In vitro kinetics of oxygen transport in erythrocyte suspension or unmodified hemoglobin solution from human and other animals

Oxygen transport behavior in erythrocyte suspension or in hemoglobin solution was studied as a potential therapeutic model for the clinical treatment of blood loss, and this can also provide physiological data with which to evaluate blood substitutes. In the present project, we examined the in vitro kinetics of hemoglobin binding to and releasing oxygen, to provide detailed oxygen-flux measurements for unmodified hemoglobin solutions and erythrocyte suspensions in human, as well as other vertebrates. An in vitro method was used, based on a widely used artificial system, with the oxygen saturation level being detected in real time. Results from this study indicated that the kinetic curves of human erythrocyte suspensions and hemoglobin solutions were either S-shaped or hyperbolic, respectively. Based on these curves, the significance of T(50) emerged in our investigation, where T(50) is defined as the time needed for 50% hemoglobin to be saturated with oxygen, and reflects the efficiency with which hemoglobin carries oxygen. This parameter may be used to diagnose blood diseases, and could be a standard for evaluating blood substitutes. In this study, we also compared the T(50) of 4 species of vertebrates, and found that it shows a distinct efficiency of oxygen binding related to species, and potentially reveals the evolutionary function of hemoglobin and its possible adaptation to the environment.     

Direct measurement of equilibrium constants for high-affinity hemoglobins

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (K(D) < 1 micro M) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and approximately 100 micro M(-1) respectively, indicate that they are not capable of facilitating oxygen transport.     

A ubiquitously expressed human hexacoordinate hemoglobin

We have identified a new human hemoglobin that we call histoglobin because it is expressed in a wide array of tissues. Histoglobin shares less than 30% identity with the other human hemoglobins, and the gene contains an intron in an unprecedented location. Spectroscopic and kinetic experiments with recombinant human histoglobin indicate that it is a hexacoordinate hemoglobin with significantly different ligand binding characteristics than the other human hexacoordinate hemoglobin, neuroglobin. In contrast to the very high oxygen affinities displayed by most hexacoordinate hemoglobins, the biophysical characteristics of histoglobin indicate that it could facilitate oxygen transport. The discovery of histoglobin demonstrates that humans, like plants, differentially express multiple hexacoordinate hemoglobins.     

Influence of hypoxia and of hypoxemia on the development of cardiac activity in zebrafish larvae

Cardiac activity and anaerobic metabolism were analyzed in zebrafish larvae raised under normoxia (PO(2) = 20 kPa) and under chronic hypoxia (PO(2) = 10 kPa) at three different temperatures (25, 28, and 31 degrees C). Heart rate increased with development and with temperature. Under normoxia, cardiac output increased significantly at high temperature (31 degrees C), but not at 28 or at 25 degrees C. Under chronic hypoxia, however, heart rate as well as cardiac output increased at all temperatures in larvae at about hatching time or shortly thereafter. Cardiac activity of larvae raised for 2 wk after fertilization with a reduced hemoglobin oxygen-carrying capacity in their blood (hypoxemia; due to the presence of CO or of phenylhydrazine in the incubation water) was not different from control animals. Whole body lactate content of these animals did not increase. Thus there was no indication of a stimulated anaerobic energy metabolism. The increase in cardiac activity observed during hypoxia suggests that at about hatching time receptors are present that sense hypoxic conditions, and this information can be used to induce a stimulation of convective oxygen transport to compensate for a reduction in bulk oxygen diffusion in the face of a reduced oxygen gradient between environmental water and tissues. Under normoxia, however, the PO(2) gradient between environmental water and tissues and diffusional oxygen transport assure sufficient oxygen supply even if hemoglobin oxygen transport in the blood is severely impaired. Thus, under normoxic conditions and with a normal metabolic rate of the tissues, convective oxygen transport is not required until approximately 2 wk after fertilization.     

Structural and functional properties of class 1 plant hemoglobins

Nonsymbiotic class 1 plant hemoglobins are induced under hypoxia. Structurally they are protein dimers consisting of two identical subunits, each containing heme iron in a weak hexacoordinate state. The weak hexacoordination of heme-iron binding to the distal histidine results in an extremely high avidity to oxygen, with a dissociation constant in the nanomolar range. This low dissociation constant is due to rapid oxygen binding resulting in protein conformational changes that slow dissociation from the heme site. Class 1 hemoglobins are characterized by an increased rate of Fe3(+) reduction which is likely mediated by cysteine residue. This cysteine can form a reversible covalent bond between two monomers as shown by mass spectrometry analysis and, in addition to its structural role, prevents the molecule from autoxidation. The structural properties of class 1 hemoglobins allow them to serve as soluble electron transport proteins in the enzymatic system scavenging nitric oxide produced in low oxygen via reduction of nitrite. During oxygenation of nitric oxide to nitrate, oxidized ferric hemoglobin is formed (methemoglobin), which can be reduced by an associated reductase. The identified candidate for this reduction is monodehydroascorbate reductase. It is suggested that hemoglobin functions as a terminal electron acceptor during the hypoxic turnover of nitrogen, the process aided by its extremely high affinity for oxygen.     

Oxygen affinities of maternal and fetal hemoglobins of the viviparous seaperch,Embiotoca lateralis

Summary The striped seaperch,Embiotoca lateralis, is a viviparous teleost. The hemoglobins of adult and fetal seaperch are both tetrameric proteins which in their native state appear to be indistinguishable from one another by electrophoresis. However, differences in the subunit structure of maternal versus fetal seaperch hemoglobins can be detected by electrophoresis in urea with a reducing agent, amino acid analyses and peptide maps of the respective proteins. Furthermore, stripped adult and fetal hemoglobins have different oxygen binding affinities at all pH's tested between pH 6.8 and 8.0. Mid-gestation fetal hemoglobin has a higher oxygen affinity than late-gestation fetal hemoglobin which in turn has a higher affinity than that of the adult hemoglobin. All three stripped hemoglobins show a similar Bohr effect (=–0.9). These data suggest that a difference in oxygen affinities exists in vivo between the adult and fetal blood of the seaperchEmbiotoca lateralis and that it can be explained in part by the presence of a structurally unique fetal hemoglobin. This report is the first to provide evidence for a mechanism of maternal-fetal oxygen transfer in a teleost fish.Abbreviations A adult - LF late-gestation fetal - MF mid-gestation fetal (hemoglobins)     

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.     

脑红蛋白和细胞红蛋白：携氧蛋白质家族2个新成员

脑红蛋白(neuroglobin, Ngb)和细胞红蛋白(cytoglobin, Cygb)是新发现的2个携氧蛋白家族的成员.脑红蛋白主要存在于脑中,而细胞红蛋白在全身各个组织都含有,它们和另外2个携氧蛋白——血红蛋白和肌红蛋白的同源性<25%,但它们在种属之间的同源性很高(>95%).脊椎动物脑红蛋白基因定位于14q24,细胞红蛋白基因定位于17q25,都含有4个外显子和3个内含子.2种蛋白在生理条件下含有6个配位键,不同于血红蛋白和肌红蛋白的5个配位键结构.这2种新蛋白和氧都具有很高的亲和力,在缺氧条件下其基因及蛋白表达都有明显的提升,对细胞的存活有一定保护作用.对于脑红蛋白和细胞红蛋白的功能研究,有助于更好地了解机体氧代谢和氧利用过程,并为临床在缺氧损伤时的治疗提供新的观点和途径.     

Regulation of Cl-dependent K transport by oxy-deoxyhemoglobin transitions in trout red cells.

The oxygenation of trout red cells opens a Cl-dependent K pathway inhibited by furosemide, and by inhibitors of the erythrocyte anion exchanger such as DIDS and niflumic acid. The trigger is the deoxy-oxy conformational change of hemoglobin. The binding of carbon monoxide to heme, which induces a similar conformational change, mimics the effect of oxygen. The possible mechanisms enabling molecular oxygen to control the transport protein are discussed. This oxygenation-activated K transport appears to play a regulatory role in the control of the extracellular K concentration.     

Solvent regulation of oxygen affinity in hemoglobin. Sensitivity of bovine hemoglobin to chloride ions

Under physiological conditions of pH (7.4) and chloride concentration (0.15 M), the oxygen affinity of bovine hemoglobin is substantially lower than that of human hemoglobin. Also, the Bohr effect is much more pronounced in bovine hemoglobin. Numerical simulations indicate that both phenomena can be explained by a larger preferential binding of chloride ions to deoxyhemoglobin in the bovine system. Also, they show that the larger preferential binding may be produced by a decreased affinity of the anions for oxyhemoglobin, thereby stressing the potential relevance of the oxy conformation in regulating the functional properties of the protein. The conformation of the amino-terminal end of the beta subunits appears to regulate the interaction of hemoglobin with solvent components. The pronounced sensitivity of the oxygen affinity of bovine hemoglobin to chloride concentration and to pH suggests that in bovine species these are the modulators of oxygen transport in vivo.     

The effect of macromolecular polyanions on the functional properties of human hemoglobin.

The binding of dextran sulphate and heparin to human hemoglobin and their effect on the properties of gas transport have been investigated. Both dextran sulphate and heparin are strongly bound by oxy-hemoglobin as well as deoxyhemoglobin and the stoichiometry of the binding (polyanion/tetrameric hemoglobin) is less than unity; sedimentation analysis gives indication for the existence of octomers. The oxygen affinity of hemoglobin is decreased, to the same extent, by both dextran sulphate and heparin. This effect is pH-dependent. In addition the polyanions affect the position and the magnitude of the Bohr effect. In the presence of dextran sulphate the recombination of hemoglobin with carbon monoxide after flash photolysis is biphasic and the fraction of quickly reacting material increases with dilution of the protein.     

Lignification of cell walls of infected cells in Casuarina glauca nodules that depend on symplastic sugar supply is accompanied by reduction of plasmodesmata number and narrowing of plasmodesmata

The oxygen protection system for the bacterial nitrogen‐fixing enzyme complex nitrogenase in actinorhizal nodules of Casuarina glauca resembles that of legume nodules: infected cells contain large amounts of the oxygen‐binding protein hemoglobin and are surrounded by an oxygen diffusion barrier. However, while in legume nodules infected cells are located in the central tissue, actinorhizal nodules are composed of modified lateral roots with infected cells in the expanded cortex. Since an oxygen diffusion barrier around the entire cortex would also block oxygen access to the central vascular system where it is required to provide energy for transport processes, here each individual infected cell is surrounded with an oxygen diffusion barrier. In order to assess the effect of these oxygen diffusion barriers on oxygen supply for energy production for transport processes, apoplastic and symplastic sugar transport pathways in C. glauca nodules were examined. The results support the idea that sugar transport to and within the nodule cortex relies to a large extent on the less energy‐demanding symplastic mechanism. This is in line with the assumption that oxygen access to the nodule vascular system is substantially restricted. In spite of this dependence on symplastic transport processes to supply sugars to infected cells, plasmodesmal connections between infected cells, and to a lesser degree with uninfected cells, were reduced during the differentiation of infected cells.     

Iron transport: emerging roles in health and disease.

In the theater of cellular life, iron plays an ambiguous and yet undoubted lead role. Iron is a ubiquitous core element of the earth and plays a central role in countless biochemical pathways. It is integral to the catalysis of the redox reactions of oxidative phosphorylation in the respiratory chain, and it provides a specific binding site for oxygen in the heme binding moiety of hemoglobin, which allows oxygen transport in the blood. Its biological utility depends upon its ability to readily accept or donate electrons, interconverting between its ferric (Fe3+) and ferrous (Fe2+) forms. In contrast to these beneficial features, free iron can assume a dangerous aspect catalyzing the formation of highly reactive compounds such as cytotoxic hydroxyl radicals that cause damage to the macromolecular components of cells, including DNA and proteins, and thereby cellular destruction. The handling of iron in the body must therefore be very carefully regulated. Most environmental iron is in the Fe3+ state, which is almost insoluble at neutral pH. To overcome the virtual insolubility and potential toxicity of iron, a myriad of specialized transport systems and associated proteins have evolved to mediate regulated acquisition, transport, and storage of iron in a soluble, biologically useful, non-toxic form. We are gradually beginning to understand how these proteins individually and in concert serve to maintain cellular and whole body homeostasis of this crucial yet potentially harmful metal ion. Furthermore, studies are increasingly implicating iron and its associated transport in specific pathologies of many organs. Investigation of the transport proteins and their functions is beginning to unravel the detailed mechanisms underlying the diseases associated with iron deficiency, iron overload, and other dysfunctions of iron metabolism.     

Gas exchange properties of goat hemoglobins A and C

Hypoxic or anemic goats with the A hemoglobin genotype switch to the production of hemoglobin C, resulting in a reduced blood oxygen affinity. However, the physiologic consequences of this switch are not clear. We therefore studied the gas exchange properties of the two hemoglobin types. We found that purified hemoglobins A and C have very similar oxygen affinities and H+ Bohr effects, but in the presence of CO2, the affinity of hemoglobin C is substantially less than that of hemoglobin A. That this is not a nonspecific ionic effect is suggested by identical effects of NaCl on O2 binding to the two proteins and by a 2-fold higher capacity of hemoglobin C to bind CO2. The data can be explained by a class of CO2 binding sites in the beta C chain whose affinity is much higher than that of either of the primary sites or of those in Hb A. Our results suggest that in hemoglobin C-containing red cells CO2 acts as a potent allosteric effector, analogous to the role played by 2,3-diphosphoglycerate in human red blood cells. Goat hemoglobin C may have advantages over hemoglobins A or B in O2 transport under hypoxic conditions or in anemia.     

Dynamics of oxygen unloading from sickle erythrocytes.

The objective of this work is to theoretically model oxygen unloading in sickle red cells. This has been done by combining into a single model diffusive transport mechanisms, which have been well-studied for normal red cells, and the hemoglobin polymerization process, which has been previously been studied for deoxyhemoglobin-S solutions and sickle cells in near-equilibrium situations. The resulting model equations allow us to study the important processes of oxygen delivery and polymerization simultaneously. The equations have been solved numerically by a finite-difference technique. The oxygen unloading curve for sickle erythrocytes is biphasic in nature. The rate of unloading depends in a complicated way on (a) the kinetics of hemoglobin S polymerization, (b) the kinetics of hemoglobin deoxygenation, and (c) the diffusive transport of both free oxygen and oxy-hemoglobin. These processes interact. For example, the hemoglobin S polymer interferes with the transport of both free oxygen and unpolymerized oxy-hemoglobin, and this is accounted for in the model by diffusivities which depend on the polymer and solution hemoglobin concentration. Other parameters which influence the interaction of these processes are the concentration of 2,3-diphosphoglycerate and total hemoglobin concentration. By comparing our model predictions for oxygen unloading with simpler predictions based on equilibrium oxygen affinities, we conclude that the relative rate of oxygen unloading of cells with different physical properties cannot be correctly predicted from the equilibrium affinities. To describe the unloading process, a kinetic calculation of the sort we give here is required.     

Hemoglobin-based blood substitutes and the hazards of blood radicals

Cell-free hemoglobins, chemically altered or genetically expressed in microbial host systems, have been developed as oxygen-carrying therapeutics. Sitedirected modifications are introduced and serve to stabilize the protein molecules in a tetrameric and/or a polymeric functional form. Animal studies, as well as recent clinical studies, have suggested these proteins probably deliver oxygen to tissues. However, concerns still persist regarding the interference of hemoglobin and its oxidation products with the vascular redox balance, potentially impeding its clinical usefulness. This article reviews our current understanding of heme-mediated toxicities and some of the emerging protective strategies used to overcome hemoglobin side reactions.     

Hemoglobin-based blood substitutes and the hazards of blood radicals

Cell-free hemoglobins, chemically altered or genetically expressed in microbial host systems, have been developed as oxygen-carrying therapeutics. Sitedirected modifications are introduced and serve to stabilize the protein molecules in a tetrameric and/or a polymeric functional form. Animal studies, as well as recent clinical studies, have suggested these proteins probably deliver oxygen to tissues. However, concerns still persist regarding the interference of hemoglobin and its oxidation products with the vascular redox balance, potentially impeding its clinical usefulness. This article reviews our current understanding of heme-mediated toxicities and some of the emerging protective strategies used to overcome hemoglobin side reactions.