Determination of glycosaminoglycans (mucopolysaccharides) from tissues on the microgram scale

Biochimica et Biophysica Acta (BBA) - Specialized Section on Mucoproteins and Mucopolysaccharides Pub Date : 1964-03-02 DOI:10.1016/0926-6526(64)90045-X

C.A. Antonopoulos , S. Gardell , J.A. Szirmai , Ellen R. De Tyssonsk

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引用次数: 272

Abstract

Microgram quantities of glycosaminoglycans can be quantitatively liberated from tissues, fractionated and determined by the following procedure: (1) Digestion of the tissue sample with papain (EC 3.4.4.10). (2) Precipitation of the glycosaminoglycans as their cetylpyridinium complexes on an inert support of powdered cellulose packed in a column. (3) Fractional elution of the complexes from the column with salt solutions of increasing concentration. (4) Estimation of the amount of glycosaminoglycans in each fraction after hydrolysis followed by determination of the hexosamines by a modified Elson and Morgan method.

Experimental data are presented to validate the various steps of the procedure. The results were found to be reproducible and accurate for fractions down to 2–5 μg of hexosamine. Small differences in the solubility properties of the glycosaminoglycan-cetylpyridinium complexes could be detected and visualized by the construction of “solubility profiles”.

Application of the procedure to histological sections of the intervertebral disc and the nasal septum cartilage revealed differences in the amount and properties of glycosaminoglycans in tissue structures 100–200 μ apart. The method should be useful to study the distribution of glycosaminoglycans at the tissue level.

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组织中糖胺聚糖(粘多糖)的微克级测定

可以定量地从组织中释放出微克量的糖胺聚糖，通过以下程序进行分离和测定:(1)用木瓜蛋白酶(EC 3.4.4.10)消化组织样品。(2)将糖胺聚糖作为十六烷基吡啶配合物沉淀在柱状粉末纤维素的惰性载体上。(3)用浓度递增的盐溶液对柱上的配合物进行分级洗脱。(4)估算水解后各馏分中糖胺聚糖的含量，然后用改进的Elson和Morgan法测定己糖胺。给出了实验数据来验证该过程的各个步骤。结果重复性好，对低至2-5 μg的己糖胺含量均准确。糖胺聚糖-十六烷基吡啶配合物溶解度特性的微小差异可以通过构建“溶解度谱”来检测和可视化。将该方法应用于椎间盘和鼻中隔软骨的组织学切片，发现在相距100-200 μ的组织结构中，糖胺聚糖的数量和性质存在差异。该方法可用于研究糖胺聚糖在组织水平上的分布。

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Biochimica et Biophysica Acta (BBA) - Specialized Section on Mucoproteins and Mucopolysaccharides

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