The effects of dansylation and acetylation on the interaction between hyaluronic acid and the hyaluronic acid-binding region of cartilage proteoglycans.

The complex of hyaluronic acid (HA)-binding region, link protein, and hyaluronic acid was isolated from trypsin digests of bovine nasal cartilage proteoglycan aggregates and the HA-binding region was then purified from the complex. After dansylation of accessible amino groups, less than 15% of the HA-binding region molecules interacted with hyaluronic acid. Conversely, after acetylation with 2.5 to 5 mM [3H]acetic anhydride, about 80% of the HA-binding region molecules still exhibited binding activity which was not abolished by subsequent dansylation. Dansylation of HA-binding region in the presence of hyaluronic acid did not abolish binding activity. These results indicate that one or more amino groups in or near the binding site of HAbinding region molecules can be acetylated without loss of binding activity while substitution of the same group(s) with the bulky dansyl moiety prevents the interaction. A sample of the intact complex was dansylated to substitute accessible groups and the HA-binding region was then isolated. Portions of this HA-binding region preparation were dansylated directly with [3H]dansylCl; dansylated in the presence of hyaluronic acid with [3H]dansyl-C1 and then with unlabeled dansyl-Cl after removing the hyaluronic acid; or dansylated in the presence of hyaluronic acid with unlabeled dansyl-Cl and then with [3H]dansyl-C1 after removing the hyaluronic acid. The three samples were reduced and alkylated, digested with trypsin, and fractionated by Sephadex G-50 chromatography. Radioactive peptides eluted at different positions for samples dansylated with [3H]dansyl-C1 in the presence of hyaluronic acid compared to the samples dansylated with nonradioactive dansyl chloride in the presence of hyaluronic acid followed by 3H dansylation with no hyaluronic acid present. The results indicate that there are separate sites in the HA-binding region molecules which are protected from dansylation when both hyaluronic acid and the link protein are present or when only hyaluronic acid is present, suggesting that the HA-binding region of the proteoglycan molecule interacts with the link protein as well as with hyaluronic acid in forming proteoglycan aggregates.