We have continued studies on oxidative processes associated with neutrophil respiratory burst. We have previously partially characterized a novel reaction in which labeled tyrosine is incorporated into high molecular weight material during neutrophil activation. Preliminary studies had suggested that dityrosine was formed but we lacked the authentic compound for comparative studies. In collaboration with Dr. Lin Tsai, dityrosine has been synthesized, purified and characterized. Labeled material was then isolated from neutrophils activated with PMA in the presence of labeled tyrosine and subjected to acid hydrolysis, Dowex 50 chromatography and reversed phase HPLC. Results from these labeled tyrosine is associated with the dityrosine peak. These data indicate that dityrosine which is formed is derived from endogenous unlabeled tyrosine and is independent of labeled tyrosine incorporation into high molecular weight material. Although the chemical form of labeled tyrosine incorporated has not yet been identified, it is alkali stable and acid labile. Other studies with neutrophils have indicated that cytoplast preparations which are largely depleted of myeloperoxidase- bearing granules generate protein carbonyl groups following stimulation with PMA. These experiments demonstrate that this model can be used to further study enzyme inactivation during neutrophil respiratory burst. Preliminary studies undertaken with Dr. Donita Garland and Dr. Edward DeMoll have indicated that purified bioactive peptides may be used to compare oxidation products from in vitro and in vivo MFO systems by fast atomic bombardment mass spectroscopy analysis.
