Rapid colorimetric assay for intestinal peptide hydrolases

✍ Scribed by Chon R. Shoaf; Kurt J. Isselbacher; William D. Heizer

• Book ID: 102982120
• Publisher: Elsevier Science
• Year: 1974
• Tongue: English
• Weight: 819 KB
• Volume: 61
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(74)90334-0

✦ Synopsis

A simple two-step method is described for quantitating the release of free L-phenylalanine, L-leucine, L-methionine, or L-isoleucine from di-or polypeptides. The calorimetric assay is based on the ability of L-amino acid oxidase to catalyze the oxidation of free L-amino acid, but not of peptides. The potent metal chelator l,lO-phenanthroline, which is included in the second step of the assay, effectively inhibits peptide hydrolase activity thus permitting the assay to be carried out in two sequential steps in the same test tube with no intervening enzyme-destroying step. Although the assay is indirect and subject to interference by some chemicals, it is not affected by a large number of compounds frequently used in enzyme studies. The method was used to study the subcellular distribution of a number of peptide hydrolase activities in rat intestinal mucosa. For nine substrates, more than 80% of the total recovered activity was present in the cytoplasmic fraction while for two substrates, phenylalanylglycine and phenylalanylglycylglycine, more than 60% of the activity recovered was present in the particulate fraction.