Generation and reactivity of HNO adducts of horse skeletal myoglobin and human hemoglobin

The discovery in the 1980's of the physiological role of nitric oxide (NO) initiated many subsequent investigations into the mechanism of formation of NO in vivo and the effects of NO on human health. It also attracted attention to nitrosyl hydride (HNO), which is the product of one-electron reduction of NO in aqueous solution. HNO is an elusive compound and can only be generated in situ. It is reactive with thiols, nitric oxide, oxygen, and transition metals; therefore, analysis of its effects in vivo is complicated. To characterize its specific reactions, in vitro studies are more appropriate. This dissertation concerns the investigations of HNO reactivity with myoglobin and hemoglobin, which may give a better understanding of the physiological role of HNO.; The first chapter is a summary of recent publications (2005-2007) on HNO, including reports on the effect of HNO in cardiovascular system, the synthesis of new HNO donors, the detection of HNO in vitro, the possible physiological reactivity of HNO, and possible bio-targets of HNO.; The second chapter concerns the photolysis of HNO-Mb, and characterization of the species produced. This includes a newly-developed method of generating HNO-Mb, which yielded samples of >90% purity. Transient absorbance traces obtained during photolysis of HNO-Mb revealed a unique photolytic pathway in that photolysis caused transient formation of a Mb-Fe(III). This implies that HNO- is also transiently generated but recombines to regenerate HNO-Mb. Interestingly, the rate of this recombination is much slower than that for NO-Mb. Possible reasons for such behavior are discussed.; The third chapter concerns the methods used to determine the rate constants of the reactions of HNO with Mb-Fe(II), Mb-Fe(III), and HNO-Mb. Time course spectral changes were analyzed using isosbestic absorbance wavelengths to characterize the changes in concentration of various Mb species over the course of the reaction. These results were modeled using the computational software React for Windows; the same software was applied to calculate the rate constants of the reaction of HNO-Mb with oxygen and nitric oxide. The modeled results were confirmed by EPR and GC-MS analysis of head gas. In addition to the computational method, a direct method of comparison of the rates of the reaction of HNO with deoxy and metMb was developed.; The final chapter describes an improved method of generating of the HNO adduct of hemoglobin (HNO-Hb). Previously, HNO-Hb could only be isolated in low yields; by modifying the reduction methodology significant improvement has been found. In addition, several spectroscopic techniques were used to identify and differentiate the 1H NMR hydride peaks characteristic of the HNO adducts of the alpha and beta subunits of hemoglobin; it was shown that different ratios of subunit HNO adducts are formed during reduction or trapping methods of their generation.