The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin was investigated by accurately measuring and analyzing the oxygen equilibrium curves of human adult hemoglobin in the presence and absence of various concentrations of one or two of the following materials: chloride (Cl-), 2,3-diphosphoglycerate (DPG), and inositol hexaphosphate (IHP). Each equilibrium curve was analyzed according to the Adair equation to evaluate the four-step oxygen equilibrium constants (Adair constants) and the median oxygen pressure. The binding constants of the anions for the molecular species of hemoglobin carrying j oxygen molecules, Hb(O2)j(j=0,1,...,4), were evaluated from the dependences of the Adair constants and the median oxygen pressure on the anion concentration by introducing a model which takes the competitive binding of Cl- and DPG or IHP into account. Assumptions made in the model are: (a) the hemoglobin molecule has two oxygen-linked binding sites for Cl- which are equivalent and independent and (b) no Cl- can be bound to hemoglobin to which DPG or IHP is already bound and vice versa. Thus, we could obtain values for the intrinsic binding constants of Cl- and DPG, i.e., the constants in the absence of other competitive anions. For IHP, only the binding constants and apparent binding constants for Hb and Hb(O2)2 were obtained. Values of the Cl- binding constants and apparent binding constants for DPG and IHP, i.e., the binding constants in the presence of Cl- for Hb and Hb(O2)4, were in reasonable agreement with literature values. From the binding constants we calculated anion binding curves for Hb(O2)j(J=0,1,...,4), the number of anions bound to Hb(O2)J, And the relationship between fractional anion saturation of hemoglobin and fractional oxygen saturation. The numbers of released anions are not uniform with respect to oxygenation step. This non-uniformity is the reason for the changes in the shape of the oxygen equilibrium curve with anion concentration changes and for the non-uniform dependences of the Adair constants on anion concentration, and also results in non-linear relations between anion saturation and oxygen saturation. The anion binding constants and various binding properties of the anions derived from those constants are consistent with those observed by other investigators using different techniques, indicating that the present model describes the oxygen-linked competitive anion binding well.     

Hemoglobin vesicles containing methemoglobin and L-tyrosine to suppress methemoglobin formation in vitro and in vivo

Hemoglobin (Hb) vesicles have been developed as cellular-type Hb-based O(2) carriers in which a purified and concentrated Hb solution is encapsulated with a phospholipid bilayer membrane. Ferrous Hb molecules within an Hb vesicle were converted to ferric metHb by reacting with reactive oxygen species such as hydrogen peroxide (H(2)O(2)) generated in the living body or during the autoxidation of oxyHb in the Hb vesicle, and this leads to the loss of O(2) binding ability. The prevention of metHb formation by H(2)O(2) in the Hb vesicle is required to prolong the in vivo O(2) carrying ability. We found that a mixed solution of metHb and L-tyrosine (L-Tyr) showed an effective H(2)O(2) elimination ability by utilizing the reverse peroxidase activity of metHb with L-Tyr as an electron donor. The time taken for the conversion of half of oxyHb to metHb (T(50)) was 420 min for the Hb vesicles containing 4 g/dL (620 microM) metHb and 8.5 mM L-Tyr ((metHb/L-Tyr) Hb vesicles), whereas the time of conversion for the conventional Hb vesicles was 25 min by stepwise injection of H(2)O(2) (310 microM) in 10 min intervals. Furthermore, in the (metHb/L-Tyr) Hb vesicles, the metHb percentage did not reach 50% even after 48 h under a pO(2) of 40 Torr at 37 degrees C, whereas T(50) of the conventional Hb vesicles was 13 h under the same conditions. Moreover, the T(50) values of the conventional Hb vesicles and the (metHb/L-Tyr) Hb vesicles were 14 and 44 h, respectively, after injection into rats (20 mL/kg), confirming the remarkable inhibitory effect of metHb formation in vivo in the (metHb/L-Tyr) Hb vesicles.     

Isolated Hb Providence β82Asn and β82Asp fractions are more stable than native HbA(0) under oxidative stress conditions

We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the β-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or β-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two β-subunit variants with single amino acid mutations at βLys82→Asp (βK82D) and at βLys82→Asn (βK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its β-mutant fractions with HbAo. Relative to HbAo, both βK82N and βK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H(2)O(2). However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of β-subunits, and α-α dimer formation in the presence of excess H(2)O(2). Mass spectrometric and amino acid analysis revealed that βCys93 and βCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in βK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H(2)O(2) and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.     

Involvement of Glu G3(101)beta in the function of hemoglobin. Comparative O2 equilibrium studies of human mutant hemoglobins

The glutamyl residue at G3(101)beta of normal hemoglobin (Hb A) is one of the alpha 1 beta 2 subunit contacts which are vital to O2 binding properties of the molecule. The O2 equilibrium properties of the four mutants with different substitutions at this site are studied in order to elucidate the role of this residue. Under stripped conditions with minimum chloride the order of O2 affinity is: Hb A (Glu) much less than Hb Rush (Gln) less than or equal to Hb British Columbia (Lys) less than or equal to Hb Potomac (Asp) less than or equal to Hb Alberta (Gly). The first Adair constants, K1, for the mutant hemoglobins are greater than that for Hb A whereas the fourth, K4, are similar, indicating that the allosteric constants (L) of these mutants are greatly reduced. Therefore, the G3(101)beta residue contributes intrinsically to the strengthening of the structural constraints that are imposed upon the deoxy (T) forms but not the oxy (R) form. On addition of 0.1 M Cl- and further addition of 2,3-diphosphoglycerate or inositol hexaphosphate, their O2 affinities and cooperativities are altered, reflecting different responses to anionic ligands. Hb Rush exhibits a stronger chloride effect than Hb A and the other variants and, as a result, an increased Bohr effect and a smaller heat of oxygenation at pH 6.5. These changes are consistent with an increased positive net charge in the central cavity of Hb Rush and subsequent extra anion binding in the deoxy form. The tetramer to dimer dissociation constants are estimated to be greater than normal for Hb British Columbia and less than normal for Hb Alberta. This comparative study of the G3(101)beta mutants indicates that the size and the charge of this residue may influence the switching of two neighboring interchain hydrogen bonds that occurs during oxygenation of normal hemoglobin.     

Haptoglobin alters oxygenation and oxidation of hemoglobin and decreases propagation of peroxide-induced oxidative reactions

We compared oxygenation and anaerobic oxidation reactions of a purified complex of human hemoglobin (Hb) and haptoglobin (Hb-Hp) to those of uncomplexed Hb. Under equilibrium conditions, Hb-Hp exhibited active-site heterogeneity and noncooperative, high-affinity O(2) binding (n(1/2)=0.88, P(1/2)=0.33mm Hg in inorganic phosphate buffer at pH 7 and 25°C). Rapid-reaction kinetics also exhibited active-site heterogeneity, with a slower process of O(2) dissociation and a faster process of CO binding relative to uncomplexed Hb. Deoxygenated Hb-Hp had significantly reduced absorption at the λ(max) of 430nm relative to uncomplexed Hb, as occurs for isolated Hb subunits that lack T-state stabilization. Under comparable experimental conditions, the redox potential (E(1/2)) of Hb-Hp was found to be +54mV, showing that it is much more easily oxidized than uncomplexed Hb (E(1/2)=+125mV). The Nernst plots for Hb-Hp oxidation showed no cooperativity and slopes less than unity indicated active-site heterogeneity. The redox potential of Hb-Hp was unchanged by pH over the range of 6.4-8.3. Exposure of Hb-Hp to excess hydrogen peroxide (H(2)O(2)) produced ferryl heme, which was found to be more kinetically inert in the Hb-Hp complex than in uncomplexed Hb. The negative shift in the redox potential of Hb-Hp and its stabilized ferryl state may be central elements in the protection against Hb-induced oxidative damage afforded by formation of the Hb-Hp complex.     

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP

The cooperative O(2)-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O(2)-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O(2)-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O(2)-affinities (K(T)=0.0085mmHg(-1) and K(R)=0.011mmHg(-1)) and thus a very low cooperativity (K(R)/K(T)=1.3 and L(0)=2.4). (1)H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O(2)-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, (1)H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14ppm, originated from inter- dimeric α(1)β(2)- (or α(2)β(1)-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15-25ppm, caused by high-spin heme-Fe(II)s. Upon addition of O(2), the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O(2), but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O(2) (P(O2)) of 760mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the P(O2) of 760mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α(1)G- and β(1)G-helices, and the other weaker binding site causes the R→T transition without release of O(2). The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α(1)β(1) (or α(2)β(2)) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α(1)α(2)- (or β(1)β(2)-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O(2)-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb - [the deoxy-state]=[the T-quaternary structure]=[the low O(2)-affinity state] and [the oxy-state]=[the R-quaternary structure]=[the high O(2)-affinity state] and the O(2)-affiny of Hb being regulated by the T/R-quaternary structural transition - are no longer sustainable. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin Brigham (α(2) β(2) (Pro100Leu) )

Hemoglobin Brigham (β Pro100 to Leu) was first reported in a patient with familial erythrocytosis. Erythrocytes of an affected individual from the same family contain both HbA and Hb Brigham and exhibit elevated O(2) affinity compared with normal cells (P(50) = 23 mm Hg vs. 31 mmHg at pH 7.4 at 37°C). O(2) affinities measured for hemolysates were sensitive to changes in pH or chloride concentrations, indicating little change in the Bohr and Chloride effects. Hb Brigham was separated from normal HbA by nondenaturing cation exchange liquid chromatography, and the amino acid substitution was verified by mass spectrometry. The properties of Hb Brigham isolated from the patient's blood were then compared with those of recombinant Hb Brigham expressed in Escherichia coli. Kinetic experiments suggest that the rate constants for ligand binding and release in the high (R) and low (T) affinity quaternary states of Hb Brigham are similar to those of native hemoglobin. However, the Brigham mutation decreases the T to R equilibrium constant (L) which accelerates the switch to the R state during ligand binding to deoxy-Hb, increasing the rate of association by approximately twofold, and decelerates the switch during ligand dissociation from HbO(2) , decreasing the rate approximately twofold. These kinetic data help explain the high O(2) affinity characteristics of Hb Brigham and provide further evidence for the importance of the contribution of Pro100 to intersubunit contacts and stabilization of the T quaternary structure.     

Cyanide binding to hexacoordinate cyanobacterial hemoglobins: hydrogen-bonding network and heme pocket rearrangement in ferric H117A Synechocystis hemoglobin

The truncated hemoglobin (Hb) from the cyanobacterium Synechocystis sp. PCC 6803 is a bis-histidyl hexacoordinate complex in the absence of exogenous ligands. This protein can form a covalent cross-link between His117 in the H-helix and the heme 2-vinyl group. Cross-linking, the physiological importance of which has not been established, is avoided with the His117Ala substitution. In the present work, H117A Hb was used to explore exogenous ligand binding to the heme group. NMR and thermal denaturation data showed that the replacement was of little consequence to the structural and thermodynamic properties of ferric Synechocystis Hb. It did, however, decelerate the association of cyanide ions with the heme iron. Full complexation required hours, instead of minutes, of incubation at optical and NMR concentrations. At neutral pH and in the presence of excess cyanide, binding occurred with a first-order dependence on cyanide concentration, eliminating distal histidine decoordination as the rate-limiting step. The cyanide complex of the H117A variant was characterized for the conformational changes occurring as the histidine on the distal side, His46 (E10), was displaced. Extensive rearrangement allowed Tyr22 (B10) to insert in the heme pocket and Gln43 (E7) and Gln47 (E11) to come in contact with it. H-bond formation to the bound cyanide was identified in solution with the use of (1)H(2)O/(2)H(2)O mixtures. Cyanide binding also resulted in a change in the ratio of heme orientational isomers, in a likely manifestation of heme environment reshaping. Similar observations were made with the related Synechococcus sp. PCC 7002 H117A Hb, except that cyanide binding was rapid in this protein. In both cases, the (15)N chemical shift of bound cyanide was reminiscent of that in peroxidases and the orientation of the proximal histidine was as in other truncated Hbs. The ensemble of the data provided insight into the structural cooperativity of the heme pocket scaffold and pointed to the reactive 117 site of Synechocystis Hb as a potential determinant of biophysical and, perhaps, functional properties.     

Interaction of deoxyhemoglobin with the cytoplasmic domain of murine erythrocyte band 3

The partial pressure of oxygen constitutes an important factor in the regulation of human erythrocyte physiology, including control of cell volume, membrane structure, and glucose metabolism. Because band 3 is thought to be involved in all three processes and because binding of hemoglobin (Hb) to the cytoplasmic domain of band 3 (cdb3) is strongly oxygen-dependent, the possibility that the reversible association of deoxyhemoglobin (deoxyHb) with cdb3 might constitute an O(2)-dependent sensor that mediates O(2)-regulated changes in erythrocyte properties arises. While several lines of evidence support this hypothesis, a major opposing argument lies in the fact that the deoxyHb binding sequence on human cdb3 is not conserved. Moreover, no effect of O(2) pressure on Hb-band 3 interactions has ever been demonstrated in another species. To explore whether band 3-Hb interactions might be widely involved in O(2)-dependent regulation of erythrocyte physiology, we undertook characterization of the effect of O(2) on band 3-Hb interactions in the mouse. We report here that murine band 3 binds deoxyHb with significantly greater affinity than oxyHb, despite the lack of significant homology within the deoxyHb binding sequence. We further map the deoxyHb binding site on murine band 3 and show that deletion of the site eliminates deoxyHb binding. Finally, we identify mutations in murine cdb3 that either enhance or eliminate its affinity for murine deoxyHb. These data demonstrate that despite a lack of homology in the sequences of both murine band 3 and murine Hb, a strong oxygen-dependent association of the two proteins has been conserved.     

Blood lactate accumulation and muscle deoxygenation during incremental exercise.

Near-infrared spectroscopy (NIRS) could allow insights into controversial issues related to blood lactate concentration ([La](b)) increases at submaximal workloads (). We combined, on five well-trained subjects [mountain climbers; peak O(2) consumption (VO(2peak)), 51.0 +/- 4.2 (SD) ml. kg(-1). min(-1)] performing incremental exercise on a cycle ergometer (30 W added every 4 min up to voluntary exhaustion), measurements of pulmonary gas exchange and earlobe [La](b) with determinations of concentration changes of oxygenated Hb (Delta[O(2)Hb]) and deoxygenated Hb (Delta[HHb]) in the vastus lateralis muscle, by continuous-wave NIRS. A "point of inflection" of [La](b) vs. was arbitrarily identified at the lowest [La](b) value which was >0.5 mM lower than that obtained at the following. Total Hb volume (Delta[O(2)Hb + HHb]) in the muscle region of interest increased as a function of up to 60-65% of VO(2 peak), after which it remained unchanged. The oxygenation index (Delta[O(2)Hb - HHb]) showed an accelerated decrease from 60- 65% of VO(2 peak). In the presence of a constant total Hb volume, the observed Delta[O(2)Hb - HHb] decrease indicates muscle deoxygenation (i.e., mainly capillary-venular Hb desaturation). The onset of muscle deoxygenation was significantly correlated (r(2) = 0.95; P < 0.01) with the point of inflection of [La](b) vs., i.e., with the onset of blood lactate accumulation. Previous studies showed relatively constant femoral venous PO(2) levels at higher than approximately 60% of maximal O(2) consumption. Thus muscle deoxygenation observed in the present study from 60-65% of VO(2 peak) could be attributed to capillary-venular Hb desaturation in the presence of relatively constant capillary-venular PO(2) levels, as a consequence of a rightward shift of the O(2)Hb dissociation curve determined by the onset of lactic acidosis.     

Increased hemoglobin O2 affinity protects during acute hypoxia

Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O(2)) transport and O(2) utilization. Although decreasing hemoglobin (Hb) O(2) affinity would favor the release of O(2) to the tissues, increasing Hb O(2) affinity would augment arterial O(2) saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O(2) affinity will augment O(2) transport during severe hypoxia (10 and 5% inspired O(2)) compared with normal Hb O(2) affinity. RBC Hb O(2) affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O(2) affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O(2) (Po(2)). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po(2) at which the Hb is 50% saturated with O(2) by 12.6 mmHg. During 10 and 5% O(2) hypoxia, 5HMF increased arterial blood O(2) saturation by 35 and 48% from the vehicle group, respectively. During 5% O(2) hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po(2) was three times higher in the 5HMF group compared with the control group at 5% O(2) hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O(2) affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization.     

Encapsulation of hemoglobin inside liposomes surface conjugated with poly(ethylene glycol) attenuates their reactions with gaseous ligands and regulates nitric oxide dependent vasodilation

Acellular hemoglobin (Hb)-based O2 carriers (HBOCs) are being investigated as red blood cell (RBC) substitutes for use in transfusion medicine. However, commercial acellular HBOCs elicit both vasoconstriction and systemic hypertension which hampers their clinical use. In this study, it is hypothesized that encapsulation of Hb inside the aqueous core of liposomes should regulate the rates of NO dioxygenation and O2 release, which should in turn regulate its vasoactivity. To test this hypothesis, poly(ethylene glycol) (PEG) conjugated liposome-encapsulated Hb (PEG-LEHs) dispersions were prepared using human and bovine Hb. In this study, the rate constants for O2 dissociation, CO association, and NO dioxygenation were measured for free Hb and PEG-LEH dispersions using stopped-flow UV-visible spectroscopy, while vasoactivity was assessed in rat aortic ring strips using both endogenous and exogenous sources of NO. It was observed that PEG-LEH dispersions had lower O2 release and NO dioxygenation rate constants compared with acellular Hbs. However, no difference was observed in the CO association rate constants between free Hb and PEG-LEH dispersions. Furthermore, it was observed that Hb encapsulation inside vesicles prevented Hb dependent inhibition of NO-mediated vasodilation. In addition, the magnitude of the vasoconstrictive effects of Hb and PEG-LEH dispersions correlated with their respective rates of NO dioxygenation and O2 release. Overall, this study emphasizes the pivotal role Hb encapsulation plays in regulating gaseous ligand binding/release kinetics and the vasoactivity of Hb.     

Nuclear magnetic resonance and oxygen affinity study of cesium binding in human erythrocytes.

We investigated the interaction of the cesium ion (Cs(+)) with the anionic intracellular components of human red blood cells (RBCs); the components studied included 2,3-bisphosphoglycerate (BPG), ADP, ATP, inorganic phosphate (P(i)), carbonmonoxy hemoglobin (COHb), and RBC membranes. We used spin-lattice (T(1)) and spin-spin (T(2)) (133)Cs NMR relaxation measurements to probe Cs(+) binding, and we found that Cs(+) bound more strongly to binding sites in BPG and in RBC membranes than in any other intracellular component in RBCs at physiologic concentrations. By using James-Noggle plots, we obtained Cs(+) binding constants per binding site in BPG (66 +/- 8 M(-1)), ADP (19 +/- 1 M(-1)), ATP (25 +/- 3 M(-1)), and RBC membranes (55 +/- 2 M(-1)) from the observed T(1) values. We also studied the effect of Cs(+) on the oxygen (O(2)) affinity of purified Hb and of Hb in intact RBCs in the absence and in the presence of BPG. In the absence of BPG, the O(2) affinity of Hb decreased upon addition of Cs(+). However, in the presence of BPG, the O(2) affinity of Hb increased upon addition of Cs(+). The O(2) affinity of Cs(+)-loaded human RBCs was larger than that of Cs(+)-free cells at the same BPG level. (31)P NMR studies on the pH dependence of the interaction between BPG and Hb indicated that the presence of Cs(+) resulted in a smaller fraction of BPG available to bind to the cleft of deoxyHb. Our NMR and O(2) affinity data indicate that a strong binding site for Cs(+) in human RBCs is BPG. A partial mechanism for Cs(+) toxicity might arise from competition between Cs(+) and deoxyHb for BPG, thereby increasing oxygenation of Hb in RBCs, and thus decreasing the ability of RBCs to give up oxygen in tissues. The presence of Cs(+) at 12.5 mM in intact human RBCs containing BPG at normal concentrations did not, however, alter significantly the O(2) affinity of Hb, thus ruling out the possibility of Cs(+)-BPG interactions accounting for Cs(+) toxicity in this cell type.     

Reduction of methemoglobin via electron transfer from photoreduced flavin: restoration of O2-binding of concentrated hemoglobin solution coencapsulated in phospholipid vesicles

Ferric methemoglobin is reduced to its ferrous form by photoirradiation either by direct photoexcitation of the heme portion to induce electron transfer from the surrounding media (Sakai at al. (2000) Biochemistry 39, 14595-14602) or by an indirect electron transfer from a photochemically reduced electron mediator such as flavin. In this research, we studied the mechanism and optimal condition that facilitates photoreduction of flavin mononucleotide (FMN) to FMNH(2) by irradiation of visible light, and the succeeding reduction of concentrated metHb in phospholipid vesicles to restore its O(2) binding ability. Visible light irradiation (435 nm) of a metHb solution containing FMN and an electron donor such as EDTA showed a significantly fast reduction to ferrous Hb with a quantum yield (Phi) of 0.17, that is higher than the method of direct photoexcitation of heme (Phi = 0.006). Electron transfer from a donor molecule to metHb via FMN was completed within 30 ns. Native-PAGE and IEF electrophoresis indicated no chemical modification of the surface of the reduced Hb. Coencapsulation of concentrated Hb solution (35 g/dL) and the FMN/EDTA system in vesicles covered with a phospholipid bilayer membrane (Hb-vesicles, HbV, diameter: 250 nm) facilitated the metHb photoreduction even under aerobic conditions, and the reduced HbV restored the reversible O(2) binding property. A concentrated HbV suspension ([Hb] = 8 g/dL) was sandwiched with two glass plates to form a liquid layer with the thickness of about 10 microm (close to capillary diameter in tissue, 5 microm), and visible light irradiation (221 mW/cm(2)) completed 100% metHb photoreduction within 20 s. The photoreduced FMNH(2) reacted with O(2) to produce H(2)O(2), which was detected by the fluorescence measurement of the reaction of H(2)O(2) and p-nitrophenylacetic acid. However, the amount of H(2)O(2) generated during the photoreduction of HbV was significantly reduced in comparison with the homogeneous Hb solution, indicating that the photoreduced FMNH(2) was effectively consumed during the metHb reduction in a highly concentrated condition inside the HbV nanoparticles.     

Effects of acute hypoxia on cerebral and muscle oxygenation during incremental exercise.

To determine if fatigue at maximal aerobic power output was associated with a critical decrease in cerebral oxygenation, 13 male cyclists performed incremental maximal exercise tests (25 W/min ramp) under normoxic (Norm: 21% Fi(O2)) and acute hypoxic (Hypox: 12% Fi(O2)) conditions. Near-infrared spectroscopy (NIRS) was used to monitor concentration (microM) changes of oxy- and deoxyhemoglobin (Delta[O2Hb], Delta[HHb]) in the left vastus lateralis muscle and frontal cerebral cortex. Changes in total Hb were calculated (Delta[THb] = Delta[O2Hb] + Delta[HHb]) and used as an index of change in regional blood volume. Repeated-measures ANOVA were performed across treatments and work rates (alpha = 0.05). During Norm, cerebral oxygenation rose between 25 and 75% peak power output {Power(peak); increased (inc) Delta[O2Hb], inc. Delta[HHb], inc. Delta[THb]}, but fell from 75 to 100% Power(peak) {decreased (dec) Delta[O2Hb], inc. Delta[HHb], no change Delta[THb]}. In contrast, during Hypox, cerebral oxygenation dropped progressively across all work rates (dec. Delta[O2Hb], inc. Delta[HHb]), whereas Delta[THb] again rose up to 75% Power(peak) and remained constant thereafter. Changes in cerebral oxygenation during Hypox were larger than Norm. In muscle, oxygenation decreased progressively throughout exercise in both Norm and Hypox (dec. Delta[O2Hb], inc. Delta [HHb], inc. Delta[THb]), although Delta[O2Hb] was unchanged between 75 and 100% Power peak. Changes in muscle oxygenation were also greater in Hypox compared with Norm. On the basis of these findings, it is unlikely that changes in cerebral oxygenation limit incremental exercise performance in normoxia, yet it is possible that such changes play a more pivotal role in hypoxia.     

The reaction of hydrogen peroxide with hemoglobin induces extensive alpha-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways

Cell-free hemoglobin (Hb) enhances the oxidation-related toxicity associated with inflammation, ischemia, and hemolytic disorders. Hb is highly vulnerable to oxidative damage, and irreversible structural changes involving iron/heme oxidation, heme-adduct products, and amino acid oxidation have been reported. Specific structural features of Hb, such as unconstrained alpha-chains and molecular size, determine the efficiency of interactions between the endogenous Hb scavengers haptoglobin (Hp) and CD163. Using HPLC, mass spectrometry, and Western blotting, we show that H(2)O(2)-mediated Hb oxidation results in the formation of covalently stabilized globin multimers, with prominent intramolecular crosslinking between alpha-globin chains. These structural alterations are associated with reduced Hp binding, reduced CD163 interaction, and severely impaired endocytosis of oxidized Hb by the Hp-CD163 pathway. As a result, when exposed to oxidized Hb, CD163-positive HEK293 cells and human macrophages do not increase hemeoxygenase-1 (HO-1) expression, the physiological anti-oxidative macrophage response to Hb exposure. Failed Hb clearance, inadequate HO-1 expression, and the subsequent accumulation of oxidatively damaged Hb species might thus contribute to pathologies related to oxidative stress.     

Stoichiometry of the reaction of oxyhemoglobin with nitrite.

During the reaction of oxyhemoglobin (HbO2) with nitrite, the concentration of residual nitrite, nitrate, oxygen, and methemoglobin (Hb+) was determined successively. The results obtained at various pH values indicate the following stoichiometry for the overall reaction: 4HbO2 + 4NO2- 4H+ leads to 4Hb+ + 4NO3- + O2 + 2H2 O (Hb denotes hemoglobin monomer). NO2- binds with methemoglobin noncooperatively with a binding constant of 340 M-1 at pH 7.4 and 25 degrees C. Thus, the major part of Hb+ produced is aquomethemoglobin, not methemoglobin nitrite, when less than 2 equivalents of nitrite is used for the oxidation.     

Allosteric kinetics and equilibria of triligated, cross-linked hemoglobin.

Using modulated excitation, we have measured the forward and reverse rates of the allosteric transition between relaxed (R) and tense (T) quaternary structures for triply ligated hemoglobin (Hb), cross-linked between the alpha chains at Lys 99. Oxygen, carbon monoxide, and water were used as ligands and were studied in phosphate and low Cl- bis-Tris buffers at neutral pH. Since the cross-link prohibits disproportionation, triply ligated aquomet Hb species with ferrous beta chains were specifically isolated by isoelectric focusing. Modulated excitation provides rate pairs and therefore gives equilibrium constants between quaternary structures. To coordinate with that information, oxygen binding curves of fully ferrous and tri-aquomet Hb were also measured. L3, the equilibrium constant between three liganded R and T structures, is determined by modulated excitation to be of order unity for O2 or CO (1.1 to 1.5 for 3O2 and 0.7 for 3CO bound), while with three aquomet subunits it is much greater (> or = 23). R-->T conversion rates are similar to those found for HbA, with weak sensitivity to changes in L3. The L3 values from HbXL O2 were used to obtain a unique allosteric decomposition of the ferrous O2 binding curve in terms of KT, KR, and L3. From these values and the O2 binding curve of tri-aquomet HbXL, L3 was calculated to be 2.7 for the tri-aquomet derivative. Consistency in L3 values between equilibrium and modulated excitation data for tri-aquomet-HbXL can be achieved if the equilibrium constant for O2 binding to the alpha chains is six times lower than that for binding to the beta chains in the R state, while the cooperative properties remain homogeneous. The results are in quantitative agreement with other studies, and suggest that the principal effect of the cross-link is to decrease the R state and T state affinity of the alpha subunits with almost no change in the affinity of the beta subunits, leaving the allosteric parameters L and c unchanged.     

The swinging movement of the distal histidine residue and the autoxidation reaction for midge larval hemoglobins.

Some insects have a globin exclusively in their fast-growing larval stage. This is the case in the 4th-instar larva of Tokunagayusurika akamusi, a common midge found in Japan. In the polymorphic hemoglobin comprised of 11 separable components, hemoglobin VII (Ta-VII Hb) was of particular interest. When its ferric met-form was exposed to pH 5.0 from 7.2, the distal histidine was found to swing away from the E7 position. As a result, the iron(III) was converted from a hexacoordinate to a pentacoordinate form by a concomitant loss of the axial water ligand. The corresponding spectral changes in the Soret band were therefore followed by stopped-flow and rapid-scan techniques, and the observed first-order rate constants of k(out) = 25 s(-1) and kin = 128 s(-1) were obtained for the outward and inward movements, respectively, of the distal histidine residue in 0.1 m buffer at 25 degrees C. For O2 affinity, Ta-VII Hb showed a value of P50 = 1.7 Torr at pH 7.4, accompanied with a remarkable Bohr effect (deltaH+ = -0.58) almost equal to that of mammalian hemoglobins. We have also investigated the stability property of Ta-VII HbO2 in terms of the autoxidation rate over a wide range of pH from 4 to 11. The resulting pH-dependence curve was compared with those of another component Ta-V HbO2 and sperm whale MbO2, and described based on a nucleophilic displacement mechanism. In light of the O2 binding affinity, Bohr effect and considerable stability of the bound O2 against acidic autoxidation, we conclude that T. akamusi Hb VII can play an important role in O2 transport and storage as the major component in the larval hemolymph.