Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin.
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 after 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 nonequivalent. 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(-1) s(-1), irrespective of the type of Hp assayed. Although Hp binding does accelerate the observed rate of HbO2 autoxidation by dissociating Hb tetramers into dimers, the rate observed for these bound dimers is three- to fourfold 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 after exposure to hydrogen peroxide. Electron paramagnetic resonance 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 one another 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.
Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20852, USA.
Todd L
TL
Mollan
Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20852, USA.
Yiping
Y
Jia
Department of Chemistry, Duke University, Durham, NC 27708, USA.
Sambuddha
S
Banerjee
Hematology Division, Department of Internal Medicine, University of Texas-Houston Medical School, Houston, TX 77030, USA.
Gang
G
Wu
Department of Chemistry, Duke University, Durham, NC 27708, USA.
R Timothy
RT
Kreulen
Hematology Division, Department of Internal Medicine, University of Texas-Houston Medical School, Houston, TX 77030, USA.
Ah-Lim
AL
Tsai
Biochemistry and Cell Biology Department, Rice University, Houston, TX 77251, USA.
John S
JS
Olson
Department of Chemistry, Duke University, Durham, NC 27708, USA.
Alvin L
AL
Crumbliss
Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20852, USA. Electronic address: abdu.alayash@fda.hhs.gov.
Abdu I
AI
Alayash
D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D006242:Haptoglobins / Q000737:chemistry / Q000235:genetics*; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics; D010084:Oxidation-Reduction; D010641:Phenotype; D011108:Polymers / Q000737:chemistry
D006242:Haptoglobins; D006454:Hemoglobins; D046912:Multiprotein Complexes; D011108:Polymers; D006861:Hydrogen Peroxide
EPR; Ferryl; Free radicals; Haptoglobin; Heme loss; Hemoglobin A; Iron(IV) oxo; Protein-based radical; Redox potential; Spectroelectrochemistry; Sulfhemoglobin