| The development of a potential blood substitute has been a goal of researchers for many years. The most promising candidate, thus far, is modified hemoglobin. Many cross-linked products have been developed over recent years, and the most studied have been diaspirin cross-linked hemoglobins. This dissertation describes the analysis of several new diaspirin cross-linkers of varying length (n = number of methyl groups in the spacer) and their reactions with hemoglobin. Decreasing yields of cross-linked product were seen as the length of the reagent was increased. The highest oxygen affinity was found be suberoyl cross-linked deoxy hemoglobin, which also had significant cooperativity. Each cross-linked product exhibited increased thermal stability from HbA, with a difference in the increase seen between the n = 5 and n = 6 cross-linked hemoglobins.; A comparative investigation of the reaction pathways of two diaspirin cross-linkers (n = 2 and n = 8) was performed using the Conjugate Peak Refinement (CPR) algorithm. This calculation was designed to examine the ability of the cross-linkers to enter the central cavity of HbA where the α99 lysines are. CPR examines the pathway from the starting structure, where the cross-linker is positioned outside the central cavity, and the final structure, which positions the reagent at the α99 lysines. It was found that the short reagent, bis (3,5-dibromosalicyl) succinate (n = 2), had the lowest energy barrier when entering the cavity of deoxy HbA. There was an increased energy barrier to cross when attempting to enter the oxy conformation, indicating the difficulty with reacting at that site. The longer reagent, bis (3,5-dibromodalicyl) sebacate (n = 8), has more difficulty entering the central cavity of deoxy HbA, indicated by a high energy barrier. Its ability to reach the α99 lysines of oxy HbA is prohibited by an even higher energy barrier. The results indicate that the method may be used to eliminate reaction sites that may not be reached by a particular reagent.; Molecular dynamics calculations were run for oxy and deoxy HbA as well as three diaspirin cross-linked hemoglobins, α99xlHbA (DBSF cross-linked at the α99 lysines), P82xlHbA (DBSF cross-linked at the 082 lysines) and sebxlHbA (DBSS cross-linked HbA at the 082 lysines). The simulations were run for 200 ps and the atomic motions of the resulting trajectories were analyzed. The three cross-linked structures deviated the most from the starting crystal structure, due to the presence of the cross-link. There was no correlation seen with the flexibility of the residues in the α1β 2 interface and oxygen affinity. The overall flexibility of P82xlHbA was higher than the other structures, while oxy HbA had the lowest flexibility, which indicates it was the most stable structure. The α-chains follow the expected results for flexibility, with the low affinity structures, sebxlHbA and α99xlHbA having the lowest flexibility. The data suggest an importance in the α-chains for the regulation of oxygen affinity. |
