De Ruiter G A, Schols H A, Voragen A G, Rombouts F M
Wageningen Agricultural University, Department of Food Science, The Netherlands.
Anal Biochem. 1992 Nov 15;207(1):176-85. doi: 10.1016/0003-2697(92)90520-h.
Sulfuric acid hydrolysis according to the Saeman procedure, TFA hydrolysis, and methanolysis combined with TFA hydrolysis were compared for the hydrolysis of water-soluble uronic acid-containing polysaccharides originating from fungi, plants, and animals. The constituent sugar residues released were subsequently analyzed by either conventional GLC analysis of alditol acetates or high-performance anion-exchange chromatography with pulsed-amperometric detection. It was shown that TFA hydrolysis alone is not sufficient for complete hydrolysis. Sulfuric acid hydrolysis of these polysaccharides resulted in low recoveries of 6-deoxy-sugar residues. Best results were obtained by methanolysis combined with TFA hydrolysis. Methanolysis with 2 M HCl prior to TFA hydrolysis resulted in complete liberation of monosaccharides from pectic material and from most fungal and animal polysaccharides tested. Any incomplete hydrolysis could be assessed easily by HPAEC, by the detection of characteristic oligomeric products, which is difficult using alternative methods currently in use. Methanolysis followed by TFA hydrolysis of 20 micrograms water-soluble uronic acid containing polysaccharides and subsequent analysis of the liberated sugar residues by HPAEC allowed us to determine the carbohydrate composition of these polysaccharides rapidly and accurately in one assay without the need for derivatization.
比较了根据塞曼程序进行的硫酸水解、三氟乙酸（TFA）水解以及甲醇解与TFA水解相结合的方法，用于水解源自真菌、植物和动物的水溶性含糖醛酸多糖。随后，通过常规的糖醇乙酸酯气相色谱分析或带脉冲安培检测的高效阴离子交换色谱法，对释放出的组成糖残基进行分析。结果表明，单独的TFA水解不足以实现完全水解。这些多糖的硫酸水解导致6-脱氧糖残基的回收率较低。甲醇解与TFA水解相结合获得了最佳结果。在TFA水解之前用2 M HCl进行甲醇解，可使单糖从果胶物质以及大多数测试的真菌和动物多糖中完全释放出来。通过检测特征性低聚物产物，利用高效阴离子交换色谱法（HPAEC）可以轻松评估任何不完全水解情况，而这使用目前的其他方法则很难做到。对20微克含水溶性糖醛酸的多糖先进行甲醇解，然后进行TFA水解，随后通过HPAEC对释放出的糖残基进行分析，这使我们能够在一次测定中快速准确地确定这些多糖的碳水化合物组成，而无需进行衍生化。
