Anna Razynska

Modulation of oxygen affinity in hemoglobin by solvent components: Interaction of bovine hemoglobin with 2,3-diphosphoglycerate and monatomic anions☆

In the absence of Cl- in Hepes buffer at pH 7.4, the oxygen affinity of bovine and human hemoglobin is equally sensitive to 2,3-diphosphoglyceric acid. The low oxygen affinity measured for bovine hemoglobin at physiological salt concentration can be explained by the high affinity of Cl- anions for oxygen-linked sites that are absent in human hemoglobin. Bovine hemoglobin can discriminate between the different halogens in the sense that different halide concentrations are necessary to produce the same P50. Competition experiments indicate that the halogens interact with the same oxygen-linked sites. In agreement with the different affinities for halides, the Bohr effect of bovine hemoglobin is larger in the presence of Cl- than in that of Br- and there is good agreement between the number of protons and anions exchanged with the solvent upon oxygenation of bovine hemoglobin.

Cerebral blood flow during hypoxic hypoxia with plasma-based hemoglobin at reduced hematocrit

We determined whether cerebral blood flow (CBF) remained related to arterial O2 content (CaO2) during hypoxic hypoxia when hematocrit and hemoglobin concentration were independently varied with cell-free, tetramerically stabilized hemoglobin transfusion. Three groups of pentobarbital sodium-anesthetized cats were studied with graded reductions in arterial O2 saturation to 50%: 1) a control group with a hematocrit of 31 +/- 1% (mean +/- SE; n = 7); 2) an anemia group with a hematocrit of 21 +/- 1% that underwent an isovolumic exchange transfusion with an albumin solution (n = 8); and 3) a group transfused with an intramolecularly cross-linked hemoglobin solution to decrease hematocrit to 21 +/- 1% (n = 10). Total arterial hemoglobin concentration (g/dl) after hemoglobin transfusion (8.8 +/- 0.2) was intermediate between that of the control (10.3 +/- 0.3) and albumin (7.2 +/- 0.4) groups. Forebrain CBF increased after albumin and hemoglobin transfusion at normoxic O2 tensions to levels attained at equivalent reductions in CaO2 in the control group during graded hypoxia. Over a wide range of arterial O2 saturation and sagittal sinus PO2, CBF remained greater in the albumin group. When CBF was plotted against CaO2 for all three groups, a single relationship was formed. Cerebral O2 transport, O2 consumption, and fractional O2 extraction were constant during hypoxia and equivalent among groups. We conclude that CBF remains related to CaO2 during hypoxemia when hematocrit is reduced with and without proportional reductions in O2-carrying capacity. Thus O2 transport to the brain is well regulated at a constant level independently of alterations in hematocrit, hemoglobin concentration, and O2 saturation.We determined whether cerebral blood flow (CBF) remained related to arterial O2 content ([Formula: see text]) during hypoxic hypoxia when hematocrit and hemoglobin concentration were independently varied with cell-free, tetramerically stabilized hemoglobin transfusion. Three groups of pentobarbital sodium-anesthetized cats were studied with graded reductions in arterial O2saturation to 50%: 1) a control group with a hematocrit of 31 ± 1% (mean ± SE; n = 7); 2) an anemia group with a hematocrit of 21 ± 1% that underwent an isovolumic exchange transfusion with an albumin solution ( n = 8); and 3) a group transfused with an intramolecularly cross-linked hemoglobin solution to decrease hematocrit to 21 ± 1% ( n = 10). Total arterial hemoglobin concentration (g/dl) after hemoglobin transfusion (8.8 ± 0.2) was intermediate between that of the control (10.3 ± 0.3) and albumin (7.2 ± 0.4) groups. Forebrain CBF increased after albumin and hemoglobin transfusion at normoxic O2 tensions to levels attained at equivalent reductions in [Formula: see text] in the control group during graded hypoxia. Over a wide range of arterial O2 saturation and sagittal sinus[Formula: see text], CBF remained greater in the albumin group. When CBF was plotted against[Formula: see text] for all three groups, a single relationship was formed. Cerebral O2 transport, O2 consumption, and fractional O2 extraction were constant during hypoxia and equivalent among groups. We conclude that CBF remains related to [Formula: see text] during hypoxemia when hematocrit is reduced with and without proportional reductions in O2-carrying capacity. Thus O2 transport to the brain is well regulated at a constant level independently of alterations in hematocrit, hemoglobin concentration, and O2 saturation.

Time-resolved fluorescence of hemoglobin species.

We used time-resolved fluorescence in the pico- to nanosecond time range to monitor the presence of tetramers, dimers and monomers in carbonmonoxyhemoglobin (COHb) solutions and to investigate how their distributions change under different experimental conditions. Comparison of fluorescence lifetime computed from the atomic coordinates of COHb (Vasquez et al., 1996) with those experimentally measured allowed identification of molecular species present in the hemoglobin solution. It was possible to observe modification of the distribution of tetramers, dimers, monomers and species with disordered hemes produced by different experimental conditions. Protein concentration affected the detectable lifetimes, indicating increasing amounts of dimers and monomers at low protein concentrations, while the amount of inverted hemes was not modified. Titration with up to 1 M NaCl modified only the extent of dissociation of hemoglobin into dimers, without affecting heme inversion and monomer formation. Hyperbaric pressure increased the amounts of dimers and monomers. This is the first time that monomeric subunits of hemoglobin have been detected at neutral pH in the normal system.

Role of Nitric Oxide Scavenging in Peripheral Vasoconstrictor Response to ββ Cross-Linked Hemoglobin

Transfusion with many crosslinked hemoglobin solutions causes an increase in arterial pressure that may be mediated by scavenging of nitric oxide (NO). If so, we postulated that inhibiting synthesis of NO after hemoglobin transfusion would fail to cause vasoconstriction ordinarily seen with such inhibition. In pentobarbital anesthetized cats, we tested whether administration of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), produced peripheral vasoconstriction after isovolemic exchange transfusion with hemoglobin to the same extent as occurs with L-NAME infusion in time controls and in controls matched for reduced hematocrit (17%) with albumin transfusion. Bovine hemoglobin was treated aerobically with bis-(3,5-dibromosalicyl) fumarate to produce βbeta;-81 lysine crosslinks. Hemoglobin exchange transfusion increased mean arterial blood pressure and there was no further increase after L-NAME. In contrast, L-NAME increased pressure in the time controls and albumin controls. Hemoglobin...

Effect of temperature on oxygen affinity and anion binding of bovine hemoglobin

Measurements of oxygen binding to bovine hemoglobin have been carried out over the temperature range 15-37 degrees C at pH 7.33. The standard enthalpy of oxygenation after correction for the heat of oxygen solution and of the Bohr protons is found to be -7.1 or -7.2 kcal/mol in the presence of 0.1 M chloride or bromide, respectively. This value is well below the -14.4 kcal/mol determined for human hemoglobin under identical experimental conditions. As reported by Fronticelli et al. (C. Fronticelli, E. Bucci and A. Razynska, J. Mol. Biol. 202 (1988) 343), the preferential binding of anions by bovine hemoglobin recognizes the various halides. Measurements at various temperatures reveal that this is true only above 25 degrees C. The halide recognition and the less exothermic enthalpy of oxygenation of bovine hemoglobin are probable due to oxygen-linked hydrophobic effects that are larger in bovine than in human hemoglobin.

Adipyl crosslinked bovine hemoglobins as new models of allosteric systems.

As indicated by peptide analyses and mass spectrometry estimations, intramolecular crosslink with bis(3,5‐dibromosalicyl)adipate of bovine hemoglobin results in the formation of two main components covalently bridged across the β‐cleft. In one component the crosslink joins the β1V1‐β2K81 residues (XL‐Peak‐1), in the other the bridge is between the β1K81‐β2K81 residues (XL‐Peak‐2). Both components are tetrameric with a mass near MW = 67 kDa as estimated by gel filtration, and a hydrodynamic radius near 3.20 nm, estimated by dynamic light scattering. They have very low oxygen affinity with Pm near100 mmHg (XL‐Peak‐1) and near 70 mmHg (XL‐Peak‐2) respectively at 37° C, at neutral pH. The Bohr effect is almost absent in XL‐Peak‐1, while in XL‐Peak‐2 it is very near normal. Both systems show oxygen binding cooperativity with an index near n = 2.0. Flash photolysis kinetics of the recombination with CO could be resolved into a fast and a slow component. The amplitude of the fast rates were not concentration‐dependent. The stopped‐flow kinetics were autoaccelerating, consistent with their ligand‐binding cooperativity. All rates were very similar to those of normal hemoglobin, suggesting that the oxy‐ rather than the deoxy‐forms of the systems were affected by the crosslink. Proteins 2000;39:166–169.

Renal response to hemodilution with albumin or crosslinked bovine hemoglobin: role of nitric oxide.

The decreased hematocrit that occurs with hemodilution leads to a decrease in peripheral resistance while venous return and cardiac output increase. We determined systemic and renal responses to hemodilution with a solution of albumin or a crosslinked hemoglobin-based oxygen carrier (XLHb) and the effect of inhibition of NO synthesis on the responses to albumin. Clearance experiments were done on anesthetized rats to determine mean arterial pressure (MAP), glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and sodium excretion before and after isovolemic exchange transfusion (2 ml per 100 gm body weight) with either (1) 5% albumin (n = 5), (2) 5% albumin plus N omega-nitro-L-arginine methyl ester (L-NAME, 3.5 mg/kg; n = 6), or (3) 6% XLHb (n = 7) and after administration of L-NAME alone (n = 4). Hematocrit decreased similarly in all exchange groups (from 42 +/- 1.0 to 29 +/- 1.3). MAP decreased with albumin exchange, increased with L-NAME, and remained unchanged with albumin+L-NAME or XLHb. GFR, ERPF, and renal blood flow increased while filtration fraction and renal resistance decreased with albumin exchange; responses were the opposite with L-NAME, and with albumin+L-NAME and XLHb these parameters remained approximately the same as control values. Red cell delivery decreased with L-NAME, albumin+L-NAME, and XLHb but remained at control levels with albumin. In conclusion, renal effects of decreased hematocrit can be offset by decreased NO availability. The similarity of results with XLHb and albumin+L-NAME is consistent with NO scavenging by hemoglobin. Increased renal vascular tone with XLHb limits oxygen delivery.

Two-photon induced fluorescence of linear alkanes; a possible intrinsic lipid probe

We measured the fluorescence emission spectra and intensity decays of the linear alkane tetradecane when excited at 300 nm by two-photon excitation. The unquenched lifetime of tetradecane in neat solution is near 4.4 ns. The emission of tetradecane centered at 210 nm is collisionally quenched by oxygen, n-propanol and water. These results suggest that aliphatic groups in non-polar environments can display good fluorescence, and that the aliphatic side chains of detergents and lipids may serve as an intrinsic fluorescent probe of micelles and bilayers.

Hemoglobin tetramers stabilized with polyaspirins.

Organic acids activated by esterification with 3,5-dibromosalicylate react preferentially either with the beta 82 lysines or the alpha 99 lysines of hemoglobin. The versatility and site specificity of these polysapirins and the usage of both human and bovine hemoglobins allowed the construction of a family of oxygen carriers with various P50 ranging from 10 to 50 mmHg. These derivatives are obtained in pure homogeneous form by column chromatography. They are stabilized tetramers where the dissociation into dimers is inhibited. The latest addition is Tri-(3,5,dibromosalicyl)-benzenetricarboxylate, which crosslinks both human and bovine hemoglobin across the beta subunits, decreasing the oxygen affinity of both proteins. The crosslinked hemoglobins have a normal Bohr effect, more expanded in the alkaline region, and are sensitive to chlorides but not to polyphosphates. Solutions of stabilized tetramers, infused into rats or cats up to 25-50% blood replacement, do not produce altered renal and cardiac function. In the cat isovolemic hemodilution increases cerebral flow in controls treated with albumin solutions, when an oxygen carrier is used the cerebral flow remains normal.

Assembly of Human Hemoglobin STUDIES WITH ESCHERICHIA COLI-EXPRESSED α-GLOBIN

The α-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of native β-chains. The functional and structural properties of the expressed α-chains were assessed in the isolated state and after assembly into a functional hemoglobin tetramer. The recombinant and native hemoglobins were essentially identical on the basis of sensitivity to effectors (Cl− and 2,3-diphosphoglycerate), Bohr effect, CO binding kinetics, dimer-tetramer association constants, circular dichroism spectra of the heme region, and nuclear magnetic resonance of the residues in the α1β1 and α1β2 interfaces. However, the nuclear magnetic resonance revealed subtle differences in the heme region of the expressed α-chain, and the recombinant human normal adult hemoglobin (HbA) exhibited a slightly decreased cooperativity relative to native HbA. These results indicate that subtle conformational changes in the heme pocket can alter hemoglobin cooperativity in the absence of modifications of quaternary interface contacts or protein dynamics. In addition to incorporation into a HbA tetramer, the α-globin refolds and incorporates heme in the absence of the partner β-chain. Although the CO binding kinetics of recombinant α-chains were the same as that of native α-chains, the ellipticity of the Soret circular dichroism spectrum was decreased and CO binding kinetics revealed an additional faster component. These results show that recombinant α-chain assumes alternating conformations in the absence of β-chain and indicate that the isolated α-chain exhibits a higher degree of conformational flexibility than the α-chain incorporated into the hemoglobin tetramer. These findings demonstrate the utility of the expressed α-globin as a tool for elucidating the role of this chain in hemoglobin structure-function relationships.