Extraction of cartilage protein-polysaccharides with inorganic salt solutions.
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ABSTRACT:
SUBMITTER: Mason RM
PROVIDER: S-EPMC1177500 | biostudies-other | 1973 Mar
REPOSITORIES: biostudies-other
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Project description:1. Protein-polysaccharides of chondroitin sulphate were extracted from fresh laryngeal cartilage at pH6.8 by two procedures. Procedure I consisted of brief low-speed homogenization in 0.15m (iso-osmotic) sodium acetate and procedure II consisted of longer homogenization followed by prolonged extraction in 10% calcium chloride solution. 2. The protein-polysaccharides in both extracts were isolated and purified by precipitation with 9-aminoacridine hydrochloride. They were free from serum proteins, collagen and nucleic acids and also of degradative enzymes. The absence of such enzymes was shown by viscosity measurements on solutions of protein-polysaccharides incubated for up to 24hr. at pH4 and 6.8. 3. Mannose, glucose or fucose were not detected by paper chromatography and only traces of sialic acid were present. 4. The yield with procedure II was twice that with procedure I and the products differed in their protein and glucosamine contents. 5. Hyaluronic acid was unlikely to have been precipitated at an acid pH, so the glucosamine was attributed to keratan sulphate, as serum proteins were absent. There was no free keratan sulphate in the preparation. 6. Both preparations were heterogeneous in the ultracentrifuge, showing at least three components.
| S-EPMC1184779 | biostudies-other
Project description:Protein aggregation is broadly important in diseases and in formulations of biological drugs. Here, we develop a theoretical model for reversible protein-protein aggregation in salt solutions. We treat proteins as hard spheres having square-well-energy binding sites, using Wertheim's thermodynamic perturbation theory. The necessary condition required for such modeling to be realistic is that proteins in solution during the experiment remain in their compact form. Within this limitation our model gives accurate liquid-liquid coexistence curves for lysozyme and ? IIIa-crystallin solutions in respective buffers. It provides good fits to the cloud-point curves of lysozyme in buffer-salt mixtures as a function of the type and concentration of salt. It than predicts full coexistence curves, osmotic compressibilities, and second virial coefficients under such conditions. This treatment may also be relevant to protein crystallization.
| S-EPMC4450416 | biostudies-literature
Project description:1. Protein-polysaccharides of chondroitin 4-sulphate were extracted with neutral calcium chloride from pig laryngeal cartilage that was not completely homogenized. The protein-polysaccharides were purified by precipitation with 9-aminoacridine. On zone electrophoresis in compressed glass fibre at pH7.2 it was separated into two fractions, although two distinct zones were not obtained. These fractions, which had already been shown to differ in their antigenic determinants, also differed considerably in amino acid composition, total protein, hexose and glucosamine contents. 2. The fraction of higher mobility contained approx. 2% of protein and only traces of glucosamine. Serine and glycine accounted for over half the total amino acid residues, but aromatic, basic and sulphur-containing amino acids were not detected. The weight-average molecular weight, determined by sedimentation, was 230000. 3. Assuming that there was the same sequence of neutral sugars at the linkage points as in PP-L fraction (protein-polysaccharide light fraction), the approximate molar ratio of hexose to serine suggested that most of the serine residues were linked to chondroitin sulphate chains. Support for this was derived from the agreement between the weight-average molecular weight of the chondroitin sulphate-peptide after proteolysis, and the chain weight calculated from its serine content. The chain weight based on the serine content of the fraction of higher electrophoretic mobility was approximately similar. 4. In contrast, the fraction of lower electrophoretic mobility resembled PP-L fraction in its amino acid composition, protein and glucosamine contents. The presence of glucosamine, together with the higher hexose content, suggested that this fraction contained some keratan sulphate. 5. The relatively low molecular weight of the fraction of higher mobility enabled it to be extracted without complete disintegration of the cartilage. The unlikelihood of its being produced by autolytic enzymes is discussed.
| S-EPMC1270417 | biostudies-other
Project description:Protein-polysaccharides of knee-joint cartilage of 9-month-old pigs were extracted sequentially with neutral iso-osmotic sodium acetate after five repeated homogenizations. One-third of the uronic acid originally present in the tissue was brought into solution, about half being in the first extract. The protein-polysaccharides, which were purified by precipitation with 9-aminoacridine, were heterogeneous in size on gel chromatography. The smallest (retarded by 6% agarose) were the most easily extracted since they were most prevalent in the initial extracts and absent from later ones, whereas the proportion of larger molecules increased progressively in successive extracts. Nevertheless a small proportion of the largest molecules (excluded from Sepharose 2B) was present even in the first extract. None of the protein-polysaccharide preparations contained hydroxyproline, and the analyses of their constituent sugars were the same, although there was a progressive increase in the protein content and in the glucosamine/galactosamine molar ratio of successive extracts. In each preparation this molar ratio was invariably greater in larger than in smaller molecules separated by gel filtration. From galactosamine/pentose molar ratios it appeared that the chondroitin sulphate chains were on average about 29 disaccharide units in length in the protein-polysaccharides of each extract, although gel-chromatography and cetylpyridinium chloride elution profiles showed that a somewhat higher proportion of shorter chondroitin sulphate chains occurred in the larger protein-polysaccharides. In the last extract, where the largest molecules predominated, about half could be reversibly dissociated by urea, whereas this had no effect on the protein-polysaccharides of earlier extracts even though these contained some large molecules.
| S-EPMC1176565 | biostudies-other
Project description:This study assessed the potential of pre-hydration treatment with aqueous solutions (electrolysed [cathodic water; CW] and non-electrolysed) prepared from four different inorganic ion combinations: 1 mM CaCl2, 1 µm CaCl2 and 1 mM MgCl2 (CaMg, hereafter), 1 mM MgCl2 and 1 mM NaCl to invigorate controlled deteriorated (CDd) Brassicaoleracea (cabbage) and Lactucasativa (lettuce) seeds by assessing germination, vigour and biochemical markers (electrolyte leakage, lipid peroxidation products, protein carbonylation, and defence and germination associated enzymes) of oxidative stress. Additionally, the possible effects of pH of electrolysed CaMg and NaCl solutions were assessed. The inorganic salt solutions were applied to fresh seeds and seeds deteriorated to 75% viability (P75), 50% viability (P50) and 25% viability (P25); deionised water served as control. The pre-hydration treatment did not enhance normal seedling production in cabbage. However, Ca-containing and CW hydration treatments (CaCl2 CW, CaMg and CaMg CW [6.5], MgCl2 CW, NaCl CW and NaCl CW [6.5]) promoted normal seedling production of CDd lettuce seeds, while seedling vigour was enhanced by CaMg, CaMg CW (6.5), NaCl CW and NaCl CW (6.5) in CDd cabbage seeds, and CaCl2, CaCl2 CW, CaMg, CaMg CW (6.5), MgCl2 CW, NaCl CW and NaCl CW (6.5) in CDd lettuce seeds. The supplementation of Ca, a component of the ionised solutes, and/or the reducing potential of CW contributed to increased normal seedling production in lettuce seeds irrespective of the pH of treatment solutions or degree of deterioration. Overall, the pre-hydration treatments enhanced endogenous antioxidants leading to reduced levels of electrolyte leakage, lipid peroxidation, protein carbonylation, and enhanced germination enzyme activities in lettuce seeds. The study concluded that pre-hydration with selected inorganic salt solutions can invigorate debilitated lettuce seeds.
| S-EPMC7569860 | biostudies-literature
Project description:In this study, we examine the temperature dependence of free energetics of nanotube association using graphical processing unit-enabled all-atom molecular dynamics simulations (FEN ZI) with two (10,10) single-walled carbon nanotubes in 3 m NaI aqueous salt solution. Results suggest that the free energy, enthalpy and entropy changes for the association process are all reduced at the high temperature, in agreement with previous investigations using other hydrophobes. Via the decomposition of free energy into individual components, we found that solvent contribution (including water, anion, and cation contributions) is correlated with the spatial distribution of the corresponding species and is influenced distinctly by the temperature. We studied the spatial distribution and the structure of the solvent in different regions: intertube, intratube and the bulk solvent. By calculating the fluctuation of coarse-grained tube-solvent surfaces, we found that tube-water interfacial fluctuation exhibits the strongest temperature dependence. By taking ions to be a solvent-like medium in the absence of water, tube-anion interfacial fluctuation shows similar but weaker dependence on temperature, while tube-cation interfacial fluctuation shows no dependence in general. These characteristics are discussed via the malleability of their corresponding solvation shells relative to the nanotube surface. Hydrogen bonding profiles and tetrahedrality of water arrangement are also computed to compare the structure of solvent in the solvent bulk and intertube region. The hydrophobic confinement induces a relatively lower concentration environment in the intertube region, therefore causing different intertube solvent structures which depend on the tube separation. This study is relevant in the continuing discourse on hydrophobic interactions (as they impact generally a broad class of phenomena in biology, biochemistry, and materials science and soft condensed matter research), and interpretations of hydrophobicity in terms of alternative but parallel signatures such as interfacial fluctuations, dewetting transitions, and enhanced fluctuation probabilities at interfaces.
| S-EPMC4445429 | biostudies-literature
Project description:1. Protein-polysaccharides from pig laryngeal cartilage extracted by two procedures described in the preceding paper (Tsiganos & Muir, 1969) were shown to consist of macromolecules of various sizes as assessed by gel filtration in 4% and 6% agarose. 2. A larger proportion of the smaller molecules was present in the preparation obtained by brief extraction in iso-osmotic sodium acetate (procedure I) than in that obtained by more prolonged extraction in 10% (w/v) calcium chloride (procedure II). 3. Two fractions were separated by gel filtration in 6% agarose and by electrophoresis in compressed glass fibre. These fractions differed in chemical composition and in antigenic determinants. The gel-retarded fraction R and that of higher electrophoretic mobility possessed the same single antigen, whereas the gel-excluded fraction E and the slower electrophoretic fraction contained all the antigens of the starting material including that of fraction R. 4. Five N-terminal amino acid residues were identified in preparation I and fraction E, only two of which were present in fraction R. 5. The relative proportions of gel-excluded and gel-retarded fractions did not change when solutions of high ionic strength, urea or guanidine hydrochloride were used for elution. 6. The differences in chemical and amino acid composition between fractions R and E showed that the latter was not a simple aggregate of the former. Fraction E contained more basic and aromatic amino acids, and some methionine and cystine; the last two were absent from fraction R. Hydroxyproline was not detected in either fraction. 7. The number of glycosidic linkages in both fractions was estimated by alkaline beta-elimination. Appreciable amounts of threonine as well as serine were destroyed in both fractions. An average chain length for chondroitin sulphate was calculated from the galactosamine content of both fractions and the amounts of hydroxy amino acid destroyed. Average chain lengths were also calculated from the xylose and galactosamine content of each fraction. Each independent method gave a value of approximately 28 disaccharide units for the chain length in both fractions and hence their difference in size could not be explained by differences in the length of carbohydrate chains. 8. All fractions contained glucosamine, which was attributed to keratan sulphate. Content of both protein and keratan sulphate increased with the size of the macromolecules. 9. It is suggested, from these results, that chondroitin sulphate-protein complexes normally exist as a heterogeneous population of macromolecules in cartilage, and that keratan sulphate is involved in the formation of larger molecules.
| S-EPMC1184780 | biostudies-other
Project description:Protein-polysaccharides of femoral articular cartilage from pigs of ages 9 months and 5 weeks were compared after extraction at pH6.8 with iso-osmotic sodium acetate followed by 0.63m-calcium acetate. The cartilage from the younger animals had a higher moisture content and contained considerably larger amounts of protein-polysaccharide, but less than half as much collagen/g. dry weight, than cartilage from the older pigs. There was notably less keratan sulphate in the fractions from the less mature animals. After gel filtration on 6% agarose, elution profiles of the calcium acetate extracts were similar to those of the sodium acetate extracts of the same tissue. Chemical analyses, however, showed that in both age-groups the extraction procedure had achieved a sequential solubilization of protein-polysaccharides in that the initial extracts contained a higher proportion of keratan sulphate than those that were extracted subsequently. Both extracts from the older animals contained up to 25% of a relatively small protein-polysaccharide that was retarded on 6% agarose and that had a lower protein content and less keratan sulphate than the larger protein-polysaccharides. In contrast, in extracts from the less mature cartilage only about 5% of the protein-polysaccharides were small enough to be retarded by 6% agarose, suggesting that the small components may not be precursors of the larger. The average length of chondroitin sulphate chains, as calculated from the analytical data, was the same in the smaller protein-polysaccharides as in the larger.
| S-EPMC1184977 | biostudies-other
Project description:The dynamics of water exhibits anomalous behavior in the presence of different electrolytes. Recent experiments [Kim JS, Wu Z, Morrow AR, Yethiraj A, Yethiraj A (2012) J Phys Chem B 116(39):12007-12013] have found that the self-diffusion of water (Dw) can either be enhanced or suppressed around CsI and NaCl, respectively, relative to that of neat water. Here we show that unlike classical empirical potentials, ab initio molecular dynamics simulations successfully reproduce the qualitative trends observed experimentally. These types of phenomena have often been rationalized in terms of the "structure-making" or "structure-breaking" effects of different ions on the solvent, although the microscopic origins of these features have remained elusive. Rather than disrupting the network in a significant manner, the electrolytes studied here cause rather subtle changes in both structural and dynamical properties of water. In particular, we show that water in the ab initio molecular dynamics simulations is characterized by dynamic heterogeneity, which turns out to be critical in reproducing the experimental trends.
| S-EPMC3948301 | biostudies-literature
Project description:Promising strategies for cartilage regeneration rely on the encapsulation of mesenchymal stromal cells (MSCs) in a hydrogel followed by an injection into the injured joint. Preclinical and clinical data using MSCs embedded in a collagen gel have demonstrated improvements in patients with focal lesions and osteoarthritis. However, an improvement is often observed in the short or medium term due to the loss of the chondrocyte capacity to produce the correct extracellular matrix and to respond to mechanical stimulation. Developing novel biomimetic materials with better chondroconductive and mechanical properties is still a challenge for cartilage engineering. Herein, we have designed a biomimetic chemical hydrogel based on silylated collagen-mimetic synthetic peptides having the ability to encapsulate MSCs using a biorthogonal sol-gel cross-linking reaction. By tuning the hydrogel composition using both mono- and bi-functional peptides, we succeeded in improving its mechanical properties, yielding a more elastic scaffold and achieving the survival of embedded MSCs for 21 days as well as the up-regulation of chondrocyte markers. This biomimetic long-standing hybrid hydrogel is of interest as a synthetic and modular scaffold for cartilage tissue engineering.
| S-EPMC8293055 | biostudies-literature