Jaques L B, Sue T K, McDuffie N M, Wice S M
Can J Physiol Pharmacol. 1977 Oct;55(5):1179-89. doi: 10.1139/y77-161.
Heparin (Hep), hyaluronic acid, chondroitins (sulfate) A, B, and C, and heparins (sulfate) A, B, C, and D were subjected to microelectrophoresis in barbital-agarose gel, fixed with cetylpyridinium chloride and stained with toluidine blue. The optical densities of the resulting bands were compared with optical densities obtained upon reaction with azure A in aqueous solution and with the carbazole reagent. A linear relation was obtained between optical density and concentration of purified sulfated mucopolysaccharide (SMP). Less than 1 microgram of Hep and 2 microgram of other SMPs are required for measurement by electrophoresis, while about 30 microgram of each is required with the carbazole reagent. The optical density of a mixture of SMPs was equal to the sum of the densities for the individual SMPs upon microelectrophoresis. It was demonstrated that the individual SMPs in mixtures were distinguishabed by reaction with specific enzymes and by changes in migration in agarose with barbital, phthalate, ethylenediamine, or propanediamine buffers, permitting ready demonstration and quantitation of various SMP species. Examples are shown of the application of the procedure to measure the total SMPs and individual SMPs in tissue extracts. The method is sensitive, reproducible, flexible, and measures quantities 1/30th of those measured colorimetrically, yet is relatively unaffected by protein, carbohydrate, or inorganic electrolytes.
将肝素（Hep）、透明质酸、软骨素（硫酸盐）A、B和C以及硫酸乙酰肝素A、B、C和D在巴比妥-琼脂糖凝胶中进行微电泳，用十六烷基氯化吡啶固定并用甲苯胺蓝染色。将所得条带的光密度与在水溶液中与天青A反应以及与咔唑试剂反应后获得的光密度进行比较。纯化的硫酸化粘多糖（SMP）的光密度与浓度之间呈线性关系。通过电泳测量需要少于1微克的肝素和2微克的其他SMP，而使用咔唑试剂时每种大约需要30微克。微电泳时，SMP混合物的光密度等于各个SMP光密度之和。结果表明，混合物中的各个SMP可通过与特定酶反应以及在含有巴比妥、邻苯二甲酸盐、乙二胺或丙二胺缓冲液的琼脂糖中迁移的变化来区分，从而便于对各种SMP种类进行展示和定量。展示了该方法在测量组织提取物中总SMP和单个SMP方面的应用实例。该方法灵敏、可重复、灵活，测量的量是比色法测量量的1/30，但相对不受蛋白质、碳水化合物或无机电解质的影响。
