Development of a peroxidase-coupled fluorometric assay for lysyl oxidase

Lysyl oxidase catalyzes the oxidation of peptidyl lysine in elastin and collagen and also acts upon nonpeptidyl amines, although the enzyme becomes slowly inactivated while processing nonpeptidyl substrates. In spite of this complexity, it has been possible to devise a continuously monitored peroxidase-coupled fluorometric assay for the oxidation of simple amines by lysyl oxidase. In the present study, optimal assay conditions have been explored and found to include assay temperatures of 50 to 60°C, the presence of urea in the assay, and the use of diaminopentane as substrate. Although the assay is subject to interference by contaminating macromolecules in enzyme fractions, a linear assay response to enzyme concentration is obtained with highly purified lysyl oxidase with a limiting sensitivity of 0.3 μg of enzyme per assay.