Significance: The worldwide blood shortage has generated a significant demand for alternatives to whole blood and packed red blood cells for use in transfusion therapy. One such alternative involves the use of acellular recombinant hemoglobin (Hb) as an oxygen carrier. Recent Advances: Large amounts of recombinant human Hb can be expressed and purified from transgenic Escherichia coli. The physiological suitability of this material can be enhanced using protein-engineering strategies to address specific efficacy and toxicity issues. Mutagenesis of Hb can (i) adjust dioxygen affinity over a 100-fold range, (ii) reduce nitric oxide (NO) scavenging over 30-fold without compromising dioxygen binding, (iii) slow the rate of autooxidation, (iv) slow the rate of hemin loss, (v) impede subunit dissociation, and (vi) diminish irreversible subunit denaturation. Recombinant Hb production is potentially unlimited and readily subjected to current good manufacturing practices, but may be restricted by cost. Acellular Hb-based O 2 carriers have superior shelf-life compared to red blood cells, are universally compatible, and provide an alternative for patients for whom no other alternative blood products are available or acceptable. Critical Issues: Remaining objectives include increasing Hb stability, mitigating iron-catalyzed and iron-centered oxidative reactivity, lowering the rate of hemin loss, and lowering the costs of expression and purification. Although many mutations and chemical modifications have been proposed to address these issues, the precise ensemble of mutations has not yet been identified. Future Directions: Future studies are aimed at selecting various combinations of mutations that can reduce NO scavenging, autooxidation, oxidative degradation, and denaturation without compromising O 2 delivery, and then investigating their suitability and safety in vivo. Antioxid. Redox Signal. 18, 2314-2328.

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 biosynthesis in erythrocyte precursors involves several steps. The correct ratios and concentrations of normal alpha (a) and beta (b) globin proteins must be expressed; apoproteins must be folded correctly; heme must be synthesized and incorporated into these globins rapidly; and the individual a and b subunits must be rapidly and correctly assembled into heterotetramers. These events occur on a large scale in vivo, and dysregulation causes serious clinical disorders such as thalassemia syndromes. Recent work has implicated a conserved erythroid protein known as Alpha-Hemoglobin Stabilizing Protein (AHSP) as a participant in these events. Current evidence suggests that AHSP enhances a subunit stability and diminishes its participation in harmful redox chemistry. There is also evidence that AHSP facilitates one or more early-stage post-translational hemoglobin biosynthetic events. In this review, recent experimental results are discussed in light of several current models describing globin subunit folding, heme uptake, assembly, and denaturation during hemoglobin synthesis. Particular attention is devoted to molecular interactions with AHSP that relate to a chain oxidation and the ability of a chains to associate with partner b chains. Antioxid. Redox Signal. 12, 219-232.

Background: ␣-Hemoglobin stabilizing protein (AHSP) facilitates hemoglobin production. Results: AHSP preferentially binds to ferric versus ferrous ␣ subunits and induces reversible structural alterations within seconds of binding. Conclusion: AHSP exerts its effects by stabilizing a ferric ␣ folding intermediate and inhibiting its participation in hemoglobin assembly. Significance: AHSP is a molecular chaperone for ferric ␣-globin.

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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.

In the last several years, significant work has been done studying hemoglobin (Hb) oxidative reactions and clearance mechanisms using both in vitro and in vivo model systems. One active research area involves the study of molecular chaperones and other proteins that are thought to mitigate the toxicity of acellular Hb. For example, the plasma protein haptoglobin (Hp) and the pre-erythroid protein alpha-hemoglobin-stabilizing protein (AHSP) bind to acellular Hb and alpha-subunits of Hb, respectively, to reduce these adverse effects. Moreover, there has been significant work studying hemopexin and alpha-1 microglobulin, both of which are thought to be involved with hemin degradation. These studies have coincided with the timely publication of the first crystal structure of the Hb-Hp complex. In constructing this Forum, we have invited a number of researchers in the area of Hb and myoglobin (Mb) redox biochemistry, as well as those who have contributed fundamentally to our knowledge of Hp function. Our goal has been to update this critically important research area, because we believe that it will ultimately impact the practice of transfusion medicine in a number of important ways.

Alpha hemoglobin stabilizing protein (AHSP) reversibly binds nascent ␣ globin to maintain its native structure and facilitate its incorporation into hemoglobin A. Previous studies indicate that some naturally occurring human ␣ globin mutations may destabilize the protein by inhibiting its interactions with AHSP. However, these mutations could also affect hemoglobin A production through AHSP-independent effects, including reduced binding to ␤ globin. We analyzed 6 human ␣ globin variants with altered AHSP contact surfaces. Alpha globin amino acid substitutions H103Y, H103R, F117S, and P119S impaired interactions with both AHSP and ␤ globin. These mutations are destabilizing in biochemical assays and are associated with microcytosis and anemia in humans. By contrast, K99E and K99N ␣ globins bind ␤ globin normally but exhibit attenuated binding to AHSP. These mutations impair protein folding and expression in vitro and appear to be mildly destabilizing in vivo. In Escherichia coli and erythroid cells, ␣ globin K99E stability is rescued on coexpression with AHSP mutants in which binding to the abnormal globin chain is restored. Our results better define the biochemical properties of some ␣ globin variants and support the hypothesis that AHSP promotes ␣ globin chain stability during human erythropoiesis. (Blood. 2009;113:5961-5969)