The Development of Two Fluorimetric Assays for the Determination of Pyroglutamyl Aminopeptidase Type-II Activity

• Published in: Analytical biochemistry Vol. 250; no. 1; pp. 1 - 9
• Main Authors: Gallagher, Seán P., O'Leary, Rhona M., O'Connor, Brendan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.07.1997
• Subjects: Aminopeptidases - metabolism; Animals; Cattle; Coumarins - analysis; Coumarins - metabolism; Cyclization; Dipeptidyl Peptidase 4 - metabolism; Oligopeptides - metabolism; Pyrrolidonecarboxylic Acid - analogs & derivatives; Spectrometry, Fluorescence
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1997.2195

Two fluorimetric assays for the determination of pyroglutamyl aminopeptidase type-II activity have been developed. The assays are based on hydrolysis of the quenched-fluorimetric substrate <Glu-His-Pro-7-amino-4-methylcoumarin. Following the removal of the N-terminal <Glu by pyroglutamyl aminopeptidase type-II, liberation of 7-amino-4-methylcoumarin from the metabolite His-Pro-7-amino-4-methylcoumarin is catalyzed by one of two methods: (i) the addition of partially purified bovine serum dipeptidyl aminopeptidase type-IV or (ii) by incubating the reaction mixture for up to 2 h at 80°C, thus promoting the nonenzymatic cyclization of His-Pro-7-amino-4-methylcoumarin to cyclo His-Pro and free 7-amino-4-methylcoumarin. Pyroglutamyl aminopeptidase type-II from bovine brain is used to establish appropriate assay conditions. These fluorimetric assays offer expeditious alternatives to the existing radiolabeled thyrotropin-releasing hormone assays for the determination of PAPII activity.