Finlayson S D, Moore P A, Johnston J R, Berry D R
Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, Great Britain.
Anal Biochem. 1990 May 1;186(2):233-5. doi: 10.1016/0003-2697(90)90072-h.
Two methods for specifically detecting maltase, alpha-glucosidase, or isomaltase activity in electrophoresis gels are described. Both systems couple the formation of glucose by enzyme action on maltose or isomaltose to the generation of a colored product. System A uses an agarose overlay which contains substrate, glucose oxidase, peroxidase, 2,4-dichlorophenol, and 4-L-amino-phenazone. A purple color is produced at the site of enzyme activity. No hazardous chemicals are used at any stage. The stain is simple, rapid, sensitive, and inexpensive and does not interfere with subsequent protein staining. However, the stain is not permanent. System B was developed to give a permanent stain. The gel is overlaid with agarose containing substrate, glucose oxidase, phenazine methosulfate, and nitroblue tetrazolium. Glucose production results in the nitroblue tetrazolium being oxidized to an insoluble formazan with a dark blue color. This stain is also sensitive, rapid, and inexpensive but does use hazardous chemicals and if overstaining occurs this can interfere with subsequent protein staining. Neither system inactivates the localized enzymes which can be recovered from the gel if desired.
本文描述了两种在电泳凝胶中特异性检测麦芽糖酶、α-葡萄糖苷酶或异麦芽糖酶活性的方法。两种系统均将酶作用于麦芽糖或异麦芽糖生成葡萄糖的过程与有色产物的生成相偶联。系统A使用含有底物、葡萄糖氧化酶、过氧化物酶、2,4-二氯苯酚和4-L-氨基苯腙的琼脂糖覆盖物。在酶活性部位产生紫色。在任何阶段都不使用危险化学品。该染色方法简单、快速、灵敏且成本低廉，并且不干扰后续的蛋白质染色。然而，该染色不是永久性的。系统B的开发目的是提供永久性染色。凝胶用含有底物、葡萄糖氧化酶、硫酸吩嗪甲酯和硝基蓝四唑的琼脂糖覆盖。葡萄糖的产生导致硝基蓝四唑被氧化成不溶性的甲臜，呈深蓝色。这种染色方法也灵敏、快速且成本低廉，但确实使用了危险化学品，并且如果过度染色，会干扰后续的蛋白质染色。如果需要，两种系统都不会使局部酶失活，这些酶可以从凝胶中回收。
