CO-MP4, a polyethylene glycol-conjugated haemoglobin derivative and carbon monoxide carrier that reduces myocardial infarct size in rats.

BACKGROUND AND PURPOSE: MP4 (Hemospan) is a Hb-based oxygen therapeutic agent, based on polyethylene-glycol (PEG) conjugation to Hb, undergoing clinical trials as an oxygen carrier. This study describes the functional interaction between MP4 and carbon monoxide (CO), as a CO delivery agent, and the effects of CO-MP4 on myocardial infarct size following ischaemia and reperfusion in rats. EXPERIMENTAL APPROACH: Kinetic measurements of CO-MP4 binding were used to evaluate the effects of PEG modification on Hb subunit structure/function and to calculate CO-MP4 equilibrium constants. CO transport by CO-MP4 was shown by ligand (O2/CO) partitioning between MP4 and red blood cell (RBC)-Hb. Pharmacological effects of CO-MP4 were studied on myocardial infarction in rats. KEY RESULTS: CO binding kinetics show primary structural/functional effects on beta chains in MP4, with alpha chains maintaining the ability to undergo tertiary conformational transition. CO confers long-term, room-temperature stability and is able to rapidly re-equilibrate between MP4 and RBCs. In a rat model of myocardial infarct, in contrast to oxy-MP4, CO-MP4 reduced infarct size when administered prior to the induction of ischaemia. CONCLUSIONS AND IMPLICATIONS: MP4 PEGylation chemistry modifies the individual function of Hb subunits, but results in an overall CO equilibrium constant similar to that for unmodified Hb. CO-MP4 is able to deliver CO to the circulation and reduces ischaemia/reperfusion injury in rats, providing the first evidence for this drug as a CO therapeutic agent.

Site-specific PEGylation of hemoglobin at Cys-93(beta): correlation between the colligative properties of the PEGylated protein and the length of the conjugated PEG chain.

Increasing the molecular size of acellular hemoglobin (Hb) has been proposed as an approach to reduce its undesirable vasoactive properties. The finding that bovine Hb surface decorated with about 10 copies of PEG5K per tetramer is vasoactive provides support for this concept. The PEGylated bovine Hb has a strikingly larger molecular radius than HbA (1). The colligative properties of the PEGylated bovine Hb are distinct from those of HbA and even polymerized Hb, suggesting a role for the colligative properties of PEGylated Hb in neutralizing the vasoactivity of acellular Hb. To correlate the colligative properties of surface-decorated Hb with the mass of the PEG attached and also its vasoactivity, we have developed a new maleimide-based protocol for the site-specific conjugation of PEG to Hb, taking advantage of the unusually high reactivity of Cys-93(beta) of oxy HbA and the high reactivity of the maleimide to protein thiols. PEG chains of 5, 10, and 20 kDa have been functionalized at one of their hydroxyl groups with a maleidophenyl moiety through a carbamate linkage and used to conjugate the PEG chains at the beta-93 Cys of HbA to generate PEGylated Hbs carrying two copies of PEG (of varying chain length) per tetramer. Homogeneous preparations of (SP-PEG5K)(2)-HbA, (SP-PEG10K)(2)-HbA, and (SP-PEG20K)(2)-HbA have been isolated by ion exchange chromatography. The oxygen affinity of Hb is increased slightly on PEGylation, but the length of the PEG-chain had very little additional influence on the O(2) affinity. Both the hydrodynamic volume and the molecular radius of the Hb increased on surface decoration with PEG and exhibited a linear correlation with the mass of the PEG chain attached. On the other hand, both the viscosity and the colloidal osmotic pressure (COP) of the PEGylated Hbs exhibited an exponential increase with the increase in PEG chain length. In contrast to the molecular volume, viscosity, and COP, the vasoactivity of the PEGylated Hbs was not a direct correlate of the PEG chain length. There appeared to be a threshold for the PEG chain length beyond which the protection against vasoactivity is decreased. These results suggest that the modulation of the vasoactivity of Hb by PEG could be a function of the surface shielding afforded by the PEG, the latter being a function of the disposition of the PEG chain on the protein surface, which in turn is a function of the length of the PEG chain. Thus, the biochemically homogeneous PEGylated Hbs described in the present study, surface-decorated with PEG chains of appropriate size, could serve as potential candidates for Hb-based oxygen carriers.

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 PO(2) drop down the capillary was calculated using finite-element analysis. RBCs, unmodified hemoglobin (HbA(0)), cross-linked hemoglobin (alpha alpha-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 alpha alpha-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.

Haemoglobin-based oxygen carriers.

Haemoglobin-based oxygen carriers are being developed for use in blood replacement therapies, either for perioperative haemodilution or for resuscitation from haemorrhagic blood loss. There is a high demand for these products because of risks associated with blood transfusions and pending worldwide blood shortages. Development of these products has required new technologies in protein engineering; since the haemoglobin is cell-free in solution, the molecule must be modified to be retained within blood circulation. Three classes of haemoglobin are under development: intramolecular cross-linked, intermolecular polymerised and surface conjugated with polyethylene glycol. Two products based on cross-linking chemistry have been discontinued because of serious adverse events and/or increased mortality rate in Phase III clinical trials. Three products based on polymerisation chemistry are in ongoing Phase III clinical trials. A new product based on surface conjugation is in preclinical evaluation. Although cross-linked and polymerised products have shown to be safe in preclinical and early Phase I/II clinical trials, they have had difficulty in proving efficacy. The primary adverse effect for the majority of cross-linked or polymerised products is a haemodynamic response, leading to increased vascular resistance to blood flow. The physiological mechanisms are still incompletely understood, so that safety and efficacy cannot be completely dissociated. New understandings on the mode of action of these products will help to define their utility and application. New products are under development, designed specifically to maximise blood flow and tissue perfusion and therefore, oxygenation.

Vascular resistance and the efficacy of red cell substitutes in a rat hemorrhage model.

We have compared polyethylene glycol-modified bovine hemoglobin (PEG-Hb; high O2 affinity, high viscosity, high oncotic pressure) and human hemoglobin cross-linked between the alpha-chains (alpha alpha-Hb; low O2 affinity, low viscosity, low oncotic pressure) with a non-O2-carrying plasma expander (pentastarch, high viscosity and oncotic pressure) after a 50% (by volume) exchange transfusion followed by a severe (60% of blood volume) hemorrhage. Mean arterial pressure and systemic vascular resistance rose significantly in the alpha alpha-Hb but not in the PEG-Hb animals. Two-hour survival was greater in the PEG-Hb animals (93%) than in control (35%), pentastarch (8%), or alpha alpha-Hb (6%) animals. In the PEG-Hb animals, there was no disturbance of acid-base balance, significantly less accumulation of lactic acid, and higher cardiac output than in the other groups. The data suggest that the rise in vascular resistance that follows alpha alpha-Hb exchange transfusion offsets the additional O2 transport provided by the cell-free hemoglobin. When resistance does not rise, as with PEG-Hb, even relatively small amounts of cell-free hemoglobin appear to be a very effective blood replacement.

Arterial blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide.

Changes in mean arterial pressure were monitored in rats following 50% isovolemic exchange transfusion with solutions of chemically modified hemoglobins. Blood pressure responses fall into three categories: 1) an immediate and sustained increase, 2) an immediate yet transient increase, or 3) no significant change either during or subsequent to exchange transfusion. The reactivities of these hemoglobins with nitric monoxide (.NO) were measured to test the hypothesis that different blood pressure responses to these solutions result from differences in .NO scavenging reactions. All hemoglobins studied exhibited a value of 30 microM-1 s-1 for both .NO bimolecular association rate constants and the rate constants for .NO-induced oxidation in vitro. Only the .NO dissociation rate constants and, thus, the equilibrium dissociation constants varied. Values of equilibrium dissociation constants ranged from 2 to 14 pM and varied inversely with vasopressor response. Hemoglobin solutions that exhibited either transient or no significant increase in blood pressure showed tighter .NO binding affinities than hemoglobin solutions that exhibited sustained increases. These results suggest that blood pressure increases observed upon exchange transfusion with cell-free hemoglobin solutions can not be the result of .NO scavenging reactions at the heme, but rather must be due to alternative physiologic mechanisms.

Hemoglobin-oxygen equilibrium curves measured during enzymatic oxygen consumption.

A rapid, new method to measure hemoglobin-oxygen equilibrium curves is described using the protocatechuic acid/protocatechuic acid 3,4-dioxygenase system [C. Bull and D.P. Ballou (1981) J. Biol. Chem. 256, 12673-12680] to deoxygenate hemoglobin solutions enzymatically. The reaction is followed by simultaneous measurements of hemoglobin spectra using a diode array spectrophotometer and oxygen tensions using a polarographic O2 microelectrode. Multicomponent analysis allows the determination of fractions of oxyhemoglobin, deoxyhemoglobin, and high-spin and low-spin methemoglobins in each spectrum collected as the reaction proceeds. Fractional saturation as a function of oxygen partial pressure is calculated as the ratio of oxyhemoglobin to oxy- plus deoxyhemoglobin. Several advantages are offered by this method: (i) Hemoglobin-O2 binding curves are obtained rapidly and reproducibly; (ii) the speed of the reaction limits methemoglobin formation by autooxidation; (iii) there is no gas-liquid interface, eliminating protein denaturation at the surface; and (iv) direct calculations of fractional saturation are made using spectral analysis, thus avoiding the assumption of a linear transition between deoxy- and oxyhemoglobin.

Blood volume and cardiac index in rats after exchange transfusion with hemoglobin-based oxygen carriers.

We have measured plasma volume and cardiac index in rats after 50% isovolemic exchange transfusion with human hemoglobin cross-linked between the alpha-chains with bis(3,5-dibromosalicyl)fumarate (alpha alpha Hb) and with bovine hemoglobin modified with polyethylene glycol (PEGHb). alpha alpha Hb and PEGHb differ in colloid osmotic pressure (23.4 and 118.0 Torr, respectively), oxygen affinity (oxygen half-saturation pressure of hemoglobin = 30.0 and 10.2 Torr, respectively), viscosity (1.00 and 3.39 cP, respectively), and molecular weight (64,400 and 105,000, respectively). Plasma volume was measured by Evans blue dye dilution modified for interference by plasma hemoglobin. Blood volumes in PEGHb-treated animals were significantly elevated (74.0 +/- 3.5 ml/kg) compared with animals treated with alpha alpha Hb (49.0 +/- 1.2 ml/kg) or Ringer lactate (48.0 +/- 2.0 ml/kg) or with controls (58.2 +/- 1.9 ml/kg). Heart rate reduction after alpha alpha Hb exchange is opposite to that expected with blood volume contraction, suggesting that alpha alpha Hb may have a direct myocardial depressant action. The apparently slow elimination of PEGHb during the 2 h after its injection is a consequence of plasma volume expansion: when absolute hemoglobin (concentration x plasma volume) is compared for PEGHb and alpha alpha Hb, no difference in their elimination rates is found. These studies emphasize the need to understand blood volume regulation when the effects of cell-free hemoglobin on hemodynamic measurements are evaluated.

Colloid osmotic properties of modified hemoglobins: chemically cross-linked versus polyethylene glycol surface-conjugated.

Colloid osmotic pressures of hemoglobin solutions containing unmodified, intramolecularly cross-linked, intermolecularly polymerized, or polyethylene glycol (PEG) surface-conjugated hemoglobin have been measured to determine their macromolecular solution properties. Tetrameric and polymeric hemoglobins show nearly ideal solution behavior: whereas, hemoglobins conjugated to PEG have significantly higher colloid osmotic activity and exhibit solution non-ideality. From these studies, the average calculated molecular weights are 65.300 +/- 3500 for unmodified and intramolecularly cross-linked hemoglobin tetramers, 156,000 for ring-opened raffinose polymerized human hemoglobin, 97,000 for pyridoxalated human hemoglobin conjugated to a carboxy-PEG polymer, and 117,000 for bovine hemoglobin conjugated to a methoxy-PEG polymer. The calculated radius of gyration for tetrameric hemoglobins is 2.9 +/- 0.2 nm compared to 4.9 nm for the polymerized hemoglobin, and 7.2 and 14.1 nm for the human and bovine PEG-conjugated hemoglobins, respectively. Exclusion volumes are calculated to be 823 +/- 148 nm3 for tetramers, 4000 nm3 for polymers, and 13,000 nm3 and 94,000 nm3 for human and bovine PEG-conjugated hemoglobins, respectively. These studies show that polyethylene glycol conjugated to surface amino groups greatly increases the effective macromolecular size of hemoglobin in solution.

Subunit dissociations in natural and recombinant hemoglobins.

A precise and rapid procedure employing gel filtration on Superose-12 to measure the tetramer-dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val-6(beta) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the alpha 1 beta 2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.

Effects of bacterial endotoxin on human cross-linked and native hemoglobins.

Previous investigations have demonstrated that hemoglobin (Hb) is a binding protein for bacterial endotoxin (lipopolysaccharide, LPS) and that the structure and biological activity of LPS are altered in the presence of Hb. In the present study, the influence of LPS on the structure of native human HbA0 and covalently cross-linked Hb (alpha alpha Hb) was studied by analyzing the absorption and circular dichroic spectra of Hb in the wavelength region of 200-650 nm. Incubation of oxyHb with each of several LPSs resulted in a decrease in the intensity of the major Soret band at 414 nm with a shift in the maximum peak to 410 nm, decreases in the intensities of the major visible region peaks at 541 and 577 nm, and the appearance of increased absorbance in the visible region in the range of 630 nm. The resultant spectra are characteristic of methemoglobin formation. These spectral changes were time-dependent and LPS-concentration-dependent. Production of methemoglobin was prominent with chemically modified, partially deacetylated rough LPS, and was observed to a lesser extent both with native, complete rough and with native smooth LPSs. The influence of LPS on the absorption spectrum of methemoglobin also was directly tested. The conversion of methemoglobin to hemichrome in the presence of LPS was demonstrated and was shown to be reversible. Analysis of circular dichroic spectra of Hb demonstrated LPS-induced spectral changes in the visible and Soret regions consistent with the production of a substantial quantity of metHb, but did not demonstrate any alteration in the far-UV region (210-240 nm). Moreover, Hb oxygen affinity was only slightly altered after incubation with any of several LPSs. In conclusion, analyses of absorption and circular dichroic spectra reveal the potential of LPS to produce a facilitated oxidation of both alpha alpha-cross-linked human Hb and native human HbA0, without substantial changes in the secondary structure of the globin.

Oxidative-stress response in vascular endothelial cells exposed to acellular hemoglobin solutions.

We investigated the effect of different hemoglobins on the activation of endothelial heme oxygenase (HO), an inducible "stress" protein, which is responsible for heme catabolism, and we determined whether the propensity of hemoglobins to autoxidize correlates with endothelial heme uptake and cell injury. Porcine aortic endothelial cells were incubated for 6 h in the presence of 60 microM unmodified hemoglobin A0 (HbA0), hemoglobin cross-linked between the alpha-chains with bis-(3,5-dibromosalicyl)fumarate (alpha alpha Hb), or cyanomet-alpha alpha-hemoglobin (CNmet alpha alpha Hb). Endothelial HO activity augmented 4.1-fold in the presence of alpha alpha Hb, 2.7-fold with HbA0, and 1.8-fold with CNmet alpha alpha Hb over the control value. Deferoxamine, but not catalase or dimethylthiourea, partially attenuated the HO induction produced by alpha alpha Hb. The rates of methemoglobin formation exhibited a linear relationship over the time of incubation (r = 0.94), and the apparent rate constant was 1.8-fold higher for alpha alpha Hb (0.023 h-1) than for HbA0 (0.013 h-1). Endothelial heme content and lactate dehydrogenase (LDH) release, an index of cell injury, were also higher in alpha alpha Hb compared with HbA0 and CNmet alpha alpha Hb groups (P < 0.05). Deferoxamine but not catalase markedly reduced the release of LDH induced by alpha alpha Hb, whereas dimethylthiourea provided only a partial cytoprotection. These studies suggest that 1) the higher rate of oxidation of alpha alpha Hb contributes to the augmented endothelial HO activity, and 2) both heme release and iron-mediated oxygen radical formation are major contributors to endothelial oxidative stress and cytotoxicity generated by the cross-linked hemoglobin.

A recombinant human hemoglobin with asparagine-102(beta) substituted by alanine has a limiting low oxygen affinity, reduced marginally by chloride.

A recombinant (r) mutant hemoglobin (Hb) with Asn-102(beta) replaced by an Ala (N102A(beta)) has been prepared by PCR amplification of a mutagenic DNA fragment and expression of the recombinant protein in yeast. The side chain of Asn-102(beta) is part of an important region of the alpha 1 beta 2 interface that undergoes large structural changes in the transition between the deoxy and oxy conformations. Three natural mutant Hbs with neutral substitutions of Thr, Ser, or Tyr at this site have low oxygen affinities because a hydrogen bond between Asn-102(beta) and Asp-94(alpha) in normal HbA was considered to be absent in these mutants, thereby destabilizing the oxy conformation in favor of the deoxy conformation. This proposal has been tested by expression of an rHb containing alanine at position 102(beta); alanine was chosen because its methyl side chain cannot participate in hydrogen bond formation, yet it is small enough not to disrupt the subunit interface. The nature of the desired replacement was established by sequencing the entire mutated beta-globin gene as well as the tryptic peptide containing the substitution. Further characterization by SDS-PAGE, isoelectric focusing, HPLC analysis, mass spectrometry, amino acid analysis, and sequencing of the mutant tryptic peptide confirmed the purity of the rHb. Its oxygen binding curve (2.4 mM in heme) in the absence of chloride showed that it had a very low oxygen affinity with a P50 of 42 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)

The autoxidation of alpha alpha cross-linked hemoglobin: a possible role in the oxidative stress to endothelium.

The aim of the present study was to investigate the role of hemoglobin autoxidation in the induction of endothelial heme oxygenase (HO), an inducible "stress" protein which is responsible for heme catabolism. Porcine aortic endothelial cells were incubated for six hours in the presence of 60 microM unmodified hemoglobin (HbA0), hemoglobin cross-linked between the alpha chains with bis-(3,5-dibromosalicyl) fumarate (alpha alpha Hb) or cyanomet-alpha alpha-hemoglobin (CNmet alpha alpha Hb). Microsomal HO content increased 4.1-fold in the presence of alpha alpha Hb, 2.7-fold with HbA0 and 1.8-fold with CNmet alpha alpha Hb over the control value. The rates of methemoglobin formation exhibited a linear relationship over the time of incubation (r = 0.94) and the apparent rate constant was 1.8-fold higher for alpha alpha Hb (0.023 h-1) than HbA0 (0.013 h-1). In addition, a linear relationship was obtained by plotting the rates of autoxidation of hemoglobins versus the HO activity (r = 0.99). When cells were incubated with 100% methemoglobin, HO activity increased 5.0-fold and 4.7-fold for HbA0 and alpha alpha Hb, respectively. Intracellular heme concentration, measured after 24 hours of incubation, was also significantly greater in the presence of alpha alpha Hb (52.6% over baseline) compared to HbA0 (10.8%) and CNmet alpha alpha Hb (15.3%) groups (p < 0.05). However, lactate dehydrogenase (LDH) release, measured as an index of endothelial cell injury, increased in all the hemoglobins examined: alpha alpha Hb, 33.8 +/- 1.1 U/l; HbA0, 38.5 +/- 3.5 U/l; CNmet alpha alpha Hb, 41.9 +/- 4.0 U/l; (control group, 19.4 +/- 2.8 U/l). We conclude that: 1) the higher rate of oxidation of alpha alpha Hb renders the molecule more susceptible to induce endothelial oxidative stress (HO induction); 2) the accelerated methemoglobin formation is directly correlated to intracellular HO content and endothelial heme uptake; 3) persistent cell injury suggests that other factors besides heme release may contribute to the hemoglobin-mediated cytotoxicity.

Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine.

Site-directed mutagenesis of an important subunit contact site, Asp-99(beta), by a Lys residue (D99K(beta)) was proven by sequencing the entire beta-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(beta) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the alpha- and beta-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments.

Assessment of hemoglobin-dependent neurotoxicity: alpha-alpha crosslinked hemoglobin.

Adult human hemoglobin A0 (HbA0) has been shown to be neurotoxic, and we wish to report on similar studies conducted using a modified hemoglobin, which has been crosslinked between the alpha subunits (alpha-alpha Hb). Cortical cell cultures were prepared from fetal Swiss-Webster mice at 15-16 days gestation. Mature cultures (days in vitro, 12-16) were exposed to alpha-alpha Hb in a defined medium for 24-48 hours at 37 degrees C. Low micromolar amounts of alpha-alpha Hb were neurotoxic in a concentration-dependent fashion. This toxicity was attenuated by the antioxidants Trolox and U-74500A and by the iron chelator deferoxamine. The hemoglobin-binding protein, haptoglobin, also completely blocked alpha-alpha Hb-dependent neurotoxicity. The latter result was unexpected because complex formation between alpha-alpha Hb and haptoglobin was not detected using assays of haptoglobin fluorescence and hemoglobin peroxidase activity.

A comparison of rates of heme exchange: site-specifically cross-linked versus polymerized human hemoglobins.

The stability of the heme-globin interaction of chemically modified human hemoglobin (Hb) was tested by measuring rates of heme loss from methemoglobin. Heme transfer from methemoglobin to human serum albumin was measured by rapid-scanning spectrophotometry, and the resulting absorption matrices were analyzed by singular value decomposition. Unmodified human HbA0, hemoglobin cross-linked between beta subunits with either 2-nor-2-formylpyridoxal 5'-phosphate or 3,5-(dibromosalicyl)fumarate (DBBF), hemoglobin cross-linked between alpha subunits with DBBF, and pyridoxalated hemoglobin polymerized with either glycolaldehyde or glutaraldehyde were tested. Initial rates were evaluated by fitting the time courses to a biexponential equation using a matrix least squares curve-fitting algorithm. Reaction rates fell into two classes: (1) HbA0 and the site-specifically cross-linked hemoglobins, with biphasic rates of heme loss of 0.02 and 0.004 min-1, and (2) polymerized hemoglobins, with 10-20-fold higher rates at 0.5 and 0.03 min-1. The total fitted amplitudes of the reaction depended upon the specific modification: beta beta-cross-linked Hbs < alpha alpha-cross-linked Hb approximately glycolaldehyde polymerized Hb < glutaraldehyde polymerized Hb < HbA0.

Encapsulation of hemoglobin in non-phospholipid vesicles.

The efficiency of encapsulating hemoglobin in non-phospholipid liposomes by rapidly mixing hemoglobin with lipids heated above their solid-liquid phase transition temperature was examined. Human hemoglobin was mixed at 55-60 degrees C with a lipid solution containing polyoxyethylene-2 cetyl ether and cholesterol (molar ratio, 3:1) at 60-65 degrees C. Repeated mixing was carried out through a high-shear orifice, followed by rapid cooling and additional mixing. Lipid vesicles were heterogeneous in size, with diameters from approximately 300 nm to 10 microns. The non-encapsulated aqueous phase was removed by centrifugation, and total hemoglobin was determined spectrophotometrically. Encapsulation efficiency was calculated as the percentage of hemoglobin associated with the liposome phase (i.e., encapsulated) as a function of hemoglobin concentration and the aqueous:lipid hydration ratio. Hemoglobin concentrations were varied from 1 to 10 nM (in heme). Aqueous:lipid ratios of 8:1 and 4:1 were tested. Percent encapsulation varied from 13-30%, with the greatest efficiency, i.e., 30%, at a 4:1 hydration ratio of hemoglobin:lipid at 5.6 mM hemoglobin.