The adsorption of prothrombin to phosphatidylserine multilayers quantitated by ellipsometry

We investigated by means of an automated ellipsometer the adsorption of prothrombin from a buffer solution by multilayers of 14:0/14:0- and 18:1/18:1-phosphatidylserine (PS) stacked on chromium slides. In this instrument thickness and refractive index of the adsorbed phospholipid and proteins are monitored continuously. Two equations are derived to relate the mass of stacked phospholipids and the mass of protein adsorbed to the thickness and refractive index. These equations are based upon the Lorentz-Lorenz relation among the molar refractivities, refractive indices, and the densities of binary mixtures. Experimental validation of these equations is performed by measuring stacked multilayers of known mass of phosphatidylserine and the adsorption of [125I] albumin and [3H]prothrombin on these multilayers. Using these equations we measured the dissociation constants Kd and the number of binding sites nb of prothrombin. Values of Kd = 0.15 x 10(-8) M and nb = 122 molecules of PS/molecule of prothrombin were observed for di C14:0 PS and values of Kd = 0.45 x 10(-8) M and nb = 54 molecules of PS/molecule of prothrombin for di C18:1 PS. These data compare well to data obtained by other methods available in the literature.     

Adsorption of bovine prothrombin to spread phospholipid monolayers

The interaction of bovine prothrombin with phospholipids was measured, using as the lipid source monolayers spread at the air-buffer interface. Fluorescence spectroscopy was implemented to determine the equilibrium concentration of free prothrombin in the aqueous subphase of the protein-monolayer suspensions, in a continuous assay system. The increase in surface pressure (pi) from the protein-monolayer adsorption was also measured and, with values of the adsorbed protein concentration (c[s]), was used to calculate dc(s)/d(pi). At a particular phosphatidylserine (PS) content of liquid-expanded (LE) phosphatidylcholine (PC)/PS monolayers, dc(s)/d(pi) was independent of the initial surface pressure (pi[i]), when this latter value exceeded 30 mN/m. However, dc(s)/d(pi) varied significantly with the relative PS content of the monolayer. Values of the equilibrium dissociation constants calculated from the concentration dependence of delta(pi) indicated that the affinity of prothrombin for LE monolayers was higher at higher PS contents and lower packing densities. The affinity of prothrombin for liquid-condensed (LC) PC/PS monolayers was found to be much weaker relative to LE monolayers of similar phospholipid composition. This approach, employing spread monolayers to study prothrombin-phospholipid binding, coupled with a simple and accurate method to determine the free protein concentration in protein-monolayer suspensions, offers significant advantages for the investigation of protein-membrane interaction. The equilibrium characteristics that describe the interaction of prothrombin with the different phospholipid monolayers under various conditions also provide support for previous results which indicated that hydrophobic interactions are involved in the adsorption of vitamin K-dependent coagulation and anticoagulation proteins to model membrane systems.     

The pH dependence of calcium adsorption onto anionic phospholipid monolayers

The adsorption of ⁴⁵Ca to monolayers of phosphatidylinositol and dicetylphosphoric acid has been measured as a function of subphase pH with simultaneous recordings of surface pressure and interfacial potential. Below pH 3 little calcium was adsorbed and the films are assumed to be unionized. With acid subphases between pH 3 and 6.5 adsorption of calcium occurred initially, but it was then gradually lost due to an ageing process in the films. This time dependent change in the properties of the film was independent of the presence of Ca²⁺, but was dependent on the H⁺ concentration in the subphase; it was however not due to an acid hydrolysis of the monolayer. Ca²⁺ was permanently adsorbed at pH values above 6.5 with an increasing affinity up to pH 11.     

Shielding of phospholipid monolayers from phospholipase C hydrolysis by alpha-lactalbumin adsorption

α-Lactalbumin interacts more strongly with lecithin and cardiolipin monolayers at pH 3～4 than at pH 7 to 10. At physiological pH this protein does not penetrate monolayers of DPPC and cardiolipin above pressures of 30 dynes/cm. Enzymatic hydrolysis of these monolayers by phospholipase C (Clostridium Welchii) is inhibited partially or totally when α-lactalbumin is injected in the subphase prior to the enzyme injection.     

The adsorption of Pseudomonas aeruginosa exotoxin A to phospholipid monolayers is controlled by pH and surface potential.

The interaction of Pseudomonas aeruginosa exotoxin A (ETA) with lipid monolayers was studied by measuring the variation in surface pressure. ETA adsorbs to the monolayer, occupying an average area of approximately 4.6 nm2 per molecule, up to a maximum density of one molecule per 28 nm2 of lipid film, which corresponds roughly to the cross-sectional area of the toxin. This suggests that ETA molecules adsorb until they contact each other, but insert only a small portion into the lipid film. The kinetic process could be described by a Langmuir adsorption isotherm. The apparent association and dissociation rate constants were determined, as were their dependence upon toxin concentration, membrane composition, pH, and ionic strength. Two parameters were found to be paramount for this interaction: pH and surface potential of the lipid. It appears that ETA binding occurs only in a conformational state induced by low pH and is promoted by an electrostatic interaction between a positively charged region of the protein and the negative charge of acidic phospholipids. On the basis of a simple model, the salient features of ETA involved in its adsorption were derived: 1) the existence of a conformational state induced by the protonation of a group with pK 4.5 +/- 0.2; 2) a positive charge of 1.9 +/- 0.3 e.u. able to interact with the surface potential of the membrane; 3) the fraction of potential experienced by the protein in the activated state that precedes binding, approximately 80%; 4) the intrinsic adsorption and desorption rate constants, k(a)0 = (4.8 +/- 0.3) x 10(3) M(-1) s(-1) and k(d)0 = (4.4 +/- 0.4) x 10(-4) s(-1). These rate constants are independent of pH and lipid and buffer composition, and provide a dissociation constant Kd approximately 90 nM.     

The interaction of meso-tetraphenylporphyrin with phospholipid monolayers

In this article, we investigate the interaction of meso-tetraphenylporphyrin (TPP) with phospholipid monolayers. Pure TPP molecules form films at the air-water interface with large extension of aggregation, which is confirmed by UV-vis spectra of transferred monolayers. For mixed films of TPP with dipalmitoyl phosphatidyl choline (DPPC) or dipalmitoyl phosphatidyl glycerol (DPPG), on the other hand, aggregation is only significant at high surface pressures or high concentrations of TPP (above 0.1 molar ratio). This was observed via Brewster angle microscopy (BAM) for the Langmuir films and UV-vis spectroscopy for transferred layers onto solid substrates. TPP indeed causes the DPPC and DPPG monolayers to expand, especially at the liquid-expanded to liquid-condensed phase transition for DPPC. The effects from TPP cannot be explained using purely geometrical considerations, as the area per TPP molecule obtained from the isotherms is at least twice the expected value from the literature. Therefore, interaction between TPP and DPPC or DPPG should be cooperative, so that more phospholipid molecules are affected than just the first neighbors to a TPP molecule.     

The insertion of D-beta-hydroxybutyrate apodehydrogenase into phospholipid monolayers and phospholipid vesicles

The strong interaction of D-beta-hydroxybutyrate dehydrogenase with phospholipid monomolecular films is demonstrated by the surface pressure increase of a film compressed up to 33 mN/m. Although the D-beta-hydroxybutyrate apodehydrogenase was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged monolayers such as those made from diphosphatidylglycerol. The weakest interaction was found with phosphatidylcholine, which is the reactivating phospholipid for the enzyme. These interactions were dependent on the phospholipid chain length, ionic strength, and pH. At basic pH the apoenzyme lost its specificity for negatively charged phospholipids, suggesting the deprotonation of a cationic amino acid residue of the enzyme polypeptide chain. The charge effects are in agreement with results obtained using phospholipid vesicles. Beside the electrostatic interactions, the influence of phospholipid chain length and the ionic strength indicate that D-beta-hydroxybutyrate apodehydrogenase penetrates into the hydrophobic part of the lipid interface.     

Adsorption of cytochrome c to phospholipid monolayers studied by reflection spectroscopy

The uptake of cytochrome c by charged and neutral lipid monolayers was studied by using reflection spectroscopy. The method was shown to be a very sensitive and useful technique in studies of lipid-protein interactions. It was found that cytochrome c is preferentially bound to monolayers of negatively charged monolayers in the solid phase. Polarized light under oblique incidence was used to determine the average orientation of chromophores in cytochrome c bound to lipid monolayer. The transition moments of chromophore are oriented parallel to the monolayer plane.     

The adsorption of phloretin to lipid monolayers and bilayers cannot be explained by langmuir adsorption isotherms alone.

Phloretin and its analogs adsorb to the surfaces of lipid monolayers and bilayers and decrease the dipole potential. This reduces the conductance for anions and increases that for cations on artificial and biological membranes. The relationship between the change in the dipole potential and the aqueous concentration of phloretin has been explained previously by a Langmuir adsorption isotherm and a weak and therefore negligible contribution of the dipole-dipole interactions in the lipid surface. We demonstrate here that the Langmuir adsorption isotherm alone is not able to properly describe the effects of dipole molecule binding to lipid surfaces--we found significant deviations between experimental data and the fit with the Langmuir adsorption isotherm. We present here an alternative theoretical treatment that takes into account the strong interaction between membrane (monolayer) dipole field and the dipole moment of the adsorbed molecule. This treatment provides a much better fit of the experimental results derived from the measurements of surface potentials of lipid monolayers in the presence of phloretin. Similarly, the theory provides a much better fit of the phloretin-induced changes in the dipole potential of lipid bilayers, as assessed by the transport kinetics of the lipophilic ion dipicrylamine.     

Interfacial behavior of phospholipid monolayers revealed by mesoscopic simulation

A mesoscopic model with molecular resolution is presented for dipalmitoyl phosphatidylcholine (DPPC) and palmitoyl oleoyl phosphatidylcholine (POPC) monolayer simulations at the air-water interface using many-body dissipative particle dynamics (MDPD). The parameterization scheme is rigorously based on reproducing the physical properties of water and alkane and the interfacial property of the phospholipid monolayer by comparison with experimental results. Using much less computing cost, these MDPD simulations yield a similar surface pressure-area isotherm as well as similar pressure-related morphologies as all-atom simulations and experiments. Moreover, the compressibility modulus, order parameter of lipid tails, and thickness of the phospholipid monolayer are quantitatively in line with the all-atom simulations and experiments. This model also captures the sensitive changes in the pressure-area isotherms of mixed DPPC/POPC monolayers with altered mixing ratios, indicating that the model is promising for applications with complex natural phospholipid monolayers. These results demonstrate a significant improvement of quantitative phospholipid monolayer simulations over previous coarse-grained models.     

Kinetic regulation of the binding of prothrombin to phospholipid membranes

A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K _D = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K _D values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K _D of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K _D in the 10^?8 M range and (ii) a kinetically rapid, low-affinity interaction with K _D in the 10^?6 M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K _D values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface.     

Lupus antibody bivalency is required to enhance prothrombin binding to phospholipid

Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid-binding proteins including prothrombin. We have proposed that LA propagates coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall. A murine monoclonal anti-prothrombin Ab and three of three LA IgGs enhanced prothrombin binding to 75:25 phosphatidyl choline:phosphatidyl serine vesicles measured by either ultracentrifugation or right-angle light scattering. The assembly of prothrombin and LA IgG on phospholipid vesicles was estimated by surface plasmon resonance. The on rates for prothrombin and LA IgG were approximately the same as the on rate for prothrombin alone. In contrast, the off rates for prothrombin and LA IgG were 2- to 3-fold slower than the off rate for prothrombin. LA IgG bivalency was required for enhanced prothrombin binding to phospholipid vesicles, as Fab of the LA IgGs did not influence prothrombin binding at concentrations up to 40 microM. Modeling of the interactions of prothrombin, LA IgG and phospholipid vesicles indicated that augmentation of prothrombin binding to phospholipid vesicles by LA IgG could be accounted for by the bivalency of the LA IgG and the elevated microenvironmental concentration of prothrombin on the surface of phospholipid vesicles.     

Penetration of furosemide into phospholipid monolayers

Furosemide is a surface-active anion and it tends to displace lipid monolayers from the surface at positive polarizations lowering their potential stability range. The efficiency of the penetration and the displacement increases with decreasing surface pressure of the monolayer. Lower capacitance at a wider potential range corresponds to higher surface pressure. Monolayers with higher capacitances are indeed more readily penetrated and displaced as demonstrated by further increase in their capacitance and increase in their proton conductance. Furosemide raises the capacitance of the monolayer in the stable region due to intercalation between the head groups thus reducing the thickness of the hydrocarbon layer. In pure PC monolayer about 10% increase in capacitance is observed in the presence of 6 X 10(-4)M furosemide. The effect of furosemide becomes more pronounced with increasing sphingomyelin content in the mixed monolayers. The monolayer of PE is more condensed and its capacitance is lower (approximately 1.45 microF/cm2) and is stable in a wider potential range than that of PC. It is less affected by furosemide and concentrations higher than 10(-3) M are required to narrow the stability range and to increase the capacitance.     

Penetration of furosemide into phospholipid monolayers

Summary Furosemide is a surface-active anion and it tends to displace lipid monolyaers from the surface at positive polarizations lowering their potential stability range. The efficiency of the penetration and the displacement increases with decreasing surface pressure of the monolayer. Lower capacitance at a wider potential range corresponds to higher surface pressure. Monolayers with higher capacitances are indeed more readily penetrated and displaced as demonstrated by further increase in their capacitance and increase in their proton conductance. Furosemide raises the capacitance of the monolayer in the stable region due to intercalation between the head groups thus reducing the thickness of the hydrocarbon layer. In pure PC monolayer about 10% increase in capacitance is observed in the presence of 6×10^–4 m furosemide. The effect of furosemide becomes more pronounced with increasing sphingomyelin content in the mixed monolayers. The monolayer of PE is more condensed and its capacitance is lower (1.45 F/cm²) and is stable in a wider potential range than that of PC. It is less affected by furosemide and concentrations higher than 10^–3 m are required to narrow the stability range and to increase the capacitance.     

Interactions of phospholipid monolayers with carbohydrates

Surface pressure studies of phospholipid monomolecular films of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) formed at an air/water interface have been made and the effects on the films studied when various carbohydrates are present in the subphase. The results obtained show that at a given temperature, the area per molecule of DPPC increases with increasing concentration of the carbohydrate in the subphase. The carbohydrate which has the greatest expanding effect on the phospholipid monolayer is glycerol, followed in turn by trehalose, sucrose, glucose, raffinose, and inositol. The mechanism of monolayer expansion by glycerol is different from that observed in other carbohydrates, as the following experiments demonstrate. Below the phase transition temperature of DPPC, the area per molecule of DPPC at a pressure of 12.5 dyn/cm is the same with and without glycerol in the subphase. However, when the monolayer is heated to a temperature above the phase transition temperature for DPPC, the area/molecule on glycerol is considerably greater than the area/molecule on water at the same surface pressure. Cooling the monolayer back to the lower temperature produces an area/molecule of DPPC which is identical on both water and glycerol subphases. Glycerol therefore has no effect on the low-temperature (condensed) monolayers but causes expansion of the high-temperature (expanded) monolayers. By contrast with glycerol, both trehalose and sucrose interact with the DPPC monolayer producing an increased area/molecule over that observed on water, both with low-temperature (condensed) monolayers and with the high-temperature (expanded) monolayers. The efficiency of these carbohydrates at expanding the monolayer films (with the exception of glycerol) shows a strong correlation with their ability to stabilize membrane structure and function at low water contents.     

Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers.

A technique for the production of supported phospholipid bilayers by adsorption and fusion of small unilamellar vesicles to supported phospholipid monolayers on quartz is described. The physical properties of these supported bilayers are compared with those of supported bilayers which are prepared by Langmuir-Blodgett deposition or by direct vesicle fusion to plain quartz slides. The time courses of vesicle adsorption, fusion and desorption are followed by total internal reflection fluorescence microscopy and the lateral diffusion of the lipids in the adsorbed layers by fluorescence recovery after photobleaching. Complete supported bilayers can be formed with phosphatidylcholine vesicles at concentrations as low as 35 microM. However, the adsorption, fusion and desorption kinetics strongly depend on the used lipid, NaCl and Ca2+ concentrations. Asymmetric negatively charged supported bilayers can be produced by incubating a phosphatidylcholine monolayer with vesicles composed of 80% phosphatidylcholine and 20% phosphatidylglycerol. Adsorbed vesicles can be removed by washing with buffer. The measured fluorescence intensities after washing are consistent with single supported bilayers. The lateral diffusion experiments confirm that continuous extended bilayers are formed by the monolayer-fusion technique. The measured lateral diffusion coefficient of NBD-labeled phosphatidylethanolamine is (3.6 +/- 0.5) x 10(-8) cm2/s in supported phosphatidylcholine bilayers, independent of the method by which the bilayers were prepared.     

Influence of vitamin C on alcohol binding to phospholipid monolayers

The simple model of the biological membrane is provided by well-controlled lipid monolayers at the air-water interface. The Maxwell displacement current technique (MDC) provides novel approach to conformation study of the membrane models. The effect of alcohols is interaction with membrane molecules, mainly with the lipid head group and consequent changes in physical-chemical properties of the membrane. The aim of study is to detect changes in structural, electrical and mechanical properties of dipalmitoyl-phosphatidylcholine (DPPC) monolayer on the subphase of methanol-water and ethanol-water mixtures before and after addition of antioxidant agent, vitamin C. Monolayers properties are investigated by a surface pressure analysis (including mechanical properties evaluation) and the Maxwell displacement current measurement, the dipole moment projection calculation. Surface pressure-area isotherms show similar behaviour of the DPPC monolayer on alcohol-water mixtures independently on presence of vitamin C. Binding/adsorption process induces change of electron density distribution across monolayer and thus the molecular dipole moment. We observe small or negligible binding of methanol molecules on oxygen bonds of DPPC. Thus the antioxidant, vitamin C, has no significant effect. For ethanol-water mixtures is observed recovery of electrical properties in presence of antioxidant agent. We suppose that vitamin C regulates DPPC-ethanol molecules interaction.     

Modulation of human prothrombin activation on phospholipid vesicles and platelets using monoclonal antibodies to prothrombin fragment 2

Prothrombin contains two kringle domains that are removed during activation to the blood clotting enzyme alpha-thrombin. By analogy with other kringle-containing proteins the prothrombin kringles may play a role in the protein-protein interactions necessary for prothrombin activation. Four monoclonal antibodies to prothrombin kringle 2 have been produced against human prothrombin, and a fifth monoclonal antibody was produced against a synthetic peptide consisting of amino acid residues 216-231 of kringle 2. Each antibody was tested for its ability to block prothrombin activation by factor Xa. In the presence of phosphatidylcholine/phosphatidylserine vesicles and factor Va, two of the antibodies, alpha HII-3 and alpha HII-4, inhibited prothrombin activation at a 90 and 50% level, respectively. Two other monoclonal antibodies (alpha HII-6 and alpha HII-7) and the antipeptide antibody (alpha HII-5) had no effect on prothrombin activation. When factor Xa was the catalyst alone, antibody alpha HII-3 lost the ability to inhibit prothrombin activation whereas antibody alpha HII-4 again partially inhibited the reaction. When human platelets were the reaction surface, the patterns of inhibition by the anti-fragment 2 antibodies were identical to that observed with phospholipid vesicles. These data suggest a role for prothrombin fragment 2 in activation, possibly by mediating the interaction of substrate prothrombin with factor Xa or factor Va on the phospholipid surface.