52] Hydrolysis of aromatic amides as assay for carboxylesterases-amidases

Abstract

Publisher Summary Most carboxylic ester hydrolases also hydrolyze aromatic amides. Normally, it is not possible to assay the hydrolysis of these amides by the titration of the released acid because the released amine will itself serve as a titrant. Actually the rate of the cleavage of amides by carboxylesterases–amidases is usually low, requiring more sensitive methods. A number of assays are described in this chapter that are useful for the determination of the hydrolysis of aromatic amides both by cell fractions and by purified enzymes. First assay involves the diazotation of aniline or aniline derivatives. This procedure can be used with substrates that yield aniline or p -phenetidine. It cannot be applied to substrates producing 2,6-dimethylaniline or 2-chloro-6-methylaniline. Second assay is direct photometric assay for the hydrolysis of butanilicaine,4 acetanilide,5 or 4-nitroacetanilide. Third assay is fluorometric assay for the hydrolysis of acetanilide, phenacetin, or paracetamol. The direct fluorometric method can be used for clear enzyme solutions.