The dependence of Fluorescein-PE fluorescence intensity on lipid bilayer state. Evaluating the interaction between the probe and lipid molecules

The degree of dependence of a lipid bilayer's surface properties on its conformational state is still an unresolved question. Surface properties are functions of molecular organization in the complex interfacial region. In the past, they were frequently measured using fluorescence spectroscopy. Since a fluorescent probe provides information on its local environment, there is a need to estimate the effect caused by the probe itself. In this paper, we address this question by calculating how lipid head-group orientation effects the fluorescence intensity of Fluorescein-PE (a probe that is sensitive to surface potential). In the theoretical model assumed the lipid bilayer state and the interactions between the charged fluorescent probe and the surrounding lipid molecules was evaluated. The results of this theoretical analysis were compared with experimentally obtained data. A lipid bilayer formed from DPPC was chosen as the experimental system, since it exhibits all the major conformational states within a narrow temperature range of 30 degrees C-45 degrees C. Fluorescein-PE fluorescence intensity depends on local pH, which in turn is sensitive to local electrostatic potential in the probe's vicinity. This local electrostatic potential is generated by lipid head-group dipole orientation. We have shown that the effect of the probe on lipid bilayer properties is limited when the lipid bilayer is in the gel phase, whereas it is more pronounced when the membrane is liquid-crystalline. This implies that Fluorescein-PE is a good reporter of local electrostatic fields when the lipid bilayer is in the gel phase, and is a poor reporter when the membrane is in the liquid-crystalline state.     

Characterization of the fluorophore 4-heptadecyl-7-hydroxycoumarin: a probe for the head-group region of lipid bilayers and biological membranes

The fluorophore 4-heptadecyl-7-hydroxycoumarin was used as a probe to study the properties of phospholipid bilayers at the lipid-water interface. To this end, the steady-state fluorescence anisotropy, the differential polarized phase fluorometry, and the emission lifetime of the fluorophore were measured in isotropic viscous medium, in lipid vesicles, and in the membrane of vesicular stomatitis virus. In the isotropic medium (glycerol), the probe showed an increase in the steady-state fluorescence anisotropy with a decrease in temperature, but the emission lifetime was unaffected by the change in temperature. In glycerol, the observed and predicted values for maximum differential tangents of the probe were identical, indicating that in isotropic medium 4-heptadecyl-7-hydroxycoumarin is a free rotator. Nuclear magnetic resonance and differential scanning calorimetric studies with lipid vesicles containing 1-2 mol % of the fluorophore indicated that the packaging density of the choline head groups was affected in the presence of the probe with almost no effect on the fatty acyl chains. The fluorophore partitioned equally well in the gel and liquid-crystalline phase of the lipids in the membrane, and the phase transition of the bilayer lipids was reflected in the steady-state fluorescence anisotropy of the probe. The presence of cholesterol in the lipid vesicles had a relatively small effect on the dynamics of lipids in the liquid-crystalline state, but a significant disordering effect was noted in the gel state. One of the most favorable properties of the probe is that its emission lifetime was unaffected by the physical state of the lipids or by the temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of type I collagen surface density on fibroblast spreading, motility, and contractility

We examine the relationships of three variables (projected area, migration speed, and traction force) at various type I collagen surface densities in a population of fibroblasts. We observe that cell area is initially an increasing function of ligand density, but that above a certain transition level, increases in surface collagen cause cell area to decline. The threshold collagen density that separates these two qualitatively different regimes, approximately 160 molecules/ microm(2), is approximately equal to the cell surface density of integrin molecules. These results suggest a model in which collagen density induces a qualitative transition in the fundamental way that fibroblasts interact with the substrate. At low density, the availability of collagen binding sites is limiting and the cells simply try to flatten as much as possible by pulling on the few available sites as hard as they can. The force per bond under these conditions approaches 100 pN, approximately equal to the force required for rupture of integrin-peptide bonds. In contrast, at high collagen density adhesion, traction force and motility are limited by the availability of free integrins on the cell surface since so many of these receptors are bound to the surface ligand and the force per bond is very low.     

Effect of substrate physical state on the activity of acid cholesteryl ester hydrolase

The effects of the physicochemical properties of the substrate vehicle on the activity of acid cholesteryl ester hydrolase (ACEH; EC 3.1.1.13) isolated from rat liver lysosomes have been studied. In particular, the influence of the physical state of the neutral lipid core of substrate emulsion particles on the enzymatic activity has been probed in the light of previous studies on the clearance of cholesteryl esters (CE) from lipid-loaded cells which indicated that inclusions that are in the isotropic (liquid) state can be hydrolyzed faster than those in the anisotropic (liquid-crystalline) state. In the present study, such lipid inclusions were isolated from cultured cells and used as substrates for the hydrolase. No appreciable difference between the hydrolysis rates of isotropic and anisotropic inclusions was observed; the Vmax values were 93.0 +/- 6.7 and 84.0 +/- 3.3 nmol CE/mg.h, respectively. To elucidate the factors which affect the activity of ACEH, model inclusions were prepared by sonication and used as substrates. The physical state of these models was varied in a systematic way by changes of droplet composition and incubation temperature. The rate of hydrolysis was found to be insensitive to the physical state of the core of the model inclusions in good agreement with the results obtained with cellular inclusions. However, the activity of ACEH is sensitive to such interfacial properties of the lipid droplets as surface area available to the enzyme, net surface charge and surface solubility of the substrate CE molecules. The enzymatic activity is also sensitive to the amount of free cholesterol present in the emulsion droplets. The interfacial concentration and molecular packing of substrate CE molecules in the droplet surface significantly affect the hydrolytic activity of ACEH.     

Rhodamine B-labeled thyroid hormone forms high molecular weight aggregates in solution: a possible source of artifacts in binding experiments

The fluorescence intensity of rhodamine B-labeled thyroid hormone is a nonlinear function of probe concentration. The concentration dependence was analyzed in terms of a model with continuous aggregation of the probe molecules to give very high molecular weight species. The apparent association constant was 3.65 microM-1 at 25 degrees C, pH 7.2. Studies at different temperatures showed apparent delta H = 6.8 kcal/mol and delta S = -7.3 cal/mol/degree for association. Based on their absorbance spectra, these aggregates seem to be linear, with the rhodamine chromophores slanted with respect to the long axis.     

Calcium binding by phosphatidylserine headgroups. Deuterium NMR study.

The binding of calcium to headgroup deuterated 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphoserine (POPS) was investigated by using deuterium magnetic resonance in pure POPS membranes and in mixed 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS 5:1 (m:m) bilayers. Addition of CaCl2 to pure POPS bilayers led to two component spectra attributed, respectively, to liquid-crystallin POPS (less than 15 kHz) and POPS molecules in the calcium-induced dehydrated phase (cochleate) (approximately 120 kHz). The liquid-crystalline component has nearly disappeared at a Ca2+ to POPS ratio of 0.5, indicating that, under such conditions, most of the POPS molecules are in the precipitated cochleate phase. After dilution of the POPS molecules in zwitterionic POPC membranes (POPC/POPS 5:1 m:m), single component spectra characteristic of POPS in the liquid-crystalline state were observed in the presence of Molar concentrations of calcium ions (Ca2+ to POPS ratio greater than 50), showing that the amount of dehydrated cochleate PS-Ca2+ phase, if any, was low (less than 5%) under such conditions. Deuterium NMR data obtained in the 15-50 degrees C temperature range with the mixed PC/PS membranes, either in the absence or the presence of Ca2+ ions, indicate that the serine headgroup undergoes a temperature-induced conformational change, independent of the presence of Ca2+. This is discussed in relation to other headgroup perturbations such as that observed upon change of the membrane surface charge density.     

A neutron solution scattering study of the structure of annexin-V and its binding to lipid vesicles.

Low-angle neutron solution scattering has been used to study the structure of annexin-V and its interaction with small single-bilayer vesicles consisting of phosphatidylserine and phosphatidylcholine at a 33:66 (mol:mol) ratio. There was no evidence for a change in the state of aggregation of annexin-V, which remained as a monomer in the presence of 3 mM-free calcium. The only difference between presence and absence of free calcium was the increase of the radius of gyration, from 19(+/- 0.4) A to 22(+/- 0.4) A in 2H2O buffer and from 19.7(+/- 1.2) A to 22.2(+/- 1.2) A in H2O buffer. The relative molecular weight, outer radius and average surface area per lipid of vesicles alone were respectively 2.5(+/- 0.5) x 10(6), 127 A and 90(+/- 19) A2. These parameters were not modified in the presence of free calcium, which testified to the absence of vesicle coalescence. The calcium-dependent binding of annexin-V was essentially interfacial and therefore did not alter significantly the structural characteristics of the vesicles. At saturation, 80(+/- 10) annexin-V molecules were bound per vesicle, the available area per molecule being 2500(+/- 300) A2 thus covering approximately 28 lipid head groups. The protein shell was approximately 35 A thick. The apparent dissociation constant was probably less than 1 nM. These data contribute to a more accurate definition of annexin-V as a possible probe of those cytodynamic events involving exposure of sequestered membrane aminophospholipids.     

Interaction energies in lectin-induced erythrocyte aggregation

Two N-acetylgalactosamine-reactive lectins, Helix pomatia (HPA) and Dolichos biflorus (DBA), were used to study the energies involved in cell-cell interactions through the specific binding of these lectins to their membrane receptors on genotype AO human erythrocytes (red blood cells) (RBCs). The energy required to dissociate a unit of aggregated membrane area (gamma d) of two RBCs bridged by lectin molecules was determined from the shear force needed to dissociate two-cell aggregates in a flow channel. When HPA were used as bridging molecules, gamma d (0.4 X 10(-4) to 3.8 X 10(-4) dyn/cm) was proportional to the density (D = 175 to 1,060 molecules/micron 2) of HPA molecules bound on the RBC membrane. A similar gamma d/D ratio was also obtained for DBA. These results indicate that the number of lectin molecules bound on the interface plays an important role in determining the energy required for cell-cell dissociation. The aggregation energy per unit membrane area (gamma a) in lectin-induced aggregates was calculated from the degree of encapsulation of a lectin-bound, heat-sphered human RBC by a normal discoid RBC. A minimum of approximately 1,800 HPA molecules/micron 2 on the spheres was required to form stable aggregates with the RBC. By using spheres having a surface HPA density of 1,830 to 2,540 molecules/micron 2, or 1.1-1.5 X 10(12) combining sites/cm2, the gamma a value for HPA-induced aggregation was found to be 2.2 X 10(-3) dyn/cm. This higher value of gamma a than gamma d has been explained on the basis of several differences in aggregation and disaggregation processes. The gamma a value for DBA-induced aggregation was not obtainable by the sphere encapsulation method because of the relative low D values. A comparison of the present results with the published value of the free energy change of 5 kcal/mol for the interactions of HPA and DBA with their ligands suggests that only a small fraction of the lectin molecules bound to RBC surface participate in the bridging of adjacent cells.     

Aggregation, lipid exchange, and metastable phases of dimyristoylphosphatidylethanolamine vesicles

A new fluorescent lipid analogue, bimanephosphatidylcholine, has been synthesized for use in lipid bilayers. This probe is well suited as an energy-transfer donor with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine as the acceptor. Dimyristoylphosphatidylethanolamine vesicles are prepared by sonication at pH 9 and characterized by electron microscopy and other methods. Resonance energy transfer between separately labeled donor and acceptor vesicles is monitored during HCl-induced aggregation to determine the kinetics of lipid randomization. Light scattering is also monitored to measure the kinetics of aggregation. The light scattering shows a marked reversal with NaOH while the energy transfer does not, indicating lipid exchange during a reversibly aggregated state; the extent of energy transfer suggests that only lipids in the outer monolayers exchange. The gel to liquid-crystalline phase transition temperature in HCl-treated vesicles is found to be 47 degrees C with diphenylhexatriene. The initial sonicated dispersion does not show a sharp phase transition. In vesicles labeled with both donor and acceptor probes, a small, irreversible increase in energy transfer is obtained upon lowering and then restoring the pH. These results suggest a metastable phase in the sonicated vesicles containing a randomized distribution of lipid and probes within the bilayers; the thermodynamically favored phase, whose formation is triggered by the pH shock, contains domains within which the probe lipids are more highly concentrated.     

Effect of oligosaccharides and their monosaccharide mixtures on the stability of proteins: a scaled particle study

Experimental results of RNase-A stabilization by sugar osmolytes show that the change in the Gibbs free energy (ΔGD) associated with the equilibrium, N (native) state ? D (denatured) state of the protein in the presence of equimolar mixture of monosaccharides is larger than that of the corresponding oligosaccharides at a given temperature and pH. However, at the molar scale, ΔGD obtained in the presence of an oligosaccharide is much higher as compared with ΔGD obtained using individual monosaccharide. We used scaled particle theory (SPT) to explain these experimental observations. The effective length, called Tolman's length that describes the curvature correlations to a surface area or surface tension and in turn contributes to the change in free energy, is discussed. Tolman's length is higher for corresponding monomer mixture than the oligosaccharide molecules. Based on SPT analysis, a geometrical model is proposed for clustering of monosaccharides in the mixture due to high particle density. The cluster is presumed to have weak interaction among them due to larger hydrodynamic radius than that of the bonded molecules of oligosaccharides.     

Phase behaviour of lipid X

The phase behaviour of aqueous dispersions of lipid X, a precursor of bacterial lipopolysaccharides has been investigated by a variety of physico-chemical techniques. The results are consistent with the presence of disk-shaped micelles with an average diameter of 13 +/- 1.8 nm. The critical micellar concentration in water and physiological saline is 4 x 10(-5) M. Consistent with the formation of micelles in water and physiological saline is the finding that lipid X is in the liquid-crystalline state at temperatures higher than 0 degrees C. The packing and the dynamics of lipid X are characteristic of micelles. Close to the polar group the hydrocarbon chains are significantly more mobile and disordered than in the corresponding region of lipid bilayers. From monolayer studies an estimate of the molecular area of lipid X is derived; under physiological conditions the area/molecule is about 0.50 nm2 at 30 mN/m indicating that lipid X has a wedge-like shape. The two pK values of the primary phosphate group of lipid X are pK1 approximately 1.3 and pK2 = 8.2. At pH values less than 7, the area/molecule decreases, i.e. the packing of the lipid X molecules becomes tighter, and there is also a decrease in the solubility of lipid X. As is characteristic of charged lipids, the state of aggregation (phase behaviour) of lipid X depends on pH, the ionic strength and the nature of the counterion.     

Incorporation of acylated antibody into planar lipid multilayers: characterization and cell binding

Multiple (up to 14) layers of lipid were deposited onto an alkylated glass surface by dialysis of egg phosphatidylcholine (PC) and deoxycholate mixed micelles in the presence of alkylated glass coverslips. The amount of lipid associated with the coverslips was measured by using radioactive PC. It was found that the number of PC molecules in the multilayer increased with increasing initial lipid concentration in the dialysis mixture. Inclusion of cholesterol resulted in a significant increase in the amount of total lipid deposited in the multilayer. However, the PC/cholesterol ratio was up to 2-fold higher in the multilayers than in the liposomes present in the same dialysis bag. In addition, mouse monoclonal anti-H2Kk antibody which had previously been derivatized with palmitic acid could be readily incorporated into the lipid multilayer during dialysis. Measurements of lateral mobility with the fluorescence recovery after photobleaching technique on fluorescently labeled lipid or antibody in the multilayer showed that the lipid molecules diffused rapidly while the antibodies were essentially immobile. Lymphoma cells such as RDM4 cells expressing surface H2Kk glycoproteins could rapidly bind to the antibody-containing multilayers. The binding was blocked by free antibody or by goat anti-mouse immunoglobulin G, indicating the immunospecificity of the binding. Cell binding to the multilayer also exhibited a threshold dependence on the antibody density of the multilayer. A lower threshold was found for cells expressing a higher surface density of H2Kk. This system may be useful for model studies of cellular recognition.     

Thermodynamics of β-Sheet Formation in Polyglutamine

The role of β-sheets in the early stages of protein aggregation, specifically amyloid formation, remains unclear. Interpretations of kinetic data have led to a specific model for the role of β-sheets in polyglutamine aggregation. According to this model, monomeric polyglutamine, which is intrinsically disordered, goes through a rare conversion into an ordered, metastable, β-sheeted state that nucleates aggregation. It has also been proposed that the probability of forming the critical nucleus, a specific β-sheet conformation for the monomer, increases with increasing chain length. Here, we test this model using molecular simulations. We quantified free energy profiles in terms of β-content for monomeric polyglutamine as a function of chain length. In accord with estimates from experimental data, the free energy penalties for forming β-rich states are in the 10-20 kcal/mol range. However, the length dependence of these free energy penalties does not mirror interpretations of kinetic data. In addition, although homodimerization of disordered molecules is spontaneous, the imposition of conformational restraints on polyglutamine molecules does not enhance the spontaneity of intermolecular associations. Our data lead to the proposal that β-sheet formation is an attribute of peptide-rich phases such as high molecular weight aggregates rather than monomers or oligomers.     

Lipophorin structure analyzed by in vitro treatment with lipases.

Adult Manduca sexta high density lipophorin (HDLp-A) is composed of three apolipoproteins (apoLp-I, -II, and -III) and 52% lipid. The flight-specific low density lipophorin (LDLp) contains 62% lipid and is associated with several additional molecules of apoLp-III. The amount of phospholipid remains constant in lipophorin (140 mol/mol of lipophorin), while the diacylglycerol content varies between different lipophorin species (310 mol/mol HDLp up to 1160 mol/mol LDLp). Both lipophorin particles were enzymatically depleted of phospholipid or diacylglycerol by in vitro incubation with either phospholipase A2 or triacylglycerol lipase. Albumin was used to remove free fatty acids generated during the reaction. Treatment with phospholipase A2 removed all phospholipids (except sphingomyelin) and the resulting particles were stable. Triacylglycerol lipase hydrolyzed large fractions of diacylglycerol. The resulting particles were smaller in size, higher in density, and devoid of apoLp-III. The particles retained apoLp-I and -II and the other lipid components, including a substantial amount of diacylglycerol. Structural integrity of diacylglycerol-depleted lipophorin was confirmed by electron microscopical analysis. When treated with both phospholipase A2 and triacylglycerol lipase, lipophorin precipitated. From these results we conclude that: 1) all phospholipid and apoLp-III are located at the surface of lipophorin, whereas diacylglycerol is partitioned between the sublayers and the surface of the particle; 2) both diacylglycerol and phospholipid play a role in stabilizing lipophorin in the aqueous medium; and 3) lipophorin can be extensively unloaded and still retain its basic structure, a necessary feature for its function as a reusable lipid shuttle.     

Conformation of phosphatidylcholine in neat and cholesterol-containing liquid-crystalline bilayers. Application of a novel method.

The conformation of phosphatidylcholine in liquid-crystalline bilayers was studied with a novel, high-resolution method employing phosphatidylcholine species containing pyrenyl moieties in both acyl chains of variable length. Analysis of the intramolecular pyrene-pyrene collision data obtained for 30 such species in terms of a simple geometrical model showed that the sn-1 acyl chain penetrates, on the average, 0.84 +/- 0.11 methylene units (0.8 A) deeper into the bilayer than the sn-2 chain at 22 degrees C. A similar value was obtained at 37 degrees C. Since the penetration difference of the sn-1 and sn-2 acyl chains is inherently coupled to the conformation of the glycerol moiety, these data mean that the glycerol moiety of phosphatidylcholine is, on the average, only moderately tilted with respect to the bilayer plane in the liquid-crystalline state. This contrasts the perpendicular orientation observed previously for phosphatidylcholine crystals [Pearson, R. H., & Pascher, I. (1979) Nature 281, 499-501]. Importantly, addition of 50 mol % cholesterol, which is known to reduce dramatically the interactions between phosphatidylcholine molecules in bilayers, had only a small effect on the penetration difference of the acyl chains, strongly suggesting that the conformation of phosphatidylcholine in the liquid-crystalline state is determined largely by intramolecular, rather than intermolecular, interactions.     

Free-radical label--new approach to the study of dynamics of lipid systems

A new method of EPR-spectroscopy--the recombination of free radicals appearing as a result of indirect radiolysis of biological molecules after a low temperature irradiation--is applied to the study of molecular dynamics of phosphatidylcholine dimyristoyl in mass and in the structure of liposomes above and below the transition temperature. It was shown, that the mobility of lipid molecules in crystalline liposomes is higher than in the structure of liquid-crystalline liposomes. The addition of cholesterol in liposome membranes decreases the lateral molecular motion of lipids in crystalline and liquid-crystalline state, in the latter case the effect of cholesterol addition is more pronounced. The activation energy for the displacement of the fragments of lipid molecules and the lipid molecule as a whole was estimated, and it was shown, that lipid matrix possesses a high degree of heterogeneity.     

Electrohydrodynamic instabilities and segregation of polysaccharides in capillary polymer solution electrophoresis

During capillary electrophoresis of negatively charged polysaccharides in polymer solutions as sieving media, concentration fluctuations develop due to electrohydrodynamic instabilities caused by polarization of the polyelectrolytic chains. This leads to deviations from electroneutrality far beyond the Debye layer and segregation of the initially homogeneous sample solution into aggregated sample‐rich domains as verified by epifluorescence videomicroscopy imaging. As a result, anomalous and irregular peak profiles are obtained impeding the characterization of such complex sample mixtures. This effect appears at an electric field strength threshold value that depends on the molecular weight of the solute polymer molecules, pH, type and concentration of the polymer solution sieving media, and buffering conditions. The magnitude increases with increasing field strength and amount of sample injected. The aggregation onset, as evaluated by the value of the threshold potential, is affected by the charge density of the sample polymer molecules and Debye screening effects and investigated through variation of pH and ionic strength, respectively. Exchange of a simple base buffer component for small and multiply charged organic bases markedly increases the electric field strength necessary to trigger the electrohydrodynamic instabilities. Ultimately, the threshold value could be increased more than seven times by addition of an oppositely charged aminodextran polymer, thereby decreasing the analysis time. © 1999 John Wiley & Sons, Inc. Biopoly 49: 515–524, 1999     

Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 microg/ml SP-A and 0, 1.64, or 5 mM CaCl(2). In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca(2+). Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures (pi approximately 5 mN/m) and the domains grew in size with increasing pi. Above 25 mN/m, the domain size decreased with increasing pi. The amount of observable dark phase was maximal at 18% of the total film area at pi approximately 25 mN/m, then decreased to approximately 3% at pi approximately 40 mN/m. The addition of 0.16 microg/ml SP-A with 0 or 1.64 mM Ca(2+) in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to approximately 25% between pi of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca(2+) and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca(2+). PSLE films were spread on a subphase containing 0.16 microg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca(2+), TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca(2+) resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

Charge effects on folded and unfolded proteins

We develop a theory for the effects of charge on the stabilization of globular proteins. The folding process is modeled as occurring through a fictitious intermediate state along a two-part thermodynamic pathway in which the molecule (i) increases its density and then (ii) rearranges its ionic groups to the protein surface. The equilibrium for the binding of protons in salt solutions is assumed to be driven by the electrical potential due to the charge distribution, in addition to the intrinsic binding affinity and bulk proton concentration. The potential is calculated for inside and outside a porous sphere model of the protein using the Poisson-Boltzmann relation, wherein the interior dielectric constant is taken to be a linear function of the chain density. The model predicts the slope of the titration curves for native myoglobin in agreement with experiments by Breslow and Gurd (1962). From the similar experiments on the unfolded state, and from the experiments of Privalov et al. (1986) on the intrinsic viscosity of the unfolded molecules, the theory shows that the unfolded state has a much higher density than a chain in a theta solvent and that the density increases with ionic strength. In addition, from the free energy of proton binding to the protein, we also calculate the electrostatic contributions to protein stability, a major contribution deriving from changes in ionization. We consider the example of the stability of myoglobin as a function of pH, ionic strength, and ionic groups buried in the native protein structure. We show that although maximum stability of most proteins should occur at their isoelectric point, the burial of nontitratable groups should lead to maximum stabilities at pH values other than the isoelectric point.