negative) of the hydrophobic tails with respect to the headgroup is termed ρ H. polar head polar head ρ - Z H Z H water medium center of the bilayer distance Scheme SI1. Electron density profile as a function of the distance from the bilayer center, modeled by the sum of three gaussian distributions. In summary, the best fit parameters used in this fitting procedure were: The lamellar repeating distance, d (for systems presenting Bragg peaks); the average number of bilayers per scattering domain (N) and the fraction of positionally uncorrelaed bilayers (N diff ); the Gaussian distributin center of the polar heads electron density profile (z H )

3 and its standard variation width (σ H ) ; the Gaussian distribution center and standard variation of the hydrophobic core (z C and σ C, respectively), the amplitude (always negative) of the hydrophobic tails Gaussian relative to the headgroup Gaussian (ρ C ) and the fluctuation Caillé parameter (η) related to the bilayer bending rigidity. The best-fit valus which describe the structure and the electron density of DMPC lamellar phases in water and in different dispersing media are reported in table SI1. For all systems the total lipid content was kept constant at 1% w/w. The electron density profiles of plain a DMPC bilayer in the presence of different water soluble species and bilayer components are shown in figure SI1 and SI2, respectively. For these profiles, normalization was performed with respect to the absolute value of the DMPC bilayer center, i.e. 0.2 e/å. 3,5 This choice was motivated by the following considerations: (i) ferroin is absorbed on the surface (see main text) and it is not expected to inflence the bilayer center electron density; (ii) the bilayer absorption of small water molecules should bring very little effct to the elctron density of this highly hydrophobic region; (ii) myr A carry the same chian as DMPC, therefore the bilayer interior of plain DMPC, DMPC-myr A and DMPC-myr A-STS bilayer should be very close to one another. Table SI1. Structural and electron density parameters System composition and dispersing medium d (Å) N z H (Å) σ H (Å) σ C (Å) ρ C (Å) η N diff DMPC in water DMPC + ferroin (a) in water DMPC in BZ/water DMPC + myr A in water DMPC + myra + STS in water (a) (b) (c) (d) ferroin = M MA 0.15 M, BrO 3-, 0.18 M, H 2 SO M, ferroin M DMPC : myra = 0.8 : 0.2 molar ratio DMPC : myra :STS = 0.80 : 0.15 :0.05 molar ratio

4 0.4 DMPC in H2O DMPC-MyrA DMPC-MyrA_STS electron density (e/å 3 ) distance from bilayer center (Å -1 ) Figure SI1. Comparison among the electron density profile of bilayers containing pure DMPC, DMPC + myr A (0.8/0.2 mol fraction) and DMPC + myr A + STS (0.80/0.15/0.05 mol fraction). 2. Zeta potential of extruded liposomes The surface charge of extruded liposomes (having the same composition as the samples used for the SAXS experiments) was measured by electrophoretic light scattering 6 with a Coulter DELSA 440 SX appartus (Coulter Corporation, Miami, FL, USA). Homemade hemispherical electrodes covered by a thin gold layer were used in the measurement cell. This variant was necessary in the present study, since samples containing BZ solutions would oxidize the silver electrodes typically used in this kind of instruments. The obtained Zeta potential values are reported in Table SI2 and refers to the average over 5 runs with detection at different angles (7.5, 15, 22.5 and 30 ). For liposome preparation, mixtures of dry lipids were dissolved in chloroform/methanol 2/1 and the solvent was evaporated under vacuum overnight. The resulting mixed lipid film was then swollen at room temperature with MilliQ grade water. Upon vortexing, multilamellar vesicles were obtained, which were then submitted to eight freeze/thaw cycles and subsequently downsized by extrusion through 100 nm polycarbonate

5 membranes.twenty-seven extrusions were performed with the LiposoFast apparatus, Avestin, Ottawa, Canada. Zeta potentials was measured not later than 5-6 hours after liposome prepration, since extruded suspension were a little less stable than the spontaneous dispersions used for the SAXS experiments. Table SI2. Zeta potential of liposomes* pure DMPC in water - 5 ± 2 mv DMPC in water + ferroin + 11 ± 2 mv pure DMPC in H 2SO ± 3 mv DMPC in BZ/water + 18 ± 2 mv DMPC + myr A in water + 18 ± 1 mv DMPC + myra + STS in water - 14 ± 3 mv * reproducilibily was observed to be within experimental error (by 2-3 replica measurements for each sample). It is to be noted that in the presence of BZ reactant liposomes bear a positive charge, which is attributed to the protonation of the phospate groups of DMPC in the extremely acidic ph given by H 2 SO M. 3. Cholesterol Bromination The bromination of olefins and ring-substituted compounds in polar solvents can be a rather complex mechanism depending on the substituents and on the presence of high concentration of Br - in solution. However, when the analytic concentrations of the reactants [olefin] 0 and [Br 2 ] 0 are equal, the process can be described by a second order rate law, being the addition of the second bromine atom the rate determining step. 7 Therefore, for the cholesterol bromination Br 2 + CHOL K Br 2 -CHOL (2)

6 when [Br 2 ] 0 = [CHOL] 0 the rate law can be written as d[br 2 dt = K[CHOL [Br 2 (3) and, given the stoichiometry of the reaction, relation (4) holds [CHOL] 0 [CHOL = [Br 2 ] 0 [Br 2 (4) therefore, equation (3) can be rewritten as d[br 2 dt = K[Br 2 2 (5) which, integrated between [Br 2 ] 0 and [Br 2 and 0 and t, yields the operative equation to calculate K as the slope of 1 1 = Kt+ [Br 2 [Br 2 ] 0 (6) In order to study the kinetics of equation (2) through the formula (6), we performed a series of experiments by following spectrophotometrically [Br 2 at λ = 400 nm, where the molar extinction coefficient of bromine is about 230 M -1 cm -1, and we plotted data according to equation (6). Experiments were performed in Methyl-tert-butyl ether (MTBE), in order to avoid the precipitation of Br 2 -CHOL and to work with higher concentration of cholesterol, which solubility in MTBE is higher than that in water. We mixed equal concentrations of the reactants (1.3 mm) and a typical spectorphotometric recording is reported in Figure SI2. From the fitting of experimental data a value ok K = M -1 s -1 has been found.

7 Figure SI2. Second order kinetics for the bromuration of Cholesterol. 4. Numerical simulation of the BZ reaction Below is listed the complete reaction scheme used for the numerical simulation performed using the CO.PA.SI. software for the ferroin catalyzed BZ reaction. Table SI2 shows the related kinetic constants. HOBr +Br +H Br +H O (R1) Br +HBrO +H + 2HOBr 2HBrO 2 BrO +HOBr+H (R2) (R3) Br +BrO +2H + 3 HOBr+HBrO 2 (R4) + BrO 3 +HBrO 2+H Br2O 4 +H2O (R5) Br2O 4 2BrO (R6) Fe(phen) +BrO +2H Fe(phen) +BrO +H O Fe(phen) +BrO +H Fe(phen) +HBrO (R7) (R8) Fe(phen) 3 +BrCH(COOH) 2 Fe(phen) 3 + CBr(COOH) 2+H (R9)

8 BrCH(COOH) (HOOC)CBr=C(OH) (R10) 2 2 (HOOC)CBr=C(OH) +Br Br C(COOH) +Br +H (HOOC)CBr=C(OH) +HOBr Br C(COOH) +H O 2 CBr(COOH) +H O BrCOH(COOH) +BrCH(COOH) BrCOH(COOH) Br +CO(COOH) +H (R11) (R12) (R13) (R14) Table SI2. Values of rate constants for reactions R1 R14. reaction k forward k inverse R mol -2 dm 6 s s -1 R mol -2 dm 6 s -1 R mol -1 dm 3 s -1 R4 10 mol -3 dm 9 s mol -1 dm 3 s -1 R5 48 mol -2 dm 6 s s -1 R s mol -1 dm 3 s -1 R mol -3 dm 9 s -1 R mol -2 dm 6 s -1 R9 13 mol -1 dm 3 s M -2 s -1 R s s -1 R mol -1 dm 3 s -1 R mol -1 dm 3 s -1 R mol -1 dm 3 s -1 R s -1 References [1] J.F. Nagle, S. Tristram-Nagle, Structure and interaction of lipid bilayers: role of fluctuations, in: J. Katsaras, T. Gutberlet Chapman (Eds.), Lipid Bilayers. Structure and Interactions (2001) pp Springer, Berlin. [2] G. Pabst, Global properties of biomimetic membranes: perspectives on molecular features, Biophys. Rev. Lett. 1 (2006) [3] G. Pabst, J. Katsaras, V.A. Raghunathan, M. Rappolt, Structure and interactions in the anomalous swelling regime of phospholipid bilayers, Langmuir 19 (2003)

9 [4] L. Cantù, M. Corti, E. Del Favero, M. Dubois, and Th.N. Zemb. Combined Small-Angle X-ray and Neutron Scattering Experiments for Thickness Characterization of Ganglioside Bilayers, J. Phys. Chem. B., 102 (1998) [5] H.I. Petrache, S. Tristram-Nagle, J.F. Nagle, Fluid phase structure of EPC and DMPC bilayers, Chem. Phys. Lipids 95 (1998) [6] K.H. Langley, Developments in electrophoretic laser light scattering andsome of biochemical applications, in: S.E. Harding, D.B. Sattelle, V.A. Bloomfield (Eds.), Laser Light Scattering in Biochemistry: The Royal Society of Chemistry, 1992, pp [7] K. Yates, R.S. McDonald, S.A. Shapiro, Kinetics and Mechanisms of Electrophilic Addition. I. Comparison of Second- and Third- Order Brominations. J. Org. Chem. 38 (1973)