Inactivation of creatine kinase during the interaction of indomethacin with horseradish peroxidase and hydrogen peroxide: involvement of indomethacinradicals

Creatine kinase (GK) was used as a marker molecule to examine the side effe ct of damage to tissues by indomethacin (IM). an effective drug to treat rh eumatoid arthritis and gout, with horseradish peroxidase and hydrogen perox ide (HRP-H2O2). Ih I inactivated CK during its interaction with trRP-H2O2. Under aerobic conditions, inactivation of CK significantly decreased. CK in rat heart homogenate was also inactivated by IM with HRP-H2O2. When IM was incubated with HRP-H2O2, the maximum absorption of Ih I at 280 nm rapidly decreased and a new peak at 410 nm occurred with isosbestic points at 260 a nd 312 nm. In contrast, under anaerobic conditions, the spectral change of IR I was almost absent, indicating IM was oxidized to the yellow substance bp HRP-H2O2. Adding catalase strongly inhibited the production of yellow su bstance. Sodium azide also blocked the formation of yellow substance and th e inactivation of CK. Electron spill resonance signals of IM carbon-centere d radical were detected using 2-methyl-2-nitrosopropane during thee interac tion of IM with HRP-H2O2 under anaerobic conditions. Oxygen was consumed du ring the interaction of IM with HRP-H2O2. These results suggest that IM car bon-centered radicals may rapidly react with O-2 to generate the peroxyl ra dicals. Sulfhydryl groups and tryptophane residues of CK decreased during t he interaction of IM with HRP-H2O2. Other sulfhydryl enzymes, including alc ohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, were also readily inactivated during the interaction with HRP-H2O2. Sulfhydryl enzyme s seem to be very sensitive to IM activated by HRP-H2O2. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.