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1.
The haemoglobin/nitric oxide cycle: involvement in flooding stress and effects on hormone signalling
BACKGROUND: Class 1 haemoglobins (Hbs) are induced in plant cells under hypoxic conditions. They have a high affinity for oxygen, which is two orders of magnitude lower than that of cytochrome oxidase, permitting the utilization of oxygen by the molecule at extremely low oxygen concentrations. Their presence reduces the levels of nitric oxide (NO) that is produced from nitrate ion during hypoxia and improves the redox and energy status of the hypoxic cell. SCOPE: The mechanism by which Hb interacts with NO under hypoxic conditions in plants is examined, and the effects of Hb expression on metabolism and signal transduction are discussed. CONCLUSIONS: The accumulated evidence suggests that a metabolic pathway involving NO and Hb provides an alternative type of respiration to mitochondrial electron transport under limited oxygen. Hb in hypoxic plants acts as part of a soluble, terminal, NO dioxygenase system, yielding nitrate ion from the reaction of oxyHb with NO. NO is mainly formed due to anaerobic accumulation of nitrite. The overall reaction sequence, referred to as the Hb/NO cycle, consumes NADH and maintains ATP levels via an as yet unknown mechanism. Hb gene expression appears to influence signal transduction pathways, possibly through its effect on NO, as evidenced by phenotypic changes in normoxic Hb-varying transgenic plants. Ethylene levels are elevated when Hb gene expression is suppressed, which could be a factor leading to root aerenchyma formation during hypoxic stress. 相似文献
2.
Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS‐PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P50) and cooperativity coefficient (n) were regressed from the measured oxygen‐RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC‐MS was used to measure the molecular weight of the alpha (α) and beta (β) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the α and β globin chains agreed well with the calculated theoretical mass of the α‐ and β‐ globin chains. Taken together, our results demonstrate that HPLC‐grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb‐based oxygen carriers. © 2008 American Institute of Chemical Engineers, 2009 相似文献
3.
Gerke P Börding C Zeis B Paul RJ 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2011,159(1):56-65
Hypoxia-induced haemoglobin (Hb) expression is a central regulatory mechanism in Daphnia in response to environmental hypoxia or warm temperatures. Changes in Hb concentration as well as Hb subunit composition, which modulate Hb oxygen affinity, guarantee the oxygen supply of tissues under these environmental conditions. Based on the sequenced D. pulex genome, Hb genes were related to the properties of haemolymph Hb, which included its concentration and oxygen affinity (both measured by spectrophotometry) as well as the Hb subunit composition (determined by 2-D gel electrophoresis and ESI-MS analysis). Permanent cultures of D. pulex acclimated to different oxygen conditions (normoxia and hypoxia) and temperatures (10°C, 20°C, and 24°C), showed characteristic changes in Hb concentration, subunit composition and oxygen affinity. Several subunits (Hb4, Hb7, Hb8, and Hb10) were obviously responsible for changes in oxygen affinity including those, which carry a number of hypoxia-responsive elements (HREs) upstream of the respective gene (hb4 and hb10). Analysing the effects of different oxygen- or temperature-acclimations on Hb subunit expression in D. pulex and D. magna on a common basis (Hb concentration or oxygen affinity) revealed a general pattern of oxygen and temperature effects on Hb, which implies that Hb quantity and quality are mostly influenced by the degree of tissue hypoxia. Differences between both species in the onset of hypoxia-induced differential Hb expression and Hb oxygen affinity, which are probably related to different HRE patterns and functionally important differences in the amino acid sequence of only a few subunits, cause a reduced ability of D. pulex to adjust Hb function to temperature changes in comparison to D. magna. 相似文献
4.
Torres Filho IP Terner J Pittman RN Somera LG Ward KR 《American journal of physiology. Heart and circulatory physiology》2005,289(1):H488-H495
A system is described for in vivo noninvasive measurements of hemoglobin oxygen saturation (HbO2Sat) at the microscopic level. The spectroscopic basis for the application is resonant Raman enhancement of Hb in the violet/ultraviolet region, allowing simultaneous identification of oxy- and deoxyhemoglobin with the same excitation wavelength. The heme vibrational bands are well known, but the technique has never been used to determine microvascular HbO2Sat in vivo. A diode laser light (power: 0.3 mW) was focused onto sample areas 15-30 microm in diameter. Raman spectra were obtained in backscattering geometry by using a microscope coupled to a spectrometer and a cooled detector. Calibration was performed in vitro by using glass capillaries containing blood at several Hb concentrations, equilibrated at various oxygen tensions. HbO2Sat was estimated using the Raman band intensities at 1,360 and 1,375 cm(-1). Glass capillary path length and Hb concentration had no effect on HbO2Sat estimated from Raman spectra. In vivo observations were made in blood flowing in microvessels of the rat mesentery. The Hb Raman peaks observed in oxygenated and deoxygenated blood were consistent with earlier Raman studies that used Hb solutions and isolated cells. The method allowed HbO2Sat determinations in the whole range of arterioles, venules, and capillaries. Tissue transillumination allowed diameter and erythrocyte velocity measurements in the same vessels. Raman microspectroscopy offers distinct advantages over other currently used techniques by providing noninvasive and reliable in vivo determinations of HbO2Sat in thin tissues as well as in solid organs and tissues, which are unsuitable for techniques requiring transillumination. 相似文献
5.
Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography 总被引:1,自引:0,他引:1
Sun G Palmer AF 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2008,867(1):1-7
Bovine and human hemoglobin (Hb) form the basis for many different types of Hb-based O(2) carriers (HBOCs) ranging from chemically modified Hbs to particle encapsulated Hbs. Hence, the development of a facile purification method for preparing ultrapure Hb is essential for the reliable synthesis and formulation of HBOCs. In this work, we describe a simple process for purifying ultrapure solutions of bovine and human Hb. Bovine and human red blood cells (RBCs) were lyzed, and Hb was purified from the cell lysate by anion exchange chromatography. The initial purity of Hb fractions was analyzed by SDS-PAGE. Pure Hb fractions (corresponding to a single band on the SDS-PAGE gel) were pooled together and the overall purity and identity assessed by LC-MS. LC-MS analysis yielded two peaks corresponding to the calculated theoretical molecular weight of the alpha and beta chains of Hb. The activity of HPLC pure Hb was assessed by measuring its oxygen affinity, cooperativity and methemoglobin level. These measures of activity were comparable to values in the literature. Taken together, our results demonstrate that ultrapure Hb (electrophoresis and HPLC pure) can be easily prepared via anion exchange chromatography. In general, this method can be more broadly applied to purify hemoglobin from any source of RBC. This work is significant, since it outlines a simple method for generating ultrapure Hb for synthesis and/or formulation of HBOCs. 相似文献
6.
I Hiebl R E Weber D Schneeganss J K?sters G Braunitzer 《Biological chemistry Hoppe-Seyler》1988,369(4):217-232
The primary structures of the hemoglobins Hb A, Hb A', Hb D and Hb D' of Rüppell's Griffon (Gyps rueppellii), which can fly as high as 11,300 m, are presented. The globin chains were separated on CM-Cellulose in 8M urea buffers, the four hemoglobin components by FPLC in phosphate buffers. The amino-acid sequences of five globin chains were established by automatic Edman degradation of the globin chains and of the tryptic peptides in liquid-phase and gas-phase sequenators. The sequences are compared with those of other Falconiformes. A new molecular pattern for survival at extreme altitudes is presented. For the first time four hemoglobins are found in blood of a bird; they show identical beta-chains and differ in the alpha A- and alpha D-chains by only one replacement. These four hemoglobins cause a gradient in oxygen affinities. The two main components Hb A and Hb A' differ at position alpha 34 Thr/Ile. In case of Ile as found in Hb A' an alpha 1 beta 1-interface is interrupted raising oxygen affinity compared to Hb A. In addition the hemoglobins of the A- and D-groups differ at position alpha 38 Pro or Gln/Thr (alpha 1 beta 2-interface). Expression of Gln in Hb D/D' raises the oxygen affinity of these components compared to Hb A/A' by destabilization of the deoxy-structure. The physiological advantage lies in the functional interplay of four hemoglobin components. Three levels of affinity are predicted: low affinity Hb A, Hb A' of intermediate affinity, and high affinity Hb D/D'. This cascade tallies exactly with oxygen affinities measured in the isolated components and predicts oxygen transport by the composite hemoglobins over an extended range of oxygen affinities. It is contended that the mechanisms of duplication of the alpha-genome (creating four hemoglobins) and of nucleotide replacements (creating different functional properties) are responsible for this remarkable hypoxic tolerance to 11,300 m. Based on this pattern the hypoxic tolerances of other vultures are predicted. 相似文献
7.
Leow MK 《Advances in physiology education》2007,31(2):198-201
The oxygen dissociation curve (ODC) of hemoglobin (Hb) has been widely studied and mathematically described for nearly a century. Numerous mathematical models have been designed to predict with ever-increasing accuracy the behavior of oxygen transport by Hb in differing conditions of pH, carbon dioxide, temperature, Hb levels, and 2,3-diphosphoglycerate concentrations that enable their applications in various clinical situations. The modeling techniques employed in many existing models are notably borrowed from advanced and highly sophisticated mathematics that are likely to surpass the comprehensibility of many medical and bioscience students due to the high level of "mathematical maturity" required. It is, however, a worthy teaching point in physiology lectures to illustrate in simple mathematics the fundamental reason for the crucial sigmoidal configuration of the ODC such that the medical and bioscience undergraduates can readily appreciate it, which is the objective of this basic dissertation. 相似文献
8.
Various oxidized mono/di/tri/poly saccharides were studied as potential hemoglobin (Hb) cross-linkers in order to produce oxygen carriers with high oxygen affinities (low P(50)'s) and high molecular weights (therefore lower macromolecular diffusivities compared to tetrameric Hb). Such physical properties were desired to produce polymerized hemoglobins (PolyHbs) with oxygen release profiles similar to that of human blood, as was demonstrated in work by Winslow (1). In this present study, bovine hemoglobin was cross-linked with a variety of oxidized (ring-opened) saccharides, which resulted in cross-linked Hb species ranging in size from 64 to 6400 kDa (depending on the particular oxidized saccharide used in the reaction) and P(50)'s ranging from 6 to 15 mmHg. A parallel synthetic approach was used to synthesize these carbohydrate-hemoglobin conjugates, and asymmetric flow field-flow fractionation (AFFF) coupled with multi-angle static light scattering (MASLS) was used to measure the absolute molecular weight distribution of these PolyHb dispersions. Cross-linking reactions were conducted at two pHs (6 and 8), with larger cross-linked Hb species produced at pH 8 (where hydrolysis was most likely to occur between glycosidic bonds linking adjacent saccharide rings) rather than at pH 6. The largest molecular weight species formed from these reactions consisted of Hb cross-linked with ring-opened lactose, maltose, methylglucopyranoside, sucrose, trehalose, and 15 kDa and 71 kDa dextran at high pH (pH 8). The most promising Hb cross-linker was methylglucopyranoside, which resulted in very large cross-linked Hb species, with low P(50)'s and lower methemoglobin (metHb) levels compared to the other Hb cross-linking reagents. 相似文献
9.
Quantification of cerebral oxygenation by full-spectrum near-infrared spectroscopy using a two-point method 总被引:1,自引:0,他引:1
Kusaka T Isobe K Nagano K Okubo K Yasuda S Kondo M Itoh S Hirao K Onishi S 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2002,132(1):121-132
The aim of this study was to quantify the relative concentrations of oxyhemoglobin and deoxyhemoglobin within the light path of the brain and to estimate cerebral hemoglobin (Hb) oxygen saturation using full-spectrum near-infrared spectroscopy (fsNIRS). For this purpose, we developed a novel exponential correction equation as well as a two-point spectroscopy method to estimate the relative concentrations of Hb and Hb oxygen saturation in biological tissues. The results of evaluation of measurements using an in vitro model indicated that our fsNIRS method enables accurate and non-invasive measurements of Hb content and saturation in a highly scattered medium such as the human brain. According to the results of analysis using a hypoxic piglet model, the mean cerebral Hb oxygen saturation (SbO(2)) of newborn piglets at an inspired oxygen gas concentration of 0.21 was estimated to be 63+/-4% (mean+/-S.D.). Umbilical arterial and left internal jugular venous Hb oxygen saturation were simultaneously estimated to be 96+/-2% and 52+/-11%, respectively. SbO(2) and arterial Hb oxygen saturation values had a linear relationship. The average oxygenation state of cerebral tissue is comparable with that of the cerebral vein. The results of this study showed that our method can be used to monitor Hb oxygen saturation in the neonatal brain at the bedside in an intensive care unit. 相似文献
10.
The effect of anions on the stability of different functional conformations of Hb is examined through the determination of the dependence of O(2) affinity on water activity (a(w)). The control of a(w) is effected by varying the sucrose osmolal concentration in the bathing solution according to the "osmotic stress" method. Thus, the hydration change following Hb oxygenation is determined as a function of Cl(-) and of DPG concentration. We find that only approximately 25 additional water molecules bind to human Hb during the deoxy-to-oxy conformation transition in the absence of anions, in contrast with approximately 72 that bind in the presence of more than 50 mM Cl(-) or more than 15 microM DPG. We demonstrate that the increase in the hydration change linked with oxygenation is coupled with anion binding to the deoxy-Hb. Hence, we propose that the deoxy-Hb coexists in two allosteric conformations which depend on whether anion is bound or not: the tense T-state, with low oxygen affinity and anion bound, or a new allosteric P-state, with intermediate oxygen affinity and free of bound anions. The intrinsic oxygen affinity of this unforeseen P-state and the differential binding of Cl(-), DPG, and H(2)O between states P and T and P and R are characteristics which are consistent with those expected for a putative intermediate allosteric state of Hb. These findings represent a new opportunity to explore the structure-function relationships of hemoglobin regulation. 相似文献
11.
Panagiotis Marinakis Maurizio Tamburrini Vito Carratore Guido di Prisco 《European journal of biochemistry》2003,270(19):3981-3987
The hemolysate of the Antarctic teleost Gobionotothen gibberifrons (family Nototheniidae) contains two hemoglobins (Hb 1 and Hb 2). The concentration of Hb 2 (15-20% of the total hemoglobin content) is higher than that found in most cold-adapted Notothenioidei. Unlike the other Antarctic species so far examined having two hemoglobins, Hb 1 and Hb 2 do not have globin chains in common. Therefore this hemoglobin system is made of four globins (two alpha- and two beta-chains). The complete amino-acid sequence of the two hemoglobins (Hb 1, alpha2(1)beta2(1); Hb 2, alpha2(2)beta2(2)) has been established. The two hemoglobins have different functional properties. Hb 2 has lower oxygen affinity than Hb 1, and higher sensitivity to the modulatory effect of organophosphates. They also differ thermodynamically, as shown by the effects on the oxygen-binding properties brought about by temperature variations. The oxygen-transport system of G. gibberifrons, with two functionally distinct hemoglobins, suggests that the two components may have distinct physiological roles, in relation with life style and the environmental conditions which the fish may have to face. The unique features of the oxygen-transport system of this species are reflected in the phylogeny of the hemoglobin amino-acid sequences, which are intermediate between those of other fish of the family Nototheniidae and of species of the more advanced family Bathydraconidae. 相似文献
12.
《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1997,320(11):849-855
Four human hemoglobin variants have already been described at position α 126 (H9), which is normally occupied by an aspartate: Hb Montefiore (→ Tyr), Hb Tarrant (→ Asn), Hb Fukutomi (→ Val), Hb Sassari (→ His). An additional variant, Hb West One (α126 (H9) Asp→ Gly) is herein described. Aspartate α126 (H9) is involved in a set of hydrogen bonds and salt bridges located at the C-terminal portion of the α-chains and of the C-helix of the β-chains, which are broken in the oxy conformer, providing one of the most important sources of the difference in free energy between the T- and R-state in hemoglobin. A comparative study of four of these α126 Hb variants is presented. An identical degree of alteration of the oxygen binding properties (increased oxygen affinity and decreased cooperativity) was found in all cases, when measured under standard experimental conditions (pH 7.2, 0.1 M NaCl). In contrast, the effect of L345 (a derivative of bezafibrate, which is a specific α-chain binding effector) on oxygen binding to Hb differed from one variant to another. When a bulky Tyr or His residue occupied the α126 (H9) position, little effect of L345 was observed. Conversely, when this position was occupied by a residue of smaller size (Gly or Asn), normal heterotropic effects were observed. Molecular graphic modelling indicates that two classes of three-dimensional structure modifications may occur. 相似文献
13.
血红蛋白携氧-释氧动力学研究 总被引:2,自引:0,他引:2
本文研究了鸡、家兔、鲤鱼、蟾蜍4种实验动物血红蛋白(hemoglobin,Hb)携氧-释氧动力学过程,初步建立Hb携氧-释氧动力学研究方法,并探讨Hb携氧-释氧动力学过程与动物生存环境之间的关系.结果显示:4种动物Hb携氧动力学曲线均呈"S"形曲线特征,与传统的Hb氧解离曲线(oxygen dissociation curve,ODC)相似;同时不同动物Hb携氧-释氧动力学曲线也有各自特点,如鸡Hb释氧时间长达(1 411±6)S;在Hb携氧.释氧曲线I阶段,鲤鱼上升斜率远大于家兔等.提示Hb携氧-释氧动力学曲线可反映不同动物Hb携氧效率的差异.与传统ODC参数P50相对应,由动力学曲线可得到Hb携氧动力学参数T50°T50是Hb达到50%氧饱和度所需时间,可直观反映Hb携氧效率的差异.4种实验动物Hb均有较稳定的T50,从大到小依次为:鸡、家兔、鲤鱼和蟾蜍.对Hb携氧动力学曲线与ODC综合分析,可得到Hb携氧效能参数E50,表示Hb达到50%氧饱和度所用时间与环境氧分压之间的关系,即E50(50% Sat,Xeo2,yr).E50有可能成为全面评价Hb携氧效能的综合指标. 相似文献
14.
F F Soprunov J I Benkowitsch T I Kazarinskaja 《Acta biologica et medica Germanica》1977,36(5-6):783-791
Polarographic titration in a closed cell of blood samples and concentrated Hb solutions was used to study the regulation of the release of oxygen by the red cell. The effect of temperature, polyethylenglycol and metabolites normally found in the erythrocyte was determined by spectrophotometric and polarographic methods. Some pecularities of O2-transport and release in beta-thalassemia, functional hypoxia and Cooley-disease are described. 相似文献
15.
A method for improving the efficiency of exchange transfusion to evaluate hemoglobin- (Hb) based erythrocyte substitutes is described. The method uses a continuous-flow hollow-fiber plasma separation filter to remove the erythrocytes while returning 75% of the plasma. The removed volume was replaced with a 14-g/dl solution of human Hb cross-linked between the alpha-chains with bis(3,5-dibromosalicyl)fumarate (alpha alpha Hb). Filtration of 2.76 blood vol in anesthetized swine resulted in a 95% reduction of hematocrit and produced a plasma Hb concentration of 7.63 g/dl. Hyperoncotic Hb solutions cause volume expansion, which reduces the efficiency of exchange but provides hemodynamic stability in the face of decreasing blood viscosity and subsequent intravascular volume loss with Hb redistribution. Filtration-assisted exchange transfusion is rapid, conserves valuable modified Hb, and ensures continuous adequate oxygen delivery. 相似文献
16.
Manjula BN Tsai A Upadhya R Perumalsamy K Smith PK Malavalli A Vandegriff K Winslow RM Intaglietta M Prabhakaran M Friedman JM Acharya AS 《Bioconjugate chemistry》2003,14(2):464-472
Increasing the molecular size of acellular hemoglobin (Hb) has been proposed as an approach to reduce its undesirable vasoactive properties. The finding that bovine Hb surface decorated with about 10 copies of PEG5K per tetramer is vasoactive provides support for this concept. The PEGylated bovine Hb has a strikingly larger molecular radius than HbA (1). The colligative properties of the PEGylated bovine Hb are distinct from those of HbA and even polymerized Hb, suggesting a role for the colligative properties of PEGylated Hb in neutralizing the vasoactivity of acellular Hb. To correlate the colligative properties of surface-decorated Hb with the mass of the PEG attached and also its vasoactivity, we have developed a new maleimide-based protocol for the site-specific conjugation of PEG to Hb, taking advantage of the unusually high reactivity of Cys-93(beta) of oxy HbA and the high reactivity of the maleimide to protein thiols. PEG chains of 5, 10, and 20 kDa have been functionalized at one of their hydroxyl groups with a maleidophenyl moiety through a carbamate linkage and used to conjugate the PEG chains at the beta-93 Cys of HbA to generate PEGylated Hbs carrying two copies of PEG (of varying chain length) per tetramer. Homogeneous preparations of (SP-PEG5K)(2)-HbA, (SP-PEG10K)(2)-HbA, and (SP-PEG20K)(2)-HbA have been isolated by ion exchange chromatography. The oxygen affinity of Hb is increased slightly on PEGylation, but the length of the PEG-chain had very little additional influence on the O(2) affinity. Both the hydrodynamic volume and the molecular radius of the Hb increased on surface decoration with PEG and exhibited a linear correlation with the mass of the PEG chain attached. On the other hand, both the viscosity and the colloidal osmotic pressure (COP) of the PEGylated Hbs exhibited an exponential increase with the increase in PEG chain length. In contrast to the molecular volume, viscosity, and COP, the vasoactivity of the PEGylated Hbs was not a direct correlate of the PEG chain length. There appeared to be a threshold for the PEG chain length beyond which the protection against vasoactivity is decreased. These results suggest that the modulation of the vasoactivity of Hb by PEG could be a function of the surface shielding afforded by the PEG, the latter being a function of the disposition of the PEG chain on the protein surface, which in turn is a function of the length of the PEG chain. Thus, the biochemically homogeneous PEGylated Hbs described in the present study, surface-decorated with PEG chains of appropriate size, could serve as potential candidates for Hb-based oxygen carriers. 相似文献
17.
Wiwanitkit V 《International journal of nanomedicine》2006,1(1):105-107
Hemoglobin variants in which a frameshift results in chain elongation are unusual. Hemoglobin Pakse (Hb Pakse) is an unstable hemoglobin with abnormal elongation, first described in Indochina. An alpha2-globin gene termination codon mutation, TAA -->TAT or Term -->Tyr, has been described in the pathogenesis of Hb Pakse. This abnormality causes a frameshift that elongates the alpha chain amino acids. Computer-based protein structure modeling was used in a bioinformatics analysis of the tertiary structure of these elongated amino acid sequences. The elongated part of Hb Pakse showed additional helices, which may cause the main alteration in Hb Pakse. Abnormalities in the fold structure of globin in Hb Pakse were identified, and helices additional to the normal alpha globin chains were shown in the elongated part of Hb Pakse. 相似文献
18.
The preparation of three hemoglobin tetramers containing the hemoglobin S mutation at beta 6 and an additional one at alpha 6, alpha 47, and alpha 75 is described. The effect of the substitutions in the alpha chains on polymerization was investigated by the equilibrium solubility of the gels as well as the abrupt change in oxygen affinity associated with the onset of gelation. Substitution of a histidine for aspartic acid at alpha 47 causes a marked inhibition of polymerization. This inhibition probably results from tetramers which carry the two substitutions on the same alpha beta dimer. By contrast, the introduction of a tyrosine at alpha 75 and an alanine at alpha 6 have the opposite effect and are the first examples of alpha chain mutations which potentiate the gelation of Hb S. The molecular mechanisms responsible for the effects of the mutations on the self-association of Hb S are discussed. 相似文献
19.
The minor tetrameric hemoglobin (Hb), Hb D, of chicken red blood cells self-associates upon deoxygenation. This self-association enhances the cooperativity of oxygen binding. The maximal Hill coefficient is greater than 4 at high Hb concentrations. Previous measurements at low Hb concentrations were consistent with a monomer-to-dimer equilibrium and an association constant of ~1.3-1.6 × 10(4) M(-1). Here, the Hb tetramer is considered as the monomer. However, new results indicate that the association extends beyond the dimer. We show by combination of Hb oligomer modeling and sedimentation velocity analyses that the data can be well described by an indefinite noncooperative or isodesmic association model. In this model, the deoxy Hb D associates noncooperatively to give a linear oligomeric chain with an equilibrium association constant of 1.42 × 10(4) M(-1) at 20°C for each step. The data are also well described by a monomer-dimer-tetramer equilibrium model with monomer-to-dimer and dimer-to-tetramer association constants of 1.87 and 1.03 × 10(4) M(-1) at 20°C, respectively. A hybrid recombinant Hb D was prepared with recombinant α(D)-globin and native β-globin to give a Hb D tetramer (α(2)(D)β(2)). This rHb D undergoes decreased deoxygenation-dependent self-association compared with the native Hb D. Residue glutamate 138 has previously been proposed to influence intertetramer interactions. Our results with recombinant Hb D show that Glu138 plays no role in deoxy Hb D intertetramer interactions. 相似文献
20.
Sakai H. Takeoka S. Yokohama H. Seino Y. Nishide H. Tsuchida E. 《Protein expression and purification》1993,4(6)
A simple method for obtaining a purified and concentrated hemoglobin (Hb) solution (25 g/100 ml) from human red blood cells has been established. To prevent MetHb formation during the purification procedure, Hb in red blood cells was carbonylated in advance, and then washed red blood cells were mixed with organic solvents such as diethyl ether or dichloromethane for hemolysis and removal of stroma. The Hb solution was isolated by centrifugation (1 900g) with the high removal efficiency of phospholipid (>99.8%). After the solution was heated (60°C, 1 h), the precipitates were removed by centrifugation. The purity of Hb was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing and oxygen-binding properties of the obtained Hb solution demonstrated its purity and showed no denaturation of globin. This purification procedure is applicable to large-scale production of the purified Hb. 相似文献