ABSTRACT
We report the utility of native-state mass spectrometry to detect zinc ion binding to the engineered hemoglobin rHb52. Various preparations of this recombinant hemoglobin had significantly different oxygen affinities. Detailed characterization of denatured globins did not show any difference between analyzed hemoglobin molecules. However, when solutions of intact hemoglobin pseudotetramers were analyzed by native-state electrospray mass spectrometry, a significant shift in the mass spectrum was observed, indicating labile modification of hemoglobin. Using collision-induced dissociation (CID), we found a mass gain of 63 Da located on the beta-globin. EDTA treatment of modified hemoglobin prior to the infusion removed the modification and restored the predicted oxygen affinity. Ion-trap fragmentation of the +8 charged ion of modified beta-globin showed a neutral loss of 96+/-1 Da, consistent with neutral loss of zinc sulfide. These findings indicated zinc binding to the beta-globin through a cysteine residue. Involvement of Cys93 was confirmed by kinetics of cysteine residue reactivity with dithiodipyridine and peptide mapping. Presence of zinc was confirmed by ICP-MS metal analysis.
Subject(s)
Hemoglobins/metabolism , Mass Spectrometry/methods , Zinc/metabolism , Binding Sites , Chromatography, Liquid , Edetic Acid/chemistry , Hemoglobins/chemistry , Isoelectric Focusing , Oxygen/metabolism , Peptide Mapping , Protein Engineering , Sulfhydryl Compounds/chemistry , Trypsin/metabolismABSTRACT
Administration of extracellular hemoglobin-based oxygen carriers often induces mild increases in blood pressure. In order to test whether nitric oxide (NO) scavenging is responsible for the hypertensive effect, we constructed and tested a set of recombinant hemoglobins that vary in rates of reaction with NO. The results suggest that the rapid reactions of oxy- and deoxyhemoglobin with nitric oxide are the fundamental cause of the hypertension. The magnitude of the blood-pressure effect correlates directly with the in vitro rate of NO oxidation. Hemoglobins with decreased NO-scavenging activity may be more suitable for certain therapeutic applications than those that cause depletion of nitric oxide.
