Oxygen Binding Properties Research Articles

Embryonic and Fetal Human Hemoglobins: Structures, Oxygen Binding, and Physiological Roles.

During the past two decades, significant advances have been made in our understanding of the human fetal and embryonic hemoglobins made possible by the availability of pure, highly characterized materials and novel methods, e.g., nano gel filtration, to study their properties and to correct some misconceptions. For example, whereas the structures of the human adult, fetal, and embryonic hemoglobins are very similar, it has generally been assumed that functional differences between them are due to primary sequence effects. However, more recent studies indicate that the strengths of the interactions between their subunits are very different leading to changes in their oxygen binding properties compared to adult hemoglobin. Fetal hemoglobin in the oxy conformation is a much stronger tetramer than adult hemoglobin and dissociates to dimers 70-times less than adult hemoglobin. This property may form the basis for its protective effect against malaria. A major source of the increased strength of fetal hemoglobin resides within the A-helix of its gamma subunit as demonstrated in studies with the hybrid hemoglobin Felix and related hybrids. Re-activating fetal hemoglobin synthesis in vivo is currently a major focus of clinical efforts designed to treat sickle cell anemia since it inhibits the aggregation of sickle hemoglobin. The mechanisms for both the increased oxygen affinity of fetal hemoglobin and its decreased response to DPG have been clarified. Acetylated fetal hemoglobin, which makes up 10-20% of total fetal hemoglobin, has a significantly weakened tetramer structure suggesting a similar role for other kinds of protein acetylation. Embryonic hemoglobins have the weakest tetramer and dimer structures. In general, the progressively increasing strength of the subunit interfaces of the hemoglobin family during development from the embryonic to the fetal and ultimately to the adult types correlates with their temporal appearance and disappearance in vivo, i.e., ontogeny.

The Influence of Oxidative Stress and Natural Antioxidants on Morphometric Parameters of Red Blood Cells, the Hemoglobin Oxygen Binding Capacity, and the Activity of Antioxidant Enzymes.

Using a wide range of different physical and chemical methods, it was found that the oxidative stress caused by addition of hydrogen peroxide to the incubation medium has a significant effect on the conformation of haematoporphyrin, influencing the oxygen-binding properties of haemoglobin in red blood cells. Morphofunctional characteristics of red blood cells change; in particular, we have observed the transformation of erythrocytes, their transition into echinocytes. In erythrocytes, in response to increased lipid peroxidation (LPO) antioxidant enzymes become active. The use of natural antioxidants (β-carotene and resveratrol) works towards reducting the level of oxidative processes. Resveratrol has the greatest antioxidant effect.

Improved accuracy of optode‐based oxygen consumption measurements by removal of system drift and nonlinear derivation

AbstractThis method evaluation aimed to improve the accuracy and precision of the previously published method to measure oxygen consumption using optodes with integrated temperature and salinity correction in dark incubations. Significant short‐term system drift currently requires a correction to remove the drift, thus reducing the precision of the oxygen consumption rates. Frequent nonlinear declines in oxygen concentration with time also call for improved data analysis and identification of its origin. Optodes in titanium casings (Aanderaa™ model 4330) with low oxygen binding properties showed no significant system drift in autoclaved seawater. Nonlinear oxygen dynamics fitting a quadratic polynomial occurred in 28% of 230 field samples, independent of season and water depth. Polynomial curve fit resulted in 64% higher respiration rates when derived within 1 h of the quality assured incubation, than obtained when using linear fit. Carbon substrate limitation explained the nonlinearity of oxygen decline during dark incubations. Pretreatment of the optode attached to stoppers with 0.3 mol dm−3 hydrochloric acid resulted in the most stable performance of the sensor and simultaneously provided proper cleaning of the equipment. A conservative detection limit of 0.97 μmol O2 dm−3 d−1 was calculated for the titanium optodes, matching other methods for oxygen consumption reported in the literature. Thus, we recommend the use of model 4330 optode pretreated with HCl and the derivation of initial respiration rates by a quadratic polynomial function for best accuracy and precision of oxygen consumption in oxygenated surface waters.

High- and low-affinity PEGylated hemoglobin-based oxygen carriers: Differential oxidative stress in a Guinea pig transfusion model

Hemoglobin-based oxygen carriers (HBOCs) are an investigational replacement for blood transfusions and are known to cause oxidative damage to tissues. To investigate the correlation between their oxygen binding properties and these detrimental effects, we investigated two PEGylated HBOCs endowed with different oxygen binding properties - but otherwise chemically identical - in a Guinea pig transfusion model. Plasma samples were analyzed for biochemical markers of inflammation, tissue damage and organ dysfunction; proteins and lipids of heart and kidney extracts were analyzed for markers of oxidative damage. Overall, both HBOCs produced higher oxidative stress in comparison to an auto-transfusion control group. Particularly, tissue 4-hydroxynonenal adducts, tissue malondialdehyde adducts and plasma 8-oxo-2'-deoxyguanosine exhibited significantly higher levels in comparison with the control group. For malondialdehyde adducts, a higher level in the renal tissue was observed for animals treated with the high-affinity HBOC, hinting at a correlation between the HBOCs oxygen binding properties and the oxidative stress they produce. Moreover, we found that the high-affinity HBOC produced greater tissue oxygenation in comparison with the low affinity one, possibly correlating with the higher oxidative stress it induced.

The in vitro Effect of a Magnetic Field on the Oxygen-Transport Function and the Gaseous Transmitter System in Blood

The effect of an alternating magnetic field with a magnetic flux density of 150 mT on the blood oxygen-transport function was studied. In vitro exposure of blood cells was performed following a 10-day series of in vivo exposure of the rat tail artery in combination with administration of chemical compounds that affect the formation of gaseous transmitters. In vitro exposure to a magnetic field changed the oxygen-transport function of the blood, as observed by a greater decrease in the affinity of hemoglobin for oxygen and an increase in the concentration of gaseous transmitters (nitric oxide and hydrogen sulfide). In animals to which nitroglycerin and sodium hydrosulfide were administered exposure to a magnetic field caused a shift in the oxyhemoglobin dissociation curve to the right; this effect was absent when a nonselective inhibitor of the NO synthase enzyme or an irreversible inhibitor of the cystathionine γ-lyase enzyme was added. These results suggest that the magnetic field affects the oxygen-binding properties of the blood by modifying intra-erythrocyte mechanisms that involve gaseous transmitters.

The oxygen-binding properties of hemocyanin from the mollusk Concholepas concholepas

Hemocyanins have highly conserved copper-containing active sites that bind oxygen. However, structural differences among the hemocyanins of various mollusks may affect their physicochemical properties. Here, we studied the oxygen-binding cooperativity and affinity of Concholepas concholepas hemocyanin (CCH) and its two isolated subunits over a wide range of temperatures and pH values. Considering the differences in the quaternary structures of CCH and keyhole limpet hemocyanin (KLH), we hypothesized that the heterodidecameric CCH has different oxygen-binding parameters than the homodidecameric KLH. A novel modification of the polarographic method was applied in which rat liver submitochondrial particles containing cytochrome c oxidase were introduced to totally deplete oxygen of the test solution using ascorbate as the electron donor. This method was both sensitive and reproducible. The results showed that CCH, like other hemocyanins, exhibits cooperativity, showing an inverse relationship between the oxygen-binding parameters and temperature. According to their Hill coefficients, KLH has greater cooperativity than CCH at physiological pH; however, CCH is less sensitive to pH changes than KLH. Appreciable differences in binding behavior were found between the CCH subunits: the cooperativity of CCH-A was not only almost double that of CCH-B, but it was also slightly superior to that of CCH, thus suggesting that the oxygen-binding domains of the CCH subunits are different in their primary structure. Collectively, these data suggest that CCH-A is the main oxygen-binding domain in CCH; CCH-B may play a more structural role, perhaps utilizing its surprising predisposition to form tubular polymers, unlike CCH-A, as demonstrated here using electron microscopy.

Change in morphometric and oxygen-binding properties of erythrocytes in vascular diseases patients

Change in morphometric and oxygen-binding properties of erythrocytes in vascular diseases patients

Predicting the Oxygen-Binding Properties of Platinum Nanoparticle Ensembles by Combining High-Precision Electron Microscopy and Density Functional Theory.

Many studies of heterogeneous catalysis, both experimental and computational, make use of idealized structures such as extended surfaces or regular polyhedral nanoparticles. This simplification neglects the morphological diversity in real commercial oxygen reduction reaction (ORR) catalysts used in fuel-cell cathodes. Here we introduce an approach that combines 3D nanoparticle structures obtained from high-throughput high-precision electron microscopy with density functional theory. Discrepancies between experimental observations and cuboctahedral/truncated-octahedral particles are revealed and discussed using a range of widely used descriptors, such as electron-density, d-band centers, and generalized coordination numbers. We use this new approach to determine the optimum particle size for which both detrimental surface roughness and particle shape effects are minimized.

Structure and Function of Adsorbed Hemoglobin on Silica Nanoparticles: Relationship between the Adsorption Process and the Oxygen Binding Properties.

The connection between the mechanisms of protein adsorption on nanoparticles and the structural and functional properties of the adsorbed protein often remains unclear. We investigate porcine hemoglobin adsorption on silica nanoparticles, and we analyze the structural and functional modifications of adsorbed hemoglobin by UV-vis spectrophotometry, circular dichroism, and oxygen binding measurement. The structural analysis of adsorbed hemoglobin on silica nanoparticles reveals a significant loss of secondary structure and a preservation of the heme electronic structure. However, adsorbed hemoglobin retains its quaternary structure and exhibits an enhanced oxygen affinity with cooperative binding. Moreover, the structural and functional modifications are fully reversible after complete desorption from silica nanoparticles at pH 8.7. The tunable adsorption and desorption of hemoglobin on SNPs with pH change, and the full control of hemoglobin activity by pH, temperature, and the addition of inorganic phosphate effectors opens the way to an interesting system whereby protein adsorption on nanoparticles can allow for full control over hemoglobin oxygen binding activity. Our results suggest that adsorption of hemoglobin on silica nanoparticles leads to a new structural, functional, and dynamic state with full reversibility in a way that significantly differs from protein denaturation.

Study of Erythrocyte Indices, Erythrocyte Morphometric Indicators, and Oxygen-Binding Properties of Hemoglobin Hematoporphyrin Patients with Cardiovascular Diseases

The current study investigates the functional state of erythrocytes and indices of the oxygen-binding capacity of hemoglobin in blood samples from healthy donors and from patients with coronary artery disease and myocardial infarction before and after treatment. It has been established that, in cardiovascular diseases, erythrocyte morphology and hemoglobin oxygen-transporting disorders are observed. Standard therapy does not result in the restoration of the structure and properties of erythrocytes. The authors believe that it is necessary for future therapeutic treatment to include preparations other than cardiovascular agents to enhance the capacity of hemoglobin to transport oxygen to the tissues.

Effect of hypoxia on the composition and state of lipids and oxygen-transport properties of erythrocyte haemoglobin

ABSTRACTThe aim of this study was to investigate the relationship between the composition and state of erythrocytic lipids and oxygen-binding properties of erythrocyte haemoglobin-derived haematoporphyrin under hypoxia. The results from this study showed that hypoxia causes a profound change in the composition of the lipids that form the bilayer of erythrocyte membranes, as well as in the functional characteristics of erythrocytes. The detected changes correlate with morphological reconstruction of erythrocytes and, consequently, affect the oxygen-transport properties of erythrocytes. The obtained results showed that the lipid phase plays an important role in the functioning of erythrocytes in health as well as in event of development of ischemic heart disease and other pathological processes that accompany hypoxia.

Effects of Heme Electronic Structure and Distal Polar Interaction on Functional and Vibrational Properties of Myoglobin.

We analyzed the oxygen (O2) and carbon monoxide (CO) binding properties, autoxidation reaction rate, and FeO2 and FeCO vibrational frequencies of the H64Q mutant of sperm whale myoglobin (Mb) reconstituted with chemically modified heme cofactors possessing a variety of heme Fe electron densities (ρ(Fe)), and the results were compared with those for the previously studied native [Shibata, T. et al. J. Am. Chem. Soc. 2010, 132, 6091-6098], and H64L [Nishimura, R. et al. Inorg. Chem. 2014, 53, 1091-1099], and L29F [Nishimura, R. et al. Inorg. Chem. 2014, 53, 9156-9165] mutants in order to elucidate the effect of changes in the heme electronic structure and distal polar interaction contributing to stabilization of the Fe-bound ligand on the functional and vibrational properties of the protein. The study revealed that, as in the cases of the previously studied native protein [Shibata, T. et al. Inorg. Chem. 2012, 51, 11955-11960], the O2 affinity and autoxidation reaction rate of the H64Q mutant decreased with a decrease in ρ(Fe), as expected from the effect of a change in ρ(Fe) on the resonance between the Fe(2+)-O2 bond and Fe(3+)-O2(-)-like species in the O2 form, while the CO affinity of the protein is independent of a change in ρ(Fe). We also found that the well-known inverse correlation between the frequencies of Fe-bound CO (ν(CO)) and Fe-C (ν(FeC)) stretching [Li, X.-Y.; Spiro, T. G. J. Am. Chem. Soc. 1988, 110, 6024-6033] is affected differently by changes in ρ(Fe) and the distal polar interaction, indicating that the effects of the two electronic perturbations due to the chemical modification of a heme cofactor and the replacement of nearby amino acid residues on the resonance between the two alternative canonical forms of the FeCO fragment in the protein are slightly different from each other. These findings provide a new insight for deeper understanding of the functional regulation of the protein.

Improving Oxygen Binding of Desiccated Human Red Blood Cells

Desiccating human red blood cells (RBCs) to increase their storage life has been the subject of intense research for a number of years. However, drying RBCs invariably compromises their integrity and has detrimental effects on hemoglobin function due to autoxidation. We have previously demonstrated an RBC desiccation and rehydration process that preserves RBC antigenic epitopes better than frozen RBCs. This study expands on those observations by examining what effects this desiccation process has on RBC hemoglobin function with respect to oxygen binding properties. In this paper, we examined RBCs from normal donors which were desiccated to 25% moisture content and stored dry for 2 weeks at room temperature prior to rehydration with plasma followed by structural and functional studies. Our data showed that approximately 98% of the RBCs were intact upon rehydration based on hemolysis assays. Oxygen dissociation curves for the desiccated/rehydrated RBCs showed a left shift compared to fresh RBCs (pO2 = 17 mmHg vs. 26 mmHg, respectively). The desiccated/rehydrated RBCs also showed an increase in methemoglobin compared to fresh RBCs (4.5% vs 0.9%, respectively). 2,3-Diphosphoglycerate concentration of the desiccated/rehydrated RBCs was reduced by 20%. In conclusion, although this RBC dehydration process preserves RBC integrity and hemoglobin oxygen binding properties better than most other dehydration techniques described so far, further optimization and long-term studies are needed to make this procedure acceptable for human transfusion.

The effects of hypercapnia on the West Coast rock lobster (Jasus lalandii) through acute exposure to decreased seawater pH — Physiological and biochemical responses

The cold water palinurid Jasus lalandii (“West Coast rock lobster”) is a commercially important crustacean in South Africa and Namibia and inhabits the Benguela Current Eastern Boundary System. This habitat is characterised by strong upwelling events in summer and algal blooms with their subsequent decay in autumn. Upwelling can lead to acute hypercapnia whereas the algal decay is associated with acute hypercapnic hypoxia. Both types of hypercapnic events could become more frequent and severe in the future due to ongoing climate change. The aim of the present study was, however, to study the capability and mechanisms of response in J. lalandii to hypercapnia exclusively. Accordingly, the following research questions were formulated: 1) To what extent is haemocyanin oxygen-binding affinity of adult J. lalandii pH-sensitive? 2) Can adult male J. lalandii respond swiftly to drastic changes in pH? 3) What physiological mechanisms facilitate a potential response to a drastically declining pH, i.e. acute hypercapnia? These questions were answered by analysing 1) the pH sensitivity of the haemocyanin's oxygen binding properties and 2) in vivo changes in the acid–base balance of adult J. lalandii during acute exposure to hypercapnia (pH7.4). Results showed the following: 1) Haemocyanin displays a strong Bohr shift (whole haemolymph: ΔlogP50/ΔpH=−1.17; dialysed haemolymph: ΔlogP50/ΔpH=−0.84) in response to lowering of pH. 2) Acute hypercapnia leads to a decline in extracellular pH within the initial 1.5h of exposure. 3) Thereafter, active compensation becomes apparent as the bicarbonate levels start to increase, with complete compensation reached after 5h of exposure (+2.3mmoll−1; +48%). 3) This bicarbonate increase is reversed when returning lobsters to normocapnia (pH7.9). 4) Levels of molecular modulators of haemocyanin oxygen affinity (Ca2+, Mg2+ and l-lactate) do not change during acute exposure to hypercapnia.Our results show the capability of adult J. lalandii to rapidly and fully compensate the experienced extracellular acidosis, protecting oxygen carrying capacity of haemocyanin and ensuring an outward gradient of CO2. The West Coast rock lobster is therefore well equipped for its habitat where these hypercapnic events are known to occur frequently.

Oxygen selective iron and cobalt–metalloporphyrin polymers – Extraordinary selectivity at low temperature

Oxygen binding properties of a recent class of porphyrin-based porous organic polymers (POPs) were investigated. POPs containing iron (Fe-POP) and cobalt (Co-POP) were synthesized, characterized and evaluated. Adsorption studies under ambient and cryogenic conditions (91–303 K) were studied. Interestingly, these POPs showed selective adsorption of O2 over N2 of up to 1.3 at 273 K, and most significantly, the oxygen uptake was found to be stable and reversible over the range of temperatures studied. Furthermore, the polymers also exhibited a molecular sieving effect, which showed increasing nitrogen exclusion between 110 K and 160 K and led to an extraordinary O2:N2 selectivity of up to 14.7 at 91 K. The high oxygen loading (13 wt% at 1 atm) together with the high selectivity suggests great potential for use of these materials in hybrid adsorption–cryogenic air separation systems.

Functional characterisation of the haemoglobins of the migratory notothenioid fish Dissostichus eleginoides

This study addresses the primary structure, the oxygen-binding properties and the CO-rebinding kinetics of the haemoglobins of the Patagonian toothfish Dissostichus eleginoides. D. eleginoides belongs to the family Nototheniidae, the most diversified of the suborder Notothenioidei, mostly exhibiting an Antarctic distribution. Some of its features are typical of Antarctic species, some are not. For instance, D. eleginoides appears not to have functional antifreeze glycoproteins (consistent with its non-Antarctic distribution). In contrast, it has a major and a minor haemoglobin (similar to many Antarctic notothenioids), and their very low oxygen affinity does not follow the trend of other non-Antarctic notothenioids and appears typical of cold-adapted species. Moreover, the amino-acid sequence reveals high identity with the globins of Antarctic notothenioids, arguing in favour of a common origin within notothenioids, and indicates that the primary structure of the major and minor haemoglobins has undergone modifications only to a limited extent. The ligand-rebinding kinetics of the major haemoglobin of D. eleginoides indicate a strong stabilisation of the quaternary T state at lower pH values.

Respiratory Properties of Hemocyanin From Wild and Aquacultured Penaeid Shrimp and the Effects of Chronic Exposure to Hypoxia.

Properties of hemocyanins vary greatly among crustaceans due to environmental conditions, lifestyle, and genetic variation. These properties can also be modified to maintain aerobic respiration in response to ambient hypoxia, as experienced by both aquacultured and wild populations of penaeid shrimp. Under normoxic conditions, hemocyanin concentrations were significantly higher (P < 0.001) in aquacultured Pacific whiteleg shrimp, Litopenaeus vannamei (10.3 g/100 ml ± 0.23 SEM, n = 49), compared to those in individuals of wild-caught L. vannamei (7.0 g/100 ml ± 0.52 SEM, n = 10), wild Farfantepenaeus aztecus (7.10 g/100 ml ± 0.48 SEM, n = 28), and wild Litopenaeus setiferus (8.0 g/100 ml ± 0.22 SEM, n = 37). Oxygen affinity of hemocyanin at 25 °C in both populations of L. vannamei was higher (Kruskal-Wallis ANOVA on ranks, P < 0.001) (aquacultured P50 = 1.47 kPa ± 0.03 SEM; wild P50 = 1.72 kPa ± 0.01 SEM at pH 7.4) than that of both Atlantic species (F. aztecus P50 = 3.94 kPa ± 0.06 SEM, L. setiferus P50 = 3.98 kPa ± 0.04 SEM at pH 7.4). The effect of l-lactate on oxygen affinity was similar among all wild groups, but significantly smaller in the aquacultured L. vannamei. Total hemocyanin concentration and oxygen binding properties were measured after exposure to 12 days and 25-31 days of hypoxia (30% air saturation). Aquacultured L. vannamei showed no change in hemocyanin concentration for up to 31 days, but both wild F. aztecus and wild L. setiferus displayed a significant increase over the same time period. No discernible change in oxygen affinity of hemocyanin was detected in any of the three species. Hypoxia tolerance appears to differ among these species of penaeid shrimp, due to either an inherent difference among the species, domestication by aquaculture, or a combination of both.

Acid–base balance and changes in haemolymph properties of the South African rock lobsters, Jasus lalandii, a palinurid decapod, during chronic hypercapnia

Few studies exist reporting on long-term exposure of crustaceans to hypercapnia. We exposed juvenile South African rock lobsters, Jasus lalandii, to hypercapnic conditions of pH 7.3 for 28 weeks and subsequently analysed changes in the extracellular fluid (haemolymph). Results revealed, for the first time, adjustments in the haemolymph of a palinurid crustacean during chronic hypercapnic exposure: 1) acid-base balance was adjusted and sustained by increased bicarbonate and 2) quantity and oxygen binding properties of haemocyanin changed. Compared with lobsters kept under normocapnic conditions (pH 8.0), during prolonged hypercapnia, juvenile lobsters increased bicarbonate buffering of haemolymph. This is necessary to provide optimum pH conditions for oxygen binding of haemocyanin and functioning of respiration in the presence of a strong Bohr Effect. Furthermore, modification of the intrinsic structure of the haemocyanin molecule, and not the presence of molecular modulators, seems to improve oxygen affinity under conditions of elevated pCO2.

Blue blood on ice: modulated blood oxygen transport facilitates cold compensation and eurythermy in an Antarctic octopod.

IntroductionThe Antarctic Ocean hosts a rich and diverse fauna despite inhospitable temperatures close to freezing, which require specialist adaptations to sustain animal activity and various underlying body functions. While oxygen transport has been suggested to be key in setting thermal tolerance in warmer climates, this constraint is relaxed in Antarctic fishes and crustaceans, due to high levels of dissolved oxygen. Less is known about how other Antarctic ectotherms cope with temperatures near zero, particularly the more active invertebrates like the abundant octopods. A continued reliance on the highly specialised blood oxygen transport system of cephalopods may concur with functional constraints at cold temperatures. We therefore analysed the octopod’s central oxygen transport component, the blue blood pigment haemocyanin, to unravel strategies that sustain oxygen supply at cold temperatures.ResultsTo identify adaptive compensation of blood oxygen transport in octopods from different climatic regions, we compared haemocyanin oxygen binding properties, oxygen carrying capacities as well as haemolymph protein and ion composition between the Antarctic octopod Pareledone charcoti, the South-east Australian Octopus pallidus and the Mediterranean Eledone moschata. In the Antarctic Pareledone charcoti at 0°C, oxygen unloading by haemocyanin was poor but supported by high levels of dissolved oxygen. However, lower oxygen affinity and higher oxygen carrying capacity compared to warm water octopods, still enabled significant contribution of haemocyanin to oxygen transport at 0°C. At warmer temperatures, haemocyanin of Pareledone charcoti releases most of the bound oxygen, supporting oxygen supply at 10°C. In warm water octopods, increasing oxygen affinities reduce the ability to release oxygen from haemocyanin at colder temperatures. Though, unlike Eledone moschata, Octopus pallidus attenuated this increase below 15°C.ConclusionsAdjustments of haemocyanin physiological function and haemocyanin concentrations but also high dissolved oxygen concentrations support oxygen supply in the Antarctic octopus Pareledone charcoti at near freezing temperatures. Increased oxygen supply by haemocyanin at warmer temperatures supports extended warm tolerance and thus eurythermy of Pareledone charcoti. Limited haemocyanin function towards colder temperatures in Antarctic and warm water octopods highlights the general role of haemocyanin oxygen transport in constraining cold tolerance in octopods.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-015-0097-x) contains supplementary material, which is available to authorized users.

Effects of heme modification on oxygen affinity and cooperativity of human adult hemoglobin

We substituted strongly electron-withdrawing trifluoromethyl ( CF3) group(s) as heme side chain(s) of human adult hemoglobin (Hb) to achieve large alterations of the heme electronic structure, in order to elucidate the relationship between the oxygen ( O2) binding properties of Hb and the electronic properties of heme peripheral side chains. The obtained results were compared with those of similar studies performed on myoglobin (Mb), e.g. (Nishimura R, Matsumoto D, Shibata T, Yanagisawa S, Ogura T, Tai H, Matsuo T, Hirota S, Neya S, Suzuki A, and Yamamoto Y. Inorg. Chem. 2014; 53: 9156–9165). These two proteins shared the common feature of a decrease in O2affinity upon the CF3substitution(s). Using the P50value, which is the partial pressure of O2required for 50% oxygenation of a protein, and the equilibrium constant ( p Ka) of the "acid-alkaline transition" in the met form of a protein as measures of the O2affinity and the electron density of heme Fe atom of the protein, respectively, a linear p Ka- log (1/P50) relationship was demonstrated for the Hb and Mb systems. The native Hb, however, deviated from the p Ka- log (1/P50) relationship, while the native Mb followed it. These results highlighted the significance of the vinyl side chains of the heme cofactor in the functional control of Hb through tertiary and quaternary structural changes upon the oxygenation of the protein.