Selective Hydrolysis of Terminal Glycosidic Bond in α-1-Acid Glycoprotein Promoted by Keggin and Wells-Dawson Type Heteropolyacids.

The reactivity of a range of Keggin and Wells-Dawson type heteropolyacids (HPAs): H PW O H SiW O , H PMo O , K P W O , and NaH W O , towards the heavily glycosylated α-1-acid glycoprotein (AGP) is reported. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) show that after incubation of the protein with HPAs at 80 °C and pH 2.8 complete hydrolysis of terminal glycosidic bond has been achieved, resulting in the removal of sialic acids with no observed destruction of the protein core or the residual glycan chains. The H NMR spectroscopy confirmed that the released sialic acids preserve intact structure upon their excision from the protein, which makes the reported method suitable for the analysis of sialic acid modifications which play an important role in numerous biological processes. The presence of other sugars was not detected by H NMR and HPAEC-PAD, suggesting that HPAs hydrolyze only the terminal glycosidic bond in the glycoprotein, resulting in the selective release of sialic acid from AGP. The kinetic results have shown that under equal temperature and pH conditions, the hydrolysis of the terminal glucosidic bond occurred faster in the presence of HPAs compared to conventional mineral acids. The observed rate constants were in the range 6,7×10 -11,9×10  min and the complete and selective excision of sialic acids could be achieved within 60 min of incubation. The Trp fluorescence and CD spectroscopy show that non-covalent interaction between HPA and protein takes place in solution which could lead to stabilization of the sialosyl cation that is formed during the glycosidic bond hydrolysis by anionic HPA cluster.