Fractionation of ahyaluronic acid preparation in a density gradient. Some properties of the hyaluronic acid.

1. Hyaluronic acid was isolated from ox synovial fluid by sedimentation equilibrium in a caesium chloride density gradient (Silpananta, Dunstone & Ogston, 1967). The product was almost free from chondroitin sulphate and from protein. 2. Its composition did not differ significantly from that of the carbohydrate part of the protein-containing material isolated by filtration. Its physicochemical properties and molecular configuration were similar, except for its viscosity, which showed markedly reduced concentration-dependence and shear-dependence. This suggests that the associated protein tends to form links between molecules of hyaluronic acid. 3. The accurate measurement of viscosity at very low velocity gradient, by use of the damping of oscillations in a Couette viscometer, is described. 4. A method is described for measuring, approximately, the thermodynamic non-ideality of a solute from the shape of its schlieren curve at sedimentation equilibrium in a density gradient. 5. A value for the partial specific volume of hyaluronic acid in dilute salt solution was calculated from its isopycnic density in a caesium chloride gradient.

The osmotic behaviour of Sephadex and its effects on chromatography.

1. Sephadex in bead form shows reversible changes of inner volume when immersed in solutions of a non-penetrating solute. These changes are in accordance with the theory of Flory (1953) for the swelling of gels. This makes possible the use of single beads for measuring the osmotic pressures of polymer solutions, up to or beyond 1kg./cm.(2). 2. Measurements of the inner volume of Sephadex G-200 by equilibrium dilution, at various concentrations of dextran 500, gave values in agreement with those obtained from the dimensions of single beads. 3. Measurements by chromatography of the inner volume of Sephadex G-200 gave values that differed slightly but significantly from those obtained by the other methods. Similar small disagreements were found between the dilution and chromatographic values for bovine serum albumin measured in the presence of various concentrations of dextran 500. The major factor that affects the dependence of the chromatographic elution volume (of dextran 500) on concentration is shown to be the change of the inner volume of the Sephadex, with dynamic factors playing a minor role. 4. Contrary to the findings of Ackers (1964), the equilibrium dilution and chromatographic methods gave closely agreeing values for the distribution coefficient, K(av.), of bovine serum albumin on Sephadex G-200; this cast doubt on his explanation of the nature of chromatographic separations on Sephadex.

Fractionation of a hyaluronic acid preparation in a density gradient. The isolation and identification of a chondroitin sulphate.

1. Following the suggestion of Nichol, Ogston & Preston (1967) that hyaluronic acid, prepared by filtration from ox synovial fluid, contains a component of high density, such material has been detected and isolated by equilibrium sedimentation in a density gradient of caesium chloride. 2. This material (fraction III) has been characterized as a chondroitin sulphate-protein complex of average molecular weight about 250000. Its amino acid pattern is characteristic of such complexes present in cartilage. It contains a proportion of glucosamine (as well as galactosamine); this is not due to contamination with hyaluronic acid. 3. Preliminary findings on fraction I (low density) and fraction II (intermediate density) suggest that these consist chiefly of protein and hyaluronic acid respectively.