Distal pocket mutants of sperm whale oxymyoglobin (oxy-Mb) were reacted with a 2.5-fold excess of hydrogen peroxide (HOOH) in phosphate buffer at pH 7.0, 37°C. We describe a mechanism composed of three distinct steps: 1) initial oxidation of oxy-to ferryl-Mb, 2) autoreduction of the ferryl intermediate to ferric metmyoglobin (metMb), and 3) reaction of metMb with an additional HOOH molecule to regenerate the ferryl intermediate creating a pseudoperoxidase catalytic cycle. Mutation of Leu-29(B10) to Phe slows the initial oxidation reaction 3-fold but has little effect on the rate of ferryl reduction to ferric met-aquo-myoglobin. In contrast, the Val-68(E11) to Phe mutation causes a small, 60%
Chemically modified hemoglobins are potential oxygen-carrying blood substitutes, but their in vivo administration has been associated with a variety of unexpected side events, including increased platelet reactivity. We studied the effects of hemoglobin A0 (HbA0) and alpha-crosslinked hemoglobin (alpha-DBBF) on platelets in vitro. Neither hemoglobin A0 nor alpha-DBBF activated platelets when added alone, but both proteins potentiated submaximal agonist-induced platelet aggregation without increasing other markers of platelet activation such as serotonin secretion. Only agonists that are known to cause release of platelet arachidonic acid (AA) were potentiated while aggregation induced by ADP, which does not release AA, was not potentiated. Blockade of the thromboxane receptor with SQ-29,548 prevented the HbA0-induced and the alpha-DBBF-induced potentiation suggesting that the AA/thromboxane signaling pathway mediates the interaction of platelets with hemoglobin.
The rapid unloading of oxygen to tissue and the prevention of subunit dissociation have been the main concerns in the search for an effective hemoglobin-based red cell substitute. The presence of redox active iron however, raises some questions about its potential to enter into reactions that mediate the formation of cytotoxic oxygen free radicals. We tested the propensity of modified hemoglobins to undergo oxidative damage by peroxide (H2O2). We found differences in their susceptibility to oxidative modification and in their ability to form the highly cytotoxic ferryl species. This protein-associated oxidant may be a physiologically important contributor to reperfusion injury. Another potential mechanism of toxicity involves the reaction of cell-free hemoglobin with endothelium derived nitric oxide (NO). Marked hypertensive responses in intact animals infused with some of these hemoglobins were reported. Cell-free hemoglobin has the potential to bind the endothelial generated NO yielding methemoglobin and nitrate, an extremely rapid reaction in vivo. We describe subsequent redox reactions between NO and methemoglobin which may further deplete NO as a biological transducer, leading to greater effects on the extent of endothelial-dependent responses. The consequences of a potential linkage between oxidative toxicity of cell-free hemoglobin and its interaction with NO is addressed.
We examined how changes in oxygen affinity brought about by different chemical modifications of hemoglobins affect their oxidation-reduction reactions. The three modified hemoglobins studied were HbA-FMDA, HbBv-FMDA, produced by the reaction of human or bovine oxyHb with fumaryl mono-dibromoaspirin; and HbA-DBBF, produced by the reaction of human deoxyHb with bis(3,5-dibromosalicyl) fumarate. Exposure of oxyHb to H2O2 causes generation of free radicals capable of cleaving dimethylsulfoxide (Me2SO) to produce formaldehyde (HCHO). Relative to the reaction rate for HbAo (630 +/- 130 M/min) the rates of HCHO formation were roughly 70% for HbA-DBBF, 50% for HbA-FMDA and 16% for HbBv-FMDA. Exposure to H2O2 also caused spectral changes at varied rates for the HBOCs analyzed. Although these rates were not directly correlated with the rates of free radical formation, addition of mannitol or thiourea slowed both the rate of spectral changes and HCHO formation. The relative ability of the ferric derivatives of the HBOCs to participate in free radical reactions was monitored by assays of non-enzymatic NADPH oxidation and aniline hydroxylation. HbBv-FMDA showed significantly slower rates than the other HBOCs in both assays. The observed differences between HBOCs in these assays indicate differences in their ability to generate or interact with free radicals.
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