Spin trapping combined with quantitative mass spectrometry defines free radical redistribution within the oxidized hemoglobin:haptoglobin complex.
Extracellular or free hemoglobin (Hb) accumulates during hemolysis, tissue damage, and inflammation. Heme-triggered oxidative reactions can lead to diverse structural modifications of lipids and proteins, which contribute to the propagation of tissue damage. One important target of Hb׳s peroxidase reactivity is its own globin structure. Amino acid oxidation and crosslinking events destabilize the protein and ultimately cause accumulation of proinflammatory and cytotoxic Hb degradation products. The Hb scavenger haptoglobin (Hp) attenuates oxidation-induced Hb degradation. In this study we show that in the presence of hydrogen peroxide (H2O2), Hb and the Hb:Hp complex share comparable peroxidative reactivity and free radical generation. While oxidation of both free Hb and Hb:Hp complex generates a common tyrosine-based free radical, the spin-trapping reaction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) yields dissimilar paramagnetic products in Hb and Hb:Hp, suggesting that radicals are differently redistributed within the complex before reacting with the spin trap. With LC-MS(2) mass spectrometry we assigned multiple known and novel DMPO adduct sites. Quantification of these adducts suggested that the Hb:Hp complex formation causes extensive delocalization of accessible free radicals with drastic reduction of the major tryptophan and cysteine modifications in the β-globin chain of the Hb:Hp complex, including decreased βCys93 DMPO adduction. In contrast, the quantitative changes in DMPO adduct formation on Hb:Hp complex formation were less pronounced in the Hb α-globin chain. In contrast to earlier speculations, we found no evidence that free Hb radicals are delocalized to the Hp chain of the complex. The observation that Hb:Hp complex formation alters free radical distribution in Hb may help to better understand the structural basis for Hp as an antioxidant protein.
Division of Internal Medicine, University Hospital, Zurich, Switzerland.
Florence
F
Vallelian
Laboratory of Physical Chemistry, ETH Zürich, Switzerland; Centro Universitario de la Defensa, carretera de Huesca, Zaragoza, Spain.
Ines
I
Garcia-Rubio
Division of Internal Medicine, University Hospital, Zurich, Switzerland; Functional Genomics Center, University of Zurich, Switzerland.
Michele
M
Puglia
Institute of Molecular Life Sciences, University of Zurich, Switzerland.
Abdullah
A
Kahraman
Division of Internal Medicine, University Hospital, Zurich, Switzerland.
Jeremy W
JW
Deuel
Division of Internal Medicine, University Hospital, Zurich, Switzerland.
Wolfgang R
WR
Engelsberger
Laboratory of Toxicology & Pharmacology, NIEHS/NIH, Research Triangle Park, NC, USA.
Ronald P
RP
Mason
Center of Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA.
Paul W
PW
Buehler
Division of Internal Medicine, University Hospital, Zurich, Switzerland; Institute of Evolutionary Medicine, University of Zurich, Switzerland. Electronic address: dominik.schaer@usz.ch.
Dominik J
DJ
Schaer
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002853:Chromatography, Liquid; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism*; D006242:Haptoglobins / Q000737:chemistry / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism; D019097:Spin Trapping; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000596:Amino Acids; D005609:Free Radicals; D006242:Haptoglobins; D006454:Hemoglobins; D006861:Hydrogen Peroxide; D010544:Peroxidases
Electron paramagnetic resonance (EPR); Haptoglobin; Hemoglobin; Mass spectrometry; Oxidation; Oxidative stress; Radical; Spin trapping