Water-soluble carbodiimide for the fluorescent measurement of the carboxyl group produced by enzyme reactions.

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.