Roth R I, Levin J
Department of Laboratory Medicine, University of California, School of Medicine, San Francisco.
J Biochem Biophys Methods. 1989 Aug-Sep;19(2-3):129-41. doi: 10.1016/0165-022x(89)90021-3.
A method is described for amplification of bands generated by chromogenic substrates following their reaction with proteases within electrophoretic gels. Chromogenic substrates, consisting of synthetic peptides containing the chromophore 4-nitroaniline (paranitroaniline, PNA), are applied directly to the surface of agarose or acrylamide gels. Protease activity within the gel results in the enzymatic amidolysis of the chromogenic substrates, releasing free PNA. The yellow PNA chromogen is then derivatized by reaction with p-dimethylaminocinnamaldehyde (DACA) to form a purple Schiff base compound. The resultant complex has a significantly higher molar absorbancy than the original PNA chromogen, thus increasing the sensitivity for low levels of amidolytic activity. The derivatized chromogen is easily visualized for photography.
本文描述了一种方法，用于在电泳凝胶中，使生色底物与蛋白酶反应后产生的条带得到扩增。由含有发色团4-硝基苯胺（对硝基苯胺，PNA）的合成肽组成的生色底物，直接施加到琼脂糖或丙烯酰胺凝胶表面。凝胶中的蛋白酶活性导致生色底物的酶促酰胺水解，释放出游离的PNA。然后，黄色的PNA色原通过与对二甲氨基肉桂醛（DACA）反应进行衍生化，形成紫色的席夫碱化合物。所得复合物的摩尔吸光度明显高于原始的PNA色原，从而提高了对低水平酰胺水解活性的检测灵敏度。衍生化的色原易于观察以便拍照。
