LACTOPEROXIDASE-CATALYZED OXIDATION OF INDOMETHACIN, A NONSTEROIDAL ANTIINFLAMMATORY DRUG, THROUGH THE FORMATION OF A FREE-RADICAL

Lactoperoxidase (LPO, EC 1.11.1.7; donor-H2O2 oxidoreductase) catalyse s the oxidation of indomethacin, a nonsteroidal antiinflammatory drug by H2O2 as measured by time-dependent decay of indomethacin extinction at 280 nm and concurrent appearance of stable oxidation product(s) at 412 nm. From a plot of log V-max against varying pH of indomethacin o xidation, involvement of an ionizable group of the enzyme having pka = 5.7 could be ascertained for controlling the oxidation process. Spect ral studies revealed that LPO-compound II oxidises indomethacin throug h one-electron transfer and is reduced to the native ferric state as s hown 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-cen tered free radical detected as a 5,5-dimethyl-l-pyrroline N-oxide (DMP O) adduct (a(N) = 15 G, a(beta)(H) = 16 G) in electron spin resonance spectroscopy. The free radical is scavenged by reaction with O-2 as sh own by O-2 consumption sensitive to the free-radical trap, DMPO. Bindi ng studies by optical difference spectroscopy indicate that indomethac in binds to LPO with an apparent K-D value of 24.5 mu M. The free ener gy change, Delta G', for the binding is -26.3 KJ mol(-1), suggesting t hat the interaction is favourable for oxidation. Indomethacin binding remains unaltered by a change of pH from 5.25 to 7.5, presumably becau se of hydrophobic interaction. The binding is competitive with resorci nol, an aromatic electron donor, showing the K-D value to be as high a s 100 mu M. We suggest that indomethacin interacts at the aromatic don or binding site and is oxidised by one-electron transfer by LPO cataly tic intermediates to stable oxidation product(s) through the formation of a free radical.