Unmodified Hemoglobin Research Articles

Thymoquinone provides structural protection of human hemoglobin against oxidative damage: Biochemical studies

Hydroxyl radicals (OH.) are one of the most active reactive oxidants recognized for their deleterious effects to cause protein oxidative damage. Thymoquinone, a monoterpene molecule abundantly present in black cumin and known for its pharmacological activities, but its activity against the OH.-induced protein oxidative damage has never been explored. This study determined the therapeutic potential of thymoquinone against OH.-induced oxidative human hemoglobin damage. Novel data demonstrated that thymoquinone provides structural protection of hemoglobin against oxidative damage. Treatment of hemoglobin with OH. induces hypochromicity at 280 and 405 nm, whereas thymoquinone reversed these hypochromic effects. In addition, OH. cause significant reduction in tryptophan fluorescence, however thymoquinone also reversed these damaging effects. Thymoquinone also reduces OH.-induced hydrophobicity and also reduces OH.-induced carbonylation. Moreover, it also inhibits thermal stabilization of OH.-hemoglobin complex. SDS-PAGE of unmodified hemoglobin showed four bands, which disappeared upon OH. treatment and these changes were also retained by thymoquinone. In conclusion, this is the first study that shows the therapeutic potential of thymoquinone against OH.-induced oxidative damage in human hemoglobin.

Analysis of protein denaturation, aggregation and post-translational modification by agarose native gel electrophoresis

Agarose native gel electrophoresis has been developed to separate proteins and protein complexes in the native state. Here, we applied this technology to analyze proteins that undergo degradation, post-translational modification or chemical/physical changes. Antibodies showed aggregation/association upon acid or heat treatment. Limited reduction of disulfide bonds resulted in non-covalent aggregation of bovine serum albumin and cleavage of only inter-chain linkages of an antibody that had no effects on its overall structure. Native agarose gel analysis showed changes in mobility of human transferrin upon Fe3+ binding. Analysis of a commercial glycated human hemoglobin A1c showed no difference in electrophoretic pattern from un-modified hemoglobin. Native agarose gel showed aggregation of a virus upon acid or heat treatment. We have extracted bands of bovine serum albumin from the agarose native gel for sodium dodecylsulfate gel electrophoresis analysis, showing degradation of aged sample. Lastly, we analyzed phosphorylation of Zap70 kinase by native gel and Western blotting. These applications should expand the utility of this native gel electrophoresis technology.

Delta beta thalassemia.

A 19-year-old woman of Lebanese ethnic origin was referred for investigation of microcytosis. Her blood count showed: red cell count 5.32 × 1012/L, hemoglobin concentration 116 g/L, mean cell volume 70 fL, mean cell hemoglobin 21.8 pg and mean cell hemoglobin concentration 312 g/L. Her blood film showed microcytosis, mild hypochromia and poikilocytosis. The poikilocytes included elliptocytes (left Image) and small numbers of irregularly contracted cells (right Image). There were only occasional target cells. Thalassemia trait was suspected and high performance liquid chromatography (HPLC) was performed. Hemoglobin A2 was normal at 2.8%, but hemoglobin F, as quantified by the Bio-Rad Variant II instrument (unmodified hemoglobin F), was increased to 11.8%; the total hemoglobin F was about 14% if acetylated F, which is not integrated by the instrument, was included. The likely diagnosis now became δβ thalassemia. This diagnosis was confirmed by Gap-PCR which showed heterozygosity for the Sicilian δβ thalassemia deletion mutation. The diagnosis of δβ thalassemia is important, particularly in a patient in the reproductive age range, since homozygosity or compound heterozygosity with β thalassemia can lead to thalassemia intermedia. It might be expected that the hemoglobin A2 would be reduced since one δ gene has been deleted but in fact it is often normal. Hemoglobin F is usually between 5 and 20%. Nothing to report.

Improving the Production of Cofactor-Containing Proteins: Production of Human Hemoglobin in Yeast.

Human hemoglobin is an essential protein, whose main function as an oxygen carrier is indispensable for life. Hemoglobin is a cofactor-containing protein with heme as prosthetic group. Same as in humans, heme is synthesized in many organisms in a complex pathway involving two cellular compartments (mitochondria and cytosol), which is tightly regulated. Red blood cells (erythrocytes) are specialized and adapted for production and transport of the hemoglobin molecules. In addition to oxygen binding, hemoglobin can participate in a variety of chemical reactions byits iron and heme and may become toxic when released from erythrocytes. Hemoglobin is a major target for the development of blood substitutes/oxygen carriers, and therefore its microbial production is attractive, as it may provide a cheap and reliable source of human hemoglobin. Significant efforts have been dedicated to this task for the last three decades. Moreover since the first generation of cell-free blood substitutes based on unmodified hemoglobin failed human trials, mutant forms became of great interest.In this chapter we summarize the existing knowledge about human hemoglobin, challenges of its microbial production, and its improvement, with a particular focus upon yeast as production host.

Bohr effect of native and chemically modified hemoglobins: Quantitative analyses based on the Wyman equation

Thirteen histidines and the α-chain terminal amino group (ACTA) make all of the contributions to the Bohr effect of human hemoglobin. The pKas of the 13 histidines in carbonmonoxy- and deoxyhemoglobin are known from 1H NMR studies. Those of ACTA are not so precisely known. We employed the Wyman equation and the 13 histidine pairs of pKas to determine the pKas of ACTA by curve-fitting to hemoglobin Bohr effect data. Using all 14 pairs of pKas as preliminary data, we employed the Wyman equation to fit the Bohr data for hemoglobin chemically modified at Cys93β with cystamine, cystine and iodoacetamide. We demonstrate quantitatively that the reduction of the Bohr effect upon chemical modification is due to three negatively contributing Bohr groups: His2β, His77β and His143β. These make twice their normal contributions to the Bohr effect in unmodified hemoglobin. We also find that the ACTA pKas increase with increasing ionic strength.

Bohr effect of human hemoglobin A: Magnitude of negative contributions determined by the equilibrium between two tertiary structures

We have measured the affinity of the CysF9[93]β sulfhydryl group of human deoxyhemoglobin and oxyhemoglobin for 5,5′-dithiobis(2-nitrobenzoate), DTNB, between pH ≈5.6 and 9 in order to understand the basis of the reported reduction of the Bohr effect induced by chemical modification of the sulfhydryl. We analyzed the results quantitatively on the basis of published data indicating that the sulfhydryl exists in two conformations that are coupled to the transition between two tertiary structures of hemoglobin in dynamic equilibrium. Our analyses show that the ionizable groups linked to the DTNB reaction have lower pKas of ionization in deoxyhemoglobin compared to oxyhemoglobin. So these ionizable groups should make negative contributions to the Bohr effect. We identify these groups as HisNA2[2]β, HisEF1[77]β and HisH21[143]β. We provide explanations for the finding that hemoglobin, chemically modified at CysF9[93]β, has a lower Bohr effect and a higher oxygen affinity than unmodified hemoglobin.

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

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

Hemoglobin-based Oxygen Carriers for Reversing Hypotension and Shock

Hemoglobin-based Oxygen Carriers for Reversing Hypotension and Shock

Structural and Functional Characterization of Glutaraldehyde-Polymerized Bovine Hemoglobin and Its Isolated Fractions

Glutaraldehyde-polymerized bovine hemoglobin (PolyHbBv, trade name Oxyglobin), is a non-site-specific modified hemoglobin-based oxygen-carrying solution, developed for use in veterinary medicine. PolyHbBv was fractionated into four distinct tetrameric and multiple polytetrameric forms ranging in molecular mass (87.2-502.3 kDa) using size exclusion chromatography (SEC) and verified by laser light scattering. We evaluated the structural modification occurring in the fractionated mixture of PolyHbBv and assessed the functionality and redox stability of each fraction in relation to the mixture as a whole. Intramolecular cross-linking evaluation as performed by MALDI-MS and SEC under dissociating conditions revealed no-site-specific tetramer stabilization within the fractions; Intermolecular cross-linking was highly correlated with lysine and histidine modification as determined by amino acid composition analysis. While native unmodified hemoglobin, HbBv, PolyHbBv, and PolyHbBv fractions (F1-F4) revealed significant methionine oxidation, modification, or both, the critical betaMet55 located in the functionally plastic domains (alpha1-beta1 interface) of HbBv was unaltered. Moreover, neither of the two betaCys93 located in the highly plastic alpha1-beta2 interface were modified in PolyHbBv or in F1-F4. Our structural analysis also revealed that the reported loss in sensitivity to chloride in PolyHbBv could not be attributed to direct alteration of chloride ion binding amino acids. Structural modification imparted by glutaraldehyde resulted in nearly identical functional characteristics of PolyHbBv and its fractions with regard to oxygen equilibrium, ligand binding, and autoxidative kinetics.

Effect of Cl- and H+ on the oxygen binding properties of glutaraldehyde-polymerized bovine hemoglobin-based blood substitutes.

Bovine hemoglobin was cross-linked with glutaraldehyde, resulting in high oxygen affinity polymeric hemoglobin dispersions of varying molecular weight distributions. High oxygen affinity acellular oxygen carriers were designed in order to exhibit oxygen release profiles closer to that of human red blood cells (RBCs), without exhibiting the inherent increased vasoactivity that occurs with low oxygen affinity acellular oxygen carriers (1, 2). Oxygen dissociation curves were measured for polymerized hemoglobin dispersions at various pH values (7.0, 7.4, and 8.0) and chloride ion concentrations. Unmodified hemoglobin showed an increase in oxygen affinity with increased chloride ion concentration and a decrease in oxygen affinity with increased pH, as was previously demonstrated in the literature (3). For glutaraldehyde-polymerized hemoglobin dispersions, the ability of the oxygen affinity to respond to changes in Bohr H+ and Cl- concentration was weakened. However, at acidic physiological pH (pH = 7), the Bohr effect was still present at high Cl- concentrations. Thus, the Bohr effect maintained some dependency on the Cl- concentration.

Hemoglobin polymerized with a naturally occurring crosslinking agent as a blood substitute: in vitro and in vivo studies.

A naturally occurring crosslinking agent, genipin, extracted from the fruits of Gardenia jasminoides ELLIS was used by our group to chemically modified biomolecules. Genipin and its related iridoid glucosides have been widely used as an antiphlogistic and cholagogue in herbal medicine. Our previous study showed that the cytotoxicity of genipin is significantly lower than glutaraldehyde. The study was to investigate the feasibility of using genipin to polymerize hemoglobin as a blood substitute. The results indicated that the rate of hemoglobin polymerization by glutaraldehyde was significantly faster than that by genipin and it readily produced polymers with molecular masses greater than 500,000 Da. It was found that the maximum degree of hemoglobin polymerization by genipin was approximately 40% if over-polymerization is to be prevented. With increasing the reaction temperature, hemoglobin concentration, and genipin-to-hemoglobin molar ratio, the duration taken to achieve the maximum degree of hemoglobin polymerization by genipin became significantly shorter. The P50 value of the unmodified hemoglobin was 9 mmHg, while that of the genipin-polymerized PLP-hemoglobin increased to 21 mmHg. It was found in a rat model that the genipin-polymerized PLP-hemoglobin resulted in a longer circulation time than the unmodified hemoglobin. In conclusion, the results of the study indicated that the genipin-polymerized hemoglobin solution has a lower oxygen affinity and a longer vascular retention time than the unmodified hemoglobin solution.

An NMR and circular dichroism study of the interaction of thiocyanate with human and cross-linked hemoglobin: identification of Lys-α-99 as a possible dissociation linked binding site

The interaction of thiocyanate with human native and cross-linked oxyhemoglobin (oxyHb), and methemoglobin (metHb) has been investigated by optical spectroscopy, circular dichroism (CD) and nuclear spin lattice relaxation rate measurements. The interaction of thiocyanate anion with human hemoglobin has been investigated by NMR measurements of the nuclear spin lattice relaxation rate of N 15 labeled thiocyanate in the presence of cyanomethemoglobin and cross-linked cyanomethemoglobin. Results show that thiocyanate is located approximately 8.9 and 6.2 Å away from the heme group in cyanomethemoglobin and cross-linked cyanomethemoblobin, respectively. These results are consistent with the binding of SCN − at the lys-α-99 in the unmodified hemoglobin. Since this site is blocked in the cross-linked hemoglobin, the binding site is different. Results show that one mole of SCN − is binding to one mole of oxyhemoglobin suggesting that binding at the lys-α-99 is linked to dissociation of the hemoglobin tetramer into dimers due to its location at the α 1 β 2 interface. Circular dichroism studies show that the interaction of thiocyanate with oxyHb decreases the optical rotation at 240 nm indicating a conformational change of the protein, which influences the electronic transitions of a number of peptide bonds or (and) a few aromatic side chains.

Redox cycling of diaspirin cross-linked hemoglobin induces G2/M arrest and apoptosis in cultured endothelial cells

AbstractIt is hypothesized that oxidative reactions of hemoglobin driven by reactive oxygen species in the vasculature lead to endothelial cell injury or death. Bovine aortic endothelial cells were incubated with diaspirin cross-linked hemoglobin (DBBF-Hb), developed as a hemoglobin-based oxygen carrier, and hydrogen peroxide (H2O2), generated by the glucose oxidase system. The low steady flux of H2O2 oxidizes the ferrous form of DBBF-Hb and drives the redox cycling of ferric and ferryl DBBF-Hb. Cells underwent rounding, swelling and detachment, and accumulated in the G2/M phase of the cell cycle. G2/M arrest preceded the onset of apoptosis as determined by increases in phosphatidylserine (PS) externalization and sub-G1 events. Redox cycling of unmodified hemoglobin also led to G2/M arrest and apoptosis. The rate and extent of DBBF-Hb oxidation correlated with the onset and extent of G2/M arrest and apoptosis and induced significant decreases in soluble reduced thiols. Earlier depletion of glutathione by pretreatment with buthionine sulfoximine rendered cells more susceptible to G2/M arrest and apoptosis. The caspase inhibitor, z-VAD-fmk, had no effect on the induction of G2/M arrest but completely inhibited the subsequent increases in PS externalization and sub-G1 events. Catalase inhibited DBBF-Hb oxidation, the loss of thiols, and the onset of G2/M arrest and apoptosis. These data support a causative role for the ferric–ferryl redox cycle in the development of endothelial cell injury.

The role of facilitated diffusion in oxygen transport by cell-free hemoglobins: implications for the design of hemoglobin-based oxygen carriers

We compared rates of oxygen transport in an in vitro capillary system using red blood cells (RBCs) and cell-free hemoglobins. The axial P o 2 drop down the capillary was calculated using finite-element analysis. RBCs, unmodified hemoglobin (HbA 0), cross-linked hemoglobin (αα-Hb) and hemoglobin conjugated to polyethylene-glycol (PEG-Hb) were evaluated. According to their fractional saturation curves, PEG-Hb showed the least desaturation down the capillary, which most closely matched the RBCs; HbA 0 and αα-Hb showed much greater desaturation. A lumped diffusion parameter, K*, was calculated based on the Fick diffusion equation with a term for facilitated diffusion. The overall rates of oxygen transfer are consistent with hemoglobin diffusion rates according to the Stokes–Einstein Law and with previously measured blood pressure responses in rats. This study provides a conceptual framework for the design of a ‘blood substitute’ based on mimicking O 2 transport by RBCs to prevent autoregulatory changes in blood flow and pressure.

Effects of hemoglobin-based oxygen-carrying solutions in anesthetized rats with acute ischemic renal failure

The effects of three hemoglobin solutions were compared with those of iso-oncotic human serum albumin in rats with ischemic renal failure and sham-operated controls. Unmodified and α-α cross-linked hemoglobins both increase mean arterial pressure and systemic vascular resistance and reduce cardiac output substantially and to a comparable extent. In contrast, o -raffinose cross-linked hemoglobin has no deleterious effect on any of these parameters. In sham-operated rats unmodified hemoglobin reduces the glomerular filtration rate (GFR) by approximately 30%, whereas neither of the two cross-linked hemoglobins has any adverse effect on GFR in this group. None of the three hemoglobin solutions exacerbated the degree to which GFR was reduced by ischemia-reperfusion injury. Also, the degree of tubular necrosis induced by ischemia-reperfusion injury was also comparable in all groups. We conclude the following: (1) o -raffinose cross-linking, but not α-α cross-linking, ameliorates the effects of unmodified hemoglobin on vascular resistance and cardiac output; (2) both forms of cross-linking reduce the nephrotoxicity exhibited by unmodified hemoglobin in sham-operated rats; and (3) none of the hemoglobin solutions exacerbate renal injury induced by ischemia-reperfusion. (J Lab Clin Med 2000;135:73-81)

P–248 - Vascular effects of unmodified hemoglobin and S-nitrosohemoglibin in rats.

P–248 - Vascular effects of unmodified hemoglobin and S-nitrosohemoglibin in rats.

Improved Blood Substitute

The authors have previously documented that appropriate chemical and pharmacologic modification of the hemoglobin molecule are required to attenuate certain pathophysiologic reactions of the reticuloendothelium. The current study further investigates the molecular responses of human coronary artery endothelial cells to a high concentration (0.4 mmol) of 1) unmodified bovine hemoglobin; and 2) an improved blood substitute that comprises hemoglobin cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione. In this study, the scavenging effect of hemoglobins toward nitric oxide (NO) was evaluated by the measurement of nitrite (NO2-) and nitrate (NO3-) formation. The pro-oxidant effect of hemoglobin on endothelial cells was examined by the measurement of intracellular reduced glutathione, and by monitoring the formation of lipid hydroperoxides and 8-iso prostaglandin F2alpha, a novel potent vasoconstrictor, which is produced by a noncyclooxygenase mechanism involving free radical catalyzed peroxidation of arachidonic acid. The inflammatory reactions of endothelial cells were evaluated by the expression of the adhesion molecule, intracellular adhesion molecule-1, and the activation of nuclear transcription factor, nuclear factor kappaB. In additional, endothelial cell responses were investigated by analysis of intracellular ionized calcium concentrations. Results indicate that unmodified hemoglobin in a concentration of 0.4 mmol/L can aggravate endothelial cell oxidative and inflammatory responses. This hemoglobin produced a significant (p < 0.01) depletion of reduced glutathione, acceleration of lipid peroxidation, and a greater influx of Ca2+. The formation of 8-iso prostaglandin F2alpha increased compared with the control cells (p < 0.01). Unmodified hemoglobin was found to be a potent scavenger of NO, great activator of nuclear factor kappaB, and a stimulator of intracellular adhesion molecule-1 expression. Contrarily, the improved blood substitute did not appear to induce oxidative stress nor to increase the intracellular Ca2+. The concentration of 8-iso prostaglandin F2alpha was similar to that in the control cells, whereas the formation of NO2-/NO3- was much lower (p < 0.05) than in the unmodified hemoglobin group. The effect of an improved blood substitute can be linked with the anti-inflammatory and cytoprotective properties of adenosine, which is used as a cross-linker and surface modifier, and the type of the chemical modification procedure that lowers hemoglobin pro-oxidant potential.

Oxygen binding by single crystals of hemoglobin: the problem of cooperativity and inequivalence of alpha and beta subunits.

Oxygen binding by the human hemoglobin tetramer in the T quaternary structure is apparently noncooperative in the crystalline state (Hill n = 1.0), as predicted by the two-state allosteric model of Monod, Wyman, and Changeux (MWC) (Mozzarelli et al., Nature 351:416-419, 1991; Rivetti et al., Biochemistry 32:2888-2906, 1993). However, cooperativity within the tetramer can be masked by a difference in affinity between the alpha and beta subunits. Indeed, analysis of the binding curves derived from absorption of light polarized along two different crystal directions, for which the projections of the alpha and beta hemes are slightly different, revealed an inequivalence in the intrinsic oxygen affinity of the alpha and beta subunits (p50(alpha) approximately 80 torr, p50(beta) approximately 370 torr at 15 degrees C) that compensates a small amount of cooperativity (Rivetti et al., Biochemistry 32:2888-2906, 1993). To further investigate this problem, we have measured oxygen binding curves of single crystals of hemoglobin (in a different lattice) in which the iron in the alpha subunits has been replaced by the non-oxygen-binding nickel(II). The Hill n is 0.90 +/- 0.06, and the p50 is slightly different for light polarized parallel to different crystal directions, indicating a very small difference in affinity between the two crystallographically inequivalent beta subunits. The average crystal p50 is 110 +/- 20 torr at 15 degrees C, close to the p50 of 80 torr observed in solution, but about threefold less than the p50 calculated by Rivetti et al. (Biochemistry 32:2888-2906, 1993) for the beta subunits of the unsubstituted tetramer. These results suggest that Rivetti et al., if anything, overestimated the alpha/beta inequivalence. They therefore did not underestimate the cooperativity within the T quaternary structure, when they concluded that it represents a small deviation from the perfectly noncooperative binding of an MWC allosteric model. Our conclusion of nearly perfect MWC behavior for binding to the T state of unmodified hemoglobin raises the question of the relevance of the large T-state cooperativity inferred for cyanide binding to partially oxidized hemoglobin (Ackers et al., Science 255:54-63, 1992).

Oxygen binding by single crystals of hemoglobin: The problem of cooperativity and inequivalence of alpha and beta subunits

Oxygen binding by the human hemoglobin tetramer in the T quaternary structure is apparently noncooperative in the crystalline state (Hill n = 1.0), as predicted by the two-state allosteric model of Monod, Wyman, and Changeux (MWC) (Mozzarelli et al., Nature 351:416–419, 1991; Rivetti et al., Biochemistry 32:2888–2906, 1993). However, cooperativity within the tetramer can be masked by a difference in affinity between the α and β subunits. Indeed, analysis of the binding curves derived from absorption of light polarized along two different crystal directions, for which the projections of the α and β hemes are slightly different, revealed an inequivalence in the intrinsic oxygen affinity of the α and β subunits (p50(α) ≅ 80 torr, p50(β) ≅ 370 torr at 15°C) that compensates a small amount of cooperativity (Rivetti et al., Biochemistry 32:2888–2906, 1993). To further investigate this problem, we have measured oxygen binding curves of single crystals of hemoglobin (in a different lattice) in which the iron in the α subunits has been replaced by the non-oxygen-binding nickel(II). The Hill n is 0.90 ± 0.06, and the p50 is slightly different for light polarized parallel to different crystal directions, indicating a very small difference in affinity between the two crystallographically inequivalent β subunits. The average crystal p50 is 110 ± 20 torr at 15°C, close to the p50 of 80 torr observed in solution, but about threefold less than the p50 calculated by Rivetti et al. (Biochemistry 32:2888–2906, 1993) for the β subunits of the unsubstituted tetramer. These results suggest that Rivetti et al., if anything, overestimated the α/β inequivalence. They therefore did not underestimate the cooperativity within the T quaternary structure, when they concluded that it represents a small deviation from the perfectly noncooperative binding of an MWC allosteric model. Our conclusion of nearly perfect MWC behavior for binding to the T state of unmodified hemoglobin raises the question of the relevance of the large T-state cooperativity inferred for cyanide binding to partially oxidized hemoglobin (Ackers et al., Science 255:54–63, 1992). © 1996 Wiley-Liss, Inc.

Role of endothelin in the cardiovascular effects of diaspirin crosslinked and stroma reduced hemoglobin

Diaspirin crosslinked hemoglobin is a resuscitative solution with excellent oxygen-carrying capacity. Diaspirin crosslinked hemoglobin produces an immediate increase in blood pressure and marked regional circulatory changes in rats and pigs. Our objective was to determine the role of endothelin in the cardiovascular actions of diaspirin crosslinked hemoglobin (modified) and (unmodified) stroma reduced hemoglobin solutions. Prospective, randomized comparison of cardiovascular effects of diaspirin crosslinked and stroma reduced hemoglobin in control rats and in rats pretreated with cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), an endothelin-A receptor antagonist. Research laboratory. Male Sprague-Dawley rats. Modified, highly purified, and heat pasteurized (diaspirin crosslinked) and unmodified (stroma reduced) hemoglobin in control (untreated) and BQ-123 (5 mg/kg/hr iv)-treated rats. Infusion of stroma reduced hemoglobin (400 mg/kg iv) in control rats produced an increase in blood pressure (43%) and total peripheral resistance (65%) without any change in heart rate, cardiac output, and stroke volume. Stroma reduced hemoglobin decreased blood flow to the kidneys and liver, increased blood flow to the heart, and had no effect on blood flow to the brain, gastrointestinal tract, spleen, musculoskeletal system, skin, and mesentery and pancreas. Infusion of stroma reduced hemoglobin in rats treated with BQ-123 (5 mg/kg/hr iv) increased the blood pressure to a similar degree when compared with control rats, but the increase in total peripheral resistance was significantly attenuated. The stroma reduced hemoglobin-induced decrease in blood flow to the kidneys and liver was significantly attenuated in BQ-123-treated rats as compared with control rats. However, the stroma reduced hemoglobin-induced increase in blood flow to the heart of BQ-123-treated rats was similar to the increase in control rats. Infusion of diaspirin crosslinked hemoglobin (400 mg/kg iv) produced increases in blood pressure (81%), cardiac output (36%), stroke volume (30%), and total peripheral vascular resistance (45%), along with increases in blood flow to the heart, spleen, gastrointestinal tract, and skin of control rats. The blood flows to the brain, kidneys, liver, musculoskeletal system, and mesentery and pancreas were not altered by diaspirin crosslinked hemoglobin in control rats. The increases in blood pressure, cardiac output, stroke volume, and total peripheral vascular resistance by diaspirin crosslinked hemoglobin were significantly blocked in BQ-123-treated rats as compared with control rats. The increases in blood flow to the heart, spleen, and skin by diaspirin crosslinked hemoglobin were significantly blocked in BQ-123-treated rats as compared with control rats. Diaspirin crosslinked hemoglobin produced an increase in the blood flow to the brain and a decrease in blood flow to the kidney and musculoskeletal system of BQ-123-treated rats as compared with control rats. Blood plasma endothelin-1-like immunoreactivity was found to be significantly increased after treatment with diaspirin crosslinked hemoglobin or stroma reduced hemoglobin. The endothelin-A receptor antagonist, BQ-123, could attenuate the systemic hemodynamic and regional circulatory effects of diaspirin crosslinked hemoglobin and stroma reduced hemoglobin. However, the increase in blood flow to the heart induced by stroma reduced hemoglobin could not be attenuated by BQ-123.