Antigenicity of protein-polysaccharide complexes from cartilage-IV: Fractionation of protein-polysaccharides from bovine nasal septa and their cross-reactivity with protein-polysaccharides from bovine heart valves

The immunological properties of protein-polysaccharide (PPL) isolated from bovine nasal septa have been compared with those of the hyaluronidase digest of the same material, uisng the techniques of passive hemagglutination and passive hemagglutination inhibition. In the presence of bovine PPL immunserum or human PPL immunserum, tanned erythrocytes coated with nondigested PPL are agglutinated at higher immunserum titers than erythrocytes coated with PPL digest. Moreover, nondigested PPL is a better inhibitor than digested PPL of the systems; bovine PPL immunserum or human PPL immunserum, versus tanned erythrocytes coated with nondigested PPL. However, hyaluronidase-digested PPL is a better inhibitor when the tanned erythrocytes are coated with digested PPL. These results indicate that the removal of part of the chondroitin sulfate chains with hyaluronidase may modify the conformation of the protein moiety of the complex and may produce a material quite different immunologically from the original one. The poor performance of hyaluronidase-digested PPL, when tested either as a coating agent or as an inhibitor against human PPL immunserum, indicates that removal of part of the chondroitin sulfate chains decreases the number or the availability of the cross-reacting determinants. Hyaluronidase-digested PPL has been fractionated on a column of DEAE-Sephadex A-50, using increasing concentrations of KCl. The fractions eluted with 0·7 M and 1·0 M KCl possess the species-specific determinants and, to a lesser extent, the cross-reacting ones and are better inhibitors than PPL or PPL digest. On the basis of these results it may be concluded that digestion of PPL with hyaluronidase produces a mixture of products in which the antigenic determinants are covered; fractionation of this mixture with a method capable of disrupting electrostatic bonds results in the appearance of two fractions having better immunological properties than the original PPL. Finally, extraction of bovine heart valves with 6 M urea produces protein-polysaccharide fractions which cross-react with bovien PPL immunserum but not with human PPL immunserum.