| The need for a safe and reliable blood substitute has long been recognized. An understanding of the molecular basis for low oxygen affinity and high cooperativity in recombinant (r) Hemoglobins (Hbs) is essential in applying protein engineering methodology to design novel rHbs as Hb-based oxygen carriers. This thesis reports studies on the structure-function relationships in the allosteric mechanism of Hb A, rHb and cross-linked rHbs. The Hb expression system developed in our laboratory enables us to construct and produce specific rHbs. Biophysical and biochemical experiments include oxygen-dissociation studies for measuring the oxygen affinity, cooperativity, and the anion and Bohr effects; kinetic studies for measuring CO-binding and dissociation constants; and 1 H nuclear magnetic resonance (NMR) spectroscopy for studying the structures and conformations of these rHbs. 1H-NMR studies suggest that the molecular basis for low-oxygen affinity in these rHbs is that they favor the T conformation even when they are still ligated. This is true for the low-oxygen-affinity rHbs with mutations) in the α1β 1 and/or α1β2 subunit interfaces studied here. Our results on the relative effect of amino acid substitution with opposite charges at β108Asn (located in the α1β1 subunit interface and in the central cavity of Hb) have shown that a change in the ionic environment has significant effects on the regulation of oxygen affinity by allosteric effectors. From a systematic study of rHbs mutated at the β108 site, rHb (βN108Q) is found to exhibit low oxygen affinity, high cooperativity, and more stable against autoxidation than other studied rHbs. rHb (αL29F, βN108Q) is shown to optimize the functions of rHb (βN108Q), i.e., low oxygen affinity, high cooperativity, stability against auto- and NO-induced oxidation, and may fulfill the requirements of an Hb-based therapeutic agent for different clinical applications. |
