Spectrophotometric Assays for l-Lysine α-Oxidase and γ-Glutamylamine Cyclotransferase

Abstract

A new assay for l-lysine α-oxidase is described. In this assay, the oxidized product generated from l-lysine is reacted with semicarbazide to form α-keto-ϵ-aminocaproate semicarbazone. Formation of the α-keto acid semicarbazone is continuously monitored spectrophotometrically at 248 nm (ϵ 10,160 ± 240 M−1 cm−1). The method was adapted to provide a new assay for γ-glutamylamine cyclotransferase. This enzyme catalyzes the conversion of many l-γ-glutamylamines to 5-oxo-l-proline and free amine. A biologically important substrate is Nϵ-(γ-l-glutamyl)-l-lysine, which is converted to 5-oxo-l-proline and l-lysine by the action of γ-glutamylamine cyclotransferase. The l-lysine generated from Nϵ-(γ-l-glutamyl)-l-lysine in an endpoint assay is converted to α-keto ϵ-aminocaproate semicarbazone in the presence of semicarbazide, excess l-lysine α-oxidase, and catalase. The methods were applied to the determination of γ-glutamylamine cyclotransferase activity of partially purified preparations of the bovine kidney enzyme and to detect γ-glutamylamine cyclotransferase activity in rat kidney and liver homogenates.