For over 20 years it has been postulated that drugs and other chemicals that are metabolized to reactive intermediates can cause toxicity by covalently altering cellular proteins. To date, however, little is known about the target proteins involved, the chemical nature of these alterations, or the mechanism by which this leads to toxicity. We have shown in the past that hemoproteins, specifically cytochrome P-450, myoglobin, and hemoglobin, can be targets for reactive metabolites of drugs and endogenous compounds. These interactions can lead to the cross-linking of the heme prosthetic group to the protein and may result in the conversion of these proteins into oxidases, which produce potentially toxic oxygen metabolites. This year we have found our canine model of cardiac ischemia and reperfusion injury studies that approximately 112 of the myoglobin heme is lost in the area of infarct injury compared to that from a non-injured section of the heart. Concomitantly, we detected the formation of abnormal 405 nm-absorbing compounds by HPLC from the infarct samples. The same abnormal products could also be formed in vitro from the normal heart sample after treatment with HOOH, a product that is known to be produced during ischemia and reperfusion injury. These results suggest that during ischemia and reperfusion, hydrogen peroxide reacts with myoglobin to form cross-linked products of heme that may contribute to the toxicity by producing toxic oxygen metabolites. We have found that a similar type of cross-linking reaction occurred with cytochromes P-450, although some isozymes, were relatively resistent to this form of enzyme alteration. This information may be ultimately used to design more stable cytochromes P-450 catalysts that can be used in synthetic chemistry or in the disposal of environmental pollutants. It was additionally discovered that the heme of hemoglobin was susceptible to the cross-linking reaction by BrCC13 and became covalently bound to the beta-chain of this protein. This modification of hemoglobin may have a role in hemolytic anemia caused by drugs or genetic diseases. Agents that could prevent the formation of cross-linked heme proteins might have important therapeutic and industrial applications in the future.
