title: Polyethylene glycol conjugation enhances the nitrite reductase activity of native and cross-linked hemoglobin.
abstract: Although stabilized hemoglobins have been evaluated as oxygen-carrying replacements for red cells in transfusions, in vivo evaluations have noted that these materials are associated with vasoactivity, a serious complication. Scavenging of endogenous nitric oxide by the deoxyheme sites of the stabilized proteins is one likely source of vasoactivity. Recent reports indicate that modification of cell-free hemoglobin derivatives with multiple chains of polyethylene glycol (PEG) suppresses vasoactivity. Gladwin and co-workers observed that the nitrite reductase activity of hemoglobin serves as a major endogenous source of nitric oxide. If PEG conjugation leads to enhanced nitrite reductase activity, this could compensate for scavenged endogenous nitric oxide. To test this possibility, the rates of conversion of nitrite ion to nitric oxide by altered hemoglobins with and without PEG were measured at 25 degrees C. Fumaryl (alpha99-alpha99) cross-linked hemoglobin reacts with nitrite with a bimolecular rate constant of 0.52 M (-1) s (-1), which is comparable to that associated with native hemoglobin (0.25 M (-1) s (-1)). Addition of PEG chains to the cross-linked hemoglobin at beta-Cys93 (alphaalpha-Hb-PEG5K 2) results in a material that produces nitric oxide much more rapidly ( k = 1.41 M (-1) s (-1)). R-State-stabilized hemoglobins with multiple PEG chains (Hb-PEG5K 2 and Hb-PEG5K 6) react 10 times faster with nitrite to produce nitric oxide than does native hemoglobin ( k = 2.5 and 2.4 M (-1) s (-1), respectively). These results, showing enhanced production of nitric oxide resulting from an increased proportion of the protein residing in the R-state, are consistent with the decrease in vasoactivity associated with PEG conjugation.
authors.affiliation: Davenport Chemical Research Laboratories, Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada.
authors.forename: Francine E
authors.initials: FE
authors.lastname: Lui
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Dong
authors.forename: Ronald
authors.initials: R
authors.lastname: Kluger
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016014:Linear Models; D015394:Molecular Structure; D009569:Nitric Oxide / Q000378:metabolism; D009572:Nitrite Reductases / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry*
chemical_list: D006454:Hemoglobins; D009569:Nitric Oxide; D011092:Polyethylene Glycols; D004228:Dithionitrobenzoic Acid; D009572:Nitrite Reductases