Pavol Balgavý

Cut-off effects in biological activities of surfactants

In the homologous series of long hydrocarbon chain surface active compounds, their various biological activities increase progressively with increasing chain length up to a critical point, beyond which the compounds cease to be active. The paper reviews several hypotheses of this cut-off effect in biological activities and experimental evidences supporting them. It is suggested that the lateral expansion of the phospholipid bilayer of biological membranes caused by the intercalation of long-chain amphiphile molecules between the phospholipid molecules and the mismatch between their hydrocarbon chain lengths results in the creation of free volume in the bilayer hydrophobic region. The elimination of the free volume via the hydrocarbon chain trans-gauche isomerisation or interdigitation results in the bilayer thickness change or in its destabilisation and formation of non-bilayer phase(s). In combination with the partition and ionisation equilibria of amphiphiles in the lipid/aqueous phase systems, the free volume predicts similar chain length and pH dependencies as observed in biological experiments. It is suggested that the free volume mechanism, in combination with other mechanisms, could be responsible for the cut-off effects in biological activities of amphiphiles.

Bilayer thickness and lipid interface area in unilamellar extruded 1,2-diacylphosphatidylcholine liposomes: a small-angle neutron scattering study.

Small-angle neutron scattering (SANS) experiments have been performed on large unilamellar liposomes prepared from 1,2-dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-phosphatidylcholine (DMPC) and 1,2-distearoylphosphatidylcholine (DSPC) in heavy water by extrusion through polycarbonate filters with 500 A pores. The neutron scattering intensity I(Q) in the region of scattering vectors Q corresponding to 0.0015 A(-2) < or = Q(2) < or = 0.0115 A(-2) was fitted using a step function model of bilayer neutron scattering length density and supposing that the liposomes are spherical and have a Gaussian distribution of radii. Using the lipid volumetric data, and supposing that the thickness of bilayer polar region equals to d(H) = 9+/-1 A and the water molecular volume intercalated in the bilayer polar region is the same as in the aqueous bulk aqueous phase, the steric bilayer thickness d(L), the lipid surface area A(L) and the number of water molecules per lipid molecule N intercalated in the bilayer polar region were obtained: d(L) = 41.58+/-1.93 A, A(L) = 57.18+/-1.00 A(2) and N = 6.53+/-1.93 in DLPC at 20 degrees C, d(L) = 44.26+/-1.42 A, A(L) = 60.01+/-0.75 A(2) and N = 7.37+/-1.94 in DMPC at 36 degrees C, and d(L) = 49.77+/-1.52 A, A(L) = 64.78+/-0.46 A(2) and N = 8.67+/-1.97 in DSPC at 60 degrees C. After correcting for area thermal expansivity alpha approximately 0.00417 K(-1), the lipid surface area shows a decrease with the lipid acyl chain length at 60 degrees C: A(L) = 67.56+/-1.18 A(2) in DLPC, A(L) = 66.33+/-0.83 A(2) in DMPC and A(L) = 64.78+/-0.46 A(2) in DSPC. It is also shown that a joint evaluation of SANS and small-angle X-ray scattering on unilamellar liposomes can be used to obtain the value of d(H) and the distance of the lipid phosphate group from the bilayer hydrocarbon region d(H1).

Cut-off Effect in Antimicrobial Activity and in Membrane Perturbation Efficiency of the Homologous Series of N,N-Dimethylalkylamine Oxides†

Abstract— The antimicrobial activity of the homologous series of N,N‐dimethylalkylamine oxides (DMAO) was found to be quasi parabolically dependent on alkyl chain length with a maximum at n ∼ 15 and n ∼ 12 for Staphylococcus aureus and Escherichia coli, respectively. The physicochemical properties of DMAOs as characterized by critical micelle concentrations, retention times of 1‐alkenes generated from DMAOs by gas‐liquid chromatography, Rm values in reversed phase chromatography, and bacterial lipid/aqueous phase partition coefficients were found to correlate with the alkyl chain length. The effect of DMAOs on the structure of the model membrane prepared from isolated lipids from Escherichia coli as detected by a spin probe method was maximal for the alkyl chain length n ∼ 10–12 coinciding with the maximum in the antimicrobial activity observed with Escherichia coli. It is suggested that the cut‐off in the DMAO antimicrobial activity is caused by the cut‐off in the DMAO perturbing effect on the membrane structure.

Areas of Monounsaturated Diacylphosphatidylcholines

We have studied the structural properties of monounsaturated diacylphosphatidylcholine lipid bilayers (i.e., diCn:1PC, where n = 14, 16, 18, 20, 22, and 24 is the number of acyl chain carbons). High-resolution x-ray scattering data were analyzed in conjunction with contrast-varied neutron scattering data using a technique we recently developed. Analyses of the data show that the manner by which bilayer thickness increases with increasing n in monounsaturated diacylphosphatidylcholines is dependent on the double bonds position. For commonly available monounsaturated diacylphosphatidylcholines, this results in the nonlinear behavior of both bilayer thickness and lipid area, whereas for diC18:1PC bilayers, lipid area assumes a maximum value. It is worthwhile to note that compared to previous data, our results indicate that lipid areas are smaller by approximately 10%. This observation highlights the need to revisit lipid areas, as they are often used in comparisons with molecular dynamics simulations. Moreover, simulators are encouraged to compare their results not only to x-ray scattering data, but to neutron data as well.

Structural changes in dipalmitoylphosphatidylcholine bilayer promoted by Ca2+ ions: a small-angle neutron scattering study.

Small-angle neutron scattering (SANS) curves of unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles in 1-60mM CaCl2 were analyzed using a strip-function model of the phospholipid bilayer. The fraction of Ca2+ ions bound in the DPPC polar head group region was determined using Langmuir adsorption isotherm. In the gel phase, at 20 degrees C, the lipid bilayer thickness, dL, goes through a maximum as a function of CaCl2 concentration (dL=54.4A at approximately 2.5mM of CaCl2). Simultaneously, both the area per DPPC molecule AL, and the number of water molecules nW located in the polar head group region decrease (DeltaAL=AL(DPPC))-AL(DPPC+Ca)=2.3A2 and Deltan=n(W(DPPC))-n(W(DPPC+Ca))=0.8mol/mol at approximately 2.5mM of CaCl2). In the fluid phase, at 60 degrees C, the structural parameters d(L), A(L), and n(W) show evident changes with increasing Ca2+ up to a concentration C(Ca)(2+) < or = 10mM. DPPC bilayers affected by the calcium binding are compared to unilamellar vesicles prepared by extrusion. The structural parameters of DPPC vesicles prepared in 60mM CaCl2 (at 20 and 60 degrees C) are nearly the same as those for unilamellar vesicles without Ca2+.

The interaction of β-adrenoceptor blocking drugs with platelet aggregation, calcium displacement and fluidization of the membrane

beta-Adrenoceptor blocking drugs interfere with adenosine diphosphate-stimulated platelet aggregation. Alprenolol, exaprolol, Kö 1124 and propranolol inhibited the aggregation, metipranolol decreased the extent and rate of aggregation significantly. Atenolol potentiated the aggregation measured by amplitude significantly. The interaction of beta-adrenoceptor blocking drugs with aggregation correlated with the displacement of calcium ions from binding sites in isolated platelets and the fluidization of the whole platelets and isolated platelet membrane as measured with electron spin resonance of the spin probe. The most potent were highly liposoluble drugs alprenolol, exaprolol, metipranolol and propranolol which increased the calcium displacement and membrane fluidity, the least active was atenolol decreasing these phenomena. The inhibition by beta-adrenoceptor blocking drugs of stimulated platelet aggregation is rather a result of unspecific than specific receptor interaction.

Effect of N-alkyl-N,N,N-trimethylammonium ions on phosphatidylcholine model membrane structure as studied by 31P-NMR

Abstract The interaction of |CnH2n+1N+(CH3)3| · I− (n = 3, 6, 9, 12, 14, 16 or 18) with egg-yolk phosphatidylcholine-water dispersions has been studied by 31P-NMR spectroscopy. It is shown that the effective anisotropy of 31P chemical shift (−Δσeff) of the lamellar phospholipid liquid-crystalline phase Lα increases with increasing concentration and alkyl chain length of the drug. Addition of | C 6 H 13 N + (CH 3 ) 3 | ·I − or |C9H19N+(CH3)3|·I− to the phospholipid-water dispersion at a molar ratio ammonium salt:phospholipid > 0.8 induces in the dispersion a structure with an effective isotropic phospholipid motion. This structure is unstable and slowly transforms into the hexagonal phase. These effects have not been observed in phospholipid-water dispersions mixed with the ammonium derivatives with the longer alkyl chains n  12, 14, 16 or 18. It is proposed that these results might explain the effects of the investigated drugs on the nerve, muscle and bacterial cells.

Small-angle neutron scattering study of the n-decane effect on the bilayer thickness in extruded unilamellar dioleoylphosphatidylcholine liposomes

Dioleoylphosphatidylcholine (DOPC) and n-decane were mixed and hydrated afterwards in an excess of heavy water at 1 wt.% of DOPC. From this dispersion, unilamellar liposomes were prepared by extrusion through polycarbonate filter with 500-A pores. Small-angle neutron scattering (SANS) was conducted on these liposomes. From the Kratky-Porod plot ln[I(Q)Q2] vs. Q2 of SANS intensity I(Q) in the range of scattering vectors Q corresponding to the interval 0.001 A(-2) < or = Q2 < or = 0.006 A(-2), the liposome bilayer radius of gyration Rg and the bilayer thickness parameter d(g) = 12(0.5)Rg were obtained. The values of d(g) indicated that the bilayer thickness is within the experimental error constant up to n-decane/DOPC approximately 0.5 molar ratio, and then increases by 2.4 +/- 1.3 A up to n-decane/DOPC = 1.2 molar ratio.

Interaction of surfactants with model and biological membranes.: II. Effect of N-alkyl-N,N,N-trimethylammonium ions on phosphatidylcholine bilayers as studied by spin probe ESR

The interaction of N-alkyl-N,N,N-trimethylammonium (CnTMA, n = 6-18) salts (iodides and/or bromides) with model membranes prepared by hydration of egg yolk phosphatidylcholine (EYPC) over aqueous salt solutions has been studied by m-doxyl stearic acid (m-DSA, m = 12 and 16) spin probe method. In disoriented EYPC bilayers the CnTMA salts decrease the orientational order parameter S33 of m-DSA evaluated from the powder pattern ESR spectra. This effect is maximal for C6TMA. In oriented EYPC bilayers prepared by the parallel-beam sputtering method and hydrated over saturated NaCl solution the order parameter S33 calculated from the angular dependence of the nitrogen hyperfine splitting is decreased in the presence of C6TMA. The order parameter S11 obtained from the angular dependence of line positions indicates deviation of m-DSA motion from axial symmetry. C6TMA increases the probability of gauche conformations of the lipid chains by about 13-14%, and decreases the effective energy difference between the trans and gauche conformations by about 420-480 J/mol, at molar ratio of EYPC/C6TMA = 2:1. The angular dependence of linewidths is analysed by employing a theory of spin relaxation based on the strong collision model for molecular reorientations. The correlation time tau 0 of the reorientation of an axis orthogonal to the doxyl ring of 16-DSA is decreased in the presence of C6TMA, while that of 12-DSA is not influenced by it. The ratio of tau 2/tau 0 is increased in the presence of C6TMA for the both spin probes. The results are explained using the free-volume model of the CnTMA-EYPC membrane interaction.

Hydrophobic thickness, lipid surface area and polar region hydration in monounsaturated diacylphosphatidylcholine bilayers: SANS study of effects of cholesterol and β-sitosterol in unilamellar vesicles

The influence of a mammalian sterol cholesterol and a plant sterol beta-sitosterol on the structural parameters and hydration of bilayers in unilamellar vesicles made of monounsaturated diacylphosphatidylcholines (diCn:1PC, n=14-22 is the even number of acyl chain carbons) was studied at 30 degrees C using small-angle neutron scattering (SANS). Recently published advanced model of lipid bilayer as a three-strip structure was used with a triangular shape of polar head group probability distribution (Kucerka et al., Models to analyze small-angle neutron scattering from unilamellar lipid vesicles, Physical Review E 69 (2004) Art. No. 051903). It was found that 33 mol% of both sterols increased the thickness of diCn:1PC bilayers with n=18-22 similarly. beta-sitosterol increased the thickness of diC14:1PC and diC16:1PC bilayers a little more than cholesterol. Both sterols increased the surface area per unit cell by cca 12 A(2) and the number of water molecules located in the head group region by cca 4 molecules, irrespective to the acyl chain length of diCn:1PC. The structural difference in the side chain between cholesterol and beta-sitosterol plays a negligible role in influencing the structural parameters of bilayers studied.