tailieunhanh - Báo cáo khoa học: Mechanism for the hydrolysis of hyaluronan oligosaccharides by bovine testicular hyaluronidase

Synthetic hyaluronan oligosaccharides with defined structures and their pyridylaminated derivatives were used to investigate the mechanism of hydrolysis of hyaluronan by bovine testicular hyaluronidase. The products of the hydrolysis were analyzed by HPLC and ion-spray mass spectroscopy (MS). | ễFEBS Journal Mechanism for the hydrolysis of hyaluronan oligosaccharides by bovine testicular hyaluronidase Ikuko Kakizaki Nobuyuki Ibori Kaoru Kojima Masanori Yamaguchi and Masahiko Endo Department of Glycotechnology Center for Advanced MedicalResearch Hirosaki University Graduate Schoolof Medicine Japan Keywords hyaluronan hydrolysis oligosaccharide testicular hyaluronidase Correspondence I. Kakizaki Department of Glycotechnology Center for Advanced Medical Research Hirosaki University Graduate Schoolof Medicine 5 Zaifu-cho Hirosaki 036-8562 Japan Fax 81 172 39 5016 Tel 81 172 39 5015 E-mail kaki@ These authors contributed equally to this work Received 18 November 2009 revised 14 January 2010 accepted 29 January 2010 doi Synthetic hyaluronan oligosaccharides with defined structures and their pyridylaminated derivatives were used to investigate the mechanism of hydrolysis of hyaluronan by bovine testicular hyaluronidase. The products of the hydrolysis were analyzed by HPLC and ion-spray mass spectroscopy MS . It was confirmed that the minimum substrate for bovine testicular hyaluronidase is the hyaluronan hexasaccharide even though it is a poor substrate that is barely cleaved even on prolonged incubation. When hyaluronan octasaccharide was the substrate increasing amounts of tetrasaccharide and hexasaccharide were produced with increasing time of incubation. Whereas disaccharide was not detectable in the reaction mixture by HPLC MS analysis revealed trace amounts. The data suggest that the enzyme generates a disaccharide intermediate from hyaluronan oligosaccharide the majority of which is transferred to the nonreducing ends of other oligosaccharides only traces being released as free disaccharide. When hyaluronan octasaccharide with an unsaturated glucuronic acid at the nonreducing end was used as a substrate only a tetrasaccharide was detected by HPLC. However MS showed that the product was a mixture of equal