Theses and Dissertations at Montana State University (MSU)
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Item Chemical interaction of nitric oxide and human hemoglobin(Montana State University - Bozeman, College of Letters & Science, 2004) Luchsinger, Benjamin Peter; Chairperson, Graduate Committee: David J. Singel; Patrik R. Callis (co-chair)In the past decade an entirely new areas of biomedical science have emerged with the recognition of the critical roles played by nitric oxide (NO) in biology. The discoveries of the role of NO in the cardiovascular systems, where it participates in regulating the tone of blood vessels and the flow of blood, raised an intriguing biochemical problem about the chemical interaction of NO and hemoglobin in blood. Historically, investigation of the interaction of NO and hemoglobin have suggested the formation of heme-Fe(II)NO hemoglobin and nitrate. Neither of these products is considered to exhibit NO-related bioactivity, thus the reaction of NO with hemoglobin was thought to quench the bioactive potential of NO. In this thesis we explore the idea that these are not the only two fates of NO in its interaction with hemoglobin. Product formation following encounters of NO and hemoglobin under various conditions of oxygenation, oxidation, and NO/heme ratios are analyzed by UV/Vis optical spectroscopy, EPR (electron paramagnetic resonance) spectroscopy, and colorimetric chemical analyses. This battery of methods enables quantification of the hemoglobin species: heme-Fe(II)NO (with distribution over the hemoglobin subunits) heme-Fe(III)NO, oxyhemoglobin, methemoglobin, deoxyhemoglobin, S-nitrosohemoglobin (SNO-hemoglobin). We present results that establish a previously unsuspected favoring of protein nitrosylation at heme and thiol positions in the reaction of oxyhemoglobin with NO. We demonstrate coupling of heme reduction/oxidation activity with thiol reactivity. We provide evidence for transfer of the NO group from heme to thiol and vice versa. We demonstrate unprecedented subunit selectivity of interactions of NO with hemoglobin. Finally, we present results that highlight the nitrite reductase activity of hemoglobin whereby nitrite, which has a significant concentration in blood, is converted to bioavailable forms of NO. Overall, the in vitro chemistry reported here provides for a complete basis for rationalizing the in vivo biochemistry required for the hemoglobin/NO system to serve as regulator of blood flow.Item Oxidation of human nitrosylhemoglobin monitored by UV-Vis and EPR spectroscopies : detection of products and intermediates(Montana State University - Bozeman, College of Letters & Science, 2005) Williams, Elizabeth Mary; Chairperson, Graduate Committee: David J. SingelOnce viewed only as a toxic free radical, nitric oxide (NO) has been established as an essential and ubiquitous signaling and regulatory molecule in biological systems. Notably, NO was identified as the endothelium-derived relaxing factor (EDRF) in the blood. NO is capable of complex redox chemistry and interaction with a host of protein families. Among these proteins is hemoglobin (Hb) which can interact with NO at the level of the heme and can bind NO at Cys93 on its â subunit to form S-nitrosylated Hb (SNO-Hb). NO bound as SNO-Hb is chemically labile and thus preserves bioavailability of NO. However, when NO reacts with oxyHb or deoxyHb NO bioavailability is quenched by conversion to nitrate or by tightly binding the heme, respectively. Therefore, the question is raised as to how NO can be EDRF in the presence of such high Hb concentrations in the blood. One way NO availability can be preserved is by exploiting the redox chemistries of both Hb and NO. Human Hb(NO)4 oxidation by K3Fe(CN)6 was studied and products and intermediates were identified by UV-Vis and EPR spectroscopies. Periodically, samples were withdrawn from the reaction mixture for nitrosylation product and/or EPR analysis. Reaction spectra converted to heme species concentration vs. time plots through leastsquares fitting of five basis spectra. These data were then utilized to generate a de minimis model of the oxidation reaction. We demonstrate that the oxidation of Hb(NO)4 by K3Fe(CN)6 1) leads to the production of SNO-Hb, 2) occurs preferentially at the â heme, and 3) proceeds through an HbFeIIINO intermediate.Item Exogenous ligand effects on S-nitrosohemoglobin formation in reactions of methemoglobin with nitric oxide(Montana State University - Bozeman, College of Letters & Science, 2009) Mellmann, Lisa Jean; Chairperson, Graduate Committee: David J. SingelBlood's function of carrying oxygen to tissues is regulated by physiological oxygen gradients that are coupled to vasoconstriction and vasodilation by nitric oxide bioactivity. However, the mechanism by which local oxygen tension utilizes nitric oxide signaling in order to regulate blood flow remains a major unanswered question in biology. Hemoglobin in red blood cells appears to be an ideal sensor, but lack of knowledge about hemoglobin's chemistry with nitric oxide creates a problem for understanding how hemoglobin induces vasodilation. A central focus of this work was to illuminate complexity and response of nitric oxide interactions with hemoglobin while showing how S-nitrosohemoglobin plays a pivotal role in this response. Electronic absorption and electron paramagnetic resonance spectroscopy were employed to observe reactions of low-spin methemoglobin species with nitric oxide. Models were developed and tested to establish the chemistry involved and the global hierarchy of reactions. De minimis models for all experiments are reported. Electron paramagnetic resonance spectra confirmed the formation of low-spin iron species that, upon the addition of nitric oxide, showed a decrease in low-spin methemoglobin species and the formation of nitrosyl hemoblobin with a beta-subunit preference. We report the yield of S-nitrosohemoglobin production in reactions of nitric oxide with various methemoglobins distinguished by axial ligand and iron spin state.