Fluorometric assay for intestinal peptidases

✍ Scribed by J.Alex Nicholson; T.J. Peters

• Book ID: 102982914
• Publisher: Elsevier Science
• Year: 1978
• Tongue: English
• Weight: 507 KB
• Volume: 87
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(78)90691-7

✦ Synopsis

A fluorometric assay for intestinal peptidases has been developed. Amino acids liberated by hydrolysis are estimated by use of L-amino-acid oxidase, peroxidase, and the fluorogenic reagent p-hydroxyphenylacetic acid, which yields a highly fluorescent compound on oxidation. During development of fluorescence. continued hydrolysis of peptides by peptidases which contaminate available preparations of L-amino-acid oxidase is prevented by the use of two inhibitors, l.lO-phenanthroline andp-hydroxymercuribenzoate.

The assay is at least 10 times more sensitive than comparable spectrophotometric methods which employ the potentially carcinogenic chromogen o-dianisidine for detection of amino acids.

MATERIALS AND METHODS

Chemicals.

Peptides, L-amino acids, L-amino-acid oxidase (Type I), horseradish peroxide (crude), p-hydroxyphenylacetic acid, p-hydroxy-