Quantitative Mass Spectrometry Defines an Oxidative Hotspot in Hemoglobin that is Specifically Protected by Haptoglobin

Abstract

The reaction of hemoglobin (Hb) with hydrogen peroxide (H(2)O(2)) results in free radicals generated at the heme iron, followed by radical transfer to the porphyrin/globin. In the present work, we employed isobaric tagging for relative and absolute quantification (iTRAQ) and a LC-MALDI-MS/MS-based proteomic approach to identify the extent of oxidative changes within tetrameric Hb and dimeric Hb-haptoglobin (Hb-Hp) complexes. Extensive oxidative modifications were found to be restricted to peptides containing alphaTyr42, betaTyr145, and betaCys93. The protein region composed of these peptides appears to define an area of oxidative activity within the Hb tetramer that extends across the critical alpha1beta2/alpha2beta1 interface. Extensive oxidative modifications occurring at betaCys93 indicate that this surface amino acid is an important end point for free radical induced protein oxidation within Hb. Conversely when Hp 1-1 or 2-2 was complexed with dissociable Hb, oxidative changes in Hp complexed dimeric Hb were prevented. This protection was not observed in a stabilized tetrameric Hb, which displays a weak binding affinity for Hp. Therefore, dimerization of Hb and Hp binding may interfere with free radical translocation and play an important role in the overall antioxidant mechanism of Hp. Interestingly, the prevention of peroxide induced Hb amino acid oxidation in purified Hb-Hp1-1 and Hb-Hp2-2 was found to be equal, indicating a phenotype independent specificity in the process of oxidative protection. Taken together, these data suggest differences in oxidative modifications resulting from peroxide induced heme emanated free radical distribution in tetrameric compared to Hp1-1/Hp2-2 stabilized dimeric Hb.

Sickle Cell Hemoglobin in the Ferryl State Promotes βCys-93 Oxidation and Mitochondrial Dysfunction in Epithelial Lung Cells (E10)

“…Amino acids residues identified by LC-MS/MS analysis in this study correlated well with previously published data (42). XICs were generated from the most abundant monoisotopic peak of each peptide isotopic profile (listed in Table 3), and the resulting ratio differences were compared.…”

Effect of Single Amino Acid Substitution on Oxidative Modifications of the Parkinson’s Disease-Related Protein, DJ-1

“…cysteine oxidation (50 -55) and carbonylation (56)). While this manuscript was being written, Pimenova et.al (57) described a method to simultaneously measure multiple oxidative protein modifications using isobaric tags for relative and absolute quantitation labeling technology. A potential drawback of labels such as isobaric tags for relative and absolute quantitation is that these reagents can produce unpredicted fragmentation patterns which confound the interpretation of MS data (58).…”

Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins

“…32 As a result of this protection, globin oxidation with subsequent protein degradation does not occur when Hb is sequestered in the Hb:Hp complex. 33 The structural stability of the complex may prevent accumulation of proinflammatory Hbdegradation products that can evade clearance by scavenger receptors. 14,34 In addition, hemin resides firmly in the Hb:Hp complex, it cannot transfer to hemin acceptors such as Hpx, lipoproteins, and albumin.…”