Haptoglobin binding stabilizes hemoglobin ferryl iron and the globin radical on tyrosine β145.
OBJECTIVE
Hemoglobin (Hb) becomes toxic when released from the erythrocyte. The acute phase protein haptoglobin (Hp) binds avidly to Hb and decreases oxidative damage to Hb itself and to the surrounding proteins and lipids. However, the molecular mechanism underpinning Hp protection is to date unclear. The aim of this study was to use electron paramagnetic resonance (EPR) spectroscopy, stopped flow optical spectrophotometry, and site-directed mutagenesis to explore the mechanism and specifically the role of specific tyrosine residues in this protection.


RESULTS
Following peroxide challenge Hb produces reactive oxidative intermediates in the form of ferryl heme and globin free radicals. Hp binding increases the steady state level of ferryl formation during Hb-catalyzed lipid peroxidation, while at the same time dramatically inhibiting the overall reaction rate. This enhanced ferryl stability is also seen in the absence of lipids and in the presence of external reductants. Hp binding is not accompanied by a decrease in the pK of ferryl protonation; the protonated ferryl species still forms, but is intrinsically less reactive. Ferryl stabilization is accompanied by a significant increase in the concentration of the peroxide-induced tyrosine free radical. EPR spectral parameters and mutagenesis studies suggest that this radical is located on tyrosine 145, the penultimate C-terminal amino acid on the beta Hb subunit.


METHODS
Hp binding decreases both the ferryl iron and free radical reactivity of Hb.


CONCLUSIONS
Hp protects against Hb-induced damage in the vasculature, not by preventing the primary reactivity of heme oxidants, but by rendering the resultant protein products less damaging.
School of Biological Sciences, University of Essex, Essex, United Kingdom. ccooper@essex.ac.uk
Chris E
CE
Cooper
Dominik J
DJ
Schaer
Paul W
PW
Buehler
Michael T
MT
Wilson
Brandon J
BJ
Reeder
Gary
G
Silkstone
Dimitri A
DA
Svistunenko
Leif
L
Bulow
Abdu I
AI
Alayash
D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D006242:Haptoglobins / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D015227:Lipid Peroxidation; D010084:Oxidation-Reduction; D011485:Protein Binding; D055550:Protein Stability; D014443:Tyrosine / Q000378:metabolism*
D005609:Free Radicals; D006242:Haptoglobins; D006454:Hemoglobins; C066317:ferryl iron; D014443:Tyrosine; D006418:Heme; D007501:Iron