Biophysical analysis of the molecular interactions between polysaccharides and mucin.

Mucoadhesive materials adhere persistently to mucosal surfaces. A mucoadhesive delivery system could therefore facilitate the controlled release of drugs and optimize their bioavailability in mucosal tissues. Polysaccharides are the most versatile class of natural polymers for transmucosal drug delivery. We used microviscosimetry to explore the mucoadhesion of a library of polysaccharide families with diverse structural characteristics as a first step toward the rational design of mucoadhesive polysaccharide-based nanoformulations. Here we show that the magnitude of deviation between the viscosity of mixed polysaccharide-mucin solutions and the corresponding individual stock solutions can indicate underlying molecular interactions. We found that nonlinear monotonic curves predicted a correlation between the magnitude of interaction and the ability of polysaccharide coils to contract in the presence of salt (i.e., chain flexibility). Charge-neutral polysaccharides such as dextran and Streptococcus thermophilus exopolysaccharide did not interact with mucin. Synchrotron small-angle X-ray scattering (SAXS) data supported the previously described structural features of mucin. Furthermore, high-q scattering data (i.e., sensitive to smaller scales) revealed that when mucin is in dilute solution (presumably in an extended conformation) in the presence of low-Mw alginate, its structure resembles that observed at higher concentrations in the absence of alginate. This effect was less pronounced in the case of high-Mw alginate, but the latter influenced the bulk properties of mucin-alginate mixtures (e.g., hydrodynamic radius and relative viscosity) more prominently than its low-Mw counterpart.

Spin-label electron paramagnetic resonance studies on the interaction of avidin with dimyristoyl-phosphatidylglycerol membranes.

The interaction of avidin--a basic protein from hen egg-white--with dimyristoyl-phosphatidylglycerol membranes was investigated by spin-label electron paramagnetic resonance spectroscopy. Phosphatidylcholines, bearing the nitroxide spin label at different positions along the sn-2 acyl chain of the lipid were used to investigate the effect of protein binding on the lipid chain-melting phase transition and acyl chain dynamics. Binding of the protein at saturating levels results in abolition of the chain-melting phase transition of the lipid and accompanying perturbation of the lipid acyl chain mobility. In the fluid phase region, the outer hyperfine splitting increases for all phosphatidylcholine spin-label positional isomers, indicating that the chain mobility is decreased by binding avidin. However, there was no evidence for direct interaction of the protein with the lipid acyl chains, clearly indicating that the protein does not penetrate the hydrophobic interior of the membrane. Selectivity experiments with different spin-labelled lipid probes indicate that avidin exhibits a preference for negatively charged lipid species, although all spin-labelled lipid species indirectly sense the protein binding. The interaction with negatively charged lipids is relevant to the use of avidin in applications such as the ultrastructural localization of biotinylated lipids in histochemical studies.

Molecular packing and intermolecular interactions in N-acylethanolamines: crystal structure of N-myristoylethanolamine.

N-Acylethanolamines elicited much interest in recent years owing to their occurrence in biological membranes under conditions of stress as well as under normal conditions. The molecular conformation, packing properties and intermolecular interactions of N-myristoylethanolamine (NMEA) have been determined by single crystal X-ray diffraction analysis. The lipid crystallized in the space group P21/a with unit cell dimensions: a=9.001, b=4.8761, c=39. 080. There are four symmetry-related molecules in the monoclinic unit cell. The molecules are organized in a tail-to-tail fashion, similar to the arrangement in a bilayer membrane. The hydrophobic acyl chain of the NMEA molecule is tilted with respect to the bilayer normal by an angle of 37 degrees. Each hydroxy group forms two hydrogen bonds, one as a donor and the other as an acceptor, with the hydroxy groups of molecules in the opposing leaflet. These O-H...O hydrogen bonds form an extended, zig-zag type network along the b-axis. In addition, the N-H and C=O groups of adjacent molecules are involved in N-H...O hydrogen bonds, which also connect adjacent molecules along the b-axis.

Interaction of N-myristoyldimyristoylphosphatidylethanolamine with dimyristoylphosphatidylcholine investigated by differential scanning calorimetry: binary phase diagram.

The temperature-composition phase diagram was derived for hydrated, binary mixtures of N-myristoyldimyristoylphosphatidylethanolamine (N-14 DMPE) and dimyristoylphosphatidylcholine by high sensitivity differential scanning calorimetry. Gel phase immiscibility was detected in mixtures containing up to 20 mol% N-14 DMPE and there was no evidence for compound formation between the two components. In the fluid phase nearly complete miscibility is indicated by the calorimetric data. These results are relevant to understanding the role of N-acylphosphatidylethanolamines in the stress combating responses of organisms and in their application to developing liposome-based drug delivery systems.

Differential scanning calorimetric studies on the thermotropic phase transitions of dry and hydrated forms of N-acylethanolamines of even chainlengths.

N-acylethanolamines (NAEs) have attracted the attention of researchers in the last two decades due to their occurrence in biological membranes under conditions of stress as well as under normal conditions. Differential scanning calorimetric studies have been carried out on dry and hydrated samples of a homologous series of N-acylethanolamines containing saturated acyl chains of even number of carbon atoms (n = 8-20). In both cases a major sharp endothermic transition was observed which occurs at the melting point for the dry NAEs whereas for the hydrated samples it occurs at considerably lower temperatures. The enthalpies and entropies corresponding to this transition could be fitted, in each case, to a straight line suggesting that the transition enthalpy and transition entropy consist of a fixed component from the polar head group and the terminal methyl group, whereas the contribution of the methylene groups, (CH2)n, is linearly proportional to the number of carbon atoms in it. The contributions of each methylene unit to the transition enthalpy and transition entropy of NAEs were found to be deltaH(inc) = 0.82 (+/-0.02) and 0.96 (+/-0.06) kcal mol(-1), and deltaS(inc) = 2.01 (+/- 0.06) and 2.37 (+/-0.17) cal mol(-1) K(-1), respectively, for the dry and hydrated samples of NAEs, whereas the end contributions arising from the head group and the terminal methyl group were determined to be deltaH(o) = -0.10 (+/-0.26) and -0.52 (+/-0.82) kcal mol(-1) and deltaS(o) = 2.12 (+/-0.71) and 3.1 (+/-2.3) cal mol(-1) K(-1), respectively, for the dry and hydrated samples of NAEs. These results are relevant to an understanding of the thermodynamics of the phase properties of NAEs in membranes.

Interaction of avidin with spin-labelled N-biotinyl phosphatidylethanolamine in a lipid membrane.

N-Biotinyl phosphatidylethanolamine spin labelled at the C-14 position of the sn-2 chain has been incorporated at a level of 1 mol% in bilayers of dimyristoyl phosphatidylcholine, and the effects on the chain mobility of binding avidin to the biotin lipid headgroup have been studied by electron spin resonance spectroscopy. In the fluid phase, avidin causes a large and selective restriction in the chain motion of the biotin lipids to which it is attached, without perturbing appreciably the mobility of the bulk lipid chains. This specific type of lipid-protein interaction is different in kind from that observed both with integral and peripheral membrane proteins and may be involved in transmembrane communication on ligand binding to lipid headgroups, as well as lateral communication (at high packing densities) between proteins with covalent lipid anchors.

Derivatised lipids in membranes. Physico-chemical aspects of N-biotinyl phosphatidylethanolamines, N-acyl phosphatidylethanolamines and N-acyl ethanolamines.

The physical properties of N-biotinyl phosphatidylethanolamines, N-acyl phosphatidylethanolamines and of N-acyl ethanolamines, in aqueous dispersions, are reviewed. Emphasis is placed on the calorimetric (i.e. chain melting) properties, the thermotropic phase behaviour, certain aspects of the structure and dynamics, and the miscibility with other membrane lipids. In the case of N-biotinyl phosphatidylethanolamines, the specific binding of avidin, and in the case of N-acyl ethanolamines, the function of the third chain, is also considered. All of these properties are relevant to the role of these rather unusual lipids in membranes.

Studies on the tryptophan residues of soybean agglutinin. Involvement in saccharide binding.

Modification of tryptophan side chains of soybean agglutinin (SBA) with N-bromosuccinimide results in a loss of the hemagglutinating and carbohydrate binding activities of the protein. One residue/subunit is probably essential for the binding activity. Modification leads to a large decrease in the fluorescence of the protein accompanied by a blue shift. Iodide ion quenching of the protein fluorescence shows that saccharide binding results in a decreased accessibility of some of the tryptophan side chains. These results strongly point towards the involvement of tryptophan residues in the active site of SBA.

Binding of porphyrins by the tumor-specific lectin, jacalin [Jack fruit (Artocarpus integrifolia) agglutinin].

Jacalin (Artocarpus integrifolia agglutinin) specifically recognizes the tumor-associated T-antigenic disaccharide structure, Gal beta13GalNAc. Porphyrins and their derivatives are currently used as photosensitizers in photodynamic therapy to treat malignant tumors. In this study, the interaction of several free base porphyrins and their metal derivatives with jacalin is investigated by absorption and fluorescence spectroscopy. Each lectin subunit was found to bind one porphyrin molecule and the association constants were estimated to be in the range of 2.4 x 10(3) M(-1) to 1.3 x 10(5) M(-1) at room temperature for the interaction of different porphyrins with jacalin. These values are in the same range as those obtained for the interaction of monosaccharides to jacalin. Both free lectin and lectin saturated with the specific saccharide were found to bind different porphyrins with comparable binding strength indicating that porphyrin binding takes place at a site different from the sugar binding site. Further, both anionic and cationic porphyrins were found to interact with the lectin with comparable affinity, clearly indicating that the charge on the porphyrin does not play any role in the binding process and that most likely the interaction is mediated by hydrophobic forces. These results suggest that jacalin and other lectins may potentially be useful for targeted delivery of porphyrins to tumor tissues in photodynamic therapy.

Further characterization of the saccharide specificity of peanut (Arachis hypogaea) agglutinin.

2-Dansylamino-2-deoxy-D-galactose (GalNDns) has been shown to bind to peanut (Arachis hypogaea) agglutinin (PNA) in a saccharide-specific manner. This binding was accompanied by a five-fold increase in the fluorescence of GalNDns. The interaction was characterized by an association constant of 0.15 mM at 15 degrees and delta H and delta S values of -57.04 kJ.mol-1 and -118.1J.mol-1.K-1, respectively. Binding of a variety of other mono-, di- and oligo-saccharides to PNA, studied by monitoring their ability to dissociate the PNA GalNDns complex, revealed that PNA interacts with several T-antigen-related structures, such as beta-D-Galp-(1----3)-D-GalNAc, beta-D-Galp-(1----3)-alpha-D-GalpNAcOMe, and beta-D-Galp-(1----3)-alpha-D-GalpNAc-(1----3)-Ser, as well as the asialo-GM1 tetrasaccharide, with comparable affinity, thus showing that this lectin does not discriminate between saccharides in which the penultimate sugar of the beta-D-Galp-(1----3)-D-GalNAc unit is the alpha or beta anomer, in contrast to jacalin (Artocarpus integrifolia agglutinin), another anti T-lectin which preferentially binds to beta-D-Galp-(1----3)-alpha-D-GalNAc and does not recognize beta-D-Galp-(1----3)-beta-D-GalNAc or the related asialo-GM1 oligosaccharide. These studies also indicated that, in the extended combining region of PNA which accommodates a disaccharide, the primary subsite (subsite A) is highly specific for D-galactose, whereas the secondary subsite (subsite B) is less specific and can accommodate various structures, such as D-galactose, 2-acetamido-2-deoxy-D-galactose, D-glucose, and 2-acetamido-2-deoxy-D-glucose.

Fluorescence and absorption spectroscopic studies on the interaction of porphyrins with snake gourd (Trichosanthes anguina) seed lectin.

The interaction of several free-base porphyrins and their corresponding copper(II) and zinc(II) derivatives with the galactose-specific lectin from snake gourd (Trichosanthes anguina) seeds has been investigated by absorption and fluorescence spectroscopic techniques. The lectin dimer contains two apparently equivalent binding sites for the porphyrins. Association constants obtained for the interaction of various porphyrins with the lectin are in the range 1.7 x 10(4)-6.2 x 10(5) M(-1), with the metalloporphyrins being seen to have higher affinity for the lectin compared with the free-base analogues. Both positively charged and negatively charged porphyrins bind to snake gourd seed lectin (SGSL) with comparable affinities, suggesting that binding occurs primarily via hydrophobic interactions. Further, binding of porphyrins is found to be largely unaffected by the presence of the sugar ligand, lactose, indicating that the binding sites for the carbohydrate and porphyrin are different. This study thus suggests that the lectin may serve as a receptor for some endogenous non-carbohydrate, hydrophobic ligand in vivo, in addition to the saccharide ligands. It also opens up the possibility of employing the T. anguina lectin in applications such as photodynamic therapy, which involve the use of porphyrins.

Thermodynamic analysis of biotin binding to avidin. A high sensitivity titration calorimetric study.

Titration calorimetric studies on the binding of biotin to avidin were performed in phosphate buffered saline, pH 7.4. From the temperature dependence of the binding enthalpy (delta H), the delta Cp value was determined. While the delta H value of -23.4 kcal/mol at 25 degrees C is in close agreement with the previously determined value of -22.5 kcal/mol (Suurkusk, J. & Wadso, I. (1972) Eur. J. Biochem. 28, 438-441), the delta Cp value of -461 cal/mol biotin/K is significantly at variance with the value of -237 cal/mol biotin/K obtained in the previous study. A comparison of the thermodynamic data obtained for the avidin-biotin system with that of the streptavidin-biotin system revealed that the higher binding affinity of avidin for biotin is due to a smaller (negative) entropy of binding.

Spin-label electron spin resonance studies on the dynamics of the different phases of N-biotinylphosphatidylethanolamines.

The chain dynamics and phase behavior of a homologous series of diacyl-N-biotinylphosphatidylethanolamines of chain lengths from C(12:0) to C(20:0) were investigated by spin-label electron spin resonance (ESR) spectroscopy using both phosphatidylcholine and N-biotinylphosphatidylethanolamine spin-label probes. Chain-melting phase transition temperatures determined from the ESR spectral measurements, for all the five lipids in the presence as well as in the absence of 1 M NaCl, correlate with the endothermic phase transition temperatures determined by differential scanning calorimetry [Swamy, M. J., Angerstein, B., & Marsh, D. (1994) Biophys. J. 66, 31-39], confirming that the latter correspond to chain-melting transitions. ESR spectra obtained in the gel phase from phosphatidylcholine probes with the spin-label near the terminal methyl of the hydrocarbon chain showed a similar degree of immobilization (as reflected by the outer hyperfine splittings) to that for spin-labels positioned close to the glycerol backbone, indicating that the biotin-lipids of chain lengths from 14 to 20 carbon atoms form interdigitated gel phases in the presence of salt, as also do those of chain lengths between 16 and 20 carbon atoms in the absence of salt. For dispersions of the C(16:0) chain length biotin-lipid in 1 M NaCl, continuous monitoring of the central ESR peak intensity as a function of temperature detects a cooperative decrease in mobility in the fluid phase at ca. 65 degrees C with a spin-label in the lipid head group and an accompanying increase in mobility at the same temperature with spin-labels positioned toward the terminal methyl of the hydrocarbon chain.(ABSTRACT TRUNCATED AT 250 WORDS)

On the metabolism of GM3 ganglioside in cultured human foreskin fibroblasts.

The metabolism of GM3 ganglioside in cultured human foreskin fibroblasts was investigated by labeling cultured cells with [1-3H]-galactose for 48 hours, followed by a 48 hour chase. More than 80% of the radioactivity associated with GM3 was found in the hexose portion of the carbohydrate chain, whereas approximately 12% of the radioactivity was observed in the sialic acid moiety. The hexose and sialic acid residues lost 42% and 53% of their initial radioactivity, respectively, during the chase period, indicating an active metabolism of these sugar residues of GM3 in growing cultures.

Spin-label studies on the anchoring and lipid-protein interactions of avidin with N-biotinylphosphatidylethanolamines in lipid bilayer membranes.

The specific binding of hen egg white avidin to phosphatidylcholine lipid membranes containing spin-labeled N-biotinylphosphatidylethanolamines (biotin-PESLs) was investigated by using ESR spectroscopy. Spin-labeled biotin-PEs were prepared with the nitroxide group at position C-5, C-8, C-10, C-12, or C-14 of the sn-2 chain and were incorporated at 1 mol % in lipid bilayer membranes of dimyristoylphosphatidylcholine. Binding of avidin produced a strong and selective restriction of the biotin-PE lipid mobility at all positions of chain labeling, as shown by the ESR spectra recorded in the fluid lipid phase. The spectral components of the fraction of the biotin-PESLs that were not complexed by avidin indicated that the mobility of the bulk membrane lipids was unperturbed by binding avidin, as demonstrated by difference spectroscopy. Comparison of the positional profiles and temperature dependences of the outer hyperfine splittings from the biotin-PESLs suggests that the C-12 and C-14 positions of the avidin-bound biotin-PEs are in register with the C-5 and C-7/C-6 positions, respectively, of the chains of the bulk membrane lipids. This indicates that the biotin-PEs are partially withdrawn from the membrane, with a vertical displacement of ca. 7-8 A, on complexation with avidin. In addition, the specific lipid-protein interaction with avidin results in a selective reduction in the rates of lipid chain motion, as shown by the increased ESR line widths. These data define the way in which avidin is anchored to lipid membranes containing biotin-PEs.

Specific surface association of avidin with N-biotinylphosphatidylethanolamine membrane assemblies: effect on lipid phase behavior and acyl-chain dynamics.

The interaction of avidin with aqueous dispersions of N-biotinylphosphatidylethanolamines, of acyl chain lengths C(14:0), C(16:0), and C(18:0), was studied by using spin-label electron spin resonance (ESR) spectroscopy, (31)P nuclear magnetic resonance ((31)P NMR) spectroscopy, differential scanning calorimetry, and chemical binding assays. In neutral buffer containing 1 M NaCl, binding of avidin is due to specific interaction with the biotinyl lipid headgroup because avidin presaturated with biotin does not bind. Saturation binding of the protein corresponds to a ratio of 50 lipid molecules per tetrameric avidin. Phospholipid probes spin-labeled at various positions between C-4 and C-14 in the sn-2 chain were used to characterize the effects of avidin binding on the lipid chain dynamics. In the fluid phase, protein binding results in a decrease of chain mobility at all positions of labeling while the flexibility gradient characteristic of a liquid-crystalline lipid phase is maintained. There is no evidence from the spin-label ESR spectra for penetration of the protein into the hydrophobic interior of the membrane. At temperatures corresponding to the gel phase, the lipid chain mobility increases on binding protein. The near constancy in mobility found with chain position, however, suggests that in the gel phase the lipid chains remain interdigitated upon binding avidin. Binding of increasing amounts of avidin results in a gradual decrease of the lipid chain-melting transition enthalpy with only small change in the transition temperature. At saturation binding, the calorimetric enthalpy is reduced to zero. (31)P NMR spectroscopy indicates that protein binding increases the surface curvature of dispersions of all three biotin lipids. The C(14:0) biotin lipid yields isotropic (31)P NMR spectra in the presence of avidin at all temperatures between 10 and 70 degrees C, in contrast to dispersions of the lipid alone, which give lamellar spectra at low temperature that become isotropic at the chain-melting temperature. In the presence of avidin, the C(16:0) and C(18:0) biotin lipids yield primarily lamellar (31)P NMR spectra at low temperature with a small isotropic component; the intensity of the isotropic component increases with temperature, and the spectra narrow and become totally isotropic at high temperature, in contrast to dispersions of the lipids alone, which give lamellar spectra in the fluid phase. The binding of avidin therefore reduces the cooperativity of the biotin lipid packing, regulates the mobility of the lipid chains, and enhances the surface curvature of the lipid aggregates. These effects may be important for both lateral and transbilayer communication in the membrane.

Thermodynamics of interdigitated phases of phosphatidylcholine in glycerol.

Comparison of the electron spin resonance spectra of phosphatidylcholines spin-labeled in the sn-2 chain at a position close to the polar region and close to the methyl terminus indicate that symmetrical saturated diacyl phosphatidylcholines with odd and even chain lengths from 13 to 20 C-atoms (and probably also 12 C-atoms) have gel phases in which the chains are interdigitated when dispersed in glycerol. The chain-length dependences of the chain-melting transition enthalpies and entropies are similar for phosphatidylcholines dispersed in glycerol and in water, but the negative end contributions are smaller for phosphatidylcholines dispersed in glycerol than for those dispersed in water: d delta Ht/dCH2 = 1.48 (1.43) kcal.mol-1, d delta St/dCH2 = 3.9 (4.0) cal.mol-1K-1, and delta H o = -12.9 (-15.0) kcal.mol-1, delta S o = -29 (-40) cal.mol-1K-1, respectively, for dispersions in glycerol (water). These differences reflect the interfacial energetics in glycerol and in water, and the different structure of the interdigitated gel phase.