Oxygen Binding Properties Research Articles

Hemoglobin-oxygen affinity changes in neonatal blood transfusions: RBC selection insights.

Despite preterm newborns often requiring blood transfusions, we have an incomplete understanding of the impact of adult packed red blood cell (pRBC) transfusions on fetal red blood cell (RBC) oxygen affinity. We investigated the influence of adult pRBC on oxygen binding in fetal RBCs obtained from the umbilical cord of preterm newborns. This included exploring the influence of the biological age of adult pRBCs on the oxygen affinity of fetal blood. Cord blood samples from preterm infants were titrated with young (Y-RBC) and old (O-RBC) adult pRBCs using an in vitro transfusion model. Parameters, including oxygen affinity (p50), hemoglobin variants, and red blood cell indices, were measured. The titration of cord blood with adult pRBCs (n = 19) resulted in a concentration-dependent decrease in p50, indicating an increased oxygen affinity. Hemoglobin variant analysis revealed a shift in composition, with a decrease in fetal hemoglobin (HbF) and an increase in adult hemoglobin (HbA) following titration. The impact of biological age was evident, as O-RBCs had a more pronounced effect on p50 values compared to Y-RBCs. Understanding the physiologic implications of transfusing preterm infants with adult pRBCs is important for optimizing transfusion practices in newborns. In an in vitro model, titrating adult pRBC into fetal blood significantly affects oxygen binding Oxygen affinity of fetal blood is significantly increased after titration of adult pRBC. Compared to older RBC subpopulations of adult pRBC, oxygen-binding properties of younger RBC subpopulations of adult pRBC more closely approximate the oxygen affinity of fetal blood. This work highlights the importance of investigating the influence of adult pRBC transfusion on fetal RBC oxygen binding. This may have implications for morbidities in the newborn period related to oxygen physiology.

Optimization and Investigation of Hemoglobin‐Loaded ZIF‐90 Metal–Organic Framework Nanoparticles as Artificial Oxygen Carriers

AbstractBlood transfusions are vital, yet limited shelf‐life and storage conditions of red blood cells (RBCs) hinder their use in emergencies. Hemoglobin‐based oxygen carriers (HBOCs) aim to address these challenges, but previous versions faced clinical setbacks due to safety concerns related to hemoglobin (Hb) extravasation. ZIF‐90, a novel metal–organic framework variant where imidazole‐2‐carboxaldehyde (2‐ICA) is bridged to Zn2+ ions, is explored. Optimizing Zn2+:2‐ICA ratios and Hb concentrations, Hb‐loaded ZIF‐90 nanoparticles (NPs) are synthesized in one‐step and under mild synthesis conditions. These NPs achieve a 25.1% yield, 7.0 mg mL−1 Hb concentration, 70.3% encapsulation efficiency (EE), and 96.4% drug loading. They exhibit a left‐shifted oxygen dissociation curve with p50 of ≈8 mmHg, indicating enhanced oxygen release at lower partial oxygen pressures as compared to native RBCs. This feature makes Hb‐loaded ZIF‐90 NPs suitable for certain medical applications including ischemia‐reperfusion injury management. Furthermore, the impact of two prominent capping agents: polyvinylpyrrolidone and polyethylene glycol (PEG) is assessed. PEG improves 2‐ICA incorporation and stabilizes the ZIF‐90 crystalline phase, albeit with reduced yield, Hb content, and EE. The findings underscore the potential of Hb‐loaded ZIF‐90 NPs as next‐generation HBOCs, offering enhanced uniformity, high Hb content, and efficient oxygen binding and release properties for medical applications.

Conjugating Hemoglobin and Albumin by Strain-Promoted Azide- Alkyne Cycloaddition.

A one-to-one conjugate of cross-linked human hemoglobin and human serum albumin results from a strain-promoted alkyne-azide cycloaddition (SPAAC) of the modified proteins. Additions of a strained alkyne-substituted maleimide to the Cys-34 thiol of human serum albumin and an azide-containing cross-link between the amino groups of each β-unit at Lys-82 of human hemoglobin provide sites for coupling by the SPAAC process. The coupled hemoglobin-albumin conjugate can be readily purified from unreacted hemoglobin. The oxygen binding properties of the two-protein bioconjugate demonstrate oxygen affinity and cooperativity that are suitable for use in an acellular oxygen carrier.

Cryopreserved red blood cells maintain allosteric control of oxygen binding when utilizing trehalose as a cryoprotectant

One of the most common life-saving medical procedures is a red blood cell (RBC) transfusion. Unfortunately, RBCs for transfusion have a limited shelf life after donation due to detrimental storage effects on their morphological and biochemical properties. Inspired by nature, a biomimetics approach was developed to preserve RBCs for long-term storage using compounds found in animals with a natural propensity to survive in a frozen or desiccated state for decades. Trehalose was employed as a cryoprotective agent and added to the extracellular freezing solution of porcine RBCs. Slow cooling (−1 °C min−1) resulted in almost complete hemolysis (1 ± 1 % RBC recovery), and rapid cooling rates had to be used to achieve satisfactory cryopreservation outcomes. After rapid cooling, the highest percentage of RBC recovery was obtained by plunging in liquid nitrogen and thawing at 55 °C, using a cryopreservation solution containing 300 mM trehalose. Under these conditions, 88 ± 8 % of processed RBCs were recovered and retained hemoglobin (14 ± 2 % hemolysis). Hemoglobin's oxygen-binding properties of cryopreserved RBCs were not significantly different to unfrozen controls and was allosterically regulated by 2,3-bisphosphoglycerate. These data indicate the feasibility of using trehalose instead of glycerol as a cryoprotective compound for RBCs. In contrast to glycerol, trehalose-preserved RBCs can potentially be transfused without time-consuming washing steps, which significantly facilitates the usage of cryopreserved transfusible units in trauma situations when time is of the essence.

Effect of Ozone on Oxygen Transport and Pro-Oxidant- Antioxidant Balance of Red Blood Cell Suspension

Introduction: Ozone affects blood oxygen transport and the pro-oxidant-antioxidant balance. However, the role of blood formed elements and gas transmitters in these processes still remains unclear. The aim of the present study was to investigate the effect of ozone on oxygen transport and the pro-oxidant-antioxidant balance in a red cell suspension. Methods: The red cell suspension was incubated with ozone at a concentration of 6 mg/l and substances affecting the synthesis of gas transmitters (nitroglycerin, sodium hydrosulfide). Parameters of blood oxygen transport and pro-oxidant-antioxidant balance were determined. Results: The effect of ozone on blood oxygen transport was found which was manifested in an increase in oxygen partial pressure and the degree of oxygenation. The index of hemoglobin –oxygen affinity p50 actual was raised and a shift of the oxyhemoglobin dissociation curve rightwards was noticed. The addition of the gas transmitter donor, nitric oxide, enhanced the effect of this gas on the parameters of oxygen transport in the erythrocyte suspension Conclusion: We found that ozone induced a change in oxygen-binding properties of the erythrocyte suspension which is a mechanism of action of the above gas on the adaptive processes in the body realized directly at the level of erythrocytes.

Structural and oxidative investigation of a recombinant high-yielding fetal hemoglobin mutant.

Human fetal hemoglobin (HbF) is an attractive starting protein for developing an effective agent for oxygen therapeutics applications. This requires that HbF can be produced in heterologous systems at high levels and in a homogeneous form. The introduction of negative charges on the surface of the α-chain in HbF can enhance the recombinant production yield of a functional protein in Escherichia coli. In this study, we characterized the structural, biophysical, and biological properties of an HbF mutant carrying four additional negative charges on each α-chain (rHbFα4). The 3D structure of the rHbFα4 mutant was solved with X-ray crystallography at 1.6Å resolution. Apart from enabling a higher yield in recombinant protein production in E. coli, we observed that the normal DNA cleavage activity of the HbF was significantly lowered, with a four-time reduced rate constant for the rHbFα4 mutant. The oxygen-binding properties of the rHbFα4 mutant were identical to the wild-type protein. No significant difference between the wild-type and rHbFα4 was observed for the investigated oxidation rates (autoxidation and H2O2-mediated ferryl formation). However, the ferryl reduction reaction indicated some differences, which appear to be related to the reaction rates linked to the α-chain.

Oxygen-Dependent Blood Processes in Beck's Sarcoidosis

The objective: to assess the state of oxygen-dependent blood processes in patients with Beck's sarcoidosis.Subjects and Methods. Parameters of blood oxygen transport function and free radical lipid oxidation, as well as the content of nitrogen monoxide and hydrogen sulfide gas transmitters were assessed in the blood of 75 patients (41 women; 34 men) with Beck's sarcoidosis, stage 2 (the pulmonary mediastinal form).Results. In the pulmonary mediastinal form of sarcoidosis versus healthy individuals, there was a decrease in the degree of blood oxygen saturation from 65.4 (60.6; 67.8) to 41.50 (36.0; 49.8), p < 0.05, %, partial oxygen pressure from 40.0 (38.0; 47.0) to 23.0 (18.3; 29.0) mmHg, p < 0.05, and blood pH increased. A decrease in the index of hemoglobin affinity to oxygen p50real (26.80 (24.70; 31.40), p < 0.05, versus healthy individuals 28.2 (27.9; 29.1) mmHg) was revealed. The level of diene conjugates increased in plasma by 86.4% (p < 0.05) and malondialdehyde increased in erythrocytes by 139.2% (p < 0.05), while the following parameters decreased: α-tocopherol (by 42.7 %, p < 0.05), retinol (by 41.9%, p < 0.05), reduced glutathione (by 30.4%, p < 0.05), and ceruloplasmin (by 22.3%, p < 0.05) in plasma. There was an increase in the concentration of nitrogen monoxide (from 15.42 (14.48; 17.71) to 17.09 (7.04; 25.09), mmol/L) and a decrease in hydrogen sulfide (from 29.01 (25.21; 37.03) to 13.08 (8.63; 20.46), mmol/L) in the blood in case of this pathology.Conclusion. There is a deterioration in the blood oxygen-binding properties in patients with Beck's sarcoidosis, stage 2, and as a consequence, tissue needs in oxygen are not adequately provided by blood circulation. There is an increase in the concentration of nitrogen monoxide and a decrease in hydrogen sulfide, which affects the oxygen transport function of blood which may be important for ensuring the processes of oxygen transfer to tissues. In this pathology, activation of lipid peroxidation and a decrease in the level of antioxidant protection factors of blood were revealed which might be important for the genesis of this disease.

Oxygen-binding properties of blood in insulin resistance with different asprosin content.

The oxygen-binding properties of blood were studied in male patients with insulin resistance (IR) with different levels of asprosin. The content of asprosin, parameters of blood oxygen transport function, as well as gas transmitters, nitrogen monoxide and hydrogen sulfide, were determined in the venous blood plasma. In the studied IR patients with increased blood asprosin content, impaired blood oxygenation was noted; IR patients with normal body weight had increased hemoglobin affinity for oxygen, while in IR patients with overweight and the 1st degree obesity, this parameter decreased. The detected increase in the concentration of nitrogen monoxide and the decrease in hydrogen sulfide may be important for the oxygen-binding properties of the blood and the development of metabolic imbalance.

Ontogeny of hemoglobin‑oxygen binding and multiplicity in the obligate air-breathing fish Arapaima gigas

The evolutionary and ontogenetic changes from water- to air-breathing result in major changes in the cardiorespiratory systems. However, the potential changes in hemoglobin's (Hb) oxygen binding properties during ontogenetic transitions to air-breathing remain poorly understood. Here we investigated Hb multiplicity and O2 binding in hemolysates and Hb components from juveniles and adults of the obligate air-breathing pirarucu (Arapaima gigas) that starts life as water-breathing hatchlings. Contrasting with previous electrophoresis studies that report one or two isoHbs in adults, isoelectric focusing (IEF) resolved the hemolysates from both stages into four major bands, which exhibited identical O2 binding properties (i.e. O2 affinities, cooperativity coefficients, and sensitivities to pH and the major organic phosphate effectors), also as compared to the cofactor-free hemolysates. Of note, the multiplicity pattern recurred upon reanalyses of the most-abundant fractions isolated from the juvenile and the adult stages, suggesting possible stabilization of different quaternary states with different isoelectric points during the purification procedure. The study demonstrates unchanged Hb-O2 binding properties during development, despite the pronounced differences in O2 availability between the two media, which harmonizes with findings based on a broader spectrum of interspecific comparisons. Taken together, these results disclose that obligate air-breathing in Arapaima is not contingent upon changes in Hb multiplicity and O2 binding characteristics.

Conversion of HbS to Hb G-Makassar By Adenine Base Editing Is Compatible with Normal Hemoglobin Function

Conversion of HbS to Hb G-Makassar By Adenine Base Editing Is Compatible with Normal Hemoglobin Function

СОСТОЯНИЕ ГОМЕОСТАЗА У КРЫС С ТЕРМИЧЕСКИМ ОЖОГОМ КОЖИ, ПОЛУЧЕННЫМ В СРЕДНЕМ ПЕРИОДЕ БЕРЕМЕННОСТИ

Background. In modern medicine, the problem of treating thermal skin burns in pregnant women remains relevant due to insufficient research of pathological processes occurring in the body of a future mother suffering from thermal injury. The aim of the research. To assess the state of homeostasis in rats with thermal skin burns in the middle period of pregnancy. Material and methods. An experimental study was carried out on 36 female outbred white rats weighing 250-350 g, which underwent thermal skin burns in the middle period of pregnancy. The processes of lipid peroxidation and the total content of nitrate / nitrites were studied, the oxygen transport function of the blood and the main biochemical parameters of the blood were assessed. Results. Thermal skin burn in rats in the middle period of pregnancy leads to the development of hypoxia, as judged by the decrease in pO2 and SO2 values, as well as a decrease in the affinity of hemoglobin for oxygen. At the same time, oxidative stress develops, and an increase in the content of nitrate / nitrites is also observed, which reflects the dysfunction of the L-arginine-NO system, causing changes in the oxygen-binding properties of the blood and forming a prooxidant-antioxidant imbalance. Changes in the main biochemical parameters of blood confirm the presence of endogenous intoxication. Conclusions. The revealed violations of homeostasis in pregnant rats with thermal skin burns indicate the important role of oxygen-dependent processes in the pathogenesis of thermal injury, which must be taken into account when developing appropriate measures aimed at eliminating violations in this pathology.

Ion Mobility Mass Spectrometry Analysis of Oxygen Affinity-Associated Structural Changes in Hemoglobin

Hemoglobin (Hb) is a major oxygen-transporting protein with allosteric properties reflected in the structural changes that accompany binding of O2. Glycated hemoglobin (GHb), which is a minor component of human red cell hemolysate, is generated by a nonenzymatic reaction between glucose and hemoglobin. Due to the long lifetime of human erythrocytes (∼120 days), GHb is widely used as a reliable biomarker for monitoring long-term glucose control in diabetic patients. Although the structure of GHb differs from that of Hb, structural changes relating to the oxygen affinity of these proteins remain incompletely understood. In this study, the oxygen-binding kinetics of Hb and GHb are evaluated, and their structural dynamics are investigated using solution small-angle X-ray scattering (SAXS), electrospray ionization mass spectrometry equipped with ion mobility spectrometry (ESI-IM-MS), and molecular dynamic (MD) simulations to understand the impact of structural alteration on their oxygen-binding properties. Our results show that the oxygen-binding kinetics of GHb are diminished relative to those of Hb. ESI-IM-MS reveals structural differences between Hb and GHb, which indicate the preference of GHb for a more compact structure in the gas phase relative to Hb. MD simulations also reveal an enhancement of intramolecular interactions upon glycation of Hb. Therefore, the more rigid structure of GHb makes the conformational changes that facilitate oxygen capture more difficult creating a delay in the oxygen-binding process. Our multiple biophysical approaches provide a better understanding of the allosteric properties of hemoglobin that are reflected in the structural alterations accompanying oxygen binding.

Structural studies of hemoglobin from two flightless birds, ostrich and turkey: insights into their differing oxygen-binding properties.

Crystal structures of hemoglobin (Hb) from two flightless birds, ostrich (Struthio camelus) and turkey (Meleagris gallopova), were determined. The ostrich Hb structure was solved to a resolution of 2.22 Å, whereas two forms of turkey Hb were solved to resolutions of 1.66 Å (turkey monoclinic structure; TMS) and 1.39 Å (turkey orthorhombic structure; TOS). Comparison of the amino-acid sequences of ostrich and turkey Hb with those from other avian species revealed no difference in the number of charged residues, but variations were observed in the numbers of hydrophobic and polar residues. Amino-acid-composition-based computation of various physical parameters, in particular their lower inverse transition temperatures and higher average hydrophobicities, indicated that the structures of ostrich and turkey Hb are likely to be highly ordered when compared with other avian Hbs. From the crystal structure analysis, the liganded state of ostrich Hb was confirmed by the presence of an oxygen molecule between the Fe atom and the proximal histidine residue in all four heme regions. In turkey Hb (both TMS and TOS), a water molecule was bound instead of an oxygen molecule in all four heme regions, thus confirming that they assumed the aqua-met form. Analysis of tertiary- and quaternary-structural features led to the conclusion that ostrich oxy Hb and turkey aqua-met Hb adopt the R-/RH-state conformation.

Structural Stability and Biophysical Properties of the Mega-Protein Erythrocruorin Are Regulated by Polyethylene Glycol Surface Coverage.

A wide variety of hemoglobin-based oxygen carriers (HBOCs) have been designed for use as red blood cell (RBC) substitutes in transfusion medicine, ex vivo organ perfusion, oxygen delivery to hypoxic tissues, and a myriad of other applications. However, hemoglobin (Hb) derived from annelids (erythrocruorins [Ecs]) comprise a natural class of HBOC, since they are larger in size (30 nm in diameter) and contain more heme groups per molecule (144 heme groups) compared to human Hb (hHb; 5 nm in diameter and 4 heme groups). The larger size of Ec compared to hHb reduces tissue extravasation from the vascular space, thus, reducing vasoconstriction, systemic hypertension, and tissue oxidative injury when used as an RBC substitute. In addition, prior research has shown that Ecs possess slower auto-oxidation rates than hHb at physiological temperature, thus, making them attractive candidates for use as RBC substitutes. Unfortunately, it was also observed that Ecs have a much lower circulatory half-life in vivo compared to other HBOCs. Hence, conjugating polyethylene glycol (PEG) to the surface of Ec was proposed as a simple strategy to increase Ec circulatory half-life. Therefore, in order to inform future in vivo studies with PEGylated Ec, we decided to investigate the structural stability and biophysical properties of variable PEG surface coverage on Ec compared to native Ec. We observed an increase in PEG-Ec diameter and molecular weight (MW) and changes to the quaternary structure, secondary structure, and surface hydrophobicity after PEGylation. There was also an increase in oxygen binding affinity, reduction in oxygen offloading rate, and increase in auto-oxidation rate for increasing PEGylation ratios. Weak dissociation of Ec was also observed after dense PEGylation caused by steric repulsion of the conjugated PEG chains. Hence, we determined an optimum Ec PEGylation ratio that resulted in a substantial size and MW increase along with preservation of oxygen binding properties. In future studies, these materials will be tested in animal models to evaluate pharmacodynamics, pharmacokinetics, tissue oxygenation, microcirculatory responses, and overall safety.

Hemoglobin: A General Review

Hemoglobin (Hb) is red blood corpuscle pigment causing red color in blood with centrally located iron atom, responsible for oxygen transport having molecular weight of a tetramer of 64,458 g/mol. The oxygen binding properties of hemoglobin are affected by pH and presence of carbon dioxide. It cause different clinical problems due to its high or low concentration in blood. Poisoning of Hb can also occur by combination of Hb with carbon monoxide resulting in formation of carboxyhemoglobin. Besides carboxyhemoglobin, sulfhemoglobin and methemoglobin are also derivatives of Hb.

Different Dosage Effects of Ozone on Oxygen Transport in Blood during in vitro Experiments

The effects of ozone on oxygen transport in the blood of experimental animals have been studied in vitro. Incubation of the blood with ozonated water at О3 concentrations of 2, 6, and 10 mg/L for 30 and 60 min caused changes in the oxygen-transport function of the blood, thus leading to increased oxygen tension and oxygenation, as well as reduced affinity of hemoglobin for oxygen. As a result, the content of the gaseous transmitters, such as nitric oxide and hydrogen sulfide, significantly increased, thus contributing to modification of the oxygen-binding properties of blood.

Black Phosphorus-Loaded Separable Microneedles as Responsive Oxygen Delivery Carriers for Wound Healing.

Oxygen carriers are attracting extensive interest in biomedical research and clinical applications such as wound healing, alternative blood transfusion, and acute trauma treatment. Great efforts have been devoted to the generation of oxygen carriers with special functions and properties to meet specific demands. Here, we present black phosphorus (BP)-loaded separable responsive microneedles (MNs) with oxygen carrying and controllable oxygen delivering ability for wound healing. Such MNs are composed of a polyvinyl acetate (PVA) backing layer and gelatin methacryloyl (GelMA) tips that are loaded with BP quantum dots (BP QDs) and hemoglobin (Hb). Taking advantage of the fast dissolvability of PVA, the backing layer soon disappears after the MNs are applied to skin and the noncytotoxic, biocompatible GelMA tips are left inside the skin. Due to the excellent photothermal effect of BP QDs and the reversible oxygen binding property of Hb, the local temperature of the skin will increase after near-infrared ray irradiation, resulting in the responsive oxygen release. Notably, the practical performance of such MNs has been demonstrated by treating the full-thickness cutaneous wounds of a type I diabetes rat model, indicating their potential value in wound healing and other related biomedical fields.

Haemoglobin-loaded metal organic framework-based nanoparticles camouflaged with a red blood cell membrane as potential oxygen delivery systems.

Transfusion of donor red blood cells (RBCs) is a crucial methodology required for the treatment of acute trauma and anaemia or for surgical procedures. Due to the many limitations of donor blood, numerous strategies have been explored to develop haemoglobin (Hb)-based oxygen carriers to be used as oxygen delivery systems. However, since free Hb suffers from a lack of stability and short circulation times in blood, an encapsulation platform is needed. Herein, we entrap Hb within a type of metal organic framework (MOF)-based nanoparticle (MOF-NP). By doing so, Hb is protected from misfolding and denaturation, which is a crucial aspect to preserve its excellent oxygen binding and releasing properties. Furthermore, the porous structure of MOF-NPs allows for the diffusion of small molecules (i.e., oxygen) in and out of the system. Our results show that the Hb-loaded MOF-NPs (MOFHb-NPs) are monodisperse and show a small hydrodynamic diameter of ∼220 nm. Importantly, the structure and functionality of the encapsulated Hb are well preserved. To achieve long circulation in the bloodstream, we functionalized MOFHb-NPs with naturally derived RBC membranes and compared the stealth properties of the membrane-coated MOFHb-NPs with our previously reported PEGylation strategy. Protein adsorption and cell uptake studies demonstrate that both coatings are able to significantly decrease the adsorption of proteins and also diminish their uptake by macrophages and endothelial cells. Furthermore, both types of coatings endow MOFHb-NPs with good biocompatibility and oxygen binding and releasing properties. Overall, this study presents a novel oxygen carrier system which might find applications as a blood surrogate.

Multiplicity and Polymorphism of Fish Hemoglobins.

The diversity of fish hemoglobins and the association with oxygen availability and physiological requirements during the life cycle has attracted scientists since the first report on multiple hemoglobin in fishes (Buhler and Shanks 1959). The functional heterogeneity of the fish hemoglobins enables many species to tolerate hypoxic conditions and exhausting swimming, but also to maintain the gas pressure in the swim bladder at large depths. The hemoglobin repertoire has further increased in various species displaying polymorphic hemoglobin variants differing in oxygen binding properties. The multiplicity of fish hemoglobins as particularly found in the tetraploid salmonids strongly contrasts with the complete loss of hemoglobins in Antarctic icefishes and illustrates the adaptive radiation in the oxygen transport of this successful vertebrate group.

Hemoglobin: Structure, Function and Allostery.

This chapter reviews how allosteric (heterotrophic) effectors and natural mutations impact hemoglobin (Hb) primary physiological function of oxygen binding and transport. First, an introduction about the structure of Hb is provided, including the ensemble of tense and relaxed Hb states and the dynamic equilibrium of Hb multistate. This is followed by a brief review of Hb variants with altered Hb structure and oxygen binding properties. Finally, a review of different endogenous and exogenous allosteric effectors of Hb is presented with particular emphasis on the atomic interactions of synthetic ligands with altered allosteric function of Hb that could potentially be harnessed for the treatment of diseases.