Mechanistic Studies of the Oxidation of Pyridoxalated Hemoglobin Polyoxyethylene Conjugate by Nitrogen Monoxide

Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), a modified human-derived hemoglobin, is currently in clinical trials as a nitrogen monoxide scavenger for the treatment of shock. Stopped-flow spectroscopy studies of the reaction between nitrogen monoxide and PHP indicate that at pH 7 the second-order rate constant is (88 ± 3) × 106 M−1 s−1, a value very similar to that for the unmodified human hemoglobin. At alkaline pH the reaction proceeds via the intermediate peroxynitrito complex PHP–FeIIIOONO, which rapidly decomposes to nitrate and the iron(III) form of PHP. The rate of decay of PHP–FeIIIOONO increases significantly with decreasing pH such that it does not accumulate in concentrations large enough to be observed spectroscopically under neutral or acidic conditions. Ion chromatographic analysis of the nitrogen-containing products of the NO•-mediated reaction of PHP shows that nitrate is formed quantitatively at both pH 7 and pH 9.