Many of the synthetic strategies to create hemoglobin based acellular blood substitutes, employ modifications that perturb the central cavity and extend the hydrodynamic volume. Cross-bridging (cross-linking), effector binding and PEGolation (attachment of polyethylene glycols) result in a wide range of Hbs having a broad spectrum of functional properties. Some of these properties such as enhanced tetramer stability and reduced oxygen affinity are needed attributes for potential blood substitutes. It is our contention that the rational design of this and the next generation of blood substitutes requires an understanding of how these modifications couple to functionally relevant domains of the protein. Four functionally important intra protein communication pathways are proposed. Experiments are being pursued to determine how central cavity modifications and PEGolation affect both key elements of these pathways and the communication between these elements. Several optical spectroscopic tools are to be used to probe the static and dynamic properties of key domains. Visible resonance Raman and near IR absorption will probe the heme and its immediate environment. Steady state front face fluorescence, time correlated single photon counting fluorescence lifetime measurements and nanosecond pulse-probe fluorescence will be used to probe tryptophans, fluorescent analogues of DPG and fluorescent probe labeled beta93. UV resonance Raman will be used both to discriminate between tryptophans and to probe interactions between the A and E helices the alpha1beta2 interface and the interactions between the FG corner and the tyrosines of the C terminus. Iron-metal and meso-proto heme hybrid forms of native, mutant, PEGolated, cross-bridged and pseudo cross-bridged Hb's will be used to create a range of tertiary structures and provide a means of probing subunit specific domains. Subunits with tryptophan replaced with 5-OH tryptophan will be used to provide subunit specificity for the fluorescence studies.
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