Ruthenium red complexation with ionic polysaccharides in dilute aqueous solutions: chirooptical evidence of stereospecific interaction.

This paper presents optical and chirooptical data on the interaction of the microscopy-staining agent ruthenium red with carboxylated polysaccharides in dilute aqueous solution. The polysaccharides used are both natural (alginate and pectate) and semisynthetic (C6-oxidized cellulose and C6-oxidized amylose). A preliminary discussion of the molecular structure and conformational features which control the interaction is presented.     

Binding site conformation dictates the color of the dye stains-all. A study of the binding of this dye to the eye lens proteins crystallins

The interaction of the cationic carbocyanine dye Stains-all (1-ethyl-2-[3-(1-ethyl-naphthol[1,2-d]thiazolin-2-ylidene)-2- methylpropenyl]naphthol[1,2-d]thiazolium bromide) with the eye lens proteins crystallins has been studied. alpha- and gamma-crystallins do not bind the dye, while beta- and delta-crystallins do, consistent with the fact that the latter two proteins bind the calcium ion. beta-Crystallin resembles parvalbumin in that it induces only the J-band of the bound dye. delta-crystallin, on the other hand, induces only the gamma-band. Analysis of the metachromasia induced in the dye by these and other proteins suggests that Stains-all is responsive to the conformational status of the region to which it binds in a protein. The J-band of the dye is activated when it binds to a globular domain, and the gamma-band is activated when it binds to a helical stretch of the protein.     

Alginate as an auxiliary bacterial membrane: binding of membrane-active peptides by polysaccharides.

The chronicity of Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients is characterized by overproduction of the exopolysaccharide alginate, in which biofilm bacteria are embedded. Alginate apparently contributes to the antibiotic resistance of bacteria in this form by acting as a diffusion barrier to positively charged antimicrobial agents. We have been investigating cationic antimicrobial peptides (CAPs) (prototypic sequence: KKAAAXAAAAAXAAWAAXAAAKKKK-NH(2), where X is any of the 20 commonly occurring amino acids) that were originally designed as transmembrane mimetic peptides. Peptides of this group above a specific hydrophobicity threshold insert spontaneously into membranes and have antibacterial activity at micromolar concentrations. While investigating the molecular basis of biofilm resistance to peptides, we found that the anionic alginate polysaccharide induces conformational changes in the most hydrophobic of these peptides typically associated with insertion of such peptides into membrane environments [Chan et al., J. Biol. Chem. (2004) vol. 279, pp. 38749-38754]. Through a combination of experiments measuring release of the fluorescent dye calcein from phospholipid vesicles, peptide interactions with vesicles in the presence and absence of alginate, and affinity of peptides for alginate as a function of net peptide core hydrophobicity, we show here that alginate offers a microenvironment that provides a protective mechanism for the encased bacteria by both binding and promoting the self-association of the CAPs. The overall results indicate that hydrophilic alginate polymers contain a significant hydrophobic compartment, and behave as an 'auxiliary membrane' for bacteria, thus identifying a unique protective role for biofilm exopolysaccharide matrices.     

Mechanism of spectral changes of a carbocyanine dye with acidic polysaccharides and oligogalacturonides

Changes in the visible spectrum of a cationic carboeyanine dye in the presence of α (1–4) linked oligomers of d-galacturonic acid have been found to be dependent on the number of uronic acid residues in the molecule. Polygalacturonic acid caused a shift in the dye spectrum that was linearly proportional to the polymer concentration. Neither mono- nor digalacturonic acid had an effect on the dye spectrum. Tri- and tetragalacturonic acid caused spectral changes which were nonlinear with respect to oligomer concentration while penta- and hexagalacturonic acid showed concentration-dependent properties similar to polygalacturonic acid.The difference spectra with polygalacturonate and other acidic polysaccharides containing one anionic site per monosaccharide residue showed two absorption maxima in the region of 550 nm and 610 nm. All of the oligomers tested (containing 3 through 6 galacturonic acid residues) yielded only a single maxima for each in the region between 650 and 670 nm. This single maxima phenomenon was also observed with acidic polysaccharides having only one anionic site for every two monosaccharide residues (hyaluronic acid and chondroitin).     

Collective sampling of intact anionic polysaccharide components and application in quantitative determination by LC-MS

Anionic polysaccharides, such as glycosaminoglycans (GAGs) and alginate, readily undergo source-induced fragmentation when analyzed by electrospray mass spectrometry with the use of high source cone voltage. The dissociation chemistry converts all components of a polysaccharide into a small set of structurally characteristic small saccharides. This chemistry enables the collective detection of a polysaccharide through the detection of one or more small saccharides. This ability, combined with the elution of polysaccharides as relatively compact bands using ion-pairing reverse phase liquid chromatography, created a unique opportunity for the development of LC–MS methods suitable for the quantitative analysis of intact anionic polysaccharides. Feasibility of this approach is demonstrated with a mixture of heparin, chondroitin sulfate A, and alginate.     

Radiation synthesis, characterization and dye adsorption of alginate-organophilic montmorillonite nanocomposite

In this paper, the preparation, characterization and dye adsorption properties of nanocomposite (calcium alginate/organophilic montmorillonite) (CA/OMMT) were investigated. A new nanocomposite consisting of alginate and OMMT was prepared by polymerization using γ-rays irradiation as initiator. Physical characteristics of CA/OMMT were studied using X-ray diffraction (XRD), infrared spectrophotometery (IR), thermal gravimetric analysis (TGA), transmission electron microscopy (TEM) and the corresponding selected area electron diffraction (SAED). Two textile dyes, acid green B and direct pink 3B, were used as model anionic dye. Factors affecting dye sorption, such as pH, sorbent concentration and temperature of each dye solution were extensively investigated. It was found from the study that the sorption of dyes by the nanocomposite is pH-dependent and maximum sorption was obtained at pH 2. The thermodynamic data showed that dye adsorption onto alginate was spontaneous, exothermic, and a physisorption reaction. On the basis of the data of the present investigation, one could conclude that the as-prepared adsorbents exhibited excellent affinity for the dye, and can be applied to treat wastewater containing anionic dyes.     

A comparison of the reactivity of alginate and pectate esters with gelatin

Aqueous solutions of highly esterified propylene glycol alginate and gelatin interact rapidly in mildly alkaline conditions to form a gel with a very high melting point. The interaction involves the formation of amide bonds between the ester and uncharged amino groups on the protein.Neither high-methoxyl pectin nor highly esterified propylene glycol pectate formed thermostable gels with gelatin, and the lack of reactivity was not due to differences between pectate and alginate in viscosity, rate of depolymerisation or rate of saponification. Pectate esters will react, however, with low molecular weight diamines in anhydrous conditions.It is suggested that the different reactivity of the uronides in water reflects differences in the geometries of their glycosidic links between monomers, and that in alginate it is the mannuronic residues that are involved in these reactions.     

Induced circular dichroism of the interaction between pinacyanol and algal alginates

The interaction between pinacyanol chloride and sodium alginate or guluronate-rich alginate is found to effect profound changes in the visible absorbance and circular dichroism spectra. Two different types of aggregates are observed depending on the relative dye/alginate concentrations. With a dye/alginate ratio at 1:1, a complex is deduced based on an analysis of Job’s method and conductometric titrations. Another complex forms at 1:10 dye/alginate ratio and only in the presence of alginate or guluronate-rich alginate. The two aggregates are in dynamic equilibrium according to the presence of isosbestic points in the visible spectra. The effects of pH and divalent cations on the spectra are studied. The 1:10 complex is damaged by addition of hydrochloric acid and divalent cations; however, at low concentration of these agents the spectra indicate conversion of the complex into the 1:1 aggregate. Models for the two complexes are proposed taking into account the preference of guluronate binding sites for chelating ions.     

Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding

Tubulointerstitial nephritis is a cardinal renal manifestation of leptospirosis. LipL32, a major lipoprotein and a virulence factor, locates on the outer membrane of the pathogen Leptospira. It evades immune response by recognizing and adhering to extracellular matrix components of the host cell. The crystal structure of Ca²⁺-bound LipL32 was determined at 2.3 Å resolution. LipL32 has a novel polyD sequence of seven aspartates that forms a continuous acidic surface patch for Ca²⁺ binding. A significant conformational change was observed for the Ca²⁺-bound form of LipL32. Calcium binding to LipL32 was determined by isothermal titration calorimetry. The binding of fibronectin to LipL32 was observed by Stains-all CD and enzyme-linked immunosorbent assay experiments. The interaction between LipL32 and fibronectin might be associated with Ca²⁺ binding. Based on the crystal structure of Ca²⁺-bound LipL32 and the Stains-all results, fibronectin probably binds near the polyD region on LipL32. Ca²⁺ binding to LipL32 might be important for Leptospira to interact with the extracellular matrix of the host cell.     

Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations

For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa.     

ALCIAN BLUE: A QUANTITATIVE AQUEOUS ASSAY FOR ALGAL ACID AND SULFATED POLYSACCHARIDES1

Alcian Blue, a cationic copper phthalocyanine dye, complexes with the anionic carboxyl and half-ester sulfate groups of acidic algal polysaccharide in aqueous solution to form an insoluble precipitate. The quantity of dye removed from solution is proportional to the quantity of polyanion in solution, and this principle forms the basis for the quantitative determination of acid and/or sulfated algal polysaccharides. The assay is linear between 0 and 100 μg/ml agar, alginic acid, carrageenan, pectin and Porphyridium aerugineum Geit. polysaccharide. In addition, the technique is used to determine the anion density of acid polysaccharides on a molar or weight equivalency basis.     

The interaction of calmodulin with the carbocyanine dye (Stains-all)

The dye "Stains-all" combines with calmodulin to yield a series of complex species whose absorption and circular dichroism spectra are sensitive to the presence of Ca2+ or Mg2+. At high dye:calmodulin ratios, the dominant complex formed is characterized by a strong absorption band at 600-650 nm, which is associated with a biphasic circular dichroism band. These spectral features are abolished in the presence of Ca2+.     

Binding of recombinant rat liver fatty acid-binding protein to small anionic phospholipid vesicles results in ligand release: a model for interfacial binding and fatty acid targeting

A number of intracellular proteins bind to negatively charged phospholipid membranes, and this interfacial binding results in a conformational change that modulates the activity of the protein. Using a fluorescent fatty acid analogue, 11-[5-(dimethylamino)naphthalenesulfonyl]undecanoic acid (DAUDA), it is possible to demonstrate the release of this ligand from recombinant rat liver FABP in the presence of phospholipid vesicles that contain a significant proportion of anionic phospholipids. The ligand release that is observed with anionic phospholipids is sensitive to the ionic strength of the assay conditions and the anionic charge density of the phospholipid at the interface, indicating that nonspecific electrostatic interactions play an important role in the process. The stoichiometric relationship between anionic phospholipid and liver FABP suggests that the liver FABP coats the surface of the phospholipid vesicle. The most likely explanation for ligand release is that interaction of FABP with an anionic membrane interface induces a rapid conformational change, resulting in a reduced affinity of DAUDA for the protein. The nature of this interaction involves both electrostatic and nonpolar interactions as maximal release of liver FABP from phospholipid vesicles with recovery of ligand binding cannot be achieved with high salt and requires the presence of a nonionic detergent. The precise interfacial mechanism that results in the rapid release of ligand from L-FABP remains to be determined, but studies with two mutants, F3W and F18W, suggest the possible involvement of the amino-terminal region of the protein in the process. The conformational change linked to interfacial binding of this protein could provide a mechanism for fatty acid targeting within the cell.     

Residue 219 impacts on the dynamics of the C-terminal region in glutathione transferase A1-1: implications for stability and catalytic and ligandin functions

The C-terminal region in class alpha glutathione transferases (GSTs) modulates the catalytic and nonsubstrate ligand binding functions of these enzymes. Except for mouse GST A1-1 (mGST A1-1), the structures of class alpha GSTs have a bulky aliphatic side chain topologically equivalent to Ile219 in human GST A1-1 (hGST A1-1). In mGST A1-1, the corresponding residue is an alanine. To investigate the role of Ile219 in determining the conformational dynamics of the C-terminal region in hGST A1-1, the residue was replaced by alanine. The substitution had no effect on the global structure of hGST A1-1 but did reduce the conformational stability of the C-terminal region of the protein. This region could be stabilized by ligands bound at the active site. The catalytic behavior of hGST A1-1 was significantly compromised by the I219A mutation as demonstrated by reduced enzyme activity, increased K(m) for the substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB), and reduced catalytic efficiencies. Inhibition studies also indicated that the binding affinities for product and substrate analogues were dramatically decreased. The affinity of the mutant for GSH was, however, only slightly increased, indicating that the G-site was unaltered by the mutation. The binding affinity and stoichiometry for the anionic dye 8-anilino-1-naphthalene sulfonate (ANS) was also not significantly affected by the I219A mutation. However, the lower DeltaC(p) for ANS binding to the mutant (-0.34 kJ/mol per K compared with -0.84 kJ/mol per K for the wild-type protein) suggests that ANS binding to the mutant results in the burial of less hydrophobic surface area. Fluorescence data also indicates that ANS bound to the mutant is more prone to quenching by water. Overall, the data from this study, together with the structural details of the C-terminal region in mGST A1-1, show that Ile219 is an important structural determinant of the stability and dynamics of the C-terminal region of hGST A1-1.     

Binding and interactions of L-BABP to lipid membranes studied by molecular dynamic simulations

Chicken liver bile acid-binding protein (L-BABP) is a member of the fatty acid-binding proteins super family. The common fold is a β-barrel of ten strands capped with a short helix-loop-helix motif called portal region, which is involved in the uptake and release of non-polar ligands. Using multiple-run molecular dynamics simulations we studied the interactions of L-BABP with lipid membranes of anionic and zwitterionic phospholipids. The simulations were in agreement with our experimental observations regarding the electrostatic nature of the binding and the conformational changes of the protein in the membrane. We observed that L-BABP migrated from the initial position in the aqueous bulk phase to the interface of anionic lipid membranes and established contacts with the head groups of phospholipids through the side of the barrel that is opposite to the portal region. The conformational changes in the protein occurred simultaneously with the binding to the membrane. Remarkably, these conformational changes were observed in the portal region which is opposite to the zone where the protein binds directly to the lipids. The protein was oriented with its macrodipole aligned in the configuration of lowest energy within the electric field of the anionic membrane, which indicates the importance of the electrostatic interactions to determine the preferred orientation of the protein. We also identified this electric field as the driving force for the conformational change. For all the members of the fatty acid-binding protein family, the interactions with lipid membranes is a relevant process closely related to the uptake, release and transfer of the ligand. The observations presented here suggest that the ligand transfer might not necessarily occur through the domain that directly interacts with the lipid membrane. The interactions with the membrane electric field that determine orientation and conformational changes described here can also be relevant for other peripheral proteins.     

Binding and interactions of L-BABP to lipid membranes studied by molecular dynamic simulations

Chicken liver bile acid-binding protein (L-BABP) is a member of the fatty acid-binding proteins super family. The common fold is a beta-barrel of ten strands capped with a short helix-loop-helix motif called portal region, which is involved in the uptake and release of non-polar ligands. Using multiple-run molecular dynamics simulations we studied the interactions of L-BABP with lipid membranes of anionic and zwitterionic phospholipids. The simulations were in agreement with our experimental observations regarding the electrostatic nature of the binding and the conformational changes of the protein in the membrane. We observed that L-BABP migrated from the initial position in the aqueous bulk phase to the interface of anionic lipid membranes and established contacts with the head groups of phospholipids through the side of the barrel that is opposite to the portal region. The conformational changes in the protein occurred simultaneously with the binding to the membrane. Remarkably, these conformational changes were observed in the portal region which is opposite to the zone where the protein binds directly to the lipids. The protein was oriented with its macrodipole aligned in the configuration of lowest energy within the electric field of the anionic membrane, which indicates the importance of the electrostatic interactions to determine the preferred orientation of the protein. We also identified this electric field as the driving force for the conformational change. For all the members of the fatty acid-binding protein family, the interactions with lipid membranes is a relevant process closely related to the uptake, release and transfer of the ligand. The observations presented here suggest that the ligand transfer might not necessarily occur through the domain that directly interacts with the lipid membrane. The interactions with the membrane electric field that determine orientation and conformational changes described here can also be relevant for other peripheral proteins.     

Biotransformation of (−)-limonene using Solanum aviculare and Dioscorea deltoidea immobilized plant cells

The biotransformation course of S-(–)-limonene by Solanum aviculare and Dioscorea deltoidea plant cells was studied using 5 different immobilization methods (entrapment into alginate, carrageenan and pectate gels, binding to polyphenyleneoxide and permeation into polyurethane foam). All techniques, with exception of permeation into pre-formed matrix, affected the product ratio. The main products were cis- and trans-carveol and carvone.     

Conformational and configurational features of acidic polysaccharides and their interactions with calcium ions: a molecular modeling investigation.

Modeling simulations have been performed on the four regular glycuronans: alpha-D-(1--->4) polygalacturonic, alpha-L-(1--->4) polyguluronic, beta-D-(1--->4) polymannuronic, and beta-D-(1--->4) polyglucuronic acids. The goal of this study was to characterize the similarities and differences in conformational and configurational behavior as well as in calcium binding in order to progress in the understanding of the physicochemical properties of the parent polysaccharides of industrial interest, namely pectin, alginate and glucuronan. This required the evaluation of the accessible conformational space for the disaccharide subunits of the four homopolymers, using the flexible residue protocol of the MM3 molecular mechanics procedure. The results were used to access the configurational statistics of representative polysaccharide chains, as well as for the determination of the regular polysaccharide helices and their conformational transitions. The surfaces of all regular helices likely to occur for each polyuronide were explored for cation binding using the GRID procedure. Both alpha-D-(1--->4) polygalacturonate and alpha-L-(1--->4) polyguluronate chains exhibit a high specificity for calcium binding, and have well-defined chelation sites. In contrast, beta-D-(1--->4) polymannuronate and beta-D-(1--->4) polyglucuronate chains do not display any stereospecificity for calcium binding. The results gathered from molecular modeling lead to a clear understanding of the different structural features that are displayed by the four ionic polymers.     

The interaction of Ca2+-binding proteins with the carbocyanine dye stains-all

The interaction of the carbocyanine dye Stains-all with the Ca²⁺-binding proteins calmodulin, troponin C, and parvalbumin has been monitored by means of absorption spectra and CD. In the absence of Ca²⁺, complexes with Stains-all of all three proteins exhibit at high dye: protein mole ratios an intense J absorption band at 600–650 nm, which is associated with a characteristic CD spectrum. In the cases of calmodulin and troponin C, the J-band is progressively lost as the dye: protein ratio decreases and is replaced by bands of the γ and β types at 450–550 nm, which likewise give rise to characteristic CD spectra. For parvalbumin, only the J-band is observed; its intensity is undiminished at the lowest dye: protein ratios examined. In the presence of excess Ca²⁺ the J-band is lost for all three proteins. For calmodulin and troponin C it is replaced by σ- and β-bands; in the case of parvalbumin the bound dye is released. A tentative model has been proposed to account for these observations.     

Absorption and circular dichroism spectra of methylene blue bound to alginate

The addition of methylene blue to certain samples of Na alginate produces a complex succession of spectrally distinguishable aggregated (metachromatic) dye species. Three of these species are active in CD; they are interpreted as aggregates of dye, but probably dimers, bound in orientations characteristic of the constituent copolymer blocks of alginate to which they are tentatively assigned. The aggregates compete with divalent metal ions and hydrogen ion for the binding sites of the polymer. Other samples of alginate give a modified succession of aggregated spectral species, which are almost devoid of CD activity. Mild treatment with acid, insufficient for hydrolysis, converts forms of alginate with CD activity into modified forms without it, the absorption spectra of which resemble those of samples originally devoid of activity. It is implied, subject to confirmation, that the chiral properties of the binding sites of the native polysaccharide are diminished or lost by acid treatment during commercial preparation.