Amplification of color yield of chromogenic substrates using p-dimethylaminocinnamaldehyde

A method is described for amplifying the color of 4-nitroaniline which is produced during enzyme catalyzed amidolysis of synthetic chromogenic substrates. The liberated chromogen is directly coupled with p-dimethylaminocinnamaldehyde to yield a colored schiffbase compound. The color complex has an absorption maximum at 570 nm and, under the reaction conditions described, has a molar absorbancy of 9.1 × 10⁴. The application of this method is presented for the enzyme urokinase using two different chromogenic substrates. A micromethod adaptation of this method is also described. This method can be used to amplify the color obtained in all enzyme assays where 4-nitroaniline is the liberated chromogen.