title: Glutathionylation induced structural changes in oxy human hemoglobin analyzed by backbone amide hydrogen/deuterium exchange and MALDI-mass spectrometry.
abstract: Glutathionyl hemoglobin, a post-translationally modified form of hemoglobin, has been reported to serve as a marker of oxidative stress in several clinical conditions. This modification causes perturbations in the hemoglobin functionality by increasing oxygen affinity and reducing cooperativity. Moreover, glutathionylation of sickle hemoglobin was reported to lead to a significant reduction in the propensity of sickling of erythrocytes. The root cause of the above functional abnormality is not known in detail, as the crystal structure of the molecule is yet to be discovered. In this study, we investigated the effects of glutathionylation on quaternary structure of hemoglobin using hydrogen/deuterium exchange (H/DX) based mass spectrometry. H/DX kinetics of nine peptides from α and β globin chains of hemoglobin were analyzed to understand the conformational change in deoxy to oxy transition of normal hemoglobin and structural perturbations associated with glutathionylation of oxy hemoglobin. Significant structural changes brought about by the glutathionylation of oxy hemoglobin were observed in the following regions of globin chains: β86-102, β1-14, α34-46, β32-41, β130-146, β115-129, β73-81. Isotope exchange kinetics monitored through mass spectrometry is a useful technique to understand structural perturbation on post-translational modification of proteins in solution phase.
authors.affiliation: Clinical Proteomics Unit, Division of Molecular Medicine, St. John's Research Institute,  St. John's National Academy of Health Sciences , 100 ft Road, Koramangala, Bangalore -560034, India.
authors.forename: Gopa
authors.initials: G
authors.lastname: Mitra
authors.forename: Monita
authors.initials: M
authors.lastname: Muralidharan
authors.forename: Sreekala
authors.initials: S
authors.lastname: Narayanan
authors.forename: Jennifer
authors.initials: J
authors.lastname: Pinto
authors.forename: Krishnamachari
authors.initials: K
authors.lastname: Srinivasan
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Mandal
mesh_terms: D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement; D004912:Erythrocytes / Q000737:chemistry; D005978:Glutathione / Q000138:chemical synthesis*; D006454:Hemoglobins / Q000138:chemical synthesis*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry; D010108:Oxyhemoglobins / Q000737:chemistry* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055542:alpha-Globins / Q000737:chemistry*; D055544:beta-Globins / Q000737:chemistry*
chemical_list: D006454:Hemoglobins; D010108:Oxyhemoglobins; D012996:Solutions; D055542:alpha-Globins; D055544:beta-Globins; C404464:glutathionyl hemoglobin; D005978:Glutathione; D010100:Oxygen