Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin

Abstract

Haptoglobin (Hp) is an abundant and conserved plasma glycoprotein, which binds acellular adult hemoglobin (Hb) dimers with high affinity and facilitates their rapid clearance from circulation following hemolysis. Humans possess three main phenotypes of Hp, designated Hp 1-1, Hp 2-1, and Hp 2-2. These variants exhibit diverse structural configurations and have been reported to be functionally non-equivalent. We have investigated the functional and redox properties of Hb-Hp complexes prepared using commercially fractionated Hp and found that all forms exhibit similar behavior. The rate of Hb dimer binding to Hp occurs with bimolecular rate constants of ~0.9 μM−1s−1, irrespective of the type of Hp assayed. Although Hp binding does accelerate the observed rate of HbO2 autooxidation by dissociating Hb tetramers into dimers, the rate observed for these bound dimers is 3- to 4-fold slower than that of Hb dimers free in solution. Co-incubation of ferric Hb with any form of Hp inhibits heme loss to below detectable levels. Intrinsic redox potentials (E1/2) of the ferric/ferrous pair of each Hb-Hp complex are similar, varying from +54 to +59 mV (vs NHE), and are essentially the same as reported by us previously for Hb-Hp complexes prepared from unfractionated Hp. All Hb-Hp complexes generate similar high amounts of ferryl Hb following exposure to hydrogen peroxide. EPR data indicate that the yields of protein-based radicals during this process are approximately 4% to 5%, and are unaffected by the variant of Hp assayed. These data indicate that the Hp fractions examined are equivalent to each other with respect to Hb binding and associated stability and redox properties, and that this result should be taken into account in the design of phenotype-specific Hp therapeutics aimed at countering Hb-mediated vascular disease.

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding

“…Although the structure of the CO form of rHb ␣(H58L)␤(WT) shows no differences in the ␣ 1 ␤ 2 interface in the tetramer, an increase in the tetramer to dimer equilibrium dissociation constant, K 4,2 , could account for the large autoxidation rate of the mutant. Adult human HbO 2 dimers autoxidize roughly 20 -30 times more rapidly than tetramers and lose hemin at much higher rates as well (17,18). As controls, we examined the gel filtration elution profiles of the reduced CO forms of HbA and rHb Kirklareli as a function of total protein concentration using the methods described by Manning et al (19).…”

“…Measurement of Tetramer to Dimer Dissociation ConstantsThe high rates of autoxidation and hemin loss for Hb Kirklareli could also be due to dissociation into dimers, which autoxidize roughly 20 times faster than tetramers and lose hemin roughly 10-fold more rapidly (17). To compare the tetramer-dimer disassociation constant (K 4,2 ) for rHb ␣(H58L)␤(WT) and HbA, we used the analytical gel filtration analysis developed by Manning et al (19 -21), (Fig.…”

Oxidized Ferric and Ferryl Forms of Hemoglobin Trigger Mitochondrial Dysfunction and Injury in Alveolar Type I Cells

“…Oxidized Hbs are generally less stable than the ferrous forms, due to unfolding of the proteins and subsequent heme loss (25 , but these expressions were lower in intensity than HO-1 content induced by the ferric form. The persistence of the ferryl iron in solutions and its radical protein together are, however, more damaging to biological molecules and tissues than the HbFe 31 (24).…”

“…Adult HbFe 21 and HbFe 31 were prepared as previously described (25). HbFe 41 was produced by addition of 10-fold molar excess of H 2 O 2 to endotoxin-free HbFe 31 in potassium phosphate buffer (pH 7.4).…”

“…HbFe 41 was produced by addition of 10-fold molar excess of H 2 O 2 to endotoxin-free HbFe 31 in potassium phosphate buffer (pH 7.4). Spectral identification and quantification of HbFe 21 and HbFe 31 were performed based on published extinction coefficients (25). The HbFe 41 was quantified using sodium sulfide (Na s S) derivatization, as previously described (26 31 , and HbFe 41 (in equimolar ratio to Hp) were then added immediately.…”