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

Abstract

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 spectrop...

Protein Associated with Hyaluronic Acid in Ox Synovial Fluid

“…However, contrary to the findings of Preston, Davies and Ogston (1965), only 50-60% of the total protein had come off the column by the time that small amounts of material containing uronic acid had begun to appear. The hyaluronic acid, which was recovered from the column by reversed flow (Preston, Davies, and Ogston 1965), still contained 13% of protein; Preston, Davies, and Ogston (1965) reported 6% of protein in hyaluronic acid recovered in this way. Table 1 shows the amino acid compositions of fractions 1, 2a (soluble), 2b (insoluble), and 3, 4, and 5.…”

“…Table 1 shows the amino acid compositions of fractions 1, 2a (soluble), 2b (insoluble), and 3, 4, and 5. Fractions 2a and 2b showed no marked differences from each other and resembled the protein analysed by Preston, Davies, and Ogston (1965). Fraction 1 differed significantly from them, particularly with respect to its contents of arginine, glutamic acid, glycine, and valine.…”

“…A column of DEAE-Sephadex A-50 (medium grade) as described by Preston, Davies, and Ogston (1965) was therefore used. The eluted protein appeared in three peaks (fractions 3, 4, and 5).…”

“…Curtain (1955) separated this residual protein from ultrafilter residue (UFR) in good yield after enzymic degradation of the hyaluronic acid, and showed that it normally consisted of two proteins which resembled serum 0(-and ,a-lipoproteins. Preston, Davies, and Ogston (1965) separated the greater part of the protein by chromatography on DEAE-Sephadex, but reported only its amino acid composition. Silpananta, Dunstone, andOgston (1967, 1968) showed that hyaluronic acid can be obtained almost free from protein by equilibrium sedimentation in a caesium chloride density gradient (hereafter called for brevity "gradient sedimentation") ; the recovery of the protein that they obtained was low and they did not report on its properties.…”

Study of hyaluronic acid flexibility by electric birefringence

“…This was followed by extensive studies by light-scattering (Laurent & Gergely, 1955;Preston et al, 1965;Cleland & Wang, 1970), which all concluded that sodium hyaluronate adopted a solution conformation that was far more rigid than the random coil, although the possibility of domains of freely coiling chains was not excluded. The chain rigidity was attributed to inter-residue hydrogenbonding, and was further supported by evidence from periodate oxidation and n.m.r.…”