Structural Perturbation of Human Hemoglobin on Glutathionylation Probed by Hydrogen鈭扗euterium Exchange and MALDI Mass Spectrometry

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• 作者：Gopa Mitra ; Monita Muralidharan ; Jennifer Pinto ; Krishnamachari Srinivasan ; Amit Kumar Mandal
• 刊名：Bioconjugate Chemistry
• 出版年：2011
• 出版时间：April 20, 2011
• 年：2011
• 卷：22
• 期：4
• 页码：785-793
• 全文大小：946K
• 年卷期：v.22,no.4(April 20, 2011)
• ISSN：1520-4812

文摘

Glutathionyl hemoglobin, an example of post-translationally modified hemoglobin, has been studied as a marker of oxidative stress in various diseased conditions. Compared to normal hemoglobin, glutathionyl hemoglobin has been found to have increased oxygen affinity and reduced cooperativity. However, detailed information concerning the structural perturbation of hemoglobin associated with glutathionylation is lacking. In the present study, we report structural changes associated with glutathionylation of deoxyhemoglobin by hydrogen/deuterium (H/D) exchange coupled to matrix assisted laser desorption ionization (MALDI) mass spectrometry. We analyzed isotope exchange kinetics of backbone amide hydrogen of eleven peptic peptides in the deoxy state of both hemoglobin and glutathionyl hemoglobin molecules. Analysis of the deuterium incorporation kinetics for both molecules showed structural changes associated with the following peptides: 伪34鈭?6, 伪1鈭?9, 尾32鈭?1, 尾86鈭?02, 尾115鈭?29, and 尾130鈭?46. H/D exchange experiments suggest that glutathionylation of hemoglobin results in a change in conformation located at the above-mentioned regions of the hemoglobin molecule. MALDI mass spectrometry based H/D exchange experiment might be a simple way of monitoring structural changes associated with post-translational modification of protein.