Coarse snapshots of oxygen-dissociation intermediates of a giant hemoglobin elucidated by determining the oxygen saturation in individual subunits in the crystalline state

Cooperative oxygen binding of hemoglobin (Hb) has been studied for over half a century as a representative example of the allostericity of proteins. The most important problem remaining to be solved is the lack of structural information on the intermediates between the oxygenated and deoxygenated forms. In order to characterize the intermediate structures, it is necessary to obtain intermediate-state crystals, determine their oxygen saturations and then determine the oxygen saturations of each of their constituent subunits, all of which are challenging issues even now. Here, intermediate forms of the 400 kDa giant Hb from the tubeworm Oligobrachia mashikoi are reported. To overcome the above problems without any artificial modifications to the protein or prosthetic groups, intermediate crystals of the giant Hb were prepared from fully oxygenated crystals by a soaking method. The oxygen saturation of the crystals was measured by in situ observation with a microspectrophotometer using thin plate crystals processed by an ultraviolet laser to avoid saturation of absorption. The oxygen saturation of each subunit was determined by occupancy refinement of the bound oxygen based on ambient temperature factors. The obtained structures reveal the detailed relationship between the structural transition and oxygen dissociation. The dimer subassembly of the giant Hb shows strong correlation with the local structural changes at the heme pockets. Although some local ternary-structural changes occur in the early stages of the structural transition, the associated global ternary-structural and quaternary-structural changes might arise at about 50% oxygen saturation. The models based on coarse snapshots of the allosteric transition support the conventional two-state model of Hbs and provide the missing pieces of the intermediate structures that are required for full understanding of the allosteric nature of Hbs in detail.

Dose- and Sex-Dependent Changes in Hemoglobin Oxygen Affinity by the Micronutrient 5-Hydroxymethylfurfural and α-Ketoglutaric Acid

5-Hydroxymethylfurfural (5-HMF) is known to increase hemoglobin oxygen affinity (Hb–O2 affinity) and to induce a left shift of the oxygen dissociation curve (ODC). It is under investigation as a therapeutic agent in sickle cell anemia and in conditions where pulmonary oxygen uptake is deteriorated or limited (e.g., various clinical conditions or altitude exposure). The combination of 5-HMF and α-ketoglutaric acid (αKG) is commercially available as a nutritional supplement. To further elucidate dose effects, ODCs were measured in vitro in venous whole blood samples of 20 healthy volunteers (10 female and 10 male) after the addition of three different doses of 5-HMF, αKG and the combination of both. Linear regression analysis revealed a strong dose-dependent increase in Hb–O2 affinity for 5-HMF (R2 = 0.887; p < 0.001) and the commercially available combination with αKG (R2 = 0.882; p < 0.001). αKG alone increased Hb–O2 affinity as well but to a lower extent. Both the combination (5-HMF + αKG) and 5-HMF alone exerted different P50 and Hill coefficient responses overall and between sexes, with more pronounced effects in females. With increasing Hb–O2 affinity, the sigmoidal shape of the ODC was better preserved by the combination of 5-HMF and αKG than by 5-HMF alone. Concerning the therapeutic effects of 5-HMF, this study emphasizes the importance of adequate dosing in various physiological and clinical conditions, where a left-shifted ODC might be beneficial. By preserving the sigmoidal shape of the ODC, the combination of 5-HMF and αKG at low (both sexes) and medium (males only) doses might be able to better maintain efficient oxygen transport, particularly by mitigating potentially deteriorated oxygen unloading in the tissue. However, expanding knowledge on the interaction between 5-HMF and Hb–O2 affinity in vitro necessitates further investigations in vivo to additionally assess pharmacokinetic mechanisms.

Right ventricular myocardial oxygen tension is reduced in monocrotaline-induced pulmonary hypertension in the rat and restored by myo-inositol trispyrophosphate

Pulmonary hypertension (PH) initially results in compensatory right ventricular (RV) hypertrophy, but eventually in RV failure. This transition is poorly understood, but may be triggered by hypoxia. Measurements of RV oxygen tension (pO2) in PH are lacking. We hypothesized that RV hypoxia occurs in monocrotaline-induced PH in rats and that myo-inositol trispyrophosphate (ITPP), facilitating oxygen dissociation from hemoglobin, can relieve it. Rats received monocrotaline (PH) or saline (control) and 24 days later echocardiograms, pressure–volume loops were obtained and myocardial pO2 was measured using a fluorescent probe. In PH mean pulmonary artery pressure more than doubled (35 ± 5 vs. 15 ± 2 in control), RV was hypertrophied, though its contractility was augmented. RV and LV pO2 was 32 ± 5 and 15 ± 8 mmHg, respectively, in control rats. In PH RV pO2 was reduced to 18 ± 9 mmHg, while LV pO2 was unchanged. RV pO2 correlated with RV diastolic wall stress (negatively) and LV systolic pressure (positively). Acute ITPP administration did not affect RV or LV pO2 in control animals, but increased RV pO2 to 26 ± 5 mmHg without affecting LV pO2 in PH. RV oxygen balance is impaired in PH and as such can be an important target for PH therapy. ITPP may be one of such potential therapies.

Recent Advances in the Physicochemical Properties and Biotechnological Application of Vitreoscilla Hemoglobin

Vitreoscilla hemoglobin (VHb), the first discovered bacterial hemoglobin, is a soluble heme-binding protein with a faster rate of oxygen dissociation. Since it can enhance cell growth, product synthesis and stress tolerance, VHb has been widely applied in the field of metabolic engineering for microorganisms, plants, and animals. Especially under oxygen-limited conditions, VHb can interact with terminal oxidase to deliver enough oxygen to achieve high-cell-density fermentation. In recent years, with the development of bioinformatics and synthetic biology, several novel physicochemical properties and metabolic regulatory effects of VHb have been discovered and numerous strategies have been utilized to enhance the expression level of VHb in various hosts, which greatly promotes its applications in biotechnology. Thus, in this review, the new information regarding structure, function and expressional tactics for VHb is summarized to understand its latest applications and pave a new way for the future improvement of biosynthesis for other products.

Treatment of sickle cell disease by increasing oxygen affinity of hemoglobin

The issue of treating sickle cell disease with drugs that increase hemoglobin oxygen affinity has come to the fore with the FDA approval in 2019 of voxelotor, the only anti-sickling drug approved since hydroxyurea in 1998. Voxelotor reduces sickling by increasing the concentration of the non-polymerizing, high oxygen affinity R (oxy) conformation of HbS. Treatment of sickle cell patients with voxelotor increases Hb levels and decreases indicators of hemolysis, but with no indication as yet that it reduces the frequency of pain episodes. Here we use the allosteric model of Monod, Wyman, and Changeux to simulate whole blood oxygen dissociation curves and red cell sickling in the absence and presence of voxelotor under the in vivo conditions of rapid oxygen pressure decreases. Our modeling agrees with experiments using a new robust assay, which shows the very large, expected decrease in sickling from the drug. The modeling indicates, however, that the increase in oxygen delivery from reduced sickling is largely offset by the increase in oxygen affinity. The net result is that the drug increases overall oxygen delivery only at the very lowest oxygen pressures. Reduction of sickling does, however, mitigate against red cell damage and explains the observed decrease in hemolysis. More importantly, our modeling of in vivo oxygen dissociation, sickling, and oxygen delivery suggests that drugs that increase fetal hemoglobin or decrease MCHC, should be more therapeutically effective than drugs that increase oxygen affinity.

Right Ventricular Myocardial Oxygen Partial Pressure Is Reduced in Monocrotaline-induced Pulmonary Hypertension in the Rat and Restored by Myo-inositol Trispyrophosphate

Pulmonary arterial hypertension (PAH) initially results in compensatory RV hypertrophy, but eventually in RV failure. This transition is poorly understood, but may be triggered by hypoxia. Measurements of RV oxygen tension (pO2) in PAH are lacking. We hypothesized that RV hypoxia occurs in monocrotaline-induced PAH in rats and that myo-inositol trispyrophosphate (ITPP), facilitating oxygen dissociation from hemoglobin, can relieve it. Rats received monocrotaline (PAH) or saline (control) and 24 days later echocardiograms, pressure-volume loops were obtained and myocardial pO2 was measured using a fluorescent probe. In PAH mean pulmonary artery pressure more than doubled (35±5 vs. 15±2 in control), RV was hypertrophied, though its contractility was augmented. RV and LV pO2 was 32±5 and 15±8 mmHg, respectively, in control rats. In PAH RV pO2 was reduced to 18±9 mmHg, while LV pO2 was unchanged. RV pO2 correlated with RV diastolic wall stress (negatively) and LV systolic pressure (positively). Acute ITPP administration did not affect RV or LV pO2 in control animals, but increased RV pO2 to 26±5 mmHg without affecting LV pO2 in PAH. RV oxygen balance is impaired in PAH and as such can be an important target for PAH therapy. ITPP may be one of such potential therapies.

The Oxygen Dissociation Curve of Blood in COVID-19

COVID-19 hinders oxygen transport to the consuming tissues by at least 2 mechanisms: In the injured lung saturation of hemoglobin is compromised, in the tissues an associated anemia reduces the volume of delivered oxygen. For the first problem increased hemoglobin oxygen affinity (left shift of the oxygen dissociation curve ODC) is of advantage, for the 2nd, however, the contrary is the case. Indeed a right shift of the ODC has been found in former studies for anemia caused by reduced cell production or hemolysis. This resulted from increased 2,3-biphosphglycerate (2,3-BPG) concentration. In 3 investigations in COVID-19, however, no change of hemoglobin affinity was detected in spite of probably high [2,3-BPG]. The most plausible cause for this finding is formation of methemoglobin, which increases the oxygen affinity and thus apparently compensates for the 2,3-BPG effect. But this "useful effect" is cancelled by the concomitant reduction of functional hemoglobin. In the largest study on COVID-19 even a clear left shift of the ODC was detected when calculated from measurements in fresh blood rather than after equilibration with gases outside the body. This additional „in vivo" left shift possibly results from various factors (e. g. concentration changes of Cl-, 2,3-BPG, ATP, lactate, nitrocompounds, glutathione, glutamate, because of time delay between blood sampling and end of equilibration, or enlarged distribution space including interstitial fluid and is useful for O2 uptake in the lungs. Under discussion for therapy are the affinity-increasing 5-hydroxymethyl-2-furfural (5-HMF), erythropoiesis stimulating substances like erythropoietin, and methylene blue against MetHb formation.