Lactoperoxidase-catalysed oxidation of indomethacin, a nonsteroidal antiinflammatory drug, through the formation of a free radical.

Lactoperoxidase (LPO, EC 1.11.1.7; donor-H2O2 oxidoreductase) catalyses the oxidation of indomethacin, a nonsteroidal antiinflammatory drug by H2O2 as measured by time-dependent decay of indo-methacin extinction at 280 nm and concurrent appearance of stable oxidation product(s) at 412 nm. From a plot of log Vmax against varying pH of indomethacin oxidation, involvement of an ionizable group of the enzyme having pka = 5.7 could be ascertained for controlling the oxidation process. Spectral studies revealed that LPO-compound II oxidises indomethacin through one-electron transfer and is reduced to the native ferric state as shown by its spectral shift from 430 nm to 412 nm through an isosbestic point at 421 nm. The one-electron oxidation product is a nitrogen-centered free radical detected as a 5,5-dimethyl-l-pyrroline N-oxide (DMPO) adduct (alpha N = 15 G, alpha H beta = 16 G) in electron spin resonance spectroscopy. The free radical is scavenged by reaction with O2 as shown by O2 consumption sensitive to the free-radical trap, DMPO. Binding studies by optical difference spectroscopy indicate that indomethacin binds to LPO with an apparent KD value of 24.5 microM. The free energy change, delta G', for the binding is -26.3 KJ mol-1, suggesting that the interaction is favourable for oxidation. Indomethacin binding remains unaltered by a change of pH from 5.25 to 7.5, presumably because of hydrophobic interaction. The binding is competitive with resorcinol, an aromatic electron donor, showing the KD value to be as high as 100 microM. We suggest that indomethacin interacts at the aromatic donor binding site and is oxidised by one-electron transfer by LPO catalytic intermediates to stable oxidation product(s) through the formation of a free radical.