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.

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Significance: The broad classes of O 2 -binding proteins known as hemoglobins (Hbs) carry out oxygenation and redox functions that allow organisms with significantly different physiological demands to exist in a wide range of environments. This is aided by allosteric controls that modulate the protein's redox reactions as well as its O 2 -binding functions. Recent Advances: The controls of Hb's redox reactions can differ appreciably from the molecular controls for Hb oxygenation and come into play in elegant mechanisms for dealing with nitrosative stress, in the malarial resistance conferred by sickle cell Hb, and in the as-yet unsuccessful designs for safe and effective blood substitutes. Critical Issues: An important basic principle in consideration of Hb's redox reactions is the distinction between kinetic and thermodynamic reaction control. Clarification of these modes of control is critical to gaining an increased understanding of Hb-mediated oxidative processes and oxidative toxicity in vivo. Future Directions: This review addresses emerging concepts and some unresolved questions regarding the interplay between the oxygenation and oxidation reactions of structurally diverse Hbs, both within red blood cells and under acellular conditions. Developing methods that control Hbmediated oxidative toxicity will be critical to the future development of Hb-based blood substitutes. Antioxid. Redox Signal. 18, 2298-2313. General PrinciplesEmergence of proteins capable of transporting O 2 I n this review, we examine the adaptive changes in the molecular controls of hemoglobin (Hb) oxygenation and oxidation that have evolved to meet the highly varied physiological and environmental demands of respiring organisms. Globins came into being during the planet's long early period of anoxia/hypoxia, and recent studies show that globins are either expressed or inducible in almost all cells (98). Studies have shown that one evolutionary pathway of Hb is that of a multipurpose domain attached to a variety of unrelated proteins, thus forming molecules with different functions (126). This pathway has allowed structurally distinct Hbs to evolve: (i) to protect against the high levels of nitrosative stress of the earth's early environment; (ii) to protect against O 2 -linked oxidation; (iii) to act as O 2 sensors that help regulate the expression of proteins during periods of hypoxia or anoxia; and (iv) to enable aerobic respiration by facilitating diffusion and/or acting as O 2 carriers (44,56,57,70,71,73,107). A common theme in the fascinating story of Hb evolution is the emergence of distinct mechanisms for controlling Hb's oxygenation and redox functions.Since increased amounts of O 2 were released in our planet's early history, O 2 toxicity brought about species extinction on a global scale. On the other hand, this ''oxygen pollution'' made possible a new biological process, that of aerobic respiration. A tremendous gain in the energy obtainable from oxidation of energy-rich metabolites was achieved when organisms evolve...

Described here is a mass spectrometry based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the slow H/D exchange reaction of the imidazole C2 proton in histidine side chains. The protocol, which involves evaluating the denaturant dependence of this slow H/D exchange reaction in proteins, allows the global and/or subglobal unfolding/refolding properties of proteins and protein-ligand complexes to be probed. The protocol is developed using several model protein systems including: ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA) II, hemoglobin, and the hemoglobin-haptoglobin protein complex. The compatibility of the protocol with conventional mass spectrometry-based proteomic sample preparation and analysis methods is also evaluated in an experiment in which the protocol is applied to proteins in a yeast cell lysate and used to detect the binding of Zn to superoxide dismutase in the yeast cell lysate sample. The yeast cell sample analyses also helped define the scope of the technique, which requires the presence of globally protected histidine residues in a protein’s three-dimensional structure for successful application.

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Background: ␣-Hemoglobin stabilizing protein (AHSP) modifies the redox properties of bound ␣-subunits. Results: Isolated hemoglobin subunits exhibit significantly different redox properties compared with HbA. A significant decrease in the reduction potential of ␣-subunits bound to AHSP results in preferential binding of ferric ␣. Conclusion: AHSP⅐␣-subunit complexes do not participate in ferric-ferryl heme redox cycling. Significance: AHSP binding to ␣-subunits inhibits subunit pseudoperoxidase activity.