USE OF HYDRAZINE TO RELEASE IN INTACT AND UNREDUCED FORM BOTH N-LINKED AND O-LINKED OLIGOSACCHARIDES FROM GLYCOPROTEINS

The use of hydrazine to release unreduced N- and O-linked oligosaccharides from glycoproteins has been investigated using several ''standard'' glycoproteins of previously defined glycosylation. It is shown that hydrazinolysis can be used to release intact N- and O-linked oligosaccharides in an unreduced form. The release of O-linked oligosaccharides occurs with a lower temperature dependence than the release of N-linked oligosaccharides, and the kinetic parameters governing release of oligosaccharides from these standard glycoproteins have been determined. These parameters allow a definition of reaction conditions under which anhydrous hydrazinolysis can be used to selectively release O-linked oligosaccharides (60-degrees-C, 5 h) or release both N- and O-linked oligosaccharides (95-degrees-C, 4 h) in high yield (>85%) from all glycoproteins investigated (n = 11). Under these reaction conditions, the recovered N- and O-linked oligosaccharides are structurally intact (as judged by 600-MHz H-1-NMR, laser-desorption mass spectrometry, HPAEC-PAD, gel filtration, and glycosidase digestion), with the possible exception of certain N- and O-acyl substituents of sialic acid. This use of mild hydrazinolysis therefore allows both the simultaneous and sequential chemical release from glycoproteins of O- and N-linked oligosaccharides in their intact unreduced form.