Polyelectrolyte films based on polysaccharides of different conformations: effects on multilayer structure and mechanical properties

Ultrathin films were prepared with cationic poly(allylamine hydrochloride) (PAH) and two anionic polysaccharides, iota- and lambda-carrageenan, of similar chemical composition but different conformations using the layer-by-layer (LbL) technique. The study of aqueous solutions of carrageenans confirms that iota-carrageenan is at room temperature in helical conformation while lambda-carrageenan is in random coil conformation. Characterization of the multilayers by ellipsometry, circular dichroism, and AFM revealed that iota-carrageenan keeps its helical conformation within the films while lambda-carrageenan chains are in random coil conformation. Investigation of the mechanical properties of the films by performing nanoindentation experiments using force spectroscopy showed clear differences between the two films based on carrageenans of different conformations.     

Physicochemical characterization of carrageenan fromGigartina teedii (Rooth) Lamouroux (Gigartinales,Rhodophyta)

The phycocolloids of female gametophytes ofGigartina teedii (Roth) Lamouroux harvested in Roscoff (Brittany, France) are a hybrid carrageenan resulting from juxtaposition of fragments of kappa-, iota-and nu-carrageenan. They represent 70% of the dry matter of the alga in summer. After alkaline transformation the proportion of iota-carrageenan increased to 76%, demonstrating the presence of nu-carrageenan. Absence of mu-carrageenan, the precursor of kappa-carrageenan, suggests that iota-carrageenan is desulfated enzymically to kappa-carrageenan.     

Solvent effects on the viscoelastic behavior of porcine submaxillary mucin

Rheological methods have been used to investigate the intermolecular interactions of porcine submaxillary mucins (PSM) in solution. PSM is a high molecular weight glycoprotein consisting of a linear, semi-flexible protein backbone to which a large number of oligosaccharides (1–5 saccharide units) are attached as side chains. Concentrated aqueous solutions of PSM containing different amounts of guanidine hydrochloride (GdnHCl) were subjected to both controlled stress and controlled strain rheological analyses. In the absence of GdnHCl, PSM solutions exhibit viscoelastic properties characteristic of a gel: the storage modulus, G′, is much larger than the loss modulus, G″, at all deformation frequencies, and the compliance is 100% recoverable at small stresses, indicative of strong intermolecular interactions. In 3.0 M aqueous GdnHCl, PSM forms a viscoelastic solution, with G″ > G′ at all frequencies and a relatively small recoverable compliance, pointing to disruption of the intermolecular interactions by the chaotropic salt. Intermediate behavior is observed in 1.5 M GdnHCl, characteristic of a marginal gel: G′ ≈ G″ and greater than 50% recoverable compliance. In dilute solution, PSM behaves viscoelastically as a typical polyelectrolyte. However, concentrated solutions are turbid, the turbidity decreasing as GdnHCl is added, indicating that extensive intermolecular association accompanies the gelation process. The results show that although PSM is secreted in nature as a viscous solution, it can form gels that are similar to those of tracheobronchial and gastric mucins, and suggest common features to the gelation mechanism, with the strength of the gel correlated with the length of the oligosaccharide side chains.     

Extreme stability of helices formed by water-soluble poly-N-substituted glycines (polypeptoids) with alpha-chiral side chains.

Poly-N-substituted glycines or "peptoids" are protease-stable peptide mimics. Although the peptoid backbone is achiral and lacks hydrogen-bond donors, substitution with alpha-chiral side chains can drive the formation of stable helices that give rise to intense CD spectra. To systematically study the solution properties and stability of water-soluble peptoid helices with alpha-chiral side chains, we have synthesized and characterized an amphipathic, 36-residue N-substituted glycine oligomer. CD was used to investigate effects of concentration and solvent environment on this helical peptoid. We saw no significant dependence of helical structure on concentration. Intense, "alpha-helix-like" CD spectra were observed for the 36-mer in aqueous, 2,2,2-trifluorethanol (TFE), and methanol solution, proving a relative insensitivity of peptoid helical structure to solvent environment. While CD spectra taken in these different solvents were fundamentally similar in shape, we did observe some interesting differences in the intensities of particular CD bands in the various solvents. For example, the addition of TFE to an aqueous solvent increases the degree of peptoid helicity, as is observed for polypeptide alpha-helices. Moreover, the helical structure of peptoids appears to be virtually unaffected by heat, even in an aqueous buffer containing 8 M urea. The extraordinary resistance of these peptoid helices to denaturation is consistent with a dominant role of steric forces in their structural stabilization. The structured polypeptoids studied here may have potential as robust mimics of helical polypeptides of therapeutic interest.     

Effect of the Change During Storage in Lipid Composition of Rice on its Amylogram

There are many reports on the deterioration during storage of rice flavor, but no work has yet been done to relate the change during storage in rheological properties with the change in lipid composition. The present study revealed that the increase in amounts of free fatty acids during rice storage resulted in the increase in maximum viscosity of amylogram, i.e., deterioration in rheological property of cooked rice. The mechanism is explained by the formation of helical structure of starch molecule with fatty acid.     

Determination of the number of cross-links in a protein gel from its mechanical and swelling properties

The relation between the chemical structure of a protein and the physical properties of a heat-set gel of that protein has been investigated. The physical properties of the gel are determined by means of mechanical experiments in which the viscoelastic properties of the gel are determined in terms of the storage shear modulus, the loss modulus and the stress-strain curve. The storage shear modulus defined the solid (elastic) character of the gel. The chemical structure of the protein and the nature of the solvent determine the nature and number of cross-links in the gel. The cross-links in gels formed by heating concentrated solutions of ovalbumin in 6M urea solutions were found to be disulfide bridges and the mechanical properties of these ovalbumin/urea gels approximated those of an ideal rubber. The latter finding enables one to calculate the number of cross-links per ovalbumin molecule from the value of the storage modulus, using the classical theory of rubber elasticity. This theory, together with the Flory-Huggins lattice model, can also be used to calculate the number of cros-links per ovalbumin molecule from the swelling behavior of ovalbumin/urea gels. The number of cross-links per ovalbumin molecule calculated from these two types of experiments are in mutual agreement and correspond with the number of thiol groups in ovalbumin. We conclude, thereforee, that theories of polymer physics can be used to relate the chemical structure of a protein to the physical properties of its gel.     

Diepoxide-triggered conformational transition of silk fibroin: formation of hydrogels

Silk fibroin hydrogels with tunable properties could be obtained from aqueous fibroin solutions (4.2 w/v %) in a short period of time. This was achieved by the addition of ethylene glycol diglycidyl ether (EGDE) into the reaction solution. Introduction of EGDE cross-links between the fibroin molecules decreases the mobility of the chains, which triggers the conformational transition from random-coil to β-sheet structure and hence fibroin gelation. Dynamic rheological measurements conducted at 50 °C show the formation of strong to weak hydrogels depending on the pH of the reaction solution. Although EGDE attacks the amino groups of fibroin and forms interstrand cross-links, β-sheets acting as physical cross-links dominate the elasticity of the hydrogels. Mechanical response of low-modulus fibroin hydrogels formed above pH 9.7 is highly nonlinear with strong strain hardening behavior (700%) arising from the alignment of the crystallizable amino acid segments.     

Minoxidil exerts different inhibitory effects on gene expression of lysyl hydroxylase 1, 2, and 3: implications for collagen cross-linking and treatment of fibrosis.

Collagen deposits in fibrotic lesions often display elevated levels of hydroxyallysine (pyridinoline) cross-links. The relation between the occurrence of pyridinoline cross-links and the irreversibility of fibrosis suggests that these cross-links contribute to the aberrant accumulation of collagen. Based on its inhibitory effect on lysyl hydroxylase activity minoxidil has been postulated to possess anti-fibrotic properties by limiting the hydroxylysine supply for hydroxyallysine cross-linking. However, to interfere with hydroxyallysine cross-linking specifically lysyl hydroxylation of the collagen telopeptide should be inhibited, a reaction predominantly catalysed by lysyl hydroxylase (LH) 2b. In this study, we demonstrate that minoxidil treatment of cultured fibroblasts reduces LH1>LH2b>LH3 mRNA levels dose-and time-dependently, but has essentially no effect on the total number of pyridinoline cross-links in the collagen matrix. Still the collagen produced in the presence of minoxidil displays some remarkable features: hydroxylation of triple helical lysine residues is reduced to 50% and lysylpyridinoline cross-linking is increased at the expense of hydroxylysylpyridinoline cross-linking. These observations can be explained by our finding that LH1 mRNA levels are the most sensitive to minoxidil treatment, corroborating that LH1 has a preference for triple helical lysine residues as substrate. In addition, the non-proportional increase in cross-links (20-fold) with respect to the decrease in lysyl hydroxylation state of the triple helix (2-fold) even suggests that LH1 preferentially hydroxylates triple helical lysine residues at the cross-link positions. We conclude that minoxidil is unlikely to serve as an anti-fibroticum, but confers features to the collagen matrix, which provide insight into the substrate specificity of LH1.     

The spatial organization of apolipoprotein A-I on the edge of discoidal high density lipoprotein particles: a mass specrometry study

The three-dimensional structure of human apoA-I on nascent, discoidal HDL particles has been debated extensively over the past 25 years. Recent evidence has demonstrated that the alpha-helical domains of apoA-I are arranged in a belt-like orientation with the long axis of the helices perpendicular to the phospholipid acyl chains on the disc edge. However, experimental information on the spatial relationships between apoA-I molecules on the disc is lacking. To address this issue, we have taken advantage of recent advances in mass spectrometry technology combined with cleavable cross-linking chemistry to derive a set of distance constraints suitable for testing apoA-I structural models. We generated highly homogeneous, reconstituted HDL particles containing two molecules of apoA-I. These were treated with a thiol-cleavable cross-linking agent, which covalently joined Lys residues in close proximity within or between molecules of apoA-I in the disc. The cross-linked discs were then exhaustively trypsinized to generate a discrete population of peptides. The resulting peptides were analyzed by liquid chromatography/mass spectrometry before and after cleavage of the cross-links, and resulting peaks were identified based on the theoretical tryptic cleavage of apoA-I. We identified at least 8 intramolecular and 7 intermolecular cross-links in the particle. The distance constraints are used to analyze three current models of apoA-I structure. The results strongly support the presence of the salt-bridge interactions that were predicted to occur in the "double belt" model of apoA-I, but a helical hairpin model containing the same salt-bridge docking interface is also consistent with the data.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing iso-branched fatty acids. 1. Differential scanning calorimetric studies

The thermotropic phase behavior of aqueous dispersions of phosphatidylcholines containing one of a series of methyl iso-branched fatty acyl chains was studied by differential scanning calorimetry. These compounds exhibit a complex phase behavior on heating which includes two endothermic events, a gel/gel transition, involving a molecular packing rearrangement between two gel-state forms, and a gel/liquid-crystalline phase transition, involving the melting of the hydrocarbon chains. The gel to liquid-crystalline transition is a relatively fast, highly cooperative process which exhibits a lower transition temperature and enthalpy than do the chain-melting transitions of saturated straight-chain phosphatidylcholines of similar acyl chain length. In addition, the gel to liquid-crystalline phase transition temperature is relatively insensitive to the composition of the aqueous phase. In contrast, the gel/gel transition is a slow process of lower cooperativity than the gel/liquid-crystalline phase transition and is sensitive to the composition of the bulk aqueous phase. The gel/gel transitions of the methyl iso-branched phosphatidylcholines have very different thermodynamic properties and depend in a different way on hydrocarbon chain length than do either the "subtransitions" or the "pretransitions" observed with linear saturated phosphatidylcholines. The gel/gel and gel/liquid-crystalline transitions are apparently concomitant for the shorter chain iso-branched phosphatidylcholines but diverge on the temperature scale with increasing chain length, with a pronounced odd/even alternation of the characteristic temperatures of the gel/gel transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence and structural characterization of versican-like proteoglycan in human vitreous

Human vitreous gel is a special type of extracellular matrix, in which interpenetrating networks of collagen fibrils and hyaluronan are found. In this study, we report that apart from significant amounts of collagen, hyaluronan and sialylated glycoproteins, it was found that the human vitreous gel also contained low amounts of versican-like proteoglycan. The concentration of versican-like proteoglycan in the whole vitreous is 0.06 mg protein/ml of vitreous gel and represents a small percentage (about 5%) of the total protein content. The versican-like proteoglycan has a molecular mass of 380 kDa, as estimated by gel chromatography. Its core protein is substituted by chondroitin sulphate side chains (average molecular weight 37 kDa), in which 6-sulphated disaccharides predominated. According to the physicochemical data, the number of chondroitin sulphate chains is likely to be 5-7 per molecule. These proteoglycan monomers form large aggregates with endogenous hyaluronan. Versican, which is able to bind lectins via its C-terminal region, may bridge or interconnect various constituents of the extracellular matrix via its terminal domains in order to stabilize large supramolecular complexes at the vitreous, contributing towards the integrity and specific properties of the tissue.     

Swelling pressure induced phase-volume transition in hybrid biopolymer gels caused by unfolding of folded crosslinks: a model

A thermodynamic model is proposed describing swelling changes and swelling transitions of hybrid gels in which domains of folded chains are chemically built in as cross-links. These folded domains can be unfolded to random coils by osmotic forces produced by the synthetic gel matrix. Uncoiling takes place if the osmotic force acting on the cross-links exceeds the critical value. By unfolding, a new interacting surface is exposed to interactions and affects the swelling pressure. The chains of the folded domains may have ionized groups. The model is based on mean-field statistical-thermodynamic treatment of swelling of polyelectrolyte gels with finite extensibility of network chains. This study is related to hybrid hydrogels with built in protein motifs. A continuous change in external variables increasing the degree of swelling of the hydrogel brings about an abrupt increase in volume (transition) of the gel. The position and magnitude of the transition depend on structural parameters of the hybrid gel, such as fraction of the folded domains in the gel, degree of ionization of chains in the domain, presence of additional chemical cross-links, or degree of dilution at gel formation. Two options for reversibility of the changes are considered: (a) unfolding is irreversible and deswelling proceeds along other curve than swelling and (b) swelling is reversible when the osmotic force decrease below the critical value. In the latter case, swelling changes are described by a closed loop with two transitions. Under certain conditions (high dilution at network formation and sufficiently high degree of ionization of chains of the folded domains), a transition appears known as the collapse transition induced by balance of hydrophobic and hydrophillic interactions. This collapse transition induces the folding transition by which the folded domains are reformed.     

A gel network constituted by rigid schizophyllan chains and nonpermanent cross-links

This work reports a gel network formed by rigid schizophyllan (SPG) chains with Borax as a cross-linking agent. The formed cross-links are non-permanent and somewhat dynamic in nature because the cross-linking reaction is governed by a complexation equilibrium. Gelation processes are traced by dynamic viscoelastic measurements to examine the effects of Borax content, SPG concentration, temperature, salt concentration, salt type, and strain. The first-order kinetic model containing three parameters, t(0) (induction time), 1/tau(c) (gelation rate), and (saturated storage modulus), is successfully applied to describe the gelation of the SPG-Borax system. Gelation occurs faster at higher Borax content, higher SPG concentration, higher salt concentration, or lower temperature. Moreover the gelation is cation-type-specific. Storage modulus is a linear function of both Borax content and SPG concentration. The linear relationship between storage modulus and Borax content can be explained by a modified ideal rubber elasticity theory with a front factor alpha to take into account the presence of ineffective cross-links and the effect of SPG chain rigidity. On the other hand, the linear dependence of storage modulus on SPG concentration could be explained on the basis of chain-chain contacting behavior of extended SPG chains. Apparent activation energy and cross-linking enthalpy are calculated to be -74.5 and -32.4 kJ/mol for the present system. Strain sweep measurements manifest that the elasticity behavior of this gel starts to deviate from Gaussian-chain network at a small strain of 10%.     

Rheological properties of cysteine-containing elastin-like polypeptide solutions and hydrogels

The rheological properties of cysteine-containing elastin-like polypeptide (Cys-ELP) solutions and Cys-ELP hydrogels are reported. The Cys-ELP solutions exhibit a surprisingly high apparent viscosity at low shear rate. The high viscosity is attributed to the formation of an interfacial cross-linked "skin" at the sample surface, rather than the bulk of the Cys-ELP solution. At higher shear rate, the interfacial cross-linked film breaks, and its influence on the viscosity of the Cys-ELP solution can be ignored. Cys-ELP hydrogels are formed by mixing Cys-ELP and hydrogen peroxide (H(2)O(2)). At fixed concentration of Cys-ELP, the gelation time can be tuned by the concentration of H(2)O(2). Cys-ELP hydrogels have the typical characteristics of covalent cross-linked networks, as the storage moduli are larger than the loss moduli and are independent of frequency in dynamic oscillatory frequency sweep experiments. The plateau moduli obtained from linear frequency sweep experiments are much lower than those estimated from the number of thiol groups along the Cys-ELP chain, indicating that only a small fraction of thiols form elastically active cross-links. From the small value of the fraction of elastically active cross-links, the Cys-ELP hydrogel is concluded to be an inhomogenous network. Under steady shear, a 2.5 wt % Cys-ELP hydrogel shear thickens at shear rates lower than that necessary for fracture.     

Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastin-like polypeptides

Genetically engineered elastin-like polypeptides consisting of Val-Pro-Gly-X-Gly repeats, where X was chosen to be Lys every 7 or 17 pentapeptides (otherwise X was Val), were synthesized and expressed in E. coli, purified, and chemically cross-linked using tris-succinimidyl aminotriacetate to produce hydrogels. Swelling experiments indicate hydrogel mass decreases by 80-90% gradually over an approximate 50 degrees C temperature range. Gels ranged in stiffness from 0.24 to 3.7 kPa at 7 degrees C and from 1.6 to 15 kPa at 37 degrees C depending on protein concentration, lysine content, and molecular weight. Changes in gel stiffness and loss angle with cross-linking formulation suggest a low-temperature gel structure that is nearly completely elastic, where force is transmitted almost exclusively through fully extended polypeptide chains and chemical cross-links, and a high-temperature gel structure, where ELP chains are contracted and force is transmitted through chemical cross-links as well as frictional contact between polypeptide chains.     

Heat capacity of folding of proteins corrected for disulfide cross-links

The heat capacities (DeltaC(p,f)) for the temperature-induced folding of proteins: barnase, lysozyme T4, papain, trypsin, ribonuclease T1, chymotrypsin, lysozyme and ribonuclease A have been calculated from the change in solvent accessible surface area between the native state and extended polypeptide chain. To visualize the effect of disulfide cross-links on molar heat capacity, loops of varying number of alanine residues and extended alanine chains with terminal cystein are modeled. The difference in DeltaC(p) values between the extended state and the loop conformation of proteins is linearly related to the number of residues in the loop. Corrections to the heat capacity of folding (DeltaC(p,f)) are applied for proteins with cross-links based on this observation. There is good correlation between corrected values of DeltaC(p,f) and experimental values.     

The structure of kappa/iota-hybrid carrageenans II. Coil-helix transition as a function of chain composition

This paper describes the effect of the kappa/iota-ratio on the physical properties of kappa/iota-hybrid carrageenans (synonyms: kappa-2, kappa-2, weak kappa, weak gelling kappa). To this end, a series of kappa/iota-hybrid carrageenans ranging from almost homopolymeric kappa-carrageenan (98 mol-% kappa-units) to almost homopolymeric-carrageenan (99 mol-% iota-units) have been extracted from selected species of marine red algae (Rhodophyta). The kappa/iota-ratio of these kappa/iota-hybrids was determined by NMR spectroscopy. Their rheological properties were determined by small deformation oscillatory rheology. The gel strength (storage modulus, G') of the kappa/iota-hybrids decreases with decreasing kappa-content. On the other hand, the gelation temperature of the kappa-rich kappa/iota-hybrids is independent of their composition. This allows one to control the gel strength independent of the gelation or melting temperature. The conformational order-disorder transition of the kappa/iota-hybrids was studied using optical rotation and high-sensitivity differential scanning calorimetry. High-sensitivity DSC showed that the total transition enthalpy of the kappa/iota-hybrids goes through a minimum at 60 mol-% kappa-units, whereas for the mixture of kappa- and iota-carrageenan, the total transition enthalpy is a linear function of the composition. With respect to the ordering capability, the kappa/iota-hybrid carrageenans seem to behave as random block copolymers with length sequence distributions truncated from the side of the small lengths. Intrinsic thermodynamic properties (e.g., transition temperature and enthalpy) of kappa- and iota-sequences in these copolymers are close to those of their parent homopolymers. The critical sequence length for kappa-sequences is 2-fold of that for iota-sequences.     

Solvent effects on the cooperative order-disorder transition of aqueous solutions of schizophyllan, a triple-helical polysaccharide

A triple helical polysaccharide schizophyllan in aqueous solution exhibited a highly cooperative transition between ordered and disordered states associated with the conformation of its side chains and nearby water molecules. The transition was followed by optical rotation and calorimetry using water containing additives such as NaOH and DMSO as solvents. The ordered state was stabilized or destabilized depending on the kind and amount of the additive employed; in particular, the addition of DMSO had a remarkable stabilizing effect. This effect was analyzed by means of a statistical mechanical theory of linear cooperative transitions, where DMSO was assumed to interact favorably with the ordered side chains. A small amount of NaOH in a solvent mixture stabilized the ordered state and made the transition curve very gradual. No molecular mechanism was elucidated to account for the role of NaOH.     

Intrastrand DNA cross-links as tools for studying DNA replication and repair: two-, three-, and four-carbon tethers between the N(2) positions of adjacent guanines

A general protocol for preparation of oligonucleotides containing intrastrand cross-links between the exocyclic amino groups of adjacent deoxyguanosines has been developed. A series of 2, 3, and 4 methylene cross-links was incorporated site-specifically into an 11-mer (5'-GGCAGGTGGTG-3', cross-linked positions are underlined) via a reaction between oligonucleotide containing 2-fluoro-O(6)-trimethylsilylethyl deoxyinosines and the appropriate diamine (ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane). These cross-linked-oligonucleotides were studied for their ability to bend DNA by the method of Koo and Crothers [Koo, H. S., and Crothers, D. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1763-1767] in which the mobility of ligated oligomers in nondenaturing polyacrylamide gels is evaluated. It was found that all cross-links induced bending (2-carbon cross-link, 30.0 +/- 4.0 deg/turn; 3-carbon cross-link, 11.7 +/- 1.6 deg/turn; 4-carbon cross-link, 7.4 +/- 1.0 deg/turn). Despite the differing extent of helical distortion exhibited by the cross-links, all appeared to be equally blocking to replication by the Escherichia coli polymerases, pol I, pol II, and pol III. In contrast, when incision of the cross-links by the E. coli UvrABC nucleotide incision complex was studied, the extent of incision of the cross-link was found to correlate closely with the degree of bending measured in the gel mobility assay, i.e., the efficiency of incision was 2-carbon > 3-carbon > 4-carbon.     

Chemical cross-linking of myosin. Disposition of the globular heads.

The interaction of a series of bifunctional reagents with skeletal muscle myosin has been studied. In the di-imido ester series dimethylmalonimidate failed to generate any cross-linked species, whereas the adipic and higher analogues gave dimers of myosin heavy chains. Analysis of free amino groups after reaction with these reagents and with the reducible species dimethyldithiobis(propionimidate) showed that no more than two to three cross-links per molecule were introduced. By contrast, the bifunctional reducible acylating agent, dithiobis(succinimidylpropionate), reacted with annihilation of about 10% of the amino groups under mild conditions that precluded the formation of intermolecularly linked species. Digestion of the intramolecularly cross-linked myosin with papain, followed by analysis of the fragments by gel electrophoresis, revealed extensive cross-linking between the globular heads of the myosin molecules. The subfragment 1 dimers regenerated subfragment 1 on reduction, as shown by the electrophoretic mobility and amino acid analysis. The extent of cross-linking, and therefore presumably the average relative orientation or freedom of the two heads, was unaffected by the addition of ADP and calcium ions. The internally cross-linked myosin retains practically its full calcium-activated adenosine triphosphatase activity, but in contrast to native myosin is soluble even at very low ionic strength. Circular dichroism measurements show that the alpha helical conformation is undisturbed in cross-linked myosin, but the sedimentation coefficient is considerably higher than that of the native protein, possibly due to freezing of the heads in a "closed" configuration. The light chaiins are not cross-linked to the heavy chains, except under extreme conditions that leads to intermolecular cross-linking and inactivation. The presence of calcium ions protects dithiobisnitrobenzoate light chains against degradation by papain.