Irreversible binding of prosthetic heme to protein: Physiological and toxicological implications.

摘要

Reactive intermediates, generated from the metabolism of drugs, environmental chemicals, and endogenous molecules, are known to play a role in a variety of toxicological processes. The precise mechanisms by which these reactive intermediates cause toxicity remain to be determined; however, the covalent alteration of hemoproteins may be involved. One particular type of hemoprotein alteration, in which the heme prosthetic group is irreversibly-bound to the apoprotein, has been suggested to participate in mechanisms of suicide inactivation, myocardial ischemia-reperfusion injury, red cell lysis, drug-induced autoimmune reactions, and processes leading to protein degradation and turnover. In order to clarify the relationship between irreversibly-bound heme and these diverse events, this thesis research has examined the conditions under which irreversibly-bound heme adducts form and characterized their formation.; The ability to detect the formation of irreversibly-bound heme adducts in biological samples is essential to an investigation of the toxicological significance of this pathway. Consequently, we have developed a sensitive and facile method, involving SDS-PAGE and enhanced chemiluminescence (ECL), to detect and quantify the formation of irreversibly-bound heme adducts of hemoproteins. This ECL assay has been used to characterize irreversibly-bound heme adducts that form as a result of oxidative modification of myoglobin by H₂O₂, as well as during the metabolism-based inactivation of neuronal nitric oxide synthase in vitro and in vivo. Furthermore, this study is the first to demonstrate that irreversibly-bound heme adducts of nitric oxide synthase can occur in vivo.; Irreversibly-bound heme adducts of myoglobin have been previously shown to possess oxidase activity. In the current study, we provide evidence that these adducts can also catalyze a peroxidase reaction, and have the ability to oxidize LDL and purified lipids. Because these reactions have the potential to be self-sustaining, they may be important to the mechanism by which this altered form of myoglobin causes cell death. Our results show that irreversibly-bound heme adducts of other hemoproteins possess peroxidase activity as well. Thus, enhanced redox cycling by these adducts may be important to many of the toxicological processes in which irreversibly-bound heme adducts have been suggested to play a role.