The nonelectrolyte permeability of planar lipid bilayer membranes

The permeability of lecithin bilayer membranes to nonelectrolytes is in reasonable agreement with Overton's rule. The is, Pd alpha DKhc, where/Pd is the permeability coefficient of a solute through the bilayer, Khc is its hydrocarbon:water partition coefficient, and D is its diffusion coefficient in bulk hydrocarbon. The partition coefficients are by far the major determinants of the relative magnitudes of the permeability coefficients; the diffusion coefficients make only a minor contribution. We note that the recent emphasis on theoretically calculated intramembranous diffusion coefficients (Dm'S) has diverted attention from the experimentally measurable and physiologically relevant permeability coefficients (Pd'S) and has obscured the simplicity and usefulness of Overton's rule.     

A computer simulation of functional group contributions to free energy in water and a DPPC lipid bilayer

A series of all-atom molecular dynamics simulations has been performed to evaluate the contributions of various functional groups to the free energy of solvation in water and a dipalmitoylphospatidylcholine lipid bilayer membrane and to the free energies of solute transfer (Delta(DeltaG(o))X) from water into the ordered-chain interior of the bilayer. Free energies for mutations of the alpha-H atom in p-toluic acid to six different substituents (-CH3, -Cl, -OCH3, -CN, -OH, -COOH) were calculated by a combined thermodynamic integration and perturbation method and compared to literature results from vapor pressure measurements, partition coefficients, and membrane transport experiments. Convergence of the calculated free energies was indicated by substantial declines in standard deviations for the calculated free energies with increased simulation length, by the independence of the ensemble-averaged Boltzmann factors to simulation length, and the weak dependence of hysteresis effects on simulation length over two different simulation lengths and starting from different initial configurations. Calculated values of Delta(DeltaG(o))X correlate linearly with corresponding values obtained from lipid bilayer transport experiments with a slope of 1.1 and from measurements of partition coefficients between water and hexadecane or decadiene, with slopes of 1.1 and 0.9, respectively. Van der Waals interactions between the functional group of interest and the acyl chains in the ordered chain region account for more than 95% of the overall potential energy of interaction. These results support the view that the ordered chain region within the bilayer interior is the barrier domain for transport and that solvation interactions within this region resemble those occurring in a nonpolar hydrocarbon.     

Interaction of human serum albumin with fatty acids. Role of anionic group studied by affinity partition.

The interaction of human serum albumin with fatty acids has been determined using the method of affinity partitioning in aqueous biphasic systems containing dextran, poly(ethylene glycol) and esters of dicarboxylic acids with poly(ethylene glycol). The difference in the partition of albumin in phase systems with and without the poly(ethylene glycol)-bound fatty acid group provides a measure of the interaction of fatty acids with the protein. The relative contribution of the polar and non-polar interaction to the binding of fatty acids to albumin has been estimated by comparing the present data with that obtained earlier using poly(ethylene glycol)-bound straight chain aliphatic hydrocarbons. In both cases, the aliphatic chain should contain a minimum of 8 carbon atoms to affect the partition of albumin and that the maximum effect is obtained with chains containing 16 carbon atoms. The effect of the polymer-bound fatty acid group is higher than the corresponding hydrocarbon only when the number of carbon atoms in it exceeds 12. The relative effect of polymer-bound 16-carbon chains, with and without a carboxyl group in the terminal position is independent of pH in the range 5--9.     

Liposomes as carriers of poorly water-soluble substrates: linear modelling of membrane systems with catalytic or binding sites of different facedness. Significance of experimental membrane partition coefficients and of kinetic and equilibrium parameters.

1. A multiphasic modelling approach to systems containing membrane-bound receptors or catalytic sites and a liposomal preparation as a substrate carrier is described. Kinetic expressions are derived for a single-substrate enzymic reaction operating at constant liposome concentration or at a fixed substrate/liposome concentration ratio. 2. The assumption that accumulation of exchangeable components into the phospholipid bilayers can be described by linear bulk-phase partition leads to simple relationships between the initial reaction rate and (a) two kinetic coefficients (V and K'm), (b) the partition coefficients of the solutes for the lipid compartments of the membrane (Pms) and liposomal preparations (P1s) and (c) the total concentrations of substrate, membrane lipid and liposomal lipid. K'm is called the effective Michaelis constant. 3. For correct estimation of the coefficients V, K', Pms and P1s extrapolation to zero lipid concentration is required. 4. The distinction is introduced between hydrophilic and hydrophobic aqueous-faced sites, lipid-faced sites and mixed sites, i.e. sites overlapping an aqueous and a lipid region. For hydrophilic aqueous-faced sites K'm is equal to the true Km and for the other types of site to Km/Ps. For lipid-faced and for mixed sites Ps corresponds to the membrane partition coefficient Pms. For binding of homologous compounds to a hydrophobic aqueous-faced binding pocket Ps is the incremental site partition coefficient Pbss, which takes into account the energetic contribution to the binding process due to the hydrophobic tail of the ligands. 5. K'm accounts for any effects due to the facedness and nature of the enzymic sites. The dependence of the systems on the size of the lipidic partition compartment(s) is expressed exclusively by a distribution function F.6. When enzyme assays are performed with a series of chemically different substrates containing the same catalytically sensitive group, independence of K'm from partition indicates a hydrophilic aqueous-faced binding site. For the low-molecular-mass members of the homologous series a linear increase in -log (K'm) with the logarithm of the partition coefficient will be observed with any of the other site types considered 7. Equilibrium relationships for binding of a ligand to a membrane-bound receptor are also derived. 8. The significance of experimental membrane partition coefficients is discussed.     

Effect of lipophilic character on the biological activity of synthetic angiotensin peptides.

[Ile5]angiotensin II (angiotensin) derivatives bearing acetyl, propionyl, butyryl, pentanoyl or hexanoyl moieties at the N-terminal amino group were synthesized. The myotropic effects in vitro (on guinea-pig ileum and rat uterus) of desamino-angiotensin and of the above compounds did not correlate with their partition coefficients in butan-1-ol/acetic acid/water. The pressor effects in vivo in rats showed a negative correlation with the partition coefficients, discouraging further attempts to raise the pressor potency of angiotensin analogues by increasing their hydrophobicity. The half-times for onset and reversal of the biological responses also did not correlate with partition coefficients, but reversal was retarded by the presence of a free amino group. It is concluded that partition between aqueous medium and the lipophilic receptor environment is not a limiting factor for angiotensin activity.     

Differences in active site structure in a family of beta-glucan endohydrolases deduced from the kinetics of inactivation by epoxyalkyl beta-oligoglucosides

The active sites of a spectrum of beta-glucan endohydrolases with distinct, but related substrate specificities have been probed using a series of epoxyalkyl beta-glycosides of glucose, cellobiose, cellotriose, laminaribiose, laminaritriose, 3O-beta-D-glucosyl-cellobiose and 4O-beta-D-glucosyl-laminaribiose with different aglycon chain lengths. The inactivation of each of the endohydrolases by these compounds results from active site-directed inhibitor action, as indicated by the dependence of the inactivation rate on pH, glycosyl chain length and linkage position, aglycon length, and the protective effect of disaccharides derived from the natural substrates. Comparisons of inhibitor specificity between a Bacillus subtilis 1,3;1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73), a Streptomyces cellulase (EC 3.2.1.4), a Schizophyllum commune cellulase (EC 3.2.1.4), a Rhizopus arrhizus 1,3-(1,3;1,4)-beta-D-glucan 3(4)-glucanohydrolase (EC 3.2.1.6), and a Nicotiana glutinosa 1,3-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) demonstrated different tolerances for glycosyl linkage positions in the inactivation process and a critical role of aglycon length reflecting differences in the active site geometry of the enzymes. For the B. subtilis endohydrolase it was concluded that the aglycon residue of the inhibitor spans the glycosyl binding subsite occupied by the 3-substituted glucosyl residue involved in the glucosidic linkage cleaved in the natural substrate. Appropriate positioning of the inhibitor epoxide group with respect to the catalytic amino acids in the active site is crucial to the inactivation step and the number of glucosyl residues in the inhibitor affects aglycon chain length specificity. The importance of this effect differs between the glucanases tested and may be related to the number of glycosyl binding subsites in the active site.     

Membrane thickness and acyl chain length

It appears reasonable to expect that the primary result of a change in the length of the acyl chains within a lipid bilayer is a similar change in the bilayer thickness. In the present communication we draw attention to the somewhat more complicated effects which are found experimentally for phosphatidylcholine bilayers as the hydrocarbon chain is varied from twelve to eighteen carbons in length. The major change in dimension which occurs with variation in acyl chain length is the area occupied per molecule rather than the bilayer thickness. The same effect is seen with solute hydrocarbon such as hexane which partition into the membrane and cause only a small variation in membrane thickness but a large increase in the molecular area of the lipid. The origin of this effect arises from the almost isotropic distribution of the additional hydrocarbon to the lipid core of the membrane.     

Transport methods for probing the barrier domain of lipid bilayer membranes.

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent.     

Mixed inhibition of β-d-glucosidase from Stachybotrys atra by substrate analogues

The binding of series of alkyl and aryl β-d-glycopyranosides and their 1-thio analogues to the active site of β-d-glucosidase from Stachybotrys atra has been investigated. The binding constants for competitive and uncompetitive inhibition clearly demonstrated the existence of a hydrophobic aglycon-binding-site. The correlations found between competitive and uncompetitive inhibition suggest that the latter type of inhibition originates from the unspecific binding of the aglycon group to the aglycon binding-site of the intermediary enzyme-glycosyl complex.     

The noneffect of a large linear hydrocarbon, squalene, on the phosphatidylcholine packing structure.

The interaction of squalene with liposomes and monolayers of dipalmitoyl phosphatidylcholine (DPL) has been studied by differential scanning calorimetry, Raman spectroscopy, and surface potential measurements. Mole ratios of squalene to DPL up to 9 to 1 were studied. In contrast to small, nonpolar molecules, which profoundly influence the structure of lipid bilayers as detected by changes in both their thermodynamic phase transition parameters and membrane fluidity, this large, nonpolar, linear hydrocarbon is devoid of such influences. It is clear from our data that a large nonpolar molecule such as squalene, having no polar group that might anchor it to the aqueous interface, cannot intercalate between the acyl chains either below or above the phase transition of DPL. This behavior is not compatible with models that treat the bilayer interior as a bulk hydrocarbon, and suggests that great caution should be exercised in extrapolating partition coefficients based on bulk hydrocarbon measurements to lipid bilayers.     

Interactions of chlorinated hydrocarbon insecticides with membranes

Chlorinated hydrocarbon insecticides quench the fluorescence of N-alkyl derivatives of carbazole. We used phospholipids with covalently attached carbazole as probes for the interactions of chlorinated hydrocarbon insecticides with lipid bilayers, the object being to understand better the toxicities of chlorinated hydrocarbons. Fluorescence quenching measurements revealed the lipid-water partition coefficients of the chlorinated hydrocarbons, their diffusion coefficients in the membranes, and the binding capacities of the membranes for the chlorinated hydrocarbons. Active insecticides were compared with inactive analogues to test whether activities correlated with chlorinated hydrocarbon-membrane interactions. Thus DDT and methoxychlor were compared with inactive DDE, and insecticidal γ-lindane was compared with three less active stereoisomers. The partition coefficients, diffusion coefficients and membrane saturation capacities did not correlate with insecticidal potency. The partition coefficients of these chlorinated hydrocarbons were larger in bilayers containing unsaturated fatty acyl chains as compared to bilayers containing saturated fatty acyl chains. Interestingly, neural membranes are known to contain a large percentage of unsaturated lipids. Our results indicate that the activities of chlorinated hydrocarbons are not a result of specific interactions of these compounds with the lipids of membranes. However, the neurotoxicity of chlorinated hydrocarbons may be amplified by selective partitioning in the unsaturated neural membranes.     

Transport of steroid hormones facilitated by serum proteins.

The affinities with steroid hormones (alpha-estradiol, ethynylestradiol, progesterone, androsterone, dehydroisoandrosterone and testosterone) were observed for Cohn's fraction IV-1 and V (albumin). It was estimated from the comparison with the binding coefficient K (protein-bound form/free form of hormone) in a 3.5% (w/v) bovine serum albumin (BSA) solution that 40-80% of bound hormone in bovine serum is the BSA-bound form. It becomes clear in a liquid membrane system consisting of a hexane source phase (I), a water phase and a hexane receiving phase (II) that the transport flux of hormone is governed primarily by the partition coefficients between the water/hexane phases. In the case of a hormone with a lower partition coefficient, the uptake process from the hexane phase (I) to the water phase is a rate-determining step in the transport system and the serum proteins accelerate the transport of hormones, while with an increase in the partition coefficient the rate-determining step changes from the uptake step to the release step from the water phase to the hexane phase (II) and the hormone transport is decelerated owing to the significant decrease of free hormone concentration in the aqueous phase by the associated with serum proteins for the system having the restricted amount of hormone in the hexane source phase.     

Transport of hydrophobic ions in erythrocyte membrane: I. Zero membrane potential properties

Summary The permeability and partition coefficients of tetraphenylarsonium (TPA) and several other organic cations were studied in the human erythrocyte using an ion-selective electrode. The permeability constant for the different cations could be explained quite well by differences in oil/water partition coefficients. No evidence for facilitated transport could be found. Binding of the organic ions occurred to both the cell membrane and to intracellular contents. Partitioning to the membrane remained relatively constant despite variation from ion intracellular binding with blood samples from different donors. TPA flux is stimulated by substoichiometric amounts of tetraphenylboron and other organic anions, suggesting an ion-pairing mechanism.     

Octanol/water partition coefficients as a model system for assessing antidotes for methylmercury(II) poisoning, and for studying mercurials with medicinal applications

1-Octanol/water partition coefficients, [HgII]octanol/[HgII]water, provide a simple but limited model system for aspects of the biological behavior of methylmercury(II) and commonly used organomercury(II) medicinal compounds. In an octanol/water system some widely studied antidotes for mercury poisoning at least partly displace the biological thiols L-cysteine and glutathione from binding to MeHgII at pH 6.9. Addition of the antidote meso-dimercaptosuccinic acid to MeHgII in the presence of glutathione results in formation of metallic mercury. For RHgII derivatives of L-cysteine and glutathione, octanol/water partition coefficients follow the order Ph greater than Et greater than Me. An exceptionally high value for diphenylmercury, compared with PhHgII derivatives of L-cysteine and glutathione, is consistent with reported results of the distribution of mercury compounds in rats. Ethylmercury(II) is partly displaced from thimerosal by L-cysteine and glutathione in the octanol/water system, indicating that the active form of thimerosal in vivo may involve binding of EtHgII to biological ligands.     

Partition coefficients of quinones and hydroquinones and their relation to biochemical reactivity

Some major effects of ring substituents on the partition coefficients of quinone headgroups are described. Attention is drawn to the large differences in partition coefficients in cyclohexane/water of the two major freely diffusing redox forms, the quinone, Q, and the hydroquinone, QH2. Methoxy substituents cause a marked increase of the cyclohexane/water partition coefficient of the hydroquinone, but this effect is absent in the quinone and is also not seen in measurements in octanol/water. The relation between partition coefficients and biochemical specificity of quinone binding sites is explored.     

Interactions in affinity partition studied using fluorescence spectroscopy.

Fluorescence titration has been used to determine the binding constant and number of binding sites for the textile triazine dye Procion Yellow HE-3G to lactate dehydrogenase from rabbit muscle (E.C. 1.1.1.27). Triazine dye was either free in solution or attached to one of the polymer carriers, polyethylene glycol or dextran. Titrations were performed in solutions of buffer, dextran, and polyethylene glycol. Aqueous two-phase systems composed of polyethylene glycol and dextran were prepared and the binding constant and number of binding sites for ligand polyethylene glycol-Procion Yellow to lactate dehydrogenase were determined in both upper and lower phases of these systems. Affinity partition of lactate dehydrogenase in a PEG-dextran system was also performed using PEG-Procion Yellow as ligand, and partition coefficients of lactate dehydrogenase showed good agreement with theoretical partition coefficients calculated from the binding constant and number of binding sites obtained from fluorescence titration. The advantage of using fluorescence titration to determine affinity of a polymer ligand for a protein is that measurement of binding strength can be made in the actual environment encountered by protein-ligand complex during the purification process.     

Hydrolysis of aryl beta-D-glucopyranosides and beta-D-xylopyranosides by an induced beta-D-glucosidase from Stachybotrys atra.

The induced beta-D-glucosidase from Stachybotrys atra hydrolyzes aryl beta-D-glucopyranosides and aryl beta-D-xylopyranosides by the same basic two-step mechanism. In the first step the aglycon group is split of with simultaneous formation of an enzyme-glycosyl complex. In the second step this intermediate complex reacts with water yeilding beta-D-glucose or beta-D-xylose. For beta-D-xyloside hydrolysis each of the two steps is partially rate-controlling, whereas for beta-D-glucoside hydrolysis the second step is rate-limiting. The enzyme is inhibited by high concentrations of substrate and the exact rate-concentration equation is a second-order equation. 1-Thio-beta-D-glycopyranosides with an aromatic aglycon inhibit the reaction in both a competitive and non-competitive way. A tentative mechanism is proposed to explain all types of inhibition. In this mechanism substrates and inhibitors with an aromatic aglycon group bind through hydrophobic forces to the 'aglycon subsite' of the intermediate enzyme-glycosyl complex. Binding of the second substrate molecule or of the inhibitor to this complex does not prevent the reaction of the glycosyl moiety with water, it only decreases the rate of the second step.     

Hydrophobic interaction determined by partition in aqueous two-phase systems. Partition of proteins in systems containing fatty-acid esters of poly(ethylene glycol).

In this report we describe a new method which is useful for measuring hydrophobic interactions between aliphatic hydrocarbon chains and proteins in aqueous environment. The method is based on partition of proteins in an aqueous two-phase system containing dextran and poly(ethylene glycol) and different fatty acid esters of poly(ethylene glycol). The partition is measured under conditions where contributions from electrostatic interactions are eliminated. The difference in partition of proteins in phase systems with and without hyrocarbon groups bound to poly(ethylene glycol), deltalog K, where K is the partition coefficient, is taken as a measure of hydrophobic interaction. Deltalog K varies with size of hydrocarbon chain and type of protein. The length of the aliphatic chain should be greater than 8 carbon atoms in order to get a measurable effect in terms of deltalog K. Bovine serum albumin, beta-lactoglobulin, hemoglobin and myoglobin have been shown to have different affinities for palmitic acid ester of poly(ethylene glycol). No hydrophobic effect could be observed for ovalbumin, cytochrome c or alpha-chymotrypsinogen A.