The objective of this project is to investigate approaches for altering the oxygen affinity of human hemoglobin in ways which should be relevant to the rationale design of clinically useful blood substitute. To this end we will use site directed mutagenesis to attempt to construct derivatives of human hemoglobin with an oxygen affinity and retention time in circulation similar to blood. The tetrameric Hb molecule will be stabilized by constructing a plasmid in which the two beta-globins are joined by a crosslinker peptide {beta1- (aa)n-beta2}. The crosslinker peptide will be designed to introduce in the Hb molecule a decreased oxygen affinity. Intermolecular crosslinker will be designed on the model of Hb Porto Alegre beta(9)Ser-Cys, in which S-S bonds are formed between Hb molecules - {Sbeta1 beta2S}-{Sbeta1 beta2S}-. We will attempt to decrease the oxygen affinity of this derivative by amino acid substitutions which should introduce the mechanism of oxygen affinity modulation by the C1 of the solvent, present in the beta-chains of bovine Hb. Alternatively site specific mutation known to decrease the oxygen affinity of natural Hb can be introduced. Combination of the intra and intermolecular crosslinks will be used to engineer octameric {beta2-(aa)n-beta1S}-{Sbeta1-(aa)n-beta2} or superpolymeric derivatives - {Sbeta1-(aa)n-beta2S}-{Sbeta1-(aa)n[beta2S}- {Sbeta1-(aa)n-beta2S}- The larger size of these hemoglobin derivatives and the increased stabilization produced by the intra and intermolecular crosslinks should greatly increase their retention time in circulation. While an isooncotic solution of Hb has a concentration of 7%, polymeric hemoglobins can be used at higher concentration, with an increase in the oxygen carrying capacity of the solutions. The proposed system will produce hemoglobin derivatives which are retained in circulation and have an oxygen delivery capacity similar to blood.
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