Biopreservation and Reversal of Oxidative Injury During Blood Storage by a Novel Curcumin-based Gel Formulation.

Blood storage lesion induces cytosolic and membrane changes driven in part by hemoglobin (Hb) oxidation reactions within red blood cells (RBCs). A novel gel formulation containing the antioxidant curcuminoids in a biocompatible solvent system was used to deliver curcumin into RBCs. Incubation of peroxide treated RBCs stored in PBS with curcumin gel led to a reduction in prooxidant ferrylHb and recovery in ATP. Curcumin treatment prevented band 3 tyrosine (Y359 and Y21) phosphorylation. RBCs stored in AS-3 solutions for 28, 35, 42 and 49 days, following a single-dose of 100µM curcuminoids at each time points, caused reduction in protein carbonylation and considerable recovery in ATP levels. Proteomic analysis revealed minimal changes in the proteomic landscape in 35 days. However, a downregulation in fibrinogen was observed in the treated samples which may reduce RBC aggregation. Additionally, we used a guinea pig model where the circulation of infused aged RBCs can be extended (approximately 10%) when treated with curcumin gel at the start of storage. Our data therefore provide mechanistic insights and supportive animal data into benefits of treating stored RBCs with a novel curcuminoid formulation based on the biopreservation of RBC membrane integrity, redox balance, and increased longevity in circulation.

Changes in hemoglobin oxidation and band 3 during blood storage impact oxygen sensing and mitochondrial bioenergetic pathways in the human pulmonary arterial endothelial cell model.

Red blood cells (RBCs) undergo metabolic, oxidative, and physiological changes during storage, collectively described as the "storage lesion." The impact of storage on oxygen homeostasis, following transfusion, is not fully understood. We show that RBC storage induces changes in oxygen binding that were linked to changes in oxygen sensing (hypoxia-inducible factor, HIF-1α) mechanisms and mitochondrial respiration in human pulmonary arterial endothelial cells (HPAECs). A decrease in oxygen affinity (P50) to approximately 20 from 30 mmHg was seen at the first week but remained unchanged for up to 42 days. This led to the suppression of HIF-1α in the first 3 weeks due to limited oxygen supplies by RBCs. Furthermore, membrane oxidative damage, band 3 alterations, and subsequent microparticle (MP) formation were also noted. Mass spectrometric analysis revealed the upregulation of transitional endoplasmic reticulum ATPase, essential for clearing ROS-damaged membrane proteins and the protein DDI1 homolog, a proteasomal shuttle chaperone. Band 3 complex proteins and superoxide dismutase were among the downregulated proteins. Mitochondrial oxygen consumption rates measured in HPAECs incubated with RBC-derived MPs (14-day and 42-day) showed a rise in maximal respiration. Intervention strategies that target intracellular hemoglobin (Hb)'s redox transitions and membrane changes may lead to the reestablishment of oxygen homeostasis in old RBCs.

Caffeic acid: an antioxidant with novel antisickling properties.

It is well documented that caffeic acid (3,4-dihydroxycinnamic acid) (CA) interacts with and inhibits the oxidative reactions of myoglobin (Mb) and hemoglobin (Hb), and this interaction underlies its antioxidative action in meat. Sickle cell hemoglobin (HbS) is known for its tendency to oxidize more readily than normal HbA in the presence of hydrogen peroxide (H2 O2 ), which leads to a more persistent and highly oxidizing ferryl Hb (HbFe4+ ). We have investigated the effects of CA on HbS oxidation intermediates, specifically on the ferric/ferryl forms. At a low concentration of H2 O2 (0.5-fold over heme), we observed a fivefold reduction in the amount of HbFe4+ accumulated in a mixture of ferric and H2 O2 solution. Higher levels of H2 O2 (onefold and twofold over heme) led to a lesser threefold and twofold reduction in the content of HbFe4+ , respectively, possibly due to the saturation of the binding sites on the Hb molecule. The most intriguing finding was that when 5-molar excess CA over heme was used, and a considerable increase in the delay time of HbS polymerization to approximately 200 s was observed. This delay in polymerization of HbS is theoretically sufficient to avoid microcapillary blockage and prevent vasoconstrictions in vivo. Mass spectrometry analysis indicated that CA was more extensively covalently bonded to ßCys93 than to ßCys112 and αCys104 . The dual antioxidant and antisickling properties of CA may be explored further to maximize its therapeutic potential in SCD.

Effects of α subunit substitutions on the oxidation of ßCys93 and the stability of sickle cell hemoglobin.

The ß subunit substitutions, F41Y and K82D, in sickle cell hemoglobin (Hb) (ßE6 V) provides significant resistance to oxidative stress by shielding ßCys93 from the oxidizing ferryl heme. We evaluated the oxidative resistance of ßCys93 to hydrogen peroxide (H2O2) in α subunit mutations in ßE6 V (at both the putative and lateral contact regions) that included (1) αH20Q/ßE6 V; (2) αH50Q/ßE6 V; (3) αH20Q/H50Q/ßE6 V; (4) αH20R/ßE6 V; and (5) αH20R/H50Q/ßE6 V. Estimation by mass spectrometry of irreversible oxidation of ßCys93 to cysteic acid (CA) was unchanged or moderately increased in the single mutants harboring a H20Q or H50Q substitution when compared to control (ßE6 V). The introduction of Arg (R) singularly or in combination with Q enhanced the pseudoperoxidative cycle by slightly decreasing the ferryl in favor of ferrous and ferric species after treatment with H2O2. Higher rates for heme loss from the ferric forms of the Q species to the receptor high affinity recombinant apomyglobin were observed in contrast to the R mutants and control. Because of their improved solubility, a combination of Q and R substitutions together with mutations carrying redox active variants (F41Y/K82D) may provide dual antioxidant and antisickling targets in the design of gene therapy-based candidates.

Antisickling Drugs Targeting ßCys93 Reduce Iron Oxidation and Oxidative Changes in Sickle Cell Hemoglobin.

Sickle cell disease is a genetic blood disorder caused by a single point mutation in the ß globin gene where glutamic acid is replaced by valine at the sixth position of the ß chain of hemoglobin (Hb). At low oxygen tension, the polymerization of deoxyHbS into fibers occurs in red blood cells (RBCs) leading to an impaired blood vessel transit. Sickle cell hemoglobin (HbS), when oxidized with hydrogen peroxide (H2O2), stays longer in a highly oxidizing ferryl (Fe4+) form causing irreversible oxidation of ßCys93 to a destabilizing cysteic acid. We have previously reported that an antisickling drug can be designed to bind specifically to ßCys93 and effectively protect against its irreversible oxidation by H2O2. Here, we report oxygen dissociation, oxidation, and polymerization kinetic reactions for four antisickling drugs (under different preclinical/clinical developmental stages) that either site-specifically target ßCys93 or other sites on the HbS molecule. Molecules that specifically bind to or modify ßCys93, such as 4,4'-di(1,2,3-triazolyl) disulfide (TD-3) and hydroxyurea (HU) were contrasted with molecules that target other sites on Hb including 5-hydroxymethyl-2-furfural (5-HMF) and L-glutamine. All reagents induced a left shift in the oxygen dissociation curve (ODC) except L-glutamine. In the presence of H2O2 (2.5:1, H2O2:heme), both TD-3 and HU reduced the ferryl heme by 22 and 37%, respectively, which corresponded to a 3- to 2-fold reduction in the levels of ßCys93 oxidation as verified by mass spectrometry. Increases in the delay times prior to polymerization of HbS under hypoxia were in the following order: TD-3 > HU > 5-HMF = L-glutamine. Designing antisickling agents that can specifically target ßCys93 may provide a dual antioxidant and antisickling therapeutic benefits in treating this disease.

Substitutions in the ß subunits of sickle-cell hemoglobin improve oxidative stability and increase the delay time of sickle-cell fiber formation.

After reacting with hydrogen peroxide (H2O2), sickle-cell hemoglobin (HbS, ßE6V) remains longer in a highly oxidizing ferryl form (HbFe4+=O) and induces irreversible oxidation of "hot-spot" amino acids, including ßCys-93. To control the damaging ferryl heme, here we constructed three HbS variants. The first contained a redox-active Tyr in ß subunits (F41Y), a substitution present in Hb Mequon; the second contained the Asp (K82D) found in the ß cleft of Hb Providence; and the third had both of these ß substitutions. Both the single Tyr-41 and Asp-82 constructs lowered the oxygen affinity of HbS but had little or no effects on autoxidation or heme loss kinetics. In the presence of H2O2, both rHbS ßF41Y and ßF41Y/K82D enhanced ferryl Hb reduction by providing a pathway for electrons to reduce the heme via the Tyr-41 side chain. MS analysis of ßCys-93 revealed moderate inhibition of thiol oxidation in the HbS single F41Y variant and dramatic 3- to 8-fold inhibition of cysteic acid formation in rHbS ßK82D and ßF41Y/K82D, respectively. Under hypoxia, ßK82D and ßF41Y/K82D HbS substitutions increased the delay time by ∼250 and 600 s before the onset of polymerization compared with the rHbS control and rHbS ßF41Y, respectively. Moreover, at 60 °C, rHbS ßK82D exhibited superior structural stability. Asp-82 also enhanced the function of Tyr as a redox-active amino acid in the rHbS ßF41Y/K82D variant. We conclude that the ßK82D and ßF41Y substitutions add significant resistance to oxidative stress and anti-sickling properties to HbS and therefore could be potential genome-editing targets.

Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles.

The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes-sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent ß-globin posttranslational modifications, including the irreversible oxidation of ßCys93 and the ubiquitination of ßLys96 and ßLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, ßCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients.

Comprehensive Biochemical and Biophysical Characterization of Hemoglobin-Based Oxygen Carrier Therapeutics: All HBOCs Are Not Created Equally.

The development of hemoglobin (Hb)-based oxygen carriers (HBOCs) has been hampered because of safety concerns in humans. Chemical and/or genetic modifications of the Hb introduce varied structural and conformational constraint on the molecule that resulted in proteins with diverse allosteric responses, nitrosative and oxidative side reactions. Here, we present for the first time a comprehensive biochemical and biophysical comparison of human, bovine, and genetically engineered HBOCs that have been tested in humans. We evaluate oxygen equilibrium and ligand binding kinetics under different experimental conditions as well as their autoxidation kinetics, redox reactions, and heme release. We determined the effects of HBOCs on cellular redox states and mitochondrial respiration. Taken together, these experiments provide a better understanding of the relationship between the structure-function and oxidative reactivity of these proteins. One can therefore select independently among these diverse properties to engineer a safe and effective HBOC with improved biochemical/biophysical characteristics.

Targeting ßCys93 in hemoglobin S with an antisickling agent possessing dual allosteric and antioxidant effects.

Sickle cell disease (SCD) is an inherited blood disorder caused by a ß globin gene mutation of hemoglobin (HbS). The polymerization of deoxyHbS and its subsequent aggregation (into long fibers) is the primary molecular event which leads to red blood cell (RBC) sickling and ultimately hemolytic anemia. We have recently suggested that HbS oxidative toxicity may also contribute to SCD pathophysiology due to its defective pseudoperoxidase activity. As a consequence, a persistently higher oxidized ferryl heme is formed which irreversibly oxidizes "hotspot" residues (particularly ßCys93) causing protein unfolding and subsequent heme loss. In this report we confirmed first, the allosteric effect of a newly developed reagent (di(5-(2,3-dihydro-1,4-benzodioxin-2-yl)-4H-1,2,4-triazol-3-yl)disulfide) (TD-1) on oxygen affinity within SS RBCs. There was a considerable left shift in oxygen equilibrium curves (OECs) representing treated SS cells. Under hypoxic conditions, TD-1 treatment of HbS resulted in an approximately 200 s increase in the delay time of HbS polymerization over the untreated HbS control. The effect of TD-1 binding to HbS was also tested on oxidative reactions by incrementally treating HbS with increasing hydrogen peroxide (H2O2) concentrations. Under these experimental conditions, ferryl levels were consistently reduced by approximately 35% in the presence of TD-1. Mass spectrometric analysis confirmed that upon binding to ßCys93, TD-1 effectively blocked irreversible oxidation of this residue. In conclusion, TD-1 appears to shield ßCys93 (the end point of radical formation in HbS) and when coupled with its allosteric effect on oxygen affinity may provide new therapeutic modalities for the treatment of SCD.

Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase-1.

Despite advances in our understanding of the oxidative pathways mediated by free hemoglobin (Hb), the precise contribution of its highly reactive redox forms to tissue and organ toxicities remains ambiguous. Heme, a key degradation byproduct of Hb oxidation, has recently been recognized as a damage-associated molecular pattern (DAMP) molecule, able to trigger inflammatory responses. Equally damaging is the interaction of the highly redox active forms of Hb with other biological molecules. We determined the kinetics of heme loss from individual Hb redox states-ferrous (Fe(2+)), ferric (Fe(3+)), and ferryl (Fe(4+))-using two different heme receptor proteins: hemopexin (Hxp), a naturally occurring heme scavenger in plasma, and a double mutant (H64Y/V86F), apomyoglobin (ApoMb), which avidly binds heme released from Hb. We show for the first time that ferric Hb (Fe(3+)) loses heme at rates substantially higher than that of ferryl Hb (Fe(4+)). This was also supported by a higher expression of heme oxygenase-1 (HO-1) when ferric Hb was added to cultured lung alveolar epithelial cells (E10). The reported cytotoxicity of Hb may therefore be attributed to a combination of accelerated heme loss from the ferric form and protein radical formation associated with ferryl Hb. Targeted therapeutic interventions can therefore be designed to curb specific oxidative pathways of Hb in hemolytic anemias and when Hb is used as an oxygen-carrying therapeutic.

Appropriate complementary feeding practices and associated factors among mothers of children age 6-23 months in Southern Ethiopia, 2015.

BACKGROUND: Poor complementary feeding of children aged 6-23 months contributes to the characteristics negative growth trends and deaths observed in developing countries. Evidences have shown that promotion of appropriate complementary feeding practices reduces the incidence of stunting and leads to better health and growth outcome. This study was aimed at assessing practices of complementary feeding and associated factors among mothers of children aged 6-23 months. METHODS: A community-based cross sectional study design was conducted among 611 mothers who had children with 6-23 months of age in the ten randomly selected Kebeles (smallest administrative unit). A multistage sampling technique was used to identify study subjects. Data were collected using pre-tested structured questionnaire. Data were entered in to Epi info version 3.5.1. Data cleaning and analysis were done using SPSS version 16. Odds ratios (ORs) with 95 % confidence interval (CI) were computed to measure the strength of association. RESULTS: The response rate was 97.6 % (611/626). The practices of timely initiation of complementary feeding, minimum meal frequency and minimum dietary diversity were 72.5, 67.3 and 18.8 % among mothers of 6-23 months aged children, respectively. The practice of appropriate complementary feeding was 9.5 %. Child's age (12-17 and 18-23 months) [Adjusted OR: 2.75 (95 % CI: 1.07 7.03), 2.64 (95 % CI: 1.06 6.74)], educational level of mother (primary and secondary and above schools) [AOR: 3.24 (1.28 8.20), 3.21 (1.1.07 9.70)], and smaller family size [AOR: 12.10 (95 % CI: 1.10 139.7)] were found to be independent predictors of appropriate complementary feeding practice of 6-23 months old children. CONCLUSION: Low appropriate complementary feeding of children aged 6-23 months was observed. Mothers who are illiterate, children age 6-11 months and families with large size were associated factors for inappropriate feeding practices. Therefore, nutritional counseling on child feeding practices were recommended.

Oxidative instability of hemoglobin E (ß26 Glu→Lys) is increased in the presence of free α subunits and reversed by α-hemoglobin stabilizing protein (AHSP): Relevance to HbE/ß-thalassemia.

When adding peroxide (H2O2), ß subunits of hemoglobin (Hb) bear the burden of oxidative changes due in part to the direct oxidation of its Cys93. The presence of unpaired α subunits within red cells and/or co-inheritance of another ß subunit mutant, HbE (ß26 Glu→Lys) have been implicated in the pathogenesis and severity of ß thalassemia. We have found that although both HbA and HbE autoxidize at initially comparable rates, HbE loses heme at a rate almost 2 fold higher than HbA due to unfolding of the protein. Using mass spectrometry and the spin trap, DMPO, we were able to quantify irreversible oxidization of ßCys93 to reflect oxidative instability of ß subunits. In the presence of free α subunits and H2O2, both HbA and HbE showed ßCys93 oxidation which increased with higher H2O2 concentrations. In the presence of Alpha-hemoglobin stabilizing protein (AHSP), which stabilizes the α-subunit in a redox inactive hexacoordinate conformation (thus unable to undergo the redox ferric/ferryl transition), Cys93 oxidation was substantially reduced in both proteins. These experiments establish two important features that may have relevance to the mechanistic understanding of these two inherited hemoglobinopathies, i.e. HbE/ß thalassemia: First, a persistent ferryl/ferryl radical in HbE is more damaging to its own ß subunit (i.e., ßCys93) than HbA. Secondly, in the presence of excess free α-subunit and under the same oxidative conditions, these events are substantially increased for HbE compared to HbA, and may therefore create an oxidative milieu affecting the already unstable HbE.

Sexual and reproductive health of young people with disability in Ethiopia: a study on knowledge, attitude and practice: a cross-sectional study.

BACKGROUND: As is common in developing countries, in Ethiopia young people with disabilities (YPWD) are more likely than the general population to be illiterate, unemployed and impoverished. They often lack equal access to information and education for reasons ranging from barriers regarding physical access to services to varied special learning needs. Very little is known about knowledge, attitude and practice (KAP) of YPWD regarding sexual and reproductive health (SRH) related issues. We, therefore, aimed to assess the KAP of 426 YPWD in Addis Ababa, Ethiopia. METHODS: A cross-sectional survey was conducted in 2012. Data were collected by trained interviewers using a structured questionnaire covering socio-demographic information, as well as information on KAP regarding SRH. RESULTS: Only 64.6% of YPWD were aware of SRH services. Radio and TV were mentioned as the main sources of information by 62.2% of the participants. 77.9% had never had a discussion about SRH topics with their parents. Even though 96.7% of the respondents had heard about HIV, 88% had poor knowledge about ways of preventing HIV. Perception of the risk of getting infected with HIV was found to be generally low in YPWD; only 21.6% believed that they were at risk of acquiring HIV. CONCLUSIONS: Our study, in general, demonstrated that there is a lack of comprehensive knowledge, appropriate practice and favorable attitude of YPWD regarding different SRH-related issues. Our findings thus clearly indicate the need for strategies and programs to raise SRH-related awareness and to help YPWD to develop the appropriate skills and attitudes needed for a healthy reproductive life.

Sickle Cell Hemoglobin in the Ferryl State Promotes ßCys-93 Oxidation and Mitochondrial Dysfunction in Epithelial Lung Cells (E10).

Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H2O2) drives a catalytic cycle that includes the following three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb; 2) autoreduction of the ferryl intermediate to ferric (metHb); and 3) reaction of metHb with an additional H2O2 molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H2O2 at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, we found more irreversibly oxidized ßCys-93in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate and impairment of cellular bioenergetics that was related to the redox state of the iron. Ferryl HbS induced a substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial colocalization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation, may be central to the evolution of vasculopathy in SCD and may suggest therapeutic modalities that interrupt heme-mediated inflammation.

Sexuality and sexual reproductive health of disabled young people in Ethiopia.

OBJECTIVES: In Ethiopia, young people with disabilities (YPWD) are often marginalized and not recognized as being sexual, and only little is known about their sexual reproductive health (SRH) status. We therefore aimed to assess the SRH status and associated factors among 426 YPWD in Addis Ababa, Ethiopia. METHODS: A cross-sectional survey was conducted in 2012. Data were collected by trained interviewers using a structured questionnaire. RESULTS: Fifty-two percent of YPWD ever had sexual intercourse. Seventy-five percent started sex between 15 and 19 years. Only 35% had used contraceptive during their first sexual encounter. Fifty-nine percent of the sexually experienced YPWD had multiple lifetime sexual partners; 19%, a casual sexual partner; and 21%, a commercial sexual partner. Only 48% consistently used condoms with their casual or commercial sexual partners. Twenty-four percent of the sexually experienced YPWD had a history of sexually transmitted infections. CONCLUSIONS: Our findings indicate that YPWD in Ethiopia are sexually active, but also highly involved in risky sexual practices. There is a need for in-depth research to better understand the determinants of risky sexual behavior and to propose preventive approaches.

Post-translational transformation of methionine to aspartate is catalyzed by heme iron and driven by peroxide: a novel subunit-specific mechanism in hemoglobin.

A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and ß subunits of adult Hb (HbA) Bristol-Alesha (ß67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H2(18)O2, we verified incorporation of (18)O into the Asp carboxyl side chain confirming the role of H2O2 in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in ß subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, ß, and γ subunits with respect to redox reactivity and may have direct implications to α/ß hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics.

Design, synthesis, and activity of 2,3-diphosphoglycerate analogs as allosteric modulators of hemoglobin O2 affinity.

Four phosphonate derivates of 2,3-diphosphoglycerate (2,3-DPG), in which the phosphate group is replaced by a methylene or difluoromethylene, were successfully synthesized for use as allosteric modulators of hemoglobin (Hb) O2 affinity. The syntheses were accomplished in four steps and the reagents were converted to their potassium salts to allow for effective binding with Hb in aqueous media. O2 equilibrium measurements of the chemically modified Hbs exhibited P50 values in the range 8.9-12.8 with Hill coefficients in the range of 1.5-2.4.