Scientific Publications by FDA Staff

Free Radic Biol Med 2014 Apr;69:265-77

Redox Properties of Human Hemoglobin in Complex with Fractionated Dimeric and Polymeric Human Haptoglobin.

Mollan TL, Jia Y, Banerjee S, Wu G, Kreulen RT, Tsai AL, Olson JS, Crumbliss AL, Alayash AI

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 approximately 0.9muM-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 +59mV (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.