Structural Perturbation of Human Hemoglobin on Glutathionylation Probed by Hydrogen−Deuterium Exchange and MALDI Mass Spectrometry

Abstract

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: α34-46, α1-29, β32-41, β86-102, β115-129, and β130-146. 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.

Glutathionylation Induced Structural Changes in Oxy Human Hemoglobin Analyzed by Backbone Amide Hydrogen/Deuterium Exchange and MALDI-Mass Spectrometry

“…In our previous communication, we described the structural changes linked to glutathionylation of the deoxy state of HbA. 10 In this study, we report glutathionylation-associated structural perturbation in the oxy state of HbA using hydrogen/ deuterium exchange (H/DX) based mass spectrometry.…”

“…10 In this study, we report glutathionylation-associated structural perturbation in the oxy state of HbA using hydrogen/ deuterium exchange (H/DX) based mass spectrometry.…”

“…Isolation of hemoglobin was done from whole blood according to the procedure described previously. 10 Glutathionyl hemoglobin was synthesized in vitro following protocol described earlier. 10 GSHb with the abundance of more than 85% was used for structural characterization.…”

“…where M 0 and M ∞ are the observed masses at zero time control experiment and for a fully deuterated molecule, respectively, 10 and N is the total number of backbone amide hydrogens in the peptide.…”

Estrutura oligomérica e dinâmica de Major Royal Jelly Protein 1 (MRJP1)/apisimina analisadas por espectrometria de massas e técnicas complementares

“…Regiões da proteína que não estão envolvidas em pontes de hidrogênio têm uma rápida cinética de troca, enquanto que regiões que estão no interior da proteína ou envolvidas em pontes de hidrogênio têm uma cinética de troca muito mais lenta [122]. Dependendo da acessibilidade do solvente e da força de ligação do hidrogênio, diferentes hidrogênios de amidas na cadeia polipeptídica (N-H) podem diferir nas taxas de trocas isotópicas (Figura 11) [123]. A troca também pode acontecer nos sítios O-H, N-H e S-H das cadeias laterais; no entanto, esses deutérios são perdidos durante a cromatografia líquida (LC), em um processo chamado backexchange.…”