MP4OX was prepared by reacting human stroma-free hemoglobin (SFH) with 2-iminothiolane and maleimide-activated PEG to produce a PEG-hemoglobin conjugate, with approximately 8 PEGs/Hb. Rates of the deoxyHb/nitrite reaction were measured by mixing deoxygenated SFH or MP4OX anaerobically with a 10-fold excess of sodium nitrite in a sealed cuvette in the presence of sodium dithionite. The reaction was monitored spectrophotometrically, and the spectra were deconvoluted using parent spectra for deoxyhemoglobin, iron-nitrosyl-hemoglobin (HbNO), and methemoglobin. Rates of the oxyHb/nitrite reaction were measured by mixing oxygenated SFH or MP4 with a 20-fold excess of nitrite. These spectra were deconvoluted using parent spectra for oxyhemoglobin, methemoglobin, and nitrite-bound methemoglobin.
Time courses for both deoxy-and oxyHb reactions showed autocatalytic properties that deviated from pseudo first-order kinetics. Therefore, the maximum reaction rates were used for comparison purposes. Fully deoxygenated SFH and MP4OX (i.e., MP4DX) reduced nitrite to NO with maximum rates of 0.022 and 0.206 ¦ÌM/s, respectively, showing a nearly 10-fold higher rate for MP4DX compared to deoxySFH. For the oxyHb/nitrite reaction, SFH and MP4OX showed maximum rates of 0.55 and 0.92 ¦ÌM/s, respectively, a nearly 2-fold enhancement for MP4OX over SFH.
Our results demonstrate that deoxygenated MP4OX is more effective at reducing nitrite to NO than unmodified Hb. In addition, oxygenated MP4OX reacts more rapidly with nitrite, which may then accelerate the release of NO from heme sites. Taken together, these enhanced nitrite reaction rates for MP4 may compensate for NO scavenging and explain, at least in part, why MP4OX does not induce vasoconstriction in vivo. The increased reaction rates of MP4OX appear to be due to R-state stabilization due to the PEG conjugation. Further studies are required to understand the extent to which MP4OX¡¯s interactions with nitrite contribute to its ability to improve perfusion and oxygenation of ischemic tissues.
This work was funded in part by Sangart Inc. and under US Army Contract No. W81XWH1020111 to Sangart.