Enhancing nitrite reductase activity of modified hemoglobin: bis-tetramers and their PEGylated derivatives

The clinical evaluation of stabilized tetrameric hemoglobin as alternatives to red cells revealed that the materials caused significant increases in blood pressure and related problems and this was attributed to the scavenging of nitric oxide and extravasation. The search for materials with reduced vasoactivity led to the report that conjugates of hemoglobin tetramers and polyethylene glycol (PEG) chains did not elicit these pressor effects. However, this material does not deliver oxygen efficiently due to its lack of cooperativity and high oxygen affinity, making it unsuitable as an oxygen carrier. It has been recently reported that PEG-conjugated hemoglobin converts nitrite to nitric oxide at a faster rate than does the native protein, which may compensate for the scavenging of nitric oxide. It is therefore important to alter hemoglobin in order to enhance nitrite reductase activity while retaining its ability to deliver oxygen. If the beneficial effect of PEG is associated with the increased size reducing extravasation, this can also be achieved by coupling cross-linked tetramers to one another, giving materials with appropriate oxygen affinity and cooperativity for use as circulating oxygen carriers. In the present study it is shown that cross-linked bis-tetramers with good oxygen delivery potential have enhanced nitrite reductase activity with k(obs) = 0.70 M(-1) s(-1) (24 degrees C), compared to native protein and cross-linked tetramers, k(obs) = 0.25 M(-1) s(-1) and k(obs) = 0.52 M(-1) s(-1), respectively, but are less active in reduction of nitrite than Hb-PEG5K(2) (k(obs) = 2.5 M(-1) s(-1)). However, conjugation of four PEG chains to the bis-tetramer (at each beta-Cys-93) produces a material with greatly increased nitrite reductase activity (k(obs) = 1.8 M(-1) s(-1)) while retaining cooperativity (P(50) = 4.1, n(50) = 2.4). Thus, PEGylated bis-tetramers combine increased size and enhanced nitrite reductase activity expected for decreased vasoactivity with characteristics of an acceptable HBOC.