De Jesús Bonilla, Walleska

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    Oxidation reactions of Lucina pectinata hemoglobins: Model system to design heme protein based blood substitutes
    (2008) De Jesús Bonilla, Walleska; López Garriga, Juan; College of Arts and Sciences - Sciences; Torres Lugo, Madeline; Cortés Figueroa, José E.; Cora, Elsa M.; Department of Chemistry; Cardona Martínez, Nelson
    Today, efforts are focused in the production of a second-generation blood substitutes that minimizes the oxidative stress and the tissue vasoactivity. The infused cell-free hemoglobin’s can act as nitric oxide (NO) scavenger inducing vasoconstriction and affecting blood pressure. Also, it can autoxide producing reactive oxygen species such as the superoxide ion and H2O2. These reactions produce the heme-Fe(IV) ferryl species, which promote hemolysis. Regarding this, Lucina pectinata hemoglobins HbI and HbII were used to explore correlations between structure and the oxidative stability in the reactions with H2O2 and NO. The HbI and HbII distal pocket active site has the conserved amino acids GlnE7 (also present in elephant and shark myoglobin), PheCD1, and PheE11. The other amino acid is PheB10 in HbI, while HbII has TyrB10. The HbI PheB10Tyr replacement was used to compare the active centers. The results indicate that the human cross-linked α-DBBF-Hb has an autoxidation rate constant (kautox) of 0.090 h-1. The autoxidation rate constant for HbI was 0.055 h-1. However, HbII and HbI PheB10Tyr have significant lower autoxidation rates of 0.003 h-1, and 0.008 h-1, respectively. The kinetic rate constant for the reaction of α-DBBF-Hb with H2O2 producing high amounts of fluorescent heme degradation products is 8.0 M-1s-1. The heme degradation for HbII (0.61 M-1s-1) and HbI PheB10Tyr (0.58 M-1s-1) is the smaller of the hemoglobins, while HbI has the higher rate constant (16.73 M-1s-1). This data suggests that TyrB10 significantly contributes to reduce the oxidation caused by H2O2. The kinetic rate constant for the oxy Hb oxidation with NO for α-DBBF-Hb and HbII is 18.95 µM-1s-1, and 2.8 µM-1s-1, and for HbI and HbI PheB10Tyr is 91.6 M-1s-1, and 49.97 M-1s-1, respectively. These reactions suggest that in addition to TyrB10, there are other factors in HbII affecting the NO entrance to the distal site leading to the slow HbII oxidation by this ligand. Furthermore, experiments with endothelial cells show that under induced oxidative stress L. pectinata hemoglobins stimulate mild apoptosis to cells when forming the ferryl species in the reactions with hydrogen peroxide. Moreover, the exposition of the hemoglobins during hypoxia increases the HIF-1α levels, thus suggesting that the cell redox signaling and death pathways are altered. This suggests novel capabilities, especially for HbII, to carry oxygen under hypoxic conditions or oxygen stress. Taken together, our results suggest that L. pectinata HbII may represent a good model for the design of future oxidative stable oxygen carrier with little or no vasoactivity.