FREE-RADICAL INACTIVATION OF RABBIT MUSCLE CREATINE-KINASE - CATALYSIS BY PHYSIOLOGICAL AND HYDROLYZED ICRF-187 (ICRF-198) IRON CHELATES

Creatine kinase is a sulfhydryl containing enzyme that is particularly susceptible to oxidative inactivation. This enzyme is potentially vul nerable to inactivation under conditions when it would be used as a di agnostic marker of tissue damage such as during cardiac ischemia/reper fusion or other oxidative tissue injury. Oxidative stress in tissues c an induce the release of iron from its storage proteins, making it an available catalyst for free radical reactions. Although creatine kinas e inactivation in a heart reperfusion model has been documented, the m echanism has not been fully described, particularly with regard to the role of iron. We have investigated the inactivation of rabbit muscle creatine kinase by hydrogen peroxide and by xanthine oxidase generated superoxide or Adriamycin radicals in the presence of iron catalysts. As shown previously, creatine kinase was inactivated by hydrogen perox ide. Ferrous iron enhanced the inactivation. In addition, micromolar l evels of iron and iron chelates that were reduced and recycled by supe roxide or Adriamycin radicals were effective catalysts of creatine kin ase inactivation. Of the physiological iron chelates studied, Fe(ATP) was an especially effective catalyst of inactivation by what appeared to be a site-localized reaction. Fe(ICRF-198), a non-physiological che late of interest because of its putative role in alleviating Adriamyci n-induced cardiotoxicity, also catalyzed the inactivation. Scavenger s tudies implicated hydroxyl radical as the oxidant involved in iron-dep endent creatine kinase inactivation. Loss of protein thiols accompanie d loss of creatine kinase activity. Reduced glutathione (GSH) provided marked protection from oxidative inactivation, suggesting that enzyme inactivation under physiological conditions would occur only after GS H depletion.