Chlorpromazine N-demethylation by hydroperoxidase activity of covalent immobilized lipoxygenase.

This work describes the application of the N-demethylase activity of immobilized soybean lipoxygenase to the oxidative degradation of xenobiotics. Previously (1) we have shown that immobilized lipoxygenase produces the oxidative degradation of CPZ in the presence of hydrogen peroxide. As a continuation of this work, here we studied the N-demethylation of CPZ by the hydroperoxidase activity of covalent immobilized soybean lipoxygenase. The obtained results clearly reveal that the immobilized system produces the N-demethylation of CPZ in the presence of hydrogen peroxide, maintaining a high level of activity in comparison with free enzyme. Additionally, the immobilized lipoxygenase shows stability higher than that of free enzyme, making feasible its use in a bioreactor operating in continuous or discontinuous mode. The results obtained in this work, together with those obtained previously by us for the oxidation of CPZ, suggest that hydroperoxidase activity of immobilized lipoxygenase may constitute a valuable tool for oxidative xenobiotics degradation or for application to synthetic processes in which a N-demethylation reaction is involved.

The use of fluorescein 5'-isothiocyanate for studies of structural and molecular mechanisms of soybean lipoxygenase.

Incubation of fluorescein 5'-isothiocyanate (FITC) with soybean lipoxygenase produces the coupling of 1 mol of fluorophore to 1 mol of enzyme. Derivatized lipoxygenase lost 40% activity through a mixed-type inhibitory mechanism. The quenching by IK of the fluorescence of FITC-labeled lipoxygenase shows that the fluorophore is located near the surface of the protein. The partial impediment of the FITC labeling when the substrate is present in the medium, together with data of the tryptic digestion, suggests that FITC is attached via the access channel of substrate to the catalytic site. Labeling does not induce appreciable modification of the enzyme specificity, suggesting that the position of substrate in the active site is not modified by the labeling. The results obtained strongly suggest that FITC labels soybean lipoxygenase specifically at a lysyl residue which contributes to fixation of the carboxylic end of the substrate to the active center. The experimental data obtained from the quenching of FITC fluorescence by NDGA reveal that this molecule interacts with the protein near the catalytic site.