Isolation and characterization of ichthyosan from tuna vitreous

Ichthyosan has been prepared from tuna vitreous. Glucuronic acid was found to account for the total uronic acid content of the macromolecule, while the hexosamine content was a mixture of N-acetyl-glucosamine and N-acetyl-galactosamine. When ichthyosan was gel filtered on Sepharose 2B or Sephacryl S-300, using sodium or calcium chloride, the elution profile of the column gave only one peak indicating no separation between glucosamine and galactosamine containing fractions. Similar results were obtained when ichthyosan was chromatographed on DEAE-cellulose using a salt gradient both in the presence and absence of 7.0 M urea. When ichthyosan was gel filtered in 4.0 M guanidine-HCL and subsequently chromatographed on DEAE-Sephacel or DEAE-cellulose, three well separated fractions were present. The two major fractions (II and III) were characterized as chondroitin and hyaluronic acid respectively; while fraction I representing about 3-5% of the total polysaccharide content of ichthyosan was identified as a keratan-like molecule. The same pattern was obtained when ichthyosan was digested with proteolytic enzymes and subsequently chromatographed on DEAE-cellulose or DEAE-Sephacel. Based on these findings it is concluded that in ichthyosan chondroitin, hyaluronate and keratan-like molecular chains are bound to proteins in non-covalent linkages.