Application of native-state electrospray mass spectrometry to identify zinc-binding sites on engineered hemoglobin

We report the utility of native-state mass spectrometry to detect zinc ion binding to the engineered hemoglobin rHb52. Various preparations of this re combinant hemoglobin had significantly different oxygen affinities. Detaile d characterization of denatured globins did not show any difference between analyzed hemoglobin molecules. However, when solutions of intact hemoglobi n pseudotetramers were analyzed by native-state electrospray mass spectrome try, a significant shift in the mass spectrum was observed, indicating labi le modification of hemoglobin. Using collision-induced dissociation (CID), we found a mass gain of 63 Da located on the p-globin, EDTA treatment of mo dified hemoglobin prior to the infusion removed the modification and restor ed the predicted oxygen affinity. Ion-trap fragmentation of the +8 charged ion of modified beta-globin showed a neutral loss of 96 +/- 1 Da, consisten t with neutral loss of zinc sulfide, These findings indicated zinc binding to the beta-globin through a cysteine residue. Involvement of Cys93 was con firmed by kinetics of cysteine residue reactivity with dithiodipyridine and peptide mapping. Presence of zinc was confirmed by ICP-MS metal analysis. (C) 2000 Academic Press.