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The analysis of intermolecular interactions in concentrated hyaluronan solutions suggest no evidence for chain-chain association.

ABSTRACT: Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to examine the influence of electrolytes (NaCl, KCl, MgCl(2), MnCl(2) and CaCl(2)) on the network and hydrodynamic properties of fluoresceinamine-labelled hyaluronan (FA-HA) at concentrations up to 10 mg/ml. Self and tracer lateral diffusion coefficients showed that in Ca(2+) and Mn(2+), FA-HA (830 kDa) was more compact than in Mg(2+), Na(+) or K(+). These results were correlated with changes in the hydrodynamic radius of HA, determined by multi-angle laser-light-scattering analysis in dilute solution, which was smaller in CaCl(2) (36 nm) than in NaCl (43 nm). The permeability of more concentrated solutions of HA (<10 mg/ml) to FITC-dextran tracers (2000 kDa) was higher in CaCl(2). The properties of HA in urea (up to 6 M) were investigated to test for hydrophobic interactions and also in ethanol/water (up to 62%, v/v). In both, there was reduced hydrodynamic size and increased permeability to FITC-dextran, suggesting increased chain flexibility, but it did not show the changes predicted if chain-chain association was disrupted by urea, or enhanced by ethanol. Oligosaccharides of HA (HA(20-26)) also had no effect on the self diffusion of high-molecular-mass FA-HA (830 kDa) solutions, or on dextran tracer diffusion, showing that there were no chain-chain interactions open to competition by short-chain segments. The results suggest that the effects of electrolytes and solvent are determined primarily by their effect on HA chain flexibility, with no evidence for association between chain segments contributing significantly to the major properties.

SUBMITTER: Gribbon P

PROVIDER: S-EPMC1221259 | biostudies-other | 2000 Aug

REPOSITORIES: biostudies-other

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Project description: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)

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The analysis of intermolecular interactions in concentrated hyaluronan solutions suggest no evidence for chain-chain association.

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