It has become apparent in recent years that free radical reactions play an important role in numerous toxicological processes. The long-term goal of this research is to understand the role of free radicals in overall protein turnover, in health and disease. The objective of this application is to test the hypothesis that: """"""""Oxygen radicals damage red cell cytoplasmic proteins, thus increasing the susceptibility of those proteins to degradation by the erythrocyte or reticulocyte proteolytic systems."""""""" The specific aims of this proposal are to determine: 1) the structural and functional damage to erythrocyte and reticulocyte cytoplasmic proteins (particularly hemoglobin) induced by various oxygen radicals; 2) the extent to which damage proteins are proteolytically degraded; 3) the functional characteristics of the proteolytic system(s); 4) the effectiveness of cellular free radical defenses. The approach to be employed will involve: 1) protein damage and degradation in intact rabbit-erythrocytes and reticulocytes, as well as cell-free extracts, directly exposed to oxygen radicals; 2) oxygen radical damage to purified hemoglobin, and selected other purified red cell cytoplasmic proteins; 3) degradation of oxygen radical-damaged purified proteins during subsequent incubation with erythrocyte or reticulocyte cell-free extracts. The methodology for protein damage and degradation will include: functional studies, optical spectra, gel electrophoresis, isoelectric focusing, HPLC-gel filtration and DEAE chromatography, amino acid analysis, fluorescamine reactivity, radiolabeling of proteins by reductive methyolation, de novo synthesis of radiolabeled reticulocyte proteins, liquid scintillation of acid-soluble and acid-precipitable radiolabels, antioxidants and radical scavengers, proteolytic inhibitors, oxygen radical generating systems (chemical, enzymatic, and radiolytic). The relationship to health concerns involves: 1) the toxicity of many redox active xenobiotics; 2) the defenses of normal and abnormal cells against oxidative stress; 3) the physiological oxidation and proteolytic degradation of cell proteins.
