Appearance of dissociable and cross-linked hemoglobins in the renal hilar lymph.

Unlike unmodified dissociable bovine hemoglobin (UHb), cross-linked hemoglobins do not dissociate into dimers, do not cross the glomerular filter, and are retained in the plasma for a longer time. Renal peritubular capillaries, which are different from the glomerulus, allow the passage of molecules as large as albumin into the renal interstitium. Cross-linked hemoglobins should pass across these capillaries, enter the renal interstitium, and drain through the renal lymphatics. The present experiments were done in anesthetized rats to determine the appearance of UHb, an intramolecularly cross-linked tetrameric hemoglobin (DECHb), and a polymerized bovine hemoglobin (PHb) of larger molecular size into the renal hilar lymph. Renal hilar lymph samples were obtained before and after an isovolemic exchange of 2 mL/100 g rat weight of a 6% solution of each hemoglobin for blood. The behavior of a 5% solution of Evan's blue-labeled albumin was also determined for comparison. After exchange, the Initial plasma concentration of each of the proteins was in excess of 20 mg/mL. UHb appeared both in urine and lymph. DECHb, PHb, and albumin were absent from the urine but appeared promptly in the renal hilar lymph and reached concentrations at least 30% that of plasma. PHb had a significantly smaller lymph clearance (in microliters per minute) and longer plasma half-time than the other nondissociable proteins. These findings indicate that DECHb and PHb, although not filtered, pass across peritubular capillaries and readily enter the renal interstitial space. The passage of the larger molecular-sized PHb may be hindered relative to the other proteins in passage across peritubular and other systemic capillaries.

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 beta-cleft. In one component the crosslink joins the beta(1)V1-beta(2)K81 residues (XL-Peak-1), in the other the bridge is between the beta(1)K81-beta(2)K81 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 near 100 mmHg (XL-Peak-1) and near 70 mmHg (XL-Peak-2) respectively at 37 degrees 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.

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.

Entropy-driven intermediate steps of oxygenation may regulate the allosteric behavior of hemoglobin.

When the oxygen binding isotherms of human, bovine and fallow deer (Dama-Dama) hemoglobins are measured at different temperatures either by optical or calorimetric techniques, analyses according to the Adair's formalism show that at least one of the intermediate steps of ligation has a positive enthalpy change, i.e., absorbs rather than emitting heat, indicating that it is entropy rather than enthalpy driven (Bucci, E., et al. 1991. Biochemistry. 30:3195-3199; Bucci, E., et al. 1993. Biochemistry. 32:3519-3526; Johnson, C., et al. 1992. Biochemistry. 31:10074-10082; Johnson, C., et al. 1995. Biophys. Chem. 59:107-117). This phenomenon is confirmed in systems in which the beta82 lysines of human hemoglobin are covalently cross-linked by acylation with dicarboxylic acids of increasing length, namely the fumaryl (four-carbon-long), adipoyl (six-carbon-long), and sebacoyl (10-carbon-long) residues. Consistently in all of the systems here reported, the enthalpy excursions are masked by compensatory entropy changes, which keep the free energy of ligand binding constant for the first three steps of oxygenation. Furthermore, the cooperativity index and the overall oxygen affinity seem to be correlated to the positive enthalpy excursions of the intermediate steps of ligation. Fumaryl-Hb (hemoglobin cross-linked with a fumaryl residue, four carbons) with the lowest absorption of heat has the highest affinity and lowest cooperativity index. Adipoyl-Hb (hemoglobin cross-linked with an adipoyl residue, six carbons) has the highest absorption of heat and the highest cooperativity index. It appears that nonuniform heat release by the intermediates of oxygenation is part of the allosteric phenomena in hemoglobin systems. There is not enough information that would allow assigning these phenomena to the interplay of the various conformations described for hemoglobin besides the classic T (Fermi et al. 1984. J. Mol. Biol. 175:159-174) and R (Shanaan. 1983. J. Mol. Biol. 171:31-59), as listed at the end of the Discussion. The possibility cannot be excluded that entropy-driven steps characterize new conformational transitions still to be described.

Alpha-subunit oxidation in T-state crystals of a sebacyl cross-linked human hemoglobin with unusual autoxidation properties.

In the crystal structure of human T-state hemoglobin with a sebacyl residue cross-linking the two beta-subunit Lys82,s (DecHb), the Fe atoms of the alpha-subunit hemes are found to be oxidized with a water molecule bound. The three-dimensional structure and heme geometries were compared to those of deoxyhemoglobin and other partially and fully oxidized hemoglobins [R. Liddington, Z. Derewenda, E. Dodson, R. Hubbard, G. Dodson, J. Mol. Biol. 228 (1992) 551]. The heme geometries of the alpha-subunits are consistent with those observed in oxidized structures. The proximal histidines of the alpha-subunits move toward the heme plane shifting the F-helix and FG-corner in a manner observed for partially oxidized human hemoglobin. This supports the hypothesis that these perturbations may precede the T- to R-state transition. Circular dichroism studies comparing DecHb and natural human hemoglobin in the deoxy and CO ligated forms confirm that the conformations of the deoxy forms are identical, but the ligated forms have slight differences in the solution structures. DecHb is found to be more resistant to autoxidation than natural hemoglobin. The time course of autoxidation of DecHb shows that it is virtually absent for the first 1500 min followed by a rapid increase. Thus, the discovery of the oxidation of the alpha-subunits in the deoxy-crystals is quite unexpected. The data confirm that ligation of the alpha-subunits precedes that of the beta-subunits. This may suggest a low ligand affinity of the alpha-diligated form of hemoglobin.

Assembly of human hemoglobin. Studies with Escherichia coli-expressed alpha-globin.

The alpha-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of native beta-chains. The functional and structural properties of the expressed alpha-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 alpha1beta1 and alpha1beta2 interfaces. However, the nuclear magnetic resonance revealed subtle differences in the heme region of the expressed alpha-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 alpha-globin refolds and incorporates heme in the absence of the partner beta-chain. Although the CO binding kinetics of recombinant alpha-chains were the same as that of native alpha-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 alpha-chain assumes alternating conformations in the absence of beta-chain and indicate that the isolated alpha-chain exhibits a higher degree of conformational flexibility than the alpha-chain incorporated into the hemoglobin tetramer. These findings demonstrate the utility of the expressed alpha-globin as a tool for elucidating the role of this chain in hemoglobin structure-function relationships.

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.

Production and characteristics of an infusible oxygen-carrying fluid based on hemoglobin intramolecularly cross-linked with sebacic acid.

Research on red cell substitutes requires the availability of oxygen-carrying fluids for physiologic experiments. This article describes the procedure for in-house production of such a fluid. It contains a hemoglobin-based oxygen carrier obtained by reacting human hemoglobin with bis-(3,5-dibromosalicyl) sebacate. This reagent produces intramolecular cross-links between the beta 82 lysines and between the alpha 99 lysines, respectively. The oxygen half-saturation pressure (P50) of the fluid is near 34 mm Hg at 37 degrees C, with a Hill's parameter of n = 2.2. The half-time of intravascular retention is near 3.0 hours in the rat and 6.5 hours in the cat. Spectrophotometric analyses of arterial and venous plasma from an infused rat reveal an efficient oxygen delivery to the tissues by the oxygen carrier. Therefore, this new cross-linked human hemoglobin can be produced in quantities sufficient for in vivo evaluation and with an oxygen affinity and cooperativity adequate for oxygen unloading in plasma.

Positive and negative cooperativities at subsequent steps of oxygenation regulate the allosteric behavior of multistate sebacylhemoglobin.

Cross-linked human hemoglobin (HbA) is obtained by reaction with bis(3,5-dibromosalicyl) sebacate. Peptide maps and crystallographic analyses confirm the presence of the 10 carbon atom long sebacyl residue cross-linking the two beta82 lysines of the beta-cleft (DecHb). The Adair's constants, obtained from the oxygen binding isotherms, show that at the first step of oxygenation normal hemoglobin and DecHb have a very similar oxygen affinity. In DecHb negative binding cooperativity is present at the second step of oxygenation, which has an affinity 27 times lower than at the first step. Positive cooperativity is present at the third binding step, whose affinity is 380 times that of the second step. The fourth binding step shows a weak negative cooperativity with an affinity one-half that of the third step. Crystals of deoxy-DecHb diffracted to 1.9 angstroms resolution. The resulting atomic coordinates are very similar to those of Fermi et al. [(1984) J. Mol.Biol. 175, 159-174] and Fronticelli et al. [(1994) J. Biol Chem. 269, 23965-23969] for deoxy-HbA. The electron density map of deoxy-DecHb indicates the presence of the 10 carbon bridge between the beta82 lysines. Molecular modeling confirms that insertion of the linker into the T structure requires only slight displacement of the two beta82 lysines. Instead, insertion of the linker into the R and R2 structures [Shaanan (1983) J. Mol. Biol. 171, 31-59; Silva et al. (1992) J. Biol. Chem. 267, 17248-17256] is hindered by serious sterical restrictions. The linker primarily affects the partially and fully liganded states of hemoglobin. The data suggest in DecHb concerted conformational changes at each step of oxygenation.

Cerebral O2 transport with hematocrit reduced by cross-linked hemoglobin transfusion.

The purpose of this study was to dissociate effects of reduced viscosity from those of low arterial O2 content (CaO2) on cerebral blood flow (CBF) during anemia. Three groups (n = 8) of pentobarbital sodium-anesthetized cats were studied: 1) a time-control group with a hematocrit of 32 +/- 1% (SE), 2) an anemia group that underwent an isovolumic exchange transfusion with albumin in a salt solution to decrease hematocrit to 18 +/- 1%, and 3) a group transfused with cell-free, tetramerically stabilized hemoglobin to decrease hematocrit equivalently to that in the albumin-transfused group. CaO2 (in ml/dl) in the hemoglobin-transfused group (11.8 +/- 0.3) and the control group (15.0 +/- 0.6) was greater than that in the albumin group (8.7 +/- 0.3). CBF (in ml.min-1.100 g-1) in the hemoglobin group (45 +/- 3) and control group (36 +/- 4) was less than that in the albumin group (60 +/- 3). Consequently, cerebral O2 transport (CaO2 x CBF) was similar in the hemoglobin, control, and albumin groups (5.3 +/- 0.3, 5.3 +/- 0.4, and 5.2 +/- 0.2 ml.min-1.100 g-1, respectively). After infusion of N omega-nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide (NO) synthase, CBF in the hemoglobin group remained lower than that in the albumin group, suggesting that NO scavenging by hemoglobin did not solely account for the lower CBF. In contrast, the neurohypophysis (posterior pituitary) exhibited substantial decreases in blood flow that were not augmented by L-NAME administration after hemoglobin transfusion and that were similar in magnitude to L-NAME alone. Thus NO scavenging by cell-free hemoglobin may be more prominent in high-flow, protein-permeable regions enriched with NO synthase. These results support the hypothesis that O2 transport to cerebrum is well regulated when CaO2 is manipulated independently of hematocrit and viscosity.

Stabilization of the tetrameric structure of human and bovine hemoglobins by pseudocrosslinking with muconic acid.

In previous studies mono-3,5-dibromosalicyl-fumarate was used to introduce an intramolecular crosslink (pseudo-crosslink) in the beta cleft between hemoglobin beta subunits. Sedimentation velocity analysis indicated that the product had a mean molecular weight indicating a tetramer with low dissociability. The product had a P50 higher than that of native hemoglobin and a plasma retention time in the rat of about 3 h, i.e., four times longer than untreated hemoglobin. However, the product contained a fraction which was rapidly eliminated in the urine and which had a short plasma half-time of about 20 min, indicating the presence of a dissociable fraction. We have attempted to further enhance the tetrameric stability of hemoglobin and prevent urine elimination by positioning a longer chain carboxylic acid than fumaric acid into the beta cleft. We reason that a longer molecule would allow for greater stabilizing interactions across the beta cleft. In the present study human and bovine hemoglobins were reacted with mono-3-5-dibromosalicyl muconate. Muconic acid is two carbons longer than fumaric acid. The products were acylated at the beta 82 (human) and beta 81 (bovine) lysines of the beta-cleft and had a low degree of dissociability. For reasons not presently understood, urine excretion was high and plasma half-time was not increased above that of untreated hemoglobin. In conclusion, it appears that only covalently crosslinked hemoglobins which are completely nondissociable tetramers escape filtration; tetramers with any degree of dissociability into dimers are filterable.

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.

Role of nitric oxide scavenging in peripheral vasoconstrictor response to beta beta 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 beta beta-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 transfusion decreased intestinal and renal blood flow, and there was no further decrease after L-NAME. In contrast, L-NAME decreased intestinal and renal blood flow in time controls and albumin controls. With L-NAME pretreatment in a separate group of cats, there was little further increase in arterial pressure or visceral vasoconstriction after hemoglobin transfusion. We conclude that the increase in arterial blood pressure after isovolemic crosslinked hemoglobin transfusion is best explained by scavenging of NO in intestinal and renal vascular beds.

Effect of intramolecular cross-links on the enthalpy and quaternary structure of the intermediates of oxygenation of human hemoglobin.

We have reported [Bucci, E., Fronticelli, C., & Grycznski, Z. (1991) Biochemistry 30, 3195-3199] that in human and bovine hemoglobins the release of heat at the subsequent steps of oxygenation is not constant. This is especially evident in the binding of the third O2 molecule, which is an endothermic event. This phenomenon was attributed to peculiar conformations of the intermediates of oxygenation, not included in the fundamental R/T transition of the system. To test this hypothesis, we have explored the effect of conformational constraints on the thermodynamics of the intermediates of oxygenation. The assumption was that intramolecular constraints would stabilize the intermediates into conformations similar to the R and T forms reducing the variability of their enthalpies. We have analyzed the temperature dependence of the oxygen binding isotherms of human hemoglobin cross-linked either between the beta 82 or between the alpha 99 lysines by bis(3,5-dibromosalicyl)fumarate. The measurements were perfomred at pH 9.0 in 0.1 M borate buffer in order to avoid thermal effects due to oxygen-linked binding of anions and protons. The data were analyzed singularly by local procedures and simultaneously using global procedures. The two cross-links had opposite effects. The cross-link between the beta-subunits decreased while that between the alpha-subunits increased the endothermic behavior of the third step of oxygenation. Also, the cross-link between the beta-subunits increased the fractional amount of the triligated species at intermediate stages of oxygenation, while that between the alpha-subunits decreased this quantity to hardly detectable values.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Intravascular retention and renal handling of purified natural and intramolecularly cross-linked hemoglobins.

Plasma half time of unmodified hemoglobin (UHb) and two intramolecularly cross-linked hemoglobins (alpha alpha XL and beta beta XL) was measured in anesthetized rats after an intravenous bolus of 20 mg.100 gm.-1 To rule out the possibility that differences among plasma half times might be caused by differences in acute effects on renal excretory function, glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured simultaneously with plasma half time. Experiments were also done to determine whether higher doses (60 to 100 mg-1.100 gm-1) of these compounds had a delayed effect (48 hours) on GFR or ERPF. Massive urinary excretion of UHb occurred; however, only 1% of the alpha alpha XL and none of the beta beta XL was excreted. Plasma half time of alpha alpha XL and beta beta XL averaged 3.3 hours, or four times longer than UHb. In no case did a decrease in GFR or ERPF occur. Instead, a transient increase in GFR, ERPF, urine flow, and systemic blood pressure was seen. Similar increases occurred after albumin administration, suggesting expansion of vascular volume as the initiating factor. Renal functions at 48 hours after 60 to 100 mg.100 gm-1 of UHb, alpha alpha XL or beta beta XL were not different from control (albumin). Intratubular hemoglobin casts or intravascular precipitates were not evident in acute or 48-hour studies. At 48 hours Perls' staining material was found in one alpha alpha XL specimen at 3 hours after administration. Perls' staining material was present in renal tubule cells in all but the albumin-treated kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine hemoglobin pseudo-crosslinked with mono(3,5-dibromosalicyl)-fumarate.

Under oxygenated conditions bovine hemoglobin reacts with mono(3,5-dibromosalicyl)-fumarate which specifically acylates the EF5 lysines in the beta-cleft of the protein. The chemical modification introduces in the molecule a pseudo-crosslink which hinders the dissociation of the hemoglobin molecule into dimers. Retention time in circulation of the chemically modified bovine hemoglobin, measured in the rat, is increased fivefold with respect to untreated bovine hemoglobin. The oxygen affinity at 37 degrees C and at pH 7.4, has a P50 = 5.4 kPa and a value of n = 1.9. Under the same experimental conditions the oxygen affinity is not sensitive to anions and polyanions whereas it is sensitive to CO2. The Bohr effect is shifted toward the alkaline pH range by 0.5-1: the maximum number of protons released is 1.5/tetramer, similar to normal bovine hemoglobin (1.8 protons/tetramer). Analysis of the binding isotherms, using the two-state Monod-Wyman-Changeux model and fixing the value of the allosteric constant L = 10(5), shows that the oxygen affinity of the T structure is not modified, and that the low oxygen affinity of the system is due to a decrease of the oxygen affinity of the R structure. Analysis using the sequential Adair model shows a modification of the overall binding constants and suggests a redistribution of the intermediate species of oxygenation.

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.

Pseudo cross-link of human hemoglobin with mono-(3,5-dibromosalicyl)fumarate.

The reaction of human oxyhemoglobin with mono(3,5-dibromosalicyl)fumarate, produces a derivative specifically acylated at the two lysines beta 82, which can be purified with a 70% yield. The oxygen affinity of this derivative at 37 degrees C at pH 7.4, 0.1 M Cl- is of 12 mm Hg, and is not affected by organic phosphate. In the presence of 5% CO2, the oxygen affinity decreases to 25 mm Hg. In all cases the cooperativity is lowered, with a value of n in the Hill plots near 2. Sedimentation velocity measurements indicate that, contrary to normal hemoglobin, this derivative fails to dissociate into dimers upon exposure to pH 5.5. The stability of the tetrameric structure is probably due to a modification of the beta-beta interface, resulting from electrostatic and hydrophobic interactions introduced in the beta cleft by the fumaryl residues. These new interactions are probably the origin of a new reverse Bohr effect group at alkaline pH. Consistent with the stabilization of the tetrameric structure, the half-time of retention of this compound in the rat is increased 4-fold with respect to that of normal hemoglobin. These characteristics cast a favorable light on the usage of this compound as an oxygen carrier in transfusional and perfusional fluids.