Correlation between Hemoglobin Oxygen Affinity and Nitrite Reductase Activity

Abstract

Plasma hemoglobin (Hb) has been associated with vasoconstriction that has been attributed to nitric oxide (NO) scavenging by heme. Evidence suggests that the reduction of nitrite to NO at deoxygenated hemes dilates blood vessels under hypoxic conditions. This reaction may be why a high oxygen affinity Hb has been shown not to constrict arterioles despite binding NO. We investigated the nitrite reductase activity of several Hb modification chemistries, including maleimidation, which reacts at Hb β93Cys residues to decrease p50. Maleimides tested include the small molecule, N‐ethylmaleimide, and maleimide‐polyethylene glycol (PEG). Using thiolation chemistry, we increased the degree of PEGylation from 2 to 4 to 8 PEGs, progressively decreasing p50. We also tested Hbs cross‐linked between α or β subunits to produce tetrameric Hbs, with varying p50s, and their maleimide‐PEGylated products. The p50 values ranged from 3 mmHg (for PEGylated ββ‐crosslinked Hb) to 33 mmHg (for αα‐crosslinked Hb). We found a direct correlation between p50 and nitrite reductase activity; Hbs with high oxygen affinity (low p50) reduced nitrite to NO at a higher rate than low affinity Hbs. With a 10‐fold excess of nitrite, the maximum rate of nitrite reduction varied from 0.46 μM/s (for PEGylated ββ‐crosslinked Hb) to 0.015 μM/s (for αα‐crosslinked Hb). Importantly, we found that NO produced by this reaction can escape heme capture and be released from Hb, and the rate of NO release is relative to the nitrite reductase activity. Together, these results suggest that oxygen affinity may be important in cell‐free Hb design, not only to target oxygen delivery, but also to optimize nitrite reductase activity.