Continuous spectrophotometric assay of conjugated bile acid hydrolase.

Abstract

Conjugated bile acid hydrolase (CBAH) refers to a class of enzymes which catalyze the cleavage of the amino acid moieties from conjugated bile acids. These enzymes are significant because of their role in providing substrates for further microbial metabolism in the gastrointestinal tract. They also are used in research laboratories for the deconjugation of bile acids prior to structural analyses. A continuous spectrophotometric assay for CBAH activity was developed using a conjugate of cholic acid and the chromophore, 5-amino-2-nitro-benzoic acid. The free chromophore is detected by virtue of its absorbance at 410 nm. The CBAH from Clostridium perfringens displayed a Km for this substrate of 120 microM. These results demonstrate that this new compound functions as an effective substrate of the enzyme and forms the basis for a convenient and rapid method to monitor CBAH activity.