Use of hydrazine to release in intact and unreduced form both N- 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 1H-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.
Patel
T
T
Oxford GlycoSystems Limited, Unit 4, Abingdon, Oxon, England.
Bruce
J
J
Merry
A
A
Bigge
C
C
Wormald
M
M
Jaques
A
A
Parekh
R
R
D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002645:Chickens; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D006834:Hydrazines / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D013552:Swine; D013696:Temperature
D006023:Glycoproteins; D006834:Hydrazines; D009844:Oligosaccharides; D010455:Peptides; C029424:hydrazine