Antinuclear antibody screening by an enzyme-linked immunosorbent assay using multiple nuclear antigens

The oxidation of aminopyrine, an N-alkyl aromatic amine, by the hydroperoxidase activity of lipoxygenase was studied. Aminopyrine gave rise to a purple color in the presence of H2O2 and lipoxygenase, the color being proportional to the aminopyrine radical cation. The H2O2/aminopyrine radical cation molar ratio was 0.5. The overall reaction was considered as an enzymic-chemical second order mechanism with substrate regeneration. From the equations, the apparent constant of the radical cation's decomposition (k'app) was evaluated under different experimental conditions. It was found to be inversely proportional to the proton concentration but unaffected by the concentration of aminopyrine. These results suggest a new comprehensive mechanism for N-demethylation, which takes into account the described presence of both nitrogen- and carbon-centered radicals and the marked effect of pH on the stability of the radical cation.