Redox reactions of hemoglobin and myoglobin: biological and toxicological implications.

Direct cytotoxic effects associated with hemoglobin (Hb) or myoglobin (Mb) have been ascribed to redox reactions (involving either one- or two-electron steps) between the heme group and peroxides. These interactions are the basis of the pseudoperoxidase activity of these hemoproteins and can be cytotoxic when reactive species are formed at relatively high concentrations during inflammation and typically lead to cell death. Peroxides relevant to biological systems include hydrogen peroxide, lipid hydroperoxides, and peroxynitrite. Reactions between Hb/Mb and peroxides form the ferryl oxidation state of the protein, analogous to compounds I and II formed in the catalytic cycle of many peroxidase enzymes. This higher oxidation state of the protein is a potent oxidant capable of promoting oxidative damage to most classes of biological molecules. Free iron, released from Hb, also has the potential to promote oxidative damage via classical "Fenton" chemistry. It has become increasingly evident that Hb/Mb redox reactions or their by-products play a critical role in the pathophysiology of some disease states. This review briefly discusses the reactions of Hb/Mb with biological peroxides, potential cytotoxicity and the impact of these interactions on modulation of cell signaling pathways regulated by these reactive species. Also discussed in this article is the role of heme-protein chemistry in relation to the toxicity of hemoproteins.

Nitric oxide-mediated heme oxidation and selective beta-globin nitrosation of hemoglobin from normal and sickle erythrocytes.

Nitric oxide (NO) has been reported to modulate the oxygen affinity of blood from sickle cell patients (SS), but not that of normal adult blood (AA), with little or no heme oxidation. However, we had found that the NO donor compounds 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO) and S-nitrosocysteine (CysNO) caused increased oxygen affinity of red cells from both AA and SS individuals and also caused significant methemoglobin (metHb) formation. Rapid kinetic experiments in which HbA(0), AA, or SS erythrocytes were mixed with CysNO or DEANO showed biphasic time courses indicative of initial heme oxidation followed by reductive heme nitrosylation, respectively. Hemolysates treated with CysNO showed by electrospray mass spectrometry a peak corresponding to a 29 mass unit increase (consistent with NO binding) of both the beta(A) and beta(S) chains but not of the alpha chains. Therapeutic use of NO in sickle cell disease may ultimately require further optimization of these competing reactions, i.e., heme reactivity (nitrosylation or oxidation) versus direct S-nitrosation of hemoglobin on the beta-globin.

Reactions of sperm whale myoglobin with hydrogen peroxide. Effects of distal pocket mutations on the formation and stability of the ferryl intermediate.

Distal pocket mutants of sperm whale oxymyoglobin (oxy-Mb) were reacted with a 2.5-fold excess of hydrogen peroxide (HOOH) in phosphate buffer at pH 7.0, 37 degreesC. We describe a mechanism composed of three distinct steps: 1) initial oxidation of oxy- to ferryl-Mb, 2) autoreduction of the ferryl intermediate to ferric metmyoglobin (metMb), and 3) reaction of metMb with an additional HOOH molecule to regenerate the ferryl intermediate creating a pseudoperoxidase catalytic cycle. Mutation of Leu-29(B10) to Phe slows the initial oxidation reaction 3-fold but has little effect on the rate of ferryl reduction to ferric met-aquo-myoglobin. In contrast, the Val-68(E11) to Phe mutation causes a small, 60% increase in the initial oxidation reaction and a much larger 2. 5-fold increase in the rate of autoreduction. Double insertion of Phe at both the B10- and E11-positions (L29F/V68F) produces a mutant with oxidation characteristics of both single mutants, slow initial oxidation, and rapid autoreduction, but an extraordinarily high affinity for O2. Replacing His-64(E7) with Gln produces 3-4-fold increases in both processes. Combining the mutation H64Q with L29F results in a myoglobin with enhanced resistance to metMb formation in the absence of antioxidant enzymes (i.e. catalase and superoxide dismutase) due to its own high pseudoperoxidase activity, which rapidly removes any HOOH produced in the initial stages of autoxidation. This double substitution occurs naturally in the myoglobin of Asian elephants, and similar multiple replacements have been used to reduce selectively the rate of nitric oxide (NO)-induced oxidation of both recombinant MbO2 and HbO2 blood substitute prototypes without altering O2 affinity.

Two distinct types of fatty acid-binding protein are expressed in heart ventricle of Antarctic teleost fishes.

This report provides the first evidence for the existence of two distinct types of fatty acid-binding protein (FABP) in cardiac tissue of vertebrates. Four species of Antarctic teleost fish (Chaenocephalus aceratus, Cryodraco antarcticus, Gobionotothen gibberifrons and Notothenia coriiceps) exhibited two FABP mRNAs of 1. 0 kb and 0.8 kb, which we have termed Hh-FABP and Had-FABP (isolated from Heart tissue, with similarity to mammalian heart-type FABP or mammalian adipose-type FABP respectively). These FABP types appear to be products of distinct genes. Both FABP transcripts were abundant in cardiac and aerobic pectoral muscle. However, relative abundance of the two types varied distinctly among other tissues such as kidney, brain, spleen and white muscle. Neither FABP type was expressed in liver or intestine. The coding regions of Hh-FABP and Had-FABP cDNAs from the same species are only approximately 60% identical with one another. However, homologues of each FABP species, which exhibit >98% identity to their respective types, were isolated from three other Antarctic teleosts. Phylogenetic analysis of aligned amino-acid sequences places Hh-FABP with other vertebrate heart-type FABPs, and Had with adipose/cutaneous FABPs. Expression of two distinct FABPs in cardiac tissue of Antarctic teleosts may be related to their ability to both utilize fatty acid as the primary metabolic fuel and to store lipid intracellularly.

Peroxynitrite-mediated heme oxidation and protein modification of native and chemically modified hemoglobins.

Peroxynitrite (ONOO-) has been shown to play a critical role in tissue reperfusion injury. We have studied the reactions of ONOO- with native and two chemically modified hemoglobins that are being developed as oxygen-carrying reperfusion agents for use in a variety of clinical conditions. Reactions of native and chemically modified oxyhemoglobins (oxyHb) at 7.4 with ONOO- lead to a rapid oxidation of the heme iron to ferric (HbFe3+) form. Addition of excess molar ratios of ONOO- to the ferryl (HbFe4+) heme protein induced a spectral change indicative of the reduction of HbFe4+ to the HbFe3+ oxidation state. No major spectral changes were noted when ONOO- was added to methemoglobin (HbFe3+) or cyanomethemoglobin (Hb3+CN-), whereas the carbonmonoxy derivative of ferrous hemoglobin (HbCO) underwent an immediate spectral change suggesting the displacement of the CO ligand and oxidation of the heme iron. Rapid mixing of ONOO- with oxyHb in the stopped-flow spectrophotometer yielded biphasic kinetic plots for the oxidation of the ferrous iron (Fe2+). Replots of the apparent rate constants for native, cross-linked and polymerized, cross-linked hemoglobins as a function of ONOO- concentration were linear, yielding a single second-order rate for all hemoglobins of between 2 to 3 x 10(4) M-1 s-1, independent of the oxygen affinities and molecular sizes of the proteins. Oxidative modifications of the protein by ONOO-, occurring primarily at the beta subunits, were observed in reaction mixtures of oxyHb and ONOO- using reverse-phase HPLC. The immuno-detection method confirms that nitration of tyrosine residues by ONOO- occurs on the hemoglobin molecule and contributes to the modifications observed. We postulate that the presence of hemoglobin in close proximity to ONOO- production sites in the vasculature can contribute to possible in vivo toxicity by a two-step mechanism involving (i) direct oxidation of the heme iron and (ii) nitration of the tyrosine residues on the molecule, leading to subsequent instability and heme loss from the protein.

Kinetic characterization of myoglobins from vertebrates with vastly different body temperatures.

Fish myoglobins are structurally distinct from the previously characterized mammalian myoglobins. Teleost fishes express generally lower levels of myoglobin than those found in mammals. Although the oxygen binding affinity is essentially the same as mammalian myoglobins, oxygen dissociation rates and carbon monoxide combination rates of the teleost myoglobins studied are significantly faster. Thus, the kinetic parameters of myoglobin from two Antarctic teleost species, measured close to their body temperature of -1 degree C, are comparable to those of mammalian myoglobins with higher body temperatures. These data suggest myoglobins from Antarctic teleosts may function at extreme environmental temperatures.

Hemoglobin and free radicals: implications for the development of a safe blood substitute.

The two major concerns in the development of cell-free hemoglobin as a blood substitute (i.e. circulatory retention and oxygen delivery) have been resolved successfully by strategic chemical or genetic modification of the protein. However, the redox reactivity of hemoglobin and its impact on the physiological processes has not been fully understood, nor has it been subject to control by design. This article reviews current research into heme-mediated toxicities that potentially constitute serious impediments to the development of a usable blood substitute.

Effects of polymerization on the oxygen carrying and redox properties of diaspirin cross-linked hemoglobin.

Human hemoglobin site specifically cross-linked with bis(3,5-dibromosalicyl)fumarate results in a low oxygen affinity hemoglobin-based red cell substitute (alpha-DBBF). Polymerization of alpha-DBBF by bis(maleoylglycylamide) polyethylene glycol (BMAA-PEG) yields poly alpha-DBBF which offers the added benefits of reduced renal clearance and increased retention in the vascular circulation. Oxygen equilibrium curves for poly alpha-DBBF are slightly left-shifted (higher O2 affinity) compared to those of alpha-DBBF; with a diminished cooperativity and a reduced Bohr effect. In rapid mixing experiments (oxygen dissociation and carbon monoxide binding), poly alpha-DBBF exhibits a several fold increase in the overall rate of deoxygenation and carbon monoxide binding kinetics over its cross-linked counterpart. The rate of nitric oxide binding to the oxidized form of poly alpha-DBBF shows little or no change compared to the intramolecularly cross-linked derivative. The reduction of cyanomet poly alpha-DBBF by dithionite is several fold faster than that of HbA0 and alpha-DBBF whereas the slow subsequent cyanide dissociation from the ferrous iron remained unchanged among all proteins. The propensity of poly alpha-DBBF for auto-oxidation is slightly enhanced over alpha-DBBF whereas the extent of oxidative modification by hydrogen peroxide is very similar. Polymerization appears to selectively modify ligand interactions and redox kinetics of the tetrameric cross-linked form which reflects a possibly more open heme pocket. The data suggests that changes in oxygenation properties of hemoglobin brought about by a given modification are not necessarily predictive of other functional changes.

Reaction of human hemoglobin HbA0 and two cross-linked derivatives with hydrogen peroxide: differential behavior of the ferryl intermediate.

Functional concerns regarding hemoglobin-based red cell substitutes have generally centered on two parameters: (a) oxygen binding and delivery properties and (b) stabilization of the hemoglobin tetramer to prevent dimerization. Strategic chemical cross-linking and site-directed mutagenesis have produced proteins that have both physiological oxygen binding characteristics and a markedly prolonged retention time in the circulation. The presence of a large amount of redoxactive iron outside the red blood cell, however, raises some concerns about the potential for toxic side effects, many involving the production or participation of oxygen free radicals. In the present study, HPLC purified human hemoglobin HbA0 and two derivatives, one cross-linked between the lysine 99 residues of the alpha subunits (alpha-DBBF) and the other between the lysine 82 residues of the beta subunits (beta-DBBF) were tested for their susceptibility to oxidation and oxidative damage caused by H2O2. We show that chemical cross-linking resulting in alpha-DBBF induces an increased tendency to form ferryl radical in the presence of H2O2 and stabilizes the radical once formed. The in vitro oxidative modification of alpha-DBBF seen here is a plausible mechanism for some of the in vivo toxicities associated with the infusion of this hemoglobin.

Functional properties of the hemoglobin from the South American snake Mastigodryas bifossatus.

The hemolysate of Mastigodryas bifossatus shows two major hemoglobins with very close isoelectric points, and four different globin chains. The stripped hemolysate exhibits a low alkaline Bohr effect (delta log P50/delta pH = -0.30 between pH 7 and 8) and a decrease of the co-operativity from 2.3 to unity when the pH increases from 6.15 to 8.5. In the presence of ATP, large changes in the oxygen affinity and co-operativity are observed. The Bohr effect rises to -0.46 and the n50 values stay at around 3 in the pH range 6-9. An increase in temperature induces a large decrease in the oxygen affinity for the stripped hemolysate. In the pH range between 7.5 and 8.5, the values of delta H in kcal/M are around 10 fold larger for the stripped protein than for the protein in the presence of ATP. Measurements of rapid kinetics of oxygen dissociation and carbon monoxide binding reflect the ATP sensitivity observed in equilibrium experiments.

Chloride masks effects of opposing positive charges in Hb A and Hb Hinsdale (beta 139 Asn-->Lys) that can modulate cooperativity as well as oxygen affinity.

In the human hemoglobin variant Hb Hinsdale, lysine is substituted for asparagine at position beta 139 (H17), which lies in the water-filled cavity that runs through the center of the molecule. This substitution adds two extra cationic residues to the excess of four cationic residues normally lining this cavity. Moo-Penn and colleagues who discovered this hemoglobin, found its oxygen affinity in 0.5 M bis-Tris buffer to be lower than that of Hb A. Their finding conflicted with our prediction that additional cationic groups lining the central cavity would destabilize the T-structure by increased electrostatic repulsion and thereby increase the oxygen affinity. We have, therefore, remeasured the ligand-binding properties of Hb Hinsdale. In chloride-free Hepes buffer, Hb Hinsdale has greatly increased oxygen affinity and lower cooperativity than Hb A. A comparison of the properties of Hb A, Hb Hinsdale, Hb Deer Lodge (beta 2 His-->Arg) and Hb Abruzzo (beta 143 His-->Arg) in 0.05 M Hepes versus 0.05 M bis-Tris buffers shows that very low chloride concentrations can significantly alter cooperativity as well as oxygen affinity. The apparent conflict between the findings of Moo-Penn and colleagues and our prediction arises from the enhanced chloride effects exhibited by Hb Hinsdale. On going from 0.05 M Hepes to 0.05 M bis-Tris at pH 7.0, log P50 values for Hb A and Hb Hinsdale are increased by 0.28 and 1.12, respectively. The Bohr effect, the kinetics of oxygen dissociation, the second-order rate constant of CO binding and the rate of CO recombination after flash photolysis were also determined for Hb Hinsdale. The enhanced chloride sensitivity of Hb Hinsdale is consistent with the allosteric mechanism of chloride interactions proposed by Perutz et al. in the accompanying paper.

Redox reactivity of modified hemoglobins with hydrogen peroxide and nitric oxide: toxicological implications.

The rapid unloading of oxygen to tissue and the prevention of subunit dissociation have been the main concerns in the search for an effective hemoglobin-based red cell substitute. The presence of redox active iron however, raises some questions about its potential to enter into reactions that mediate the formation of cytotoxic oxygen free radicals. We tested the propensity of modified hemoglobins to undergo oxidative damage by peroxide (H2O2). We found differences in their susceptibility to oxidative modification and in their ability to form the highly cytotoxic ferryl species. This protein-associated oxidant may be a physiologically important contributor to reperfusion injury. Another potential mechanism of toxicity involves the reaction of cell-free hemoglobin with endothelium derived nitric oxide (NO). Marked hypertensive responses in intact animals infused with some of these hemoglobins were reported. Cell-free hemoglobin has the potential to bind the endothelial generated NO yielding methemoglobin and nitrate, an extremely rapid reaction in vivo. We describe subsequent redox reactions between NO and methemoglobin which may further deplete NO as a biological transducer, leading to greater effects on the extent of endothelial-dependent responses. The consequences of a potential linkage between oxidative toxicity of cell-free hemoglobin and its interaction with NO is addressed.

A putative glutathione-binding site in CdZn-metallothionein identified by equilibrium binding and molecular-modelling studies.

Glutathione (GSH) has been found to form a complex with both vertebrate and invertebrate copper-metallothionein (CuMT) [Freedman, Ciriolo and Peisach (1989) J. Biol. Chem. 264, 5598-5605; Brouwer and Brouwer-Hoexum (1991) Arch. Biochem. Biophys. 290, 207-213]. In this paper we report on the interaction of GSH with CdZnMT-I and CdZnMT-II from rabbit liver and with CdMT-I from Blue crab hepatopancreas. Ultrafiltration experiments showed that all three MTs combined with GSH. The measured binding data for the three MTs could be described by a single binding isotherm. The GSH/MT stoichiometry was 1.4 +/- 0.3 and Kdiss. = 14 +/- 6 microM. Partially Zn-depleted MT does not significantly bind GSH, indicating that the GSH-binding site is located on MT's Zn-containing N-terminal domain. The putative GSH-binding site on rabbit liver MT was investigated using molecular-graphics analysis. A cleft on the MT's N-terminal domain, which has the labile Zn-2 at its base, could easily accommodate GSH. Cysteine-ligand exchange between the terminal (non-bridging) Cys-26, bound to Zn-2, and the cysteine in GSH is stereochemically possible. Based on these considerations a model of MT-GSH was built in which GSH's cysteine replaces Cys-26 as a terminal Zn-2 ligand. This complex was energy-minimized by molecular-mechanics calculations, taking into account computed partial electrostatic charges on all atoms, including Cd and Zn. These calculations showed that the MT-GSH complex was thermodynamically more stable than MT, due to favourable non-bonded, electrostatic and van der Waals interactions. Six hydrogen bonds can form between GSH and MT. The average pairwise root-mean-square deviations (RMSD) of the metals in energy-minimized MT and MT-GSH, compared with the metals in the crystal structure, were 0.0087 +/- 0.0028 nm (0.087 +/- 0.028 A) and 0.0168 +/- 0.0087 nm (0.168 +/- 0.087 A) respectively. The RMSD values for the polypeptide-backbone alpha carbons were 0.0136 +/- 0.0060 nm (0.136 +/- 0.060 A) and 0.0491 +/- 0.0380 nm (0.491 +/- 0.380 A) respectively. No other docking sites for GSH were found. The energy-minimized structure of an MT-2-mercaptoethanol complex was somewhat less stable than the native MT domain, attesting to the specificity of the MT-GSH interaction. The possible physiological significance of the MT-GSH interaction is discussed.

Nitric oxide binding to human ferrihemoglobins cross-linked between either alpha or beta subunits.

We have examined the interactions between nitric oxide (NO) and oxidized human hemoglobin, comparing the behavior of unmodified HbA0 with that of two chemically modified hemoglobins. The latter are promising red cell substitute candidates due to their lower oxygen affinity and greater stability as tetramers. The modified forms examined were HbA-DBBF, cross-linked between the alpha chains with bis(3,5-dibromosalicyl) fumarate, and HbA-FMDA, modified between the beta chains with fumaryl monodibromoaspirin. NO binding to the oxidized forms of these hemoglobins is biphasic, due to the differing reactivities of alpha and beta chains. The structural modifications result in altered rate constants for NO binding to both alpha and beta chains. The affinity of the ferric hemes for NO is not correlated with their oxygen affinities in the ferrous state. In a much slower first-order process, the ferric hemes of HbA become reduced. Faster and more heterogeneous kinetics are observed for reduction of the modified hemoglobins. These results may have physiological relevance, since endogenously produced NO is now recognized to play an important role in the relaxation of vascular smooth muscles. If present in vivo, cell-free hemoglobins exposed to NO become rapidly oxidized. Our results show that subsequent interactions of NO with ferrihemoglobin can result in redox cycling. This has the potential of depleting NO and further altering vascular tone with rates dependent on structural parameters of the ferrihemoglobin that are not determined by oxygen affinity.

Alteration of hemoglobin function by diadenosine 5',5'''-P1,P4-tetraphosphate and other alarmones.

Heat-shocked organisms are known to produce not only "heat shock proteins" but also diadenosine tetraphosphate (Ap4A) and related compounds that may act as "alarmones" that alert the cell to the onset of metabolic stress. We found that Ap4A is synthesized in chicken erythrocytes and that the Ap4A level in the whole blood of heat-stressed birds increases about 10-fold. In searching for alarmone receptors, we found that the diadenosine polyphosphates bind preferentially with high affinity to the deoxy conformation of hemoglobin in a ratio of one/tetramer. The binding affinity of this new class of effectors of hemoglobin function is directly related to the number of phosphates which bridge the nucleotide moieties, with the most dramatic in vitro effect on oxygen affinity being shown by Ap6A. Decreasing effects are brought about by diadenosine penta-, tetra-, tri-, di-, and monophosphates. The association constant for Ap4A binding to deoxygenated human hemoglobin at pH 7.25 is 26 microM-1, close to that for 2,3-diphosphoglycerate. At 100-fold excess over heme, Ap4A increases the P50 of stripped Hb A in 0.05 M HEPES buffer at pH 7.25, 20 degrees C, from 0.85 to 6.03 mm Hg. The binding, which markedly enhances the Bohr effect, involves the beta chain anion-binding site. The kinetics of both ligand binding and dissociation are affected, with a greater quantitative effect on the oxygen dissociation process. Although the low concentration of the diadenosine polyphosphates in red cells precludes a physiologically significant modulation of oxygen delivery, competition with the ATP- and NAD(P)H-binding sites on hemoglobin or regulatory enzymes may prove to be of adaptive significance.

Carbodiimide-mediated coupling of benzenepentacarboxylate to human hemoglobin: structural and functional consequences.

We have examined the covalent modification of HbA with BPC (benzenepentacarboxylate) whose carboxyl groups were activated with EDC [1-ethyl-3-(3-dimethyl- aminopropyl)-carbodiimide]. Reaction of deoxy-HbA at pH 8 with a 10-fold excess of BPC, preactivated with a 2-fold excess of EDC for 5 minutes, followed by anion-exchange chromatography, gives three components with p50 values of 1.15 (unreacted HbA), 11.7 and 7.6 mm of Hg at 20 degrees C (50 mM Bis-Tris pH 7.0). Component III does not dissociate into dimers upon dilution, but components I and II do. When deoxy-HbA is reacted at pH 6 with 10-fold BPC, preactivated with two-fold EDC for 5 minutes, the resultant HbA derivatives can be separated into three components, with p50 values at pH 7 of 14.2, 10.2 and 5.2 mm of Hg, respectively. All three components are stable tetramers. Oxygen binding by all of the covalent HbA-(BPC)x complexes is cooperative, pH sensitive, but IHP insensitive. The latter observation suggest that BPC is covalently bound to HbA's DPG/IHP binding site. This conclusion is corroborated by reversed phase HPLC analysis which shows that all five modified HbAs contain at least one modified beta chain. In addition, 4 of the 5 derivatives also contain modified alpha chains. No inter or intratetramer crosslinks are observed.

Involvement of the distal histidine in the low affinity exhibited by Hb Chico (Lys beta 66----Thr) and its isolated beta chains.

Hemoglobin (Hb) Chico (Lys beta 66----Thr at E10) has a diminished oxygen affinity (Shih, D. T.-b., Jones, R. T., Shih, M. F.-C., Jones, M. B., Koler, R. D., and Howard, J. (1987) Hemoglobin 11, 453-464). Our studies show that its P50 is about twice that of Hb A and that its cooperativity, anion, and Bohr effects between pH 7 and 8 are normal. The Bohr effect above pH 8 is somewhat reduced, indicating a small but previously undocumented involvement of the ionic bond formed by Lys beta 66 in the alkaline Bohr effect. Since the oxygen affinity of the alpha-hemes is likely to be normal, that of the beta-hemes in the tetramer is likely to be reduced by the equivalent of 1.2 kcal/mol beta-heme in binding energy. Remarkably, both initial and final stages of oxygen binding to Hb Chico are of lowered affinity relative to Hb A under all conditions examined. The isolated beta chains also show diminished oxygen affinity. In T-state Hb A, Lys(E10 beta) forms a salt bridge with one of the heme propionates, but comparison with other hemoglobin variants shows that rupture of this bridge cannot be the cause of the low oxygen affinity. X-ray analysis of the deoxy structure has now shown that Thr beta 66 either donates a hydrogen bond to or accepts one from His beta 63 via a bridging water molecule. This introduces additional steric hindrance to ligand binding to the T-state that results in slower rates of ligand binding. We measured the O2/CO partition coefficient and the kinetics of oxygen dissociation and carbon monoxide binding and found that lowered O2 and CO affinity is also exhibited by the R-state tetramers and the isolated beta chains of Hb Chico.

Oxidized dimeric Scapharca inaequivalvis. Co-driven perturbation of the redox equilibrium.

The dimeric hemoglobin isolated from Scapharca inaequivalvis, HbI, is notable for its highly cooperative oxygen binding and for the unusual proximity of its heme groups. We now report that the oxidized protein, an equilibrium mixture of a dimeric high spin aquomet form and a monomeric low spin hemichrome, binds ferrocyanide tightly which allows for internal electron transfer with the heme iron. Surprisingly, when ferricyanide-oxidized HbI is exposed to CO, its spectrum shifts to that of the ferrous CO derivative. Gasometric removal of CO leads to the oxidized species rather than to ferrous deoxy-HbI. At equilibrium, CO binds with an apparent affinity (p50) of about 10-25 mm of Hg and no cooperativity (20 degrees C, 10-50 mM buffers at pH 6.1). The kinetics of CO binding under pseudo-first order conditions are biphasic (t1/2 of 15-50 s at pH 6.1). The rates depend on protein, but not on CO concentration. The nitrite-oxidized protein is not reduced readily in the presence of CO unless one equivalent of ferrocyanide, but not of ferricyanide, is added. We infer that ferrocyanide, produced in the oxidation reaction, is tightly bound to the protein forming a redox couple with the heme iron. CO shifts the redox equilibrium by acting as a trap for the reduced heme. The equilibrium and kinetic aspects of the process have been accounted for in a reaction scheme where the internal electron transfer reaction is the rate-limiting step.

Functional properties of hemoglobin immobilized in coacervates prepared from gelatin A and polyanionic carbohydrates.

Complex coacervation is a phenomenon of phase separation that may occur in a solution of positively and negatively charged polyions. The resulting two phases are distinguished by the total concentration of both polyions, with the concentrated phase often containing vesicular structures composed of the two polyelectrolytes. We have used this phenomenon in an attempt to-prepare a hemoglobin-based red blood cell analog. Hemoglobin-containing coacervate vesicles have been prepared from gelatin A and the polyanionic carbohydrates acacia, pectin, or dextranstilfate. Hemoglobin seems to be anchored into the vesicle walls through interaction of its polyanion binding site with the negatively charged residues on the carbohydrates. Oxygen binding by the immobilized HbA is reversible and cooperative, with p50 values at 20 degrees C of 2.8, 6, and 24 mm Hg for the acacia- (pH 7.5), pectin- (pH 6.6), and dextransulfate-(pH 6.6) derived coacervates. Kinetic studies on CO binding show that the rate of CO uptake by the coacervates (t((1/2)) = 13-27 ms at 0.5 mM CO) is similar to that of human erythrocytes.The HbA-containing coacervates slowly dissolve in isotonic salt solutions (145 mM NaCl, pH 7.4), but they can be stabilized by treatment with glutaraldehyde. Oxygen binding by HbA incorporated into the stabilized coacervates derived from dextran sulfate is very similar to oxy gen binding by human red blood cells: p50 = 26 mm Hg and n = 1.89 at 37 degrees C in isotonic salt. These results show how a novel approach, based on an old concept, has led to the preparation of immobilized HbA, with functional properties similar to those of intraerythrocytic HbA.