Steady-State, Hemoglobin-Facilitated O2 Transport in Human Erythrocytes

We measured the rate of oxygen transport through thin (165 µ) films of packed erythrocytes (Hb concentration = 30 g/100 ml). Under optimal conditions steady-state O₂ diffusion was nearly three times that found when the hemoglobin was prevented from acting as a carrier molecule by carbon monoxide binding or high oxygen back pressure. After each experiment we measured hemolysis and found that it averaged less than 1%. Hemolysis could not account for the facilitation, thus proving that facilitated transport of O₂ by hemoglobin can occur in red blood cells. The rate of facilitated transport was identical for Hb solutions of equal concentration to the cells. We interpret this to mean that under the conditions of our experiments the red cell membrane offers no detectable diffusion resistance to O₂ and that the mobility of Hb in intact red cells is the same as in concentrated Hb solution.     

Study on Conformational Changes in Hemoglobin Protoporphyrin in Essential Hypertension

Changes in protoporphyrin conformation, partial pressures of O₂ and CO₂, and the mechanisms responsible for regulation of pCa and pH in erythrocytes were studied in essential hypertension (EH). Changes in protoporphyrin conformation in EH were accompanied by a decrease in the partial pressure of O₂ and an increase in the partial pressure of CO₂. This was associated with increased activities of Na⁺/H⁺-exchange and Ca²⁺-dependent K⁺-channels and with a decreased activity of Ca²⁺-ATPase. The changes in protoporphyrin conformation in EH are suggested to decrease the efficiency of O₂ metabolism in hemoglobin and increase the values of intracellular pCa and pH of erythrocytes.     

Atmospheric stability in cell culture vessels

Summary We have investigated the atmospheric stability in polystyrene and glass cell culture vessels by measuring the dissolved O₂ and CO₂ in the media of both seeded and unseeded culture vessels incubated at 37°C. There was no diffusion of either O₂ or CO₂ through glass vessels. At low partial pressures of oxygen (P_{O 2}), oxygen diffused into the polystyrene flasks at a rate of 1 to 2 mm Hg per 24 hr, and at high P_{O 2}, oxygen diffused slowly out of polystyrene flasks. CO₂ diffused out of polystyrene flasks with a half-time of 260 hr resulting in a considerable elevation in pH. In seeded polystyrene flasks with the P_{O 2} ⩽ room air, cellular oxygen consumption was masked by the inward diffusion of oxygen. In addition, the fall in pH due to metabolic CO₂ and organic acid production during cell growth in polystyrene flasks was buffered by the diffusion of CO₂ out of the vessels. Presented, in part, by Dr. Arthur Balin in partial fulfillment of the requirements for the Ph.D. This work was supported by USPHS grants AG-00378 from the National Institute of Aging and CA-14345 from the National Cancer Institute and NR 202-005 from the Office of Naval Research. A.K.B. is a trainee of the Medical Scientist Training Program, National Institutes of Health (GM 02046).     

The relationship between contents of photosynthetic metabolites and the rate of photosynthetic carbon assimilation in leaves of Amaranthus edulis L.

The relationship between the gas-exchange characteristics of attached leaves of Amaranthus edulis L. and the contents of photosynthetic intermediates was examined in response to changing irradiance and intercellular partial pressure of CO₂. After determination of the rate of CO₂ assimilation at known intercellular CO₂ pressure and irradiance, the leaf was freeze-clamped and the contents of ribulose-1,5-bisphosphate, glycerate-3-phosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, fructose-6-phosphate, triose phosphates, phosphoenolpyruvate, pyruvate, oxaloacetate, aspartate, alanine, malate and glutamate were measured. A comparison between the sizes of metabolite pools and theoretical calculations of metabolite gradients required for transport between the mesophyll and the bundle-sheath cells showed that aspartate, alanine, glycerate-3-phosphate and triose phosphates were present in sufficient quantities to support transport by diffusion, whereas pyruvate and oxaloacetate were not likely to contribute appreciably to the flux of carbon between the two cell types. The amounts of ribulose-1,5-bisphosphate were high at low intercellular partial pressures of CO₂, and fell rapidly as the CO₂-assimilation rate increased with increasing intercellular partial pressures of CO₂, indicating that bundle-sheath CO₂ concentrations fell at low intercellular partial pressures of CO₂. In contrast, the amount of phosphoenolpyruvate and of C₄-cycle intermediates declined at low intercellular partial pressures of CO₂. This behaviour is discussed in relation to the co-ordination of carbon assimilation between the Calvin and C₄ cycles.Abbreviations PEP phosphoenolpyruvate - PGA glycerate-3-phosphate - p _i intercellular CO₂ pressure - RuBP ribulose-1,5-bisphosphate - triose-P triose phosphates     

Effects of acclimation to altitude on oxygen affinity and organic phosphate concentrations in pigeon blood.

Hematocrit ratio, hemoglobin concentration and blood oxygen affinity, Bohr effect factor and Hill coefficient, adenosine triphosphate and inositol pentaphosphate (IPP) concentrations were studied in blood of adult pigeons exposed first at 140 m, and then for 3 weeks at 4000 m in an altitude chamber. At altitude, the hematocrit ratio and hemoglobin concentration significantly increased, IPP concentration decreased, and P₅₀ did not change. A lower mean red cell age and a higher hemoglobin concentration may account for the unchanged P₅₀. Adaptation to hypoxia of the tissue oxygen supply was shown by a greater blood O₂ capacitance (ΔC_HbO₂/Δ_o2) in the physiological range of oxygen partial pressures.     

Quantitative evaluation of respiration induced metabolic oscillations in erythrocytes

The changes in the partial pressures of oxygen and carbon dioxide (P_O2 and P_CO2) during blood circulation alter erythrocyte metabolism, hereby causing flux changes between oxygenated and deoxygenated blood. In the study we have modeled this effect by extending the comprehensive kinetic model by Mulquiney and Kuchel [P.J. Mulquiney, and P.W. Kuchel. Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement, Biochem. J. 1999, 342, 581–596.] with a kinetic model of hemoglobin oxy-/deoxygenation transition based on an oxygen dissociation model developed by Dash and Bassingthwaighte [R. Dash, and J. Bassingthwaighte. Blood HbO₂ and HbCO₂ dissociation curves at varied O₂, CO₂, pH, 2,3-DPG and temperature levels, Ann. Biomed. Eng., 2004, 32(12), 1676–1693.]. The system has been studied during transitions from the arterial to the venous phases by simply forcing P_O2 and P_CO2 to follow the physiological values of venous and arterial blood. The investigations show that the system passively follows a limit cycle driven by the forced oscillations of P_O2 and is thus inadequately described solely by steady state consideration. The metabolic system exhibits a broad distribution of time scales. Relaxations of modes with hemoglobin and Mg²⁺ binding reactions are very fast, while modes involving glycolytic, membrane transport and 2,3-BPG shunt reactions are much slower. Incomplete slow mode relaxations during the 60 s period of the forced transitions cause significant overshoots of important fluxes and metabolite concentrations – notably ATP, 2,3-BPG, and Mg²⁺. The overshoot phenomenon arises in consequence of a periodical forcing and is likely to be widespread in nature – warranting a special consideration for relevant systems.     

Mathematical studies of the interaction of respiratory gases with whole blood II. CO2 absorption

Some progress has been made on the problem of the interaction of respiratory gases with whole blood. A working mathematical model for the O₂−CO₂ interaction phenomena has been developed from mathematical studies of the data. The Edsall-Wyman (1958) model for CO₂ absorption is improved upon in this paper by consolidating it with the O₂ absorption model developed in paper I of this set (Bernard, S. R.,Bull. Math. Biophysics,22, 391–415, 1960). This improved model assumed the effect of O₂ on CO₂ absorption is mediated through the electrical charge possessed by the hemoglobin molecule,i.e., O₂ molecules bound to hemoglobin displace protons from the hemoglobin thereby increasing the negative charge on the hemoglobin and at the same time increasing the acidity of the solution. The model is tested against the data.     

Hemoglobin‐based oxygen carrier and convection enhanced oxygen transport in a hollow fiber bioreactor

A mathematical model was developed to study O₂ transport in a convection enhanced hepatic hollow fiber (HF) bioreactor, with hemoglobin‐based O₂ carriers (HBOCs) present in the flowing cell culture media stream of the HF lumen. In this study, four HBOCs were evaluated: PEG‐conjugated human hemoglobin (MP4), human hemoglobin (hHb), bovine hemoglobin (BvHb) and polymerized bovine hemoglobin (PolyBvHb). In addition, two types of convective flow in the HF extra capillary space (ECS) were considered in this study. Starling flow naturally occurs when both of the ECS ports are closed. If one of the ECS ports is open, forced convective flow through the ECS will occur due to the imposed pressure difference between the lumen and ECS. This type of flow is referred to as cross‐flow in this work, since some of the fluid entering the HF lumen will pass across the HF membrane and exit via the open ECS port. In this work, we can predict the dissolved O₂ concentration profile as well as the O₂ transport flux in an individual HF of the bioreactor by solving the coupled momentum and mass transport equations. Our results show that supplementation of the cell culture media with HBOCs can dramatically enhance O₂ transport to the ECS (containing hepatocytes) and lead to the formation of an in vivo‐like O₂ spectrum for the optimal culture of hepatocytes. However, both Starling flow and cross‐flow have a very limited effect on O₂ transport in the ECS. Taken together, this work represents a novel predictive tool that can be used to design or analyze HF bioreactors that expose cultured cells to defined overall concentrations and gradients of O₂. Biotechnol. Bioeng. 2009;102: 1603–1612. © 2008 Wiley Periodicals, Inc.     

Simultaneous Measurements of Steady State Chlorophyll a Fluorescence and CO(2) Assimilation in Leaves: The Relationship between Fluorescence and Photosynthesis in C(3) and C(4) Plants

Rates of CO₂ assimilation and steady state chlorophyll a fluorescence were measured simultaneously at different intercellular partial pressures of CO₂ in attached cotton (Gossypium hirsutum L. cv Deltapine 16) leaves at 25°C. Electron transport activity for CO₂ assimilation plus photorespiration was calculated for these experiments. Under light saturating (1750 microeinsteins per square meter per second) and light limiting (700 microeinsteins per square meter per second) conditions there was a good correlation between fluorescence and the calculated electron transport activity at 19 and 200 millibars O₂, and between fluorescence and rates of CO₂ assimilation at 19 millibars but not 200 millibars O₂. The values of fluorescence measured at about 220 microbars intercellular CO₂ were not greatly affected by increasing O₂ from 19 to 800 millibars. Fluorescence increased with light intensity at any one intercellular CO₂ partial pressure. But the values obtained for fluorescence, expressed as a ratio of the maximum fluorescence obtained in DCMU-treated tissue, over the same range of CO₂ partial pressure at 500 microeinsteins per square meter per second were similar to those obtained at 1000 and 2000 microeinsteins per square meter per second. There were two phases in the observed correlation between fluorescence and calculated electron transport activity: an initial inverse relationship at low CO₂ partial pressures which reversed to a positive correlation at higher values of CO₂ partial pressures. Similar results were observed in the C₃ species Helianthus annuus L., Phaseolus vulgaris L., and Brassica chinensis. In all C₄ species (Zea mays L., Sorghum bicolor L., Panicum maximum Jacq., Amaranthus edulis Speg., and Echinochloa frumentacea [Roxb.] Link) examined changes in fluorescence were directly correlated with changes in CO₂ assimilation rates. The nature and the extent to which Q (primary quencher) and high-energy state (q_E) quenching function in determining the steady state fluorescence obtained during photosynthesis in leaves is discussed.     

A Model Describing the Regulation of Ribulose-1,5-Bisphosphate Carboxylase, Electron Transport, and Triose Phosphate Use in Response to Light Intensity and CO(2) in C(3) Plants

A model of the regulation of the activity of ribulose-1,5-bisphosphate carboxylase, electron transport, and the rate of orthophosphate regeneration by starch and sucrose synthesis in response to changes in light intensity and partial pressures of CO₂ and O₂ is presented. The key assumption behind the model is that nonlimiting processes of photosynthesis are regulated to balance the capacity of limiting processes. Thus, at CO₂ partial pressures below ambient, when a limitation on photosynthesis by the capacity of rubisco is postulated, the activities of electron transport and phosphate regeneration are down-regulated in order that the rate of RuBP regeneration matches the rate of RuBP consumption by rubisco. Similarly, at subsaturating light intensity or elevated CO₂, when electron transport or Pi regeneration may limit photosynthesis, the activity of rubisco is downregulated to balance the limitation in the rate of RuBP regeneration. Comparisons with published data demonstrate a general consistency between modelled predictions and measured results.     

Predictability of PaO2 in different inert gas-oxygen environments

Recent experimentation with metabolic changes in rats exposed to thermally isoconductive environments has involved the use of inert gas-oxygen mixtures with different total pressures (PT) and inspired oxygen fractions (F₁O₂). To determine the F_IO₂ for each mixture that would result in similar arterial oxygen pressures (P _a O₂) and saturations (OS), arterial blood of dogs breathing the mixtures through a mask was analyzed for pH,^PO₂,^PCO₂, and OS. Using P_IO₂'s calculated from the alveolar gas equation as a theoretical basis, the oxygen partial pressure for the helium mixture had to be increased by 5.6% while that in argon decreased by 10.2% below the computed values to obtain P _a O₂'s acceptably similar to those resulting from air exposure. No consistent variation in pH, P _a CO₂, and OS were apparent. Based on the data presented, which were obtained under highly specialized conditions, it appears that the use of helium and argon as diluent gases may significantly affect arterial oxygen tension.     

A Mathematical Model of the Controlled Plant of the Réspiratory System

Ability to predict the dynamic response of oxygen, carbon dioxide tensions, and pH in blood and tissues to abrupt changes in ventilation is important in the mathematical modeling of the respiratory system. In this study, the controlled plant (the amount and distribution of O₂ and CO₂) of the respiratory system is modeled. Although the body tissues are divided into a finite number of “compartments” (three tissue groups), in contrast to earlier models, the blood and tissue gas tensions within each compartment are considered to be continuously distributed in time and in one spatial coordinate. The mass conservation equations for oxygen and carbon dioxide involved in the blood-tissue gas exchange are described by a set of partial differential equations which take into account convection of O₂ and CO₂ caused by the flow of blood as well as diffusion due to local tension gradients. Nonlinear algebraic equations for the dissociation curves, which take into account the Haldane and Bohr effects in blood, are used to obtain the relationships between concentrations and partial pressures. Time-variable delays caused by the arterial and venous transport of the respiratory gases are also included. The model so constructed successfully reproduced actual O₂ and CO₂ tensions in arterial blood, and in muscle venous and mixed venous blood when ventilation was abruptly changed.     

Oxygen limitation of N2 fixation in stem-girdled and nitrate-treated soybean

The effects of increasing rhizosphere pO₂on nitrogenase activity and nodule resistance to O₂diffusion were investigated in soybean plants [Glycine max (L.) Merr. cv. Harosoy 63] in which nitrogenase (EC 1.7.99.2) activities were inhibited by (a) removal of the phloem tissue at the base of the stem (stem girdling), (b) exposure of roots to 10 mM NO₃over 5 days (NO₃-treated), or (c) partial inactivation of nitrogenase activity by an exposure of nodulated roots to 100 kPa O₂(O₂-inhibitcd). In control plants and in plants which had been treated with 100 kPa O₂, increasing rhizosphere O₂concentrations in 10 kPa increments from 20 to 70 kPa did not alter the steady-state nitrogenase activity. In contrast, in plants in which nitrogenase activities were depressed by stem girdling or by exposure to NO₃, increasing rhizosphere pO₂resulted in a recovery of 57 or 67%, respectively, of the initial, depressed rates of nitrogenase activity. This suggests that the nitrogenase activity of stem-girdled and NO₃-treated soybeans was O₂-limited. For each treatment, theoretical resistance values for O₂diffusion into nodules were estimated from measured rates of CO₂exchange, assuming a respiratory quotient of 1.1 and 0 kPa of O₂in the infected cells. At an external partial pressure of 20 kPa O₂, the stem-girdled and NO₃^--treated plants displayed resistance values which were 4 to 8.6 times higher than those in the nodules of the control plants. In control and O₂-inhibited plants, increases in pO₂from 20 to 70 kPa in 10 kPa increments resulted in a 2.5- to 3.9-fold increase in diffusion resistance to O₂, and had little effect on either respiration or nitrogenase activity. In contrast, in stem-girdled and NO₃^--treated plants, increases in external pO₂had little effect on diffusion resistance to O₂, but resulted in a 2.3- to 3.2-fold increase in nodule respiration and nitrogenase activity. These results are consistent with stem-girdling and NO₃^--inhibition treatments limiting phloem supply to nodules causing an increase in diffusion resistance to O₂at 20 kPa and an apparent insensitivity of diffusion resistance to increases in external pO₂.     

Physical and morphological constraints on transport in nodules

For active nodule nitrogen fixation, O₂, N₂, and carbohydrate must be transported throughout the nodule. No quantitative analysis of these transport processes in the nodules has been presented. By invoking several simplifying assumptions, a second-order differential equation for the various gradients and concentrations in the nodule was solved. Even though the nodule can only be approximated in this analysis, it indicates clearly that intercellular gas spaces must exist in nodules for adequate O₂ distribution. To preserve low O₂ concentrations and protect the nitrogenase, these gas spaces cannot be in direct contact with the ambient atmosphere. It is hypothesized that a gas barrier exists in the cortical region of the nodule to limit O₂ diffusion. This barrier would not substantially inhibit N₂ transport. Carbohydrate transport from the vascular tissue via diffusion in the liquid phase can adequately accommodate the requirements within the nodule.     

Is Transfer of CO2 to C3-Plants Purely by Diffusion?

The dependence of the CO₂ compensation concentration on O₂ partial pressure and the dependence of differential uptake of ¹⁴CO₂ and ¹²CO₂ on CO₂ and O₂ partial pressures are analyzed in illuminated white clover (Trifolium repens L.) leaves. The data show a deviation of the photosynthetic gas exchange from ribulose bisphosphate carboxylase oxygenase kinetics at 10°C but not at 30°C. This deviation is due to an effect of CO₂ partial pressure on the ratio of photosynthesis to photorespiration which can be explained if active inorganic carbon transport is assumed.     

Influence of O2 availability on NO and N2O release by nitrification and denitrification in soils

The availability of O₂ is believed to be one of the main factors regulating nitrification and denitrification and the release of NO and N₂O. The availability of O₂ in soil is controlled by the O₂ partial pressure in the gas phase and by the moisture content in the soil. Therefore, we investigated the influence of O₂ partial pressures and soil moisture contents on the NO and N₂O release in a sandy and a loamy silt and differentiated between nitrification and denitrification by selective inhibition of nitrification with 10 Pa acetylene. At 60% whc (maximum water holding capacity) NO and N₂O release by denitrification increased with decreasing O₂ partial pressure and reached a maximum under anoxic conditions. Under anoxic conditions NO and N₂O were only released by denitrification. NO and N₂O release by nitrification also increased with decreasing O₂ partial pressure, but reached a maximum at 0.1–0.5% O₂ and then decreased again. Nitrification was the main source of NO and N₂O at O₂ partial pressures higher than 0.1–0.5% O₂. At lower O₂ partial pressures denitrification was the main source of NO and N₂O. With decreasing O₂ partial pressure N₂O release increased more than NO release, indicating that the N₂O release was more sensitive against O₂ than the NO release. At ambient O₂ partial pressure (20.5% O₂) NO and N₂O release by denitrification increased with increasing soil moisture content. The maximum NO and N₂O release was observed at soil moisture contents of 65–80% whc and 100% whc, respectively. NO and N₂O release by nitrification also increased with increasing soil moisture content with a maximum at 45–55% whc and 90% whc, respectively. Nitrification was the main source of NO and N₂O at soil moisture contents lower than 90% whc and 80% whc, respectively. Higher soil moisture contents favoured NO and N₂O release by denitrification. Soil texture had also an effect on the release of NO and N₂O. The coarse-textured sandy silt released more NO than N₂O compared with the fine-textured loamy silt. At high soil moisture contents (80–100% whc) the fine-textured soil showed a higher N₂O release by denitrification than the coarse-textured soil. We assume that the fine-textured soil became anoxic at a lower soil moisture content than the coarse-textured soil. In conclusion, the effects of O₂ partial pressure, soil moisture and soil texture were consistent with the theory that denitrification increasingly contributes to the release of NO and in particular N₂O when conditions for soil microorganisms become increasingly anoxic.     

Oxidation of lipids and membranes II: Effects of oxidants on cerebral white matter homogenates

Bovine cerebral white matter homogenates were oxidized by various oxidizing solutions of equal molarity and subsequently extracted with water. Most of the oxidants tested (K-dichromate, FeCl₃, H₂O₂, O₂, and chloroperbenzoic, ascorbic, performic, and periodic acids) rendered the various myelin constituents less extractable than the constituents of unoxidized control homogenates. KMnO₄, and to a lesser degree hemoglobin, rendered myelin constituents more extractable with water than those of the control homogenates. The findings indicate that most of the oxidants produced stabilization of the lamellar pattern, probably through cross-linking and polymerization. KMnO₄ and hemoglobin caused labilization and breakdown of the membranous structure. Proof that stabilization of membranes is caused by some oxidants and that fragmentation occurs by the action of KMnO₄ and hemoglobin was obtained by the light-scattering technique and by electron microscopy of the oxidized homogenates. Evidence obtained indicated that formation of hydrophobic end groups during oxidation favors polymerization, while prevalence of hydrophilic groups is associated with fragmentation.     

Resistance of citrus fruit to mass transport of water vapor and other gases

The resistance of oranges (Citrus sinensis L. Osbeck) and grapefruit (Citrus paradisi Macf.) to ethylene, O₂, CO₂, and H₂O mass transport was investigated anatomically with scanning electron microscope and physiologically by gas exchange measurements at steady state. The resistance of untreated fruit to water vapor is far less than to ethylene, CO₂ and O₂. Waxing partially or completely plugs stomatal pores and forms an intermittent cracked layer over the surface of fruit, restricting transport of ethylene, O₂, and CO₂, but not of water; whereas individual sealing of fruit with high density polyethylene films reduces water transport by 90% without substantially inhibiting gas exchange.

Stomata of harvested citrus fruits are essentially closed. However, ethylene, O₂ and CO₂ still diffuse mainly through the residual stomatal opening where the relative transport resistance (approximately 6,000 seconds per centimeter) depends on the relative diffusivity of each gas in air. Water moves preferentially by a different pathway, probably through a liquid aqueous phase in the cuticle where water conductance is 60-fold greater. Other gases are constrained from using this pathway because their diffusivity in liquid water is 10⁴-fold less than in air.

     

Photoinhibition of intact attached leaves of c(3) plants illuminated in the absence of both carbon dioxide and of photorespiration

When leaflets of bean and leaves of other species of C₃ plants are illuminated in the absence of CO₂ and at low O₂ partial pressure, the capacity for CO₂ assimilation at saturating light and its efficiency at low light intensities are inhibited. This photoinhibition is dependent on leaflet age and period of illumination. In young leaflets and following short exposure to these photoinhibitory conditions, some recovery of CO₂ assimilation capacity is observed immediately after treatment. Following substantial (70 to 80%) photoinhibition of CO₂ assimilation, recovery in fully expanded leaflets is observed only after 48 hours in normal air. The photoinhibition is largely prevented by providing CO₂ at partial pressures equivalent to the CO₂ compensation point, or by >210 millibars O₂ which permits internal CO₂ production by photorespiration. If leaflets are illuminated in 60 microbars CO₂ and 210 millibars O₂ (the CO₂ compensation point in air), no photoinhibition is observed. Electron transport processes and fluorescence emission associated with photosystem II are inhibited in chloroplast thylakoids isolated from leaflets after illumination in zero CO₂ and 10 millibars O₂. These studies support the hypothesis that CO₂ recycling through photorespiration is one means of effectively dissipating excess photochemical energy when CO₂ supply to illuminated leaves is limited.     

Hemoglobin crystals immersed in liquid oxygen reveal diffusion channels

Human hemoglobin (HbA) transports molecular oxygen (O₂) from the lung to tissues where the partial pressure of O₂ is lower. O₂ binds to HbA at the heme cofactor and is stabilized by a distal histidine (HisE7). HisE7 has been observed to occupy opened and closed conformations, and is postulated to act as a gate controlling the binding/release of O₂. However, it has been suggested that HbA also contains intraprotein oxygen channels for entrances/exits far from the heme. In this study, we developed a novel method of crystal immersion in liquid oxygen prior to X-ray data collection. In the crystals immersed in liquid oxygen, the heme center was oxidized to generate aquomethemoglobin. Increases of structural flexibility were also observed in regions that are synonymous with previously postulated oxygen channels. These regions also correspond to medically relevant mutations which affect O₂ affinity. The way HbA utilizes these O₂ channels could have a profound impact on understanding the relationship of HbA O₂ transport within these disease conditions. Finally, the liquid oxygen immersion technique can be utilized as a new tool to crystallographically examine proteins and protein complexes which utilize O₂ for enzyme catalysis or transport.