Role of ionic strength on the relationship of biopolymer conformation,DLVO contributions,and steric interactions to bioadhesion of Pseudomonas putida KT2442

Biopolymers produced extracellularly by Pseudomonas putida KT2442 were examined via atomic force microscopy (AFM) and single molecule force spectroscopy. Surface biopolymers were probed in solutions with added salt concentrations ranging from that of pure water to 1 M KCl. By studying the physicochemical properties of the polymers over this range of salt concentrations, we observed a transition in the steric and electrostatic properties and in the conformation of the biopolymers that were each directly related to bioadhesion. In low salt solutions, the electrophoretic mobility of the bacterium was negative, and large theoretical energy barriers to adhesion were predicted from soft-particle DLVO theory calculations. The brush layer in low salt solution was extended due to electrostatic repulsion, and therefore, steric repulsion was also high (polymers extended 440 nm from surface in pure water). The extended polymer brush layer was "soft", characterized by the slope of the compliance region of the AFM approach curves (-0.014 nN/nm). These properties resulted in low adhesion between biopolymers and the silicon nitride AFM tip. As the salt concentration increased to > or =0.01 M, a transition was observed toward a more rigid and compressed polymer brush layer, and the adhesion forces increased. In 1 M KCl, the polymer brush extended 120 nm from the surface and the rigidity of the outer cell surface was greater (slope of the compliance region = -0.114 nN/nm). A compressed and more rigid polymer layer, as well as a less negative electrophoretic mobility for the bacterium, resulted in higher adhesion forces between the biopolymers and the AFM tip. Scaling theories for polyelectrolyte brushes were also used to explain the behavior of the biopolymer brush layer as a function of salt concentration.     

Affinity-repulsion chromatography. Principle and application to lectins

The interactions of proteins with their immobilized ligands in an electrically charged microenvironment were studied. The binding of lectins to erythrocytes and to affinity matrices was used as a model system. Lectins bind and agglutinate erythrocytes in the presence of at least 10 mM NaCl or 1 mM CaCl2, but not in deionized water. The salt dependence of the agglutination process is due to the ability of salts to provide counterions neutralizing the forces of repulsion between the electrostatic charges of similar sign present on the erythrocyte cell surface and on the lectins. The same salt dependence is observed for the binding of lectins to affinity matrices. These observations are the basis of a protein separation process coined affinity-repulsion chromatography in which the electrostatic charges present, or purposely introduced, on affinity matrices are exploited and allow the elution, by electrostatic repulsion, of proteins carrying electrostatic charges of the same sign as that of the matrix. In this process, proteins are loaded on the affinity matrix in a salt solution and eluted with deionized water. Affinity-repulsion chromatography has been successfully applied here to the isolation of several lectins. Its physicochemical basis, merits, and potential applications are discussed.     

Mechanical, microstructural and solubility properties of pectin/poly(vinyl alcohol) blends

Citrus pectin was blended and cast into films with poly(vinyl alcohol) (PVOH). PVOH and pectin were miscible in all proportions. Dynamic mechanical analysis revealed that pectin controls exhibited no thermal transitions, whereas PVOH controls exhibited a glass transition temperature (T_g) over a broad temperature range commencing at about 0 °C and ending about 50 °C. A mixture of 49% pectin, 21% PVOH and 30% glycerol exhibited lower storage moduli and more flexibility than comparable mixtures of either pectin/PVOH or pectin/glycerol. Scanning electron microscopy and phase contrast optical microscopy indicated that the mixture was biphasic and a compatible composite either of PVOH in pectin or pectin in PVOH depending on which macromolecule was in excess. Elongation to break measurements revealed that pectin/PVOH films underwent a brittle to ductile transition with increasing PVOH composition. The addition of glycerol to pectin/PVOH films increased ductility significantly when films were relatively brittle. Initial moduli (IM) as a function of composition gave complex curves which exhibited either one or two local maxima depending on such factors as degree of hydrolysis and molar mass of the PVOH in addition to the moisture content of the film. Solubility studies in water revealed that, at 30 and 50 °C, only films with 30% PVOH or less were soluble. At 70 °C, all compositions were soluble but films containing pectin dissolved more rapidly than those without. The solution kinetics of pectin/PVOH films with 30% or less PVOH were approximated with zero-order kinetics and activation energies were about 3–5 kcal mol⁻¹. In general, addition of PVOH to pectin films resulted in films with more PVOH-like properties and addition of pectin to PVOH films resulted in films with more pectin-like properties.     

Thermodynamic incompatibility and complex formation in pectin/caseinate mixtures

The instability of polysaccharide/protein mixtures occurs because of either thermodynamic incompatibility or complexation. We studied which instability mechanism dominated given the external conditions. Therefore the effect of temperature, pH, and biopolymer concentration on the phase separation of pectin/caseinate mixtures was investigated. At pH > 6, thermodynamic incompatibility with spinodal decomposition was observed in pectin/caseinate mixtures resulting in the formation of water-in-water emulsions in intermediate stages of the phase separation process. The demixing rate of these emulsions and appearance of two macroscopic phases (lower phase enriched with caseinate and upper phase with pectin) was retarded when the pectin concentration increased or when the storage temperature decreased due to a higher viscosity of the mixtures at those conditions. As the pH of the mixture was lowered below 6, pectin accumulated in the caseinate-rich phase. Complexation of pectin and caseinate led to the formation of microparticles (approximately 3 microm), whose shape depends on the biopolymer concentration ratio and rate of acidification. These pectin/caseinate particles do not coalesce and are insensitive to salt addition.     

Sugar Beet Pectin–Protein Complexes

The physical structure of pectin extracted from fresh sugar beet has been examined by atomic force microscopy (AFM). The images obtained reveal that these extracts contain a mixture of pectin polysaccharides and protein–pectin complexes. The AFM data demonstrate, for the first time, that these protein–pectin complexes consist of pectin molecules with protein attached to one end of the pectin chain.     

Effect of pH on the association behavior in aqueous solutions of pig gastric mucin

In this study, dynamic light scattering (DLS), turbidity, and rheo-small angle light scattering (rheo-SALS) methods have been utilized to examine the impact of pH (1 < or = pH < or = 7) on aqueous solutions of noncommercial purified pig gastric mucin. The asymmetric flow field-flow fractionation (AFFFF) measurements established that the mucin sample has a high molecular weight and is polydisperse. DLS measurements on dilute solutions of mucin disclosed large interchain aggregates at pH 2, where the polymer has a low charge density or is uncharged. At lower or higher values of pH, mucin is charged and the tendency of forming interpolymer complexes is affected. In the semidilute concentration regime, pronounced junction zones ('lumps' of polymer) are evolved and a heterogeneous connected network is formed at pH 2, whereas the association structures are disintegrated (smaller 'lumps') at lower or higher pH values due to electrostatic repulsive interactions, and a more homogeneous network is evolved. The DLS and viscosity results at pH 1 indicate the development of a fragmented network, composed of contracted chains that are decorated by some positive charges. The effect of shear flow on the structure of semidilute solutions of mucin was investigated with the aid of rheo-SALS methods. The scattered intensity revealed a strong upturn at low values of the wave vector (q) for mucin solutions at pH 2 and pH 4, which suggests the evolution of large association domains. At these pH values, a flow-induced anisotropy in the 2D SALS patterns in the form of elliptical shapes was observed at high shear rates.     

The isolated MUC5AC gene product from human ocular mucin displays intramolecular conformational heterogeneity

Atomic force microscopy (AFM) has been used to show that human ocular mucins contain at least three distinct polymer conformations, separable by isopycnic density gradient centrifugation. In this work we have used affinity purification against the anti(mucin peptide core) monoclonal antibody 45M1 to isolate MUC5AC gene products, a major component of human ocular mucins. AFM images confirm that the affinity-purified polymers adopt distinct conformations that coidentify with two of those observed in the parent population, and further reveal that these two different conformations can be present within the same polymer. AFM images of the complexes formed after incubation of 45M1 with the parent sample reveal different rates of binding to the two MUC5AC polymer types. The variability of gene products within a mucin population was revealed by analyzing the height distributions along the polymer contour and periodicities in distances between occupied antibody binding sites. AFM analysis of mucin polymers at the single molecule level provides new information about the genetic origins of individual polymers and the contributions of glycosylation to the physicochemical properties of mucins, which can be correlated with information obtained from biochemistry, antibody binding assays, and molecular biology techniques.     

Study of the compatibility/incompatibility of gelatin/iota-carrageenan/water mixtures

The incompatibility of acid gelatin/iota-carrageenan mixtures has been studied. Both these biopolymers undergo a conformational coil-helix transition under suitable conditions of temperature and salt. The aim of this work was to study the concentration at which mixtures are incompatible and the influence of pH, salt and temperature on the phase diagram. Incompatibility occurred over a wide range of concentrations for mixtures prepared in deionized water. Compatibility was increased by increasing the pH or the salt concentration. Temperature did not greatly influence the size of the incompatible region. This is in agreement with the hypothesis that attractive electrostatic interactions lead to associative phase separation (traditionally called complex coacervation).     

Enhancement of the viscosity of mucin by serum albumin.

The interaction of serum albumin with a model epithelial mucin from pig stomach was explored by rotary viscometry. During 30 min of incubation of human serum albumin(20mg/ml) and pig gastric mucin (8mg/ml) in iso-osmotic buffers at 37 degrees C, the solution became markedly viscous. Viscosity enhancement was proportional to albumin concentration (2-40mg/ml), was most pronounced under conditions of low shear rate (less than 45S-1), and was considerably greater than the additive or multiplicative viscosity values calculated from albumin or mucin solutions measured separately. The viscous mucin-albumin complex was destroyed by high shear rates (greater than 90S-1), but slowly re-formed under zero shear conditions. Elevation of pH (7 to 9), ionic strength (0.1 to 1.0), and addition of disodium EDTA (5mM) did not cause marked or specific alterations in the viscosity of the mixture, suggesting that electrostatic interactions probably do not stabilize mucin-albumin complexes. Urea (7M) and heating (35 to 55 degrees C) caused a major increase in the viscosity of mucin and mucin-albumin mixtures, suggesting that rupture of hydrogen bonds, unfolding and partial denaturation of mucin promotes greater intertangling (possibly hydrophobic interactions) between mucin and albumin molecules. The implications of mucin-albumin interaction in diseases associated with mucus obstruction are briefly discussed.     

How to Dig Deeper? Improved Enrichment Methods for Mucin Core-1 Type Glycopeptides

Two different workflows were tested in order to develop methods that provide deeper insight into the secreted O-glycoproteome. Bovine serum samples were subjected to lectin affinity-chromatography both at the protein- and peptide-level in order to selectively isolate glycopeptides with the most common, mucin core-1 sugar. This enrichment step was implemented with either protein-level mixed-bed ion-exchange chromatography or with peptide-level electrostatic repulsion hydrophilic interaction chromatography. Both methods led to at least 65% of the identified products being glycopeptides, in comparison to ≈ 25% without the additional chromatography steps [Darula, Z., and Medzihradszky, K. F. (2009) Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum. Mol. Cell. Proteomics 8, 2515-2526]. In order to improve not only the isolation but also the characterization of the glycopeptides exoglycosidases were used to eliminate carbohydrate extensions from the directly peptide-bound GalNAc units. Consequent tandem MS analysis of the mixtures using higher-energy collision-dissociation and electron-transfer dissociation led to the identification of 124 glycosylation sites in 51 proteins. While the electron-transfer dissociation data provided the bulk of the information for both modified sequence and modification site assignment, the higher-energy collision-dissociation data frequently yielded confirmation of the peptide identity, and revealed the presence of some core-2 or core-3 oligosaccharides. More than two-thirds of the sites as well as the proteins have never been reported modified.     

Studies on Pectolytic Enzymes of Molds

The clarification of apple juice has been studied using six pectolytic enzymes produced by Coniothyrium diplodiella, endo-PG (polygalacturonase) I, II and III, exo-PG and PE (pectinesterase) I and II. Each of these six enzymes had no effect on the clarification of apple juice when acted alone, whereas mixtures of any one of endo-PGs and PEs were all able to clarify the juice. Mixtures of exo-PG and either of PEs has no effect on the clarification. Clarifying activities of PG-PE mixtures were varied with the kind of endo-PG used in each mixture and not with the kind of PE. Clarifying activity of PG-PE mixture depended on either endo-PG or PE activities when the other was kept constant.

Crude enzyme from the mold and a mixture of the four PGs and PE in the ratio of the crude enzyme had essentially identical effect on apple juice as well as on artificial pectin and pectic acid.     

Evidence for long-range electrostatic repulsion between HeLa cells

Agglutination curves obtained on addition of low molecular weight poly-l-lysines (mol. wt 4 000–23 000) to HeLa cells, show a deviation from linearity at low polymer concentration. This probably indicates the existence of a ζ-potential which has to be lowered before agglutination can take place. Experiments with dilysine support the assumption that cell surface charge is lowered on addition of low concentrations of short chain poly-l-lysines.Long poly-l-lysine molecules (mol. wts 70 000; 100 000) yield linear agglutination curves already at the lowest polymer concentrations. This might indicate that these polymers are able to bridge the original repulsion gap between HeLa cells.After removal of peripheral sialic acid by neuraminidase, linear agglutination curves are obtained with all poly-l-lysines irrespective of their chain lengths. This is interpreted as evidence for involvement of sialic acid residues in the charge organization responsible for electrostatic repulsion.The magnitude of the presumed repulsion effect is shown to vary with the cell density at the time the HeLa cells were harvested from the culture. The largest repulsion effect is obtained with cells from density inhibited cultures which also have the lowest tendency for mutual adhesion. With fast growing cells from low density cultures linear agglutination curves are obtained with short chain poly-l-lysines. This is interpreted as evidence for a strong diminishment or absence of long-range electrostatic repulsion between such cells.     

Charge substitution shows that repulsive electrostatic interactions impede the oligomerization of Alzheimer amyloid peptides

The strong pH dependence of A beta oligomerization could arise from favorable intermolecular charge-charge interactions between His and carboxylate groups, or, alternatively, by mutual electrostatic repulsion of peptide molecules. To test between these two possibilities, the pH dependence of the oligomerization of A beta and three charge substitution variants with Asp, Glu and His substituted by Ala is measured. All four peptides oligomerize, as detected by thioflavin T fluorescence, turbidity, and amyloid fibril formation; therefore, specific charge-charge interactions are nonessential for oligomerization. The strong negative correlation between net charge and oligomerization indicates that electrostatic repulsion between A beta monomers impedes their association.     

Nonideal mixing of phosphatidylserine and phosphatidylcholine in the fluid lamellar phase.

The mixing of phosphatidylserine (PS) and phosphatidylcholine (PC) in fluid bilayer model membranes was studied by measuring binding of aqueous Ca2+ ions. The measured [Ca2+]aq was used to derive the activity coefficient for PS, gamma PS, in the lipid mixture. For (16:0, 18:1) PS in binary mixtures with either (16:0, 18:1)PC, (14:1, 14:1)PC, or (18:1, 18:1)PC, gamma PS > 1; i.e., mixing is nonideal, with PS and PC clustered rather than randomly distributed, despite the electrostatic repulsion between PS headgroups. To understand better this mixing behavior, Monte Carlo simulations of the PS/PC distributions were performed, using Kawasaki relaxation. The excess energy was divided into an electrostatic term Uel and one adjustable term including all other nonideal energy contributions, delta Em. Uel was calculated using a discrete charge theory. Kirkwood's coupling parameter method was used to calculate the excess free energy of mixing, delta GEmix, hence In gamma PS,calc. The values of In gamma PS,calc were equalized by adjusting delta Em in order to find the simulated PS/PC distribution that corresponded to the experimental results. We were thus able to compare the smeared charge calculation of [Ca2+]surf with a calculation ("masked evaluation method") that recognized clustering of the negatively charged PS: clustering was found to have a modest effect on [Ca2+]surf, relative to the smeared charge model. Even though both PS and PC tend to cluster, the long-range nature of the electrostatic repulsion reduces the extent of PS clustering at low PS mole fraction compared to PC clustering at an equivalent low PC mole fraction.     

The structure of high-methoxyl sugar acid gels of citrus pectin as determined by AFM

Images of native high-methoxyl sugar acid gels (HMSAGs) were obtained by atomic force microscopy (AFM) in the Tapping Mode^™. Electronic thinning of the pectin strands to one-pixel wide allowed the pectin network to be viewed in the absence of variable strand widths related to preferentially solvated sugar. Thinned images revealed that HMSAGs of pectin comprise a partially cross-linked network, in that many of the cross-linking moieties are attached at only one end. Based on their structural similarities, aggregated pectin in water appears to be a fluid precursor of a HMSAG of pectin. Furthermore, examination of AFM images revealed that gels with ‘uniform’ distribution of strands and pores between strands had higher gel strengths than gels in which strands were non-uniformly distributed and were separated by large and small spaces.     

Global structures of high methoxyl pectin from solution and in gels

Images of high methoxyl orange pectin deposited from solution and high methoxyl sugar acid gels (HMSAG) were obtained by atomic force microscopy (AFM) in the tapping mode. For the first time, images of pectin deposited from water revealed that the transition from pectin networks to individual molecules or aggregates thereof occurred at concentrations between 6.5 and 13.1 microg/mL. At 6.5 microg/mL, shapes included rods, segmented rods, kinked rods, rings, branched molecules, and dense circular areas. At 13.1 microg/mL, all of these shapes were integrated into networks. These same structures were discernible in pectin high methoxyl sugar acid gels. Thus one might consider pectin networks in water at concentrations in excess of 10 microg/mL to be separate fluid precursors of networks in high methoxyl sugar acid gels. Examination of AFM images revealed that gels with "uniform" distribution of strands and pores between strands had higher gel strengths as measured by a penetrometer than gels in which strands were nonuniformly distributed and were separated by large and small spaces.     

Binding of divalent cations of dipalmitoylphosphatidylcholine bilayers and its effect on bilayer interaction

We have confirmed that CaCl2 swells the multilayer lattice formed by dipalmitolyphosphatidylcholine (DPPC) in an aqueous solution. Specifically, at room temperature 1 mM CaCl2 causes these lipid bilayers to increase their separation, dw, from 19 A in pure water to greater than 90 A. CaCl2 concentrations greater than 4 mM cause less swelling. We have measured the net repulsive force between the bilayers in 30 mM CaCl2 at T = 25 degrees C (below the acyl chain freezing temperature). For interbilayer separations between 30 and 90 A, the dominant repulsion between bilayers is probably electrostatic; Ca2+ binds to DPPc lecithin bilayers, imparting a charge to them. The addition of NaCl to CaCl2 solutions decreases this repulsion. For dw less than 20 A, the bilayer repulsion appears to be dominated by the "hydration forces" observed previously between both neutral and charged phospholipids. From the electrostatic repulsive force, we estimate the extent of Ca2+ binding to the bilayer surface. The desorption and bound Ca2+, apparent when bilayers are pushed together, is more rapid than one would expect if an association constant governed Ca2+ binding. The association affinity does not appear to be a fixed quantity but rather a sensitive function of ionic strength and bilayer separation.     

Polysaccharide-based polyanion--polycation--polyanion ternary systems. A preliminary analysis of interpolyelectrolyte interactions in dilute solutions

The present contribution deals with the preparation and characterization of ternary mixtures of polysaccharides with potential applications in the field of tissue engineering. Two natural polyanions, i.e., alginate and hyaluronic acid, and a polycation, a lactose-modified chitosan (chitlac), were mixed in dilute conditions. The miscibility between the three components was explored in the presence of different amounts of supporting simple salt. These analyses allowed to identify the experimental conditions avoiding polymer phase separation and leading to either solution of independent polymers or soluble nonstoichiometric interpolyelectrolyte complexes. The characterization of the interpolyelectrolyte complexes was tackled by means of viscometry, light scattering, fluorescence quenching, and energy transfer. The electrostatic interactions taking place among the different polyelectrolytes led to synergistic effects on the viscosity of the polymer mixtures which strongly depend on the ionic strength. It has been found that, starting from binary soluble complexes of alginate and chitlac, the addition of hyaluronan led to the dissolution of the complexes. At variance, the addition of alginate to a phase-separated binary mixture of hyaluronan and chitlac led to the formation of soluble complexes composed of all three polysaccharides and, eventually, to their dissolution. In addition, the results showed that at low ionic strength the overall properties of the ternary mixtures depend on their order of mixing.     

Thermoreversible gelation of aqueous mixtures of pectin and chitosan. Rheology

The synergistic interaction between pectin and chitosan in aqueous acid solution and in the gel phase has been studied by oscillatory shear measurements. Mixtures of pectin and chitosan form thermoreversible gels over a broad composition range by lowering the temperature. The value of the gelation temperature depends on the composition of the mixture, with low values for mixtures with low pectin contents. For incipient gels, a power law can describe the frequency dependence of the complex viscosity, with power law exponents close to -1. The gel evolution of pectin-chitosan mixtures upon a temperature quench below the gel point has been studied. Evidence is provided for a relation between gelation and phase separation in the process of temperature-induced gelation of pectin-chitosan mixtures. A simple model is proposed to rationalize the gelation process in these systems.     

Polymer coated liposomes for use in the oral cavity – a study of the in vitro toxicity,effect on cell permeability and interaction with mucin

In this study we investigated the in vitro toxicity, impact on cell permeability and mucoadhesive potential of polymer-coated liposomes intended for use in the oral cavity. A TR146 cell line was used as a model. The overall aim was to end up with a selection of safe polymer coated liposomes with promising mucoadhesive properties for drug delivery to the oral cavity. The following polymers were tested: chitosan, low-methoxylated pectin (LM-pectin), high-methoxylated pectin (HM-pectin), amidated pectin (AM-pectin), Eudragit, poly(N-isopropylacrylamide-co-methacrylic acid) (p(NIPAAM-co-MAA)), hydrophobically modified hydroxyethyl cellulose (HM-HEC), and hydrophobically modified ethyl hydroxyethyl cellulose (HM-EHEC). With chitosan as an exception, all the systems exhibited no significant effect on cell viability and permeability at the considered concentrations. Additionally, all the formulations showed to a varying degree an interaction with mucin (BSM type I-S); the positively charged formulations exhibited the strongest interaction, while the negatively and neutrally charged formulations displayed a moderate or low interaction. The ability to interact with mucin makes all the liposomal formulations promising for oromucosal administration. Although the chitosan-coated liposomes affected the cell viability, this formulation also influenced the cell permeability, which makes it an interesting candidate for systemic drug delivery from the oral cavity.