Application of native-state electrospray mass spectrometry to identify zinc-binding sites on engineered hemoglobin
| Autor: | Julie Lippincott, Douglas D. Lemon, Timothy J. Fattor, Izydor Apostol |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Biophysics
chemistry.chemical_element Zinc Mass spectrometry Protein Engineering Biochemistry Peptide Mapping Mass Spectrometry Hemoglobins Native state Trypsin Globin Sulfhydryl Compounds Molecular Biology Zinc ion binding Edetic Acid Chromatography Binding Sites Cell Biology Oxygen chemistry Mass spectrum Hemoglobin Isoelectric Focusing Cysteine Chromatography Liquid |
| Zdroj: | Analytical biochemistry. 284(2) |
| ISSN: | 0003-2697 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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