Charge isomers of urinary bikunin (trypsin inhibitor)

It was observed that the purified urinary bikunin (trypsin inhibitor) consisted of four major isomers with different electric charges which could be separated by HPLC using a Mono Q column. These isomers revealed the same antitrypsin activity and did not show any differences in the apparent molecular weight by SDS-PAGE, amino-acid composition, N-terminal amino-acid sequence (1-40) and C-terminal amino acid (Leu). The contents of sialic acid and uronic acid were also identical among these isomers. However, analysis of chondroitin sulfate revealed all the glycosaminoglycan chains of these isomers were undersulfated, comprising nonsulfated and 4-sulfated disaccharide units, and 4-sulfated disaccharide unit ratio varied among these isomers. After the chondroitin ABC lyase digestion, all the isomers were eluted at the same position on a Mono Q column chromatography. These results indicated that charge isomers of urinary bikunin was attributed to the difference on sulfation ratio in a glycosaminoglycan chain.