Structural analysis of glutathionyl hemoglobin using native mass spectrometry

Area of Science:

• Biochemistry
• Proteomics
• Structural Biology

Background:

• Glutathionylation is a reversible post-translational modification involving cysteine residues and oxidized glutathione (GSSG).
• This modification typically occurs under elevated oxidative stress conditions in vivo.
• In human hemoglobin, Cys93 of the β-globin chain is susceptible to glutathionylation, forming glutathionyl hemoglobin (GSHb).
• GSHb serves as a biomarker for oxidative stress in various clinical conditions like chronic renal failure, diabetes mellitus, and atherosclerosis.

Purpose of the Study:

• To investigate the structural integrity and quaternary architecture of glutathionyl hemoglobin (GSHb).
• To understand how glutathionylation affects the structural stability of human hemoglobin.

Main Methods:

• Native mass spectrometry
• Ion mobility mass spectrometry
• Molecular dynamics simulations

Main Results:

• Dissociation equilibrium constants (Kd1 for tetramer/dimer and Kd2 for dimer/monomer) increased by 1.91-fold and 3.64-fold, respectively, indicating weakened quaternary structure.
• The collision cross-section area of the tetrameric hemoglobin molecule remained unchanged after glutathionylation.
• Molecular dynamics simulations supported the experimental findings on structural integrity.

Conclusions:

• Glutathionylation significantly destabilizes the quaternary structure of human hemoglobin, promoting dissociation.
• Despite altered stability, the overall molecular size (collision cross-section) of the tetrameric GSHb remains comparable to native hemoglobin.
• These structural insights contribute to understanding the functional consequences of GSHb formation under oxidative stress.