Shear creep of gelatin gels from mammalian and piscine collagens.

This paper describes new measurements on the creep rheological behaviour of gelatin gels from both traditional mammalian and piscine sources. Measurements on a series of concentrations of gels were obtained using a high-precision controlled stress rheometer. Results for the concentration dependence of compliance are close to those expected from dynamic oscillatory measurements of gel modulus, assuming ideal elasticity. The concentration dependence of viscosity approximates power law behaviour, with eta~C( approximately 2-3), lower than the exponent expected for semi-dilute solutions. The apparent contradiction implied by this is discussed and a novel gel viscosity versus concentration state diagram presented.

Pressure cell assisted solution characterization of polysaccharides. 1. Guar gum.

To reduce time-dependent aggregation phenomena and achieve true "molecular" solution, the "pressure cell" solubilization method of Vorwerg and co-workers was applied to solutions of guar galactomannans (three samples of different molecular weights), using various heating, time, and pressure profiles. Physicochemical characterization of the guar samples before and after pressure cell treatment included measurements of intrinsic viscosity [eta] by capillary viscometry and M(w) and radius of gyration from size exclusion chromatography coupled to multiangle laser light scattering (SEC/MALLS). Heating the guar solutions (100-160 degrees C) without pressurization produced chain degradation with [eta] and M(w) values being reduced significantly, whereas this effect was reduced substantially for samples subject to initial pressurization ( approximately 5-10 bar). The constants in the Mark-Houwink-Sakurada equation, relating [eta] and M(w) were established and the characteristic ratio C(infinity) and chain persistence length L(p) were calculated using both the Burchard-Stockmayer-Fixman (BSF) method for flexible and semiflexible chains and the Hearst method more appropriate for stiffened chains. Definitive conclusions can now be drawn on the flexibility of the guar chain backbone, with L(p) approximately 4 nm from the BSF plot, in good agreement with previously published work using such geometric methods. This contrasts with the higher values obtained from extrapolation of data for polyelectrolytes with a similar backbone geometry, such as sodium carboxymethyl cellulose, to "infinite" ionic strength.

Heat-induced gelation of globular proteins: part 3. Molecular studies on low pH beta-lactoglobulin gels.

Heat-set gels and aggregates from beta-lactoglobulin (beta-Lg), one of the major globular proteins from milk, have been studied on a molecular distance scale using negative-staining transmission electron microscopy (TEM), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared spectroscopy (FTIR). The microscopy showed long linear aggregates forming in solutions at pH 2 (and sometimes 2.5) after prolonged heating. While there appeared to be no differences in aggregates formed under these conditions in H(2)O as compared with D(2)O, at all other pH and pD values, and in the presence of added salt, much shorter linear aggregates were formed. These became slightly more extended the further the pH was removed from pI. Wide-angle X-ray diffraction (WAXD) showed a diffuse beta-sheet halo at 2θ=19 degrees in patterns for both dried native and aggregated protein (irrespective of pH) with only a small change (sharpening) of this feature on heat treatment. Solution FTIR spectra, measured at pD=2, 2.5, 3, and 7, during heating, indicated shoulder development at 1612 cm(-1) in the carbonyl-stretching Amide I region diagnostic of a modest increase in intermolecular beta-sheet. In terms of the shoulder size, no distinctions could be made between acid and neutral aggregate structures. At all pHs, beta-lactoglobulin showed only limited secondary and tertiary structural changes in aggregation, in contrast to previous studies of insulin aggregation, where highly ordered crystalline fibrils were indicated. The current work has implications both in structural studies of food biopolymers and in ongoing studies of pathological protein self-assembly in disease states, such as spongiform encephalopathies.

Reduction of plasma clot stability by a novel factor XIIIa inhibitor from the Giant Amazon Leech, Haementeria ghilianii.

The blood-sucking leech, Haementeria ghilianii, has evolved a number of agents that attenuate haemostasis. Recently we have isolated a potent inhibitor of factor XIIIa, tridegin, in the salivary glands which is almost certainly involved in feeding. Addition of purified natural tridegin to plasma, prior to clotting with thrombin, results in clots that deform more readily as adjudged by the greatly reduced development of the storage modulus on application of a shear force. The increase in the storage modulus in developing plasma clots is a slow process and continues for many hours. The effect of tridegin is particularly great when the clots are permitted to age in this way, demonstrating the role of factor XIIIa in the process. The IC50 for this inhibition is 138 ng/ml. Clots formed in the presence of tridegin are also lysed more rapidly in vitro by the leech's own fibrinolytic enzyme, hementin (time for 50% lysis, 16.0 +/- 0.8 h versus 22.3 +/- 2.0 h, P < 0.05). The synergy with which these agents act together may provide lessons for therapy of thrombosis in man.

A new polysaccharide from a traditional Nigerian plant food: Detarium senegalense Gmelin.

The seed flour of an African leguminous plant, Detarium senegalense Gmelin, is used traditionally in Nigeria as a thickening agent in foods. Recent studies have shown that the detarium seed contains a large amount of water-soluble, non-starch polysaccharide (s-NSP), which suggests it has important nutritional properties. The aims of the present study were to characterise the structure and solution properties of purified s-NSP. The main monosaccharide residues of the extracted s-NSP were glucose, xylose, and galactose in the ratio of 1.39:1.00:0.52, suggesting structural similarity to the xyloglucan group of cell wall storage polysaccharides. This was confirmed by comparing the oligosaccharides released on endo-(1 --> 4)-beta-D-glucanase digestion with those obtained from tamarind seed xyloglucan. The intrinsic viscosity [eta] of a sample of the detarium polysaccharide was found to be 8.9 dl/g, indicating that the sample was of high molecular weight, a result confirmed by light scattering. Histochemical examination of detarium seed using bright field and epifluorescence microscopy showed the presence of xyloglucan in highly thickened cell walls, which were particularly prominent at the cell junctions.

Changes in clot deformability--a possible explanation for the epidemiological association between plasma fibrinogen concentration and myocardial infarction.

This study examined the rheological properties of fibrin gels formed by adding thrombin to plasma samples from 99 subjects with fibrinogen concentrations ranging from 1.45 to 4.14 g/l. A highly significant (r = 0.757; P < 0.001) inverse correlation was observed between plasma fibrinogen concentration and the extent of clot deformability as estimated from the final value of the storage modulus (G') of the fibrin gel when obtained by rheological analysis. A similarly significant correlation (r = 0.844; P < 0.001) was obtained using samples from 47 subjects in which fibrin cross-linking was blocked by addition of 0.1 mM iodoacetamide to inactivate factor XIIIa. The characteristics of the relationship between G' and fibrinogen concentration in the plasma samples was comparable with that observed when the fibrin gel was formed by adding thrombin to purified fibrinogen. These results suggest that the increased risk of myocardial infarction associated with an elevated plasma fibrinogen concentration may, in part, be explained on the basis of a decreased deformability of the fibrin clot formed.

Polysaccharide strong and weak gels.

Small deformation oscillatory shear measurements have enabled a distinction to be made between so-called "strong" and "weak" gels, in particular those formed from biologically significant polysaccharides. At small enough strains, both systems give essentially the same mechanical spectrum, with G' > G", and with both moduli largely independent of frequency. However, the deformation dependence of the two classes of materials is very different. Strong gels are essentially strain independent (linearly viscoelastic) for strains of greater than about 0.25, whereas weak gels show such a response only for strains of less than about 0.05. At large deformations strong gels will rupture and fail, and will never "heal" without melting and resetting. Conversely, weak gels will recover and can flow without fracture, giving a power law response, with an exponent approaching -1, so-called "yield stress" behavior. The rheological properties of a strong gel, agarose, derived from the Rhodophyceae (marine algae) and a weak gel xanthan, an exocellular slime exuded by bacteria of the genus Xanthomonas, are measured in vitro, and related to in vivo requirements.

Exopolysaccharides from Rhizobium meliloti YE-2 grown under different osmolarity conditions: viscoelastic properties.

The exopolysaccharides fro Rhizobium meliloti YE-2 extracted 10 days after the inoculum from culture broths and having different osmolarity values (0, 0.2, 0.4, and 0.6 M) have been investigated by means of oscillatory and steady-shear measurements on 1% solutions in 0.1M NaCl. The micro-organism produces a mixture of a galactoglucan and a succinoglycan. At low osmolarity (0 and 0.2M), the main fraction consists of galactoglucan and the viscoelastic properties of the mixture are typical of an entanglement system. At 0.4 and 0.6M, the proportion of the succinoglycan increases and the viscoelastic properties change abruptly to those typical of a "weak gel" system. Different conformations are adopted by the two exopolysaccharides in solution.

The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.

Creep measurements on gelatin gels.

The present work describes creep measurements on a series of concentrations of gelatin gels well above the critical gel concentration C0, using a high precision constant stress rheometer. Results for the concentration dependence of compliance are close to those expected both from theory and from dynamic oscillatory measurements of gel modulus. The concentration dependence of viscosity follows an approximate power law behaviour, with eta proportional C1.1. This exponent is consistent with relaxation in the sol fraction, and in regions of dangling chain attached to the gel. At concentrations closer to C0 we predict that a higher power law regime will prevail.

Influence of acetyl and pyruvate substituents on the solution properties of xanthan polysaccharide.

Xanthan, an exocellular polysaccharide produced by the plant pathogenic bacterium Xanthomonas campestris has been the subject of considerable interest in recent years because of its unusual rheological properties in solution ('weak gel') and consequent range of applications. The polymer consists of a cellulosic backbone with trisaccharide side chains linked to alternate backbone residues; acetyl and pyruvate substituents are carried in variable amounts on these side chains. In this study a series of xanthans differing in the percentage of substituent groups and in molecular weight range have been prepared by culturing a variety of different strains of X. campestris. All of the xanthans have been characterized by a range of physicochemical techniques. In particular, the intrinsic viscosities at low shear rates, and at a range of ionic strengths, have been determined and the geometric persistence lengths evaluated by the Smidsrød-Haug method. Intensity light scattering measurements have been made using the procedure of Coviello and co-workers to promote molecular dispersion. Despite significant differences in the acetyl and pyruvate contents, the molecular weight vs mean square radius behaviour of our samples did not differ substantially from each other or from those reported for other xanthan samples in the literature. The persistence length, determined by the method of Schmidt et al. (120 +/- 8 nm) was also, within experimental error, the same for all the samples measured. These values differed considerably from those calculated from the ionic strength dependence of intrinsic viscosity (the Smidsrød-Haug method) was reported by Tinland and Rinaudo and calculated for our samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Interpretation of the renaturation kinetics of gelatin solutions.

Renaturation kinetics of dilute gelatin solutions are studied by optical rotation and u.v. absorption. Comparing initial slopes of renaturation curves for concentrations below 10 mg.ml-1, the rate limiting process appears as the sum of a constant rate intramolecular nucleation and a bimolecular nucleation. To fit the whole renaturation curves, a kinetic model using Monte Carlo calculations was tested, consisting of random nucleation, rapid total propagation and slow reversion depending on the size of helical segment. Mismatch on the final renaturation extent indicates that propagation is more limited than proposed.

Submaxillary mucins. Intermolecular interactions and gel-forming potential of concentrated solutions.

The intermolecular interactions in concentrated solutions of pig submaxillary mucin (PSM) and sheep submaxillary mucin (SSM) were studied by mechanical spectroscopy. PSM and SSM were purified from detectable protein and nucleic acid by equilibrium centrifugation in a CsCl density gradient. PSM and SSM isolated in the presence of proteinase inhibitors showed distinct differences from preparations isolated in the presence of 0.2 M-NaCl alone, the latter having a carbohydrate and amino acid analysis similar to other preparations isolated by precipitation or ion-exchange techniques. Gel-filtration studies showed that preparations isolated in the presence of 0.2 M-NaCl alone were dissociated into smaller-sized glycoprotein units by 3.5 M-CsCl or 2.0 M-NaCl (SSM), pH 2.0 (PSM) or heating at 100 degrees C for 10 min (PSM and SSM). Preparations isolated in the presence of proteinase inhibitors were not dissociated by these treatments. Proteolysis fragmented all submaxillary mucin preparations into small glycopeptides of Mr 13,700 for PSM and of Mr 14,000 and 15,000 for SSM. PSM preparations when concentrated formed viscoelastic gels, as determined by mechanical spectroscopy. In contrast, SSM showed characteristics of a weak viscoelastic liquid under comparable conditions (coil overlap). PSM glycoprotein isolated in proteinase inhibitors formed weak viscoelastic gels at concentrations between 5 and 15 mg/ml. Preparations of PSM glycoprotein isolated in the presence of 0.2 M-NaCl (concentration 10-97 mg/ml) had the same overall mechanical gel structure as those preparations extracted in the presence of proteinase inhibitors. This gel structure was seen to collapse following proteolysis of both preparations or after acid treatment of the glycoprotein isolated in the presence of 0.2 M-NaCl, consistent with the breakdown in size of the polymeric glycoprotein. Treatment of PSM gel with 0.2 M-2-mercaptoethanol caused a surprising increase in gel strength, which was further markedly increased on removal of the reducing agent by dialysis. An association of reduced subunits of PSM was observed by gel filtration after removal of 0.2 M-2-mercaptoethanol. These results point to intermolecular disulphide exchange occurring on reduction of these PSM glycoprotein preparations. These results demonstrate that gel formation in PSM glycoprotein is similar to that for other gastrointestinal mucus glycoproteins from stomach to colon. Gel formation in PSM, as in other mucins, depends on polymerization of subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Mucus glycoprotein gels. Role of glycoprotein polymeric structure and carbohydrate side-chains in gel-formation.

The structure of mucus glycoprotein gels from the pig gastrointestinal tract was investigated by mechanical spectroscopy. Gastric, duodenal, and colonic mucus had the same mechanical profile, characteristic of a viscoelastic gel. The gel structure collapsed on destruction of the polymeric structure of the component glycoprotein by reduction with 0.2M mercaptoethanol or after proteolysis with papain. The progressive weakening of mechanical properties and the decrease in polymeric glycoprotein content were measured as functions of time of reduction. A linear correlation was obtained between the gel quality [defined by tan delta, the ratio of the loss modulus (G'') to the storage modulus (G')] and the proportion of polymeric to subunit glycoprotein in the mucus. Purified mucus glycoprotein, at the same concentration as that in native mucus, resulted in a gel with mechanical properties no different from those of the respective native secretion, demonstrating that the glycoprotein alone could reproduce the gel-forming properties of mucus. After proteolytic digestion, all native secretions and reconstituted mucus showed an absence of Newtonian behaviour in the frequency dependence of dynamic viscosity at low frequencies. This provided evidence that the noncovalent interactions, characteristic of the native gel matrix, were still present after proteolytic digestion when the nonglycosylated protein core accessible to proteinases had been removed. These results were interpreted to show (a) a common mechanism for gel-formation in gastric, duodenal, and colonic mucus; (b) that the polymeric structure of mucus glycoproteins confers the three-dimensional structure necessary for formation of the gel network; and (c) that noncovalent interactions which arise between the glycoprotein molecules by relatively stable interdigitation of the carbohydrate side-chains are involved in formation of the gel network.

Mechanical characterization and properties of gastrointestinal mucus gel.

Mechanical spectroscopy has been used to study the structure of mucus gel taken from the surface of the pig gastrointestinal tract. Mucus from stomach, duodenum and colon was insoluble and its mechanical properties, characteristic of a weak viscoelastic gel, were unchanged in saline, acid (pH 2) and denaturants. Small intestinal mucus gel which was of poorer quality, was disrupted following exposure to acid and denaturants. Concentration of purified glycoprotein produced gels that had mechanical spectra with the same profiles as the respective native secretion except for reconstituted small intestinal mucus which was of better quality and similar to the other native and reconstituted gels. Reduction of S-S linkages or proteolysis of all mucus gels caused a collapse of structure to give profiles typical of a viscous solution. This collapse of gel structure was shown to result from a breakdown of the covalent polymeric structure of the component glycoproteins. A linear correlation for mucus gels was observed between gel quality (as defined by tan delta) and the ratio of polymeric glycoprotein to its degraded lower molecular weight subunit. Human gastric mucus from a histologically normal stomach also had the characteristics of a weak viscoelastic gel, although that from patients with peptic ulcer disease has a significantly reduced content of polymeric glycoprotein.

Properties of gastric and duodenal mucus: effect of proteolysis, disulfide reduction, bile, acid, ethanol, and hypertonicity on mucus gel structure.

Small deformation oscillatory rheologic measurements have been used to investigate the structure of human and pig gastric mucus and pig duodenal mucus. All three secretions had viscoelastic properties characteristic of water-insoluble, viscoelastic gels. Mucus will flow and anneal if damaged, due to the making and breaking of its elastic structure, the measured lifetime of which was 10-120 min. Mucus reconstituted by concentration of the purified glycoprotein (pig gastric and duodenal mucus) had the same viscoelastic properties as the fresh mucus, giving evidence that the glycoprotein alone will reproduce the rheologic characteristics of the mucus. The structure of fresh mucus gel was unaffected by prolonged exposure to the following mucosal damaging agents: undiluted pig bile, 20 mM sodium taurocholate or 20 mM sodium glycocholate (all at pH 2, 6, and 8), HCl at pH 1, 2 M NaCl, and ethanol less than 40% (vol/vol). Higher concentrations of ethanol greater than 40% (vol/vol), caused dehydration and denaturation of mucus. Proteolysis by pepsin and other enzymes resulted in solubilization of the mucus gel with a complete change in the properties from an "elastic" gel to those of a "viscous" liquid. A similar collapse of mucus gel structure was observed after reduction of disulfide bonds in 0.2 M mercaptoethanol, but only after incubation for at least 50 min. This study demonstrates the stability of mucus to several mucosal damaging agents. It is proposed in vivo that although adherent gastroduodenal mucus allows penetration of these agents to the underlying mucosa, it can remain in situ and continue to protect against acid (with HCO3-) and pepsin, thus minimizing mucosal damage and maximizing repair.