title: Application of native-state electrospray mass spectrometry to identify zinc-binding sites on engineered hemoglobin.
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.
authors.affiliation: Baxter Hemoglobin Therapeutics, 2545 Central Avenue, Boulder, Colorado 80301, USA.
authors.forename: J
authors.initials: J
authors.lastname: Lippincott
authors.forename: T J
authors.initials: TJ
authors.lastname: Fattor
authors.forename: D D
authors.initials: DD
authors.lastname: Lemon
authors.forename: I
authors.initials: I
authors.lastname: Apostol
mesh_terms: D001665:Binding Sites; D002853:Chromatography, Liquid; D004492:Edetic Acid / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D007525:Isoelectric Focusing; D013058:Mass Spectrometry / Q000379:methods*; D010100:Oxygen / Q000378:metabolism; D010449:Peptide Mapping; D015202:Protein Engineering; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
chemical_list: D006454:Hemoglobins; D013438:Sulfhydryl Compounds; D004492:Edetic Acid; D014357:Trypsin; D015032:Zinc; D010100:Oxygen