Patel T, Bruce J, Merry A, Bigge C, Wormald M, Jaques A, Parekh R
Oxford GlycoSystems Limited, Unit 4, Abingdon, Oxon, England.
Biochemistry. 1993 Jan 19;32(2):679-93. doi: 10.1021/bi00053a037.
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.
利用几种先前已确定糖基化的“标准”糖蛋白，研究了用肼从糖蛋白中释放未还原的N - 连接和O - 连接寡糖的方法。结果表明，肼解可用于以未还原形式释放完整的N - 连接和O - 连接寡糖。O - 连接寡糖的释放比N - 连接寡糖的释放对温度的依赖性更低，并且已经确定了控制从这些标准糖蛋白中释放寡糖的动力学参数。这些参数确定了反应条件，在此条件下，无水肼解可用于从所有研究的糖蛋白（n = 11）中高产率（> 85%）地选择性释放O - 连接寡糖（60℃，5小时）或同时释放N - 连接和O - 连接寡糖（95℃，4小时）。在这些反应条件下，回收的N - 连接和O - 连接寡糖在结构上是完整的（通过600兆赫兹1H - NMR、激光解吸质谱、高效阴离子交换色谱 - 脉冲安培检测、凝胶过滤和糖苷酶消化判断），可能除了唾液酸的某些N - 和O - 酰基取代基外。因此，这种温和的肼解方法允许以完整未还原形式从糖蛋白中同时和顺序化学释放O - 连接和N - 连接寡糖。
