Reuter G, Pfeil R, Stoll S, Schauer R, Kamerling J P, Versluis C, Vliegenthart J F
Eur J Biochem. 1983 Jul 15;134(1):139-43. doi: 10.1111/j.1432-1033.1983.tb07542.x.
Improvements in the isolation procedure and the analytical equipment enabled the detection of seven novel sialic acids in bovine submandibular gland glycoprotein: N-acetyl-8-O-acetylneuraminic acid, N-acetyl-8,9-di-O-acetylneuraminic acid, N-acetyl-7,8,9-tri-O-acetylneuraminic acid, N-glycoloyl-7-O-acetylneuraminic acid, N-glycoloyl-7,9-di-O-acetylneuraminic acid, N-glycoloyl-8,9-di-O-acetylneuraminic acid, and N-glycoloyl-7,8,9-tri-O-acetylneuraminic acid. There are also indications for the presence of N-glycoloyl-8-O-acetylneuraminic acid. In addition, the sialic acids already known to occur in this tissue, namely N-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-acetyl-7,9-di-O-acetylneuraminic acid, N-glycoloylneuraminic acid, and N-glycoloyl-9-O-acetylneuraminic acid could be identified. Sialic acids were released from the mucin by mild acid hydrolysis and prefractionated on a Dowex 2X8 anion-exchange column (formate form) by elution with a 0-0.6 M gradient of formic acid. The four pools of sialic acids obtained in this way were each further fractionated by column chromatography on cellulose with n-butanol/n-propanol/water (1/2/1, v/v/v) as eluent. By this procedure, N-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-glycoloylneuraminic acid, and N-glycoloyl-9-O-acetylneuraminic acid were obtained in pure form. The other sialic acids could be enriched sufficiently in different fractions for structural identification. Analyses of sialic acids were carried out by one-dimensional and two-dimensional thin-layer chromatography, gas-liquid chromatography, and gas-liquid chromatography-mass spectrometry.
N-乙酰基-8-O-乙酰神经氨酸、N-乙酰基-8,9-二-O-乙酰神经氨酸、N-乙酰基-7,8,9-三-O-乙酰神经氨酸、N-糖基酰基-7-O-乙酰神经氨酸、N-糖基酰基-7,9-二-O-乙酰神经氨酸、N-糖基酰基-8,9-二-O-乙酰神经氨酸以及N-糖基酰基-7,8,9-三-O-乙酰神经氨酸。也有迹象表明存在N-糖基酰基-8-O-乙酰神经氨酸。此外，还能够鉴定出已知存在于该组织中的唾液酸，即N-乙酰神经氨酸、N-乙酰基-7-O-乙酰神经氨酸、N-乙酰基-9-O-乙酰神经氨酸、N-乙酰基-7,9-二-O-乙酰神经氨酸、N-糖基酰神经氨酸以及N-糖基酰基-9-O-乙酰神经氨酸。通过温和酸水解从粘蛋白中释放出唾液酸，并在Dowex 2X8阴离子交换柱（甲酸根形式）上进行预分级分离，用0 - 0.6 M的甲酸梯度洗脱。以这种方式获得的四个唾液酸组分再分别在纤维素柱上用正丁醇/正丙醇/水（1/2/1，v/v/v）作为洗脱剂进行进一步分级分离。通过该程序，获得了纯形式的N-乙酰神经氨酸、N-乙酰基-7-O-乙酰神经氨酸、N-乙酰基-9-O-乙酰神经氨酸、N-糖基酰神经氨酸以及N-糖基酰基-9-O-乙酰神经氨酸。其他唾液酸可以在不同组分中充分富集以进行结构鉴定。通过一维和二维薄层色谱、气液色谱以及气液色谱 - 质谱联用对唾液酸进行分析。
