A chromogenic assay for catecholoxidases based on the addition of L-proline to quinones

The coupling reaction between L-proline and the quinone products of the oxidation of various catechols serves as a sensitive assay for catecholoxidases. The chromogenic products, 4-N-prolyl-o-quinones, were unique and stable over the course of the reaction. The spectra of these adducts typically had two absorbance maxima, in the ranges 309-340 and 526-540 nm. Assay conditions in which the oxidation of catechols was rate limiting were developed, and initial rates of reaction, monitored spectrophotometrically at the lambda max of the adducts, showed improved initial linearity when compared with the direct spectrophotometric determination of quinone formation. The molar extinction coefficients (epsilon) of a number of adducts ranged between 5310 and 9630 M-1 cm-1, about five- to sevenfold greater than those of the corresponding quinones. Since 2 mol catechol must be oxidized to their quinones to generate 1 mol of adduct, this assay improves catecholoxidase detection sensitivity by approximately three- to fourfold compared with direct estimation of quinone formation.