The composition and physicochemical properties of hyaluronic acids prepared from ox synovial fluid and from a case of mesothelioma.

1. Materials containing hyaluronic acid have been prepared by filtration (Ogston & Stanier, 1950) from ox synovial fluid and from a protein-rich human mesothelioma fluid. The ox material has been deproteinized by treatment with chloroform and pentanol and by gradient elution on DEAE-Sephadex; several fractions were obtained by the latter method. These materials can be stored in solution at -20 degrees without change of properties. The ox material contained 21% of protein; all other preparations contained less than 6% of protein. 2. The two materials have been compared by sedimentation and viscosity and shown to be closely similar. Treatment of the ox material with neuraminidase caused no change in its viscosity behaviour. 3. Information about the molecular configuration of the ox material has been obtained from measurements of light-scattering and viscosity. The results, though consistent with a highly extended configuration, are not consistent with a linear random-coil configuration. It is tentatively suggested that the structure may have some degree of branching and of cross-linking, which give it a rigidity with respect to expansion of the molecular domain that would not be possessed by a random coil. 4. The deproteinized material recovered from DEAE-Sephadex, though polydisperse, showed unchanged average molecular weight; however, the average radius of gyration was greater than before this treatment. 5. Acidification to approx. pH3 resulted in a contraction of the structure, with only a slight degree of expansion when the pH was restored to 6.8-7.0. 6. Measurements of optical rotatory dispersion qualitatively support a structure less simple than a linear random coil. 7. Colloid osmotic pressures of mixed solutions of bovine serum albumin and of hyaluronic acid prepared by filtration from ox synovial fluid have been measured. The results agree approximately with those of Laurent & Ogston (1963) but are in quantitative disagreement with the partition measurements of Ogston & Phelps (1960). The relationships between thermodynamic quantities in a quaternary system of electrolytes are discussed in Appendix 2. 8. Refractometric measurements have been made in connexion with light-scattering measurements, as the basis for a convenient method of determining the concentrations of solutions of hyaluronic acids, and to measure the partition of sodium chloride in dialysis experiments. The theory of the last use is discussed in Appendix 1. 9. Sedimentation measurements on the ox preparation have been made up to a concentration of 1.4x10(-2)g. /ml. The form of the sedimentation coefficient-concentration relationship is discussed. The value of the sedimentation coefficient at higher concentration is the basis of an illustration of the likely effect of hyaluronic acid on the flow of water through narrow channels in connective tissue. 10. Available colorimetric methods have been shown to give low estimates for glucuronic acid when applied to highly polymerized materials, as compared with estimates by decarboxylation. A spectrophotometric titration with cetylpyridinium bromide has been shown to give estimates of carboxyl groups that agree well with those of decarboxylation when applied to preparations of hyaluronic acid under suitable conditions; the results are not affected by the presence of protein. 11. Estimates of glucosamine (Ogston, 1964) have been found to be low compared with those of total acetyl, independently of the presence of protein. The magnitude of the discrepancy is characteristically different for preparations from ox synovial fluid and from mesothelioma. 12. Sialic acid was estimated in several preparations. It is likely that this forms part of the protein. 13. Analyses of preparations for total nitrogen, amino acids, total acetyl, glucuronic acid (by decarboxylation) and ash account for at least 95.7% of the dry weight in terms of N-acetylglucosaminyl, glucuronyl, protein and metal ions. Previously published analyses of hyaluronic acids are reviewed. 14. The estimated molar ratios of glucuronic acid to glucosamine were all significantly greater than unity. 15. The analytical results are interpreted as agreeing with the physicochemical measurements in suggesting a more complex structure, for at least some hyaluronic acids, than that of an alternate linear copolymer in random-coil configuration.