Hyaluronan can be non-enzymatically linked to protein through an alkali sensitive bond

To test for the presence of hyaluronan to protein linkages, both purified and unpurified preparations of hyaluronan were blotted onto nitrocellulose, and the adsorbed hyaluronan was detected using a biotinylated probe derived from cartilage. While purified hyaluronan did not adsorb to nitrocellulose, the unpurified hyaluronan from rat fibrosarcoma (RFS) cells and chick embryos did. This adsorption appeared to require the presence of protein, since it was inhibited by treatment with proteases. Furthermore, when hyaluronan from RFS cells was subjected to conditions which break most non-covalent bonds, it retained its ability to adsorb to nitrocellulose, suggesting that this hyaluronan was covalently bound to protein which mediated its adsorption to nitrocellulose. While this bond was resistant to acidic buffers, it was readily broken by alkaline buffers. Additional experiments demonstrated that when either purified [3H] hyaluronan or oligosaccharide fragments of hyaluronan were incubated with a variety of proteins, they slowly gained the ability to adsorb to nitrocellulose. However, this process could be blocked by the addition of low molecular weight amines or by reducing the [3H] hyaluronan with NaBH4. These results are consistent with the possibility that the reducing terminal of the hyaluronan reacts with amine groups of protein to form a Schiff base which then rearranges to a stable bond. Such a bond could account for the association of hyaluronan with the surfaces of RFS cells.