Kobayashi M, Chiba Y
National Food Research Institute, Tsukuba, Japan.
Anal Biochem. 1994 Jun;219(2):189-94. doi: 10.1006/abio.1994.1256.
Fluorescent measurement of the carboxyl group was achieved using water-soluble carbodiimide, EDC (1-ethyl-3-(3- dimethylaminopropyl)carbodiimide). Detection of the active intermediate from the glucuronic acid-EDC reaction mixture using the OPA (o-phthalaldehyde) reagent described in a previous paper was greatly improved to about 4 ng/tube (17 pmol) glucuronic acid owing to a modification of the OPA cocktail. Moreover, fluorescent EDAN (N-1-ethylenediamino-naphthalene) labeling of the carboxyl group was also enabled by EDC catalysis. Quenching of the excess fluorescence of EDAN by OPA increased the sensitivity of EDC-EDAN to a level above the EDC-OPA method. In addition to the assay of enzymes concerning the uronic acid metabolism such as pectinase and pectinesterase, lipase and protease actions could be measured by the EDC-OPA and EDC-EDAN quenching methods, respectively. Thus, these two fluorescent means of detecting carboxyl groups seemed to have extensive application not only to acidic sugars but also to various carboxylic compounds regardless of their solubility.
使用水溶性碳二亚胺EDC（1-乙基-3-（3-二甲基氨基丙基）碳二亚胺）实现了羧基的荧光测量。由于对OPA混合液进行了改进，使用先前论文中描述的OPA（邻苯二甲醛）试剂检测葡萄糖醛酸-EDC反应混合物中的活性中间体，其灵敏度大大提高，达到约4 ng/管（17 pmol）葡萄糖醛酸。此外，通过EDC催化还能实现羧基的荧光EDAN（N-1-乙二胺基萘）标记。OPA对过量EDAN荧光的淬灭作用将EDC-EDAN的灵敏度提高到了高于EDC-OPA方法的水平。除了用于检测诸如果胶酶和果胶酯酶等与糖醛酸代谢相关的酶外，脂肪酶和蛋白酶的活性分别可以通过EDC-OPA和EDC-EDAN淬灭法进行测量。因此，这两种检测羧基的荧光方法似乎不仅在酸性糖方面，而且在各种羧酸化合物（无论其溶解性如何）上都有广泛的应用。
