RELEASE OF O-LINKED SUGAR CHAINS FROM GLYCOPROTEINS WITH ANHYDROUS HYDRAZINE AND PYRIDYLAMINATION OF THE SUGAR CHAINS WITH IMPROVED REACTION CONDITIONS

A method for preparation of pyridylamino (PA-) derivatives of O-linked sugar chains from glycoproteins was developed. A glycopeptide containing O-linked Gal-beta-1-3GalNAc was prepared from fetuin and treated with anhydrous hydrazine followed by N-acetylation of free amino groups. Sugar chains released were pyridylaminated with improved reaction conditions and excess reagents were removed by gel filtration. Gal-beta-1-3GaINAc-PA obtained together with PA-Gal as a by-product was quantified by HPLC. Conditions for the hydrazine treatment were investigated and the treatment at 40-degrees-C for 350 h gave the best results for releasing O-linked sugar chains. The total yield of Gal-beta-1-3GaINAc-PA from the glycopeptide was 53% under the established conditions and that of PA-Gal was 18%. The present method was applied to a glycoprotein, and the expected PA-O-linked sugar chains were obtained. Under these conditions, N-linked sugar chains were also released.