Electron paramagnetic resonance investigations of adducts of human hemoglobin
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Date
2010
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Montana State University - Bozeman, College of Letters & Science
Abstract
Hemoglobin transports oxygen to the tissues of the body. The delivery of oxygen to tissues by hemoglobin is dependent on blood flow, which is determined by vessel tension regulated by local oxygen gradients. Dilation of the blood vessels in the microcirculation of tissues under high metabolic demand is induced by the endothelium-derived relaxation factor, nitric oxide, in a process known as hypoxic vasodilation. Although the means of nitric oxide bioactivity preservation and transportation in the blood are disputed, it is clear that S-nitrosohemoglobin, a nitrosylated variant of hemoglobin, plays a pivotal role. The details surrounding S-nitrosohemoglobin formation in vivo, however, remain uncertain. Using electron paramagnetic resonance (EPR) spectroscopy, in conjunction with detailed spectral simulation and least-squares fitting, various hemoglobin species which possibly participate in the formation of S-nitrosohemoglobin were characterized. The EPR spectrum of methemoglobin-nitrite, a purported precursor to S-nitrosohemoglobin formation, was determined to be a composite spectrum arising from the presence of two species, the origin of which is proposed to lie in the differences between the distal heme pockets and histidine residues of the alpha- and beta-subunits of hemoglobin. By direct measurement of methemoglobin-nitrite by EPR spectroscopy, the weak affinity of methemoglobin for nitrite was confirmed, precluding nitrite-methemoglobin from having a direct role in physiological hypoxic vasodilation. Furthermore, the temperature dependence of the EPR spectra of the various species of neat methemoglobin was determined, as was the temperature dependence of the nitrosyl-hemoglobin (Hb(NO) 4) spectrum at high frequency. The high frequency spectrum of Hb(NO) 4 provided additional resolution of the axial and rhombic components of the spectrum, but revealed no evidence of distinct subunit spectra. Finally, synthetic routes to generate Fe(II)NO/Fe(III)-Hb hybrids have been presented, which, among other things, demonstrated that bolus addition of nitric oxide can produce similar results as the addition of time and condition dependent nitric oxide donors. Overall, this work expands the understanding of hemoglobin, specifically with regard to hemoglobin species with possible involvement in S-nitrosohemoglobin formation.