Inactivation of creatine kinase during the interaction of indomethacin with horseradish peroxidase and hydrogen peroxide: involvement of indomethacin radicals.
Creatine kinase (CK) was used as a marker molecule to examine the side effect of damage to tissues by indomethacin (IM), an effective drug to treat rheumatoid arthritis and gout, with horseradish peroxidase and hydrogen peroxide (HRP-H2O2). IM inactivated CK during its interaction with HRP-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 IM at 280 nm rapidly decreased and a new peak at 410 nm occurred with isosbestic points at 260 and 312 nm. In contrast, under anaerobic conditions, the spectral change of IM was almost absent, indicating IM was oxidized to the yellow substance by HRP-H2O2. Adding catalase strongly inhibited the production of yellow substance. Sodium azide also blocked the formation of yellow substance and the inactivation of CK. Electron spin resonance signals of IM carbon-centered radical were detected using 2-methyl-2-nitrosopropane during the interaction of IM with HRP-H2O2 under anaerobic conditions. Oxygen was consumed during the interaction of IM with HRP-H2O2. These results suggest that IM carbon-centered radicals may rapidly react with O2 to generate the peroxyl radicals. Sulfhydryl groups and tryptophane residues of CK decreased during the interaction of IM with HRP-H2O2. Other sulfhydryl enzymes, including alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, were also readily inactivated during the interaction with HRP-H2O2. Sulfhydryl enzymes seem to be very sensitive to IM activated by HRP-H2O2.
Miura
T
T
Hokkaido College of Pharmacy, Katuraoka-cho 7-1, 047-0264, Otaru, Japan. miurat@hokuyakudai.ac.jp
Muraoka
S
S
Fujimoto
Y
Y
D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002374:Catalase / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003402:Creatine Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism*; D007213:Indomethacin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000378:metabolism; D009603:Nitroso Compounds; D010101:Oxygen Consumption / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D019810:Sodium Azide / Q000494:pharmacology; D013053:Spectrophotometry; D013113:Spin Labels
D005609:Free Radicals; D009603:Nitroso Compounds; D013113:Spin Labels; D019810:Sodium Azide; D006861:Hydrogen Peroxide; D000426:Alcohol Dehydrogenase; D006735:Horseradish Peroxidase; D002374:Catalase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003402:Creatine Kinase; C034526:tert-nitrosobutane; D007213:Indomethacin