Redox Reactions of Cross-linked Haemoglobins with

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the kinetics of redox reactions of Hb with both oxygen and nitrite. Introduction ... avoid nitric oxide (NO) scavenging and concomitant increases in blood pres-.
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9 Redox Reactions of Cross-linked Haemoglobins with Oxygen and Nitrite Celia Bonaventura, Robert Henkens, Katherine D. Weaver, Abdu I. Alayash and Alvin L. Crumbliss

Abstract Redox reactions of haemoglobin (Hb) with oxygen can initiate a cascade of oxidative reactions that appear to underlie the adverse side reactions observed when cell-free Hbs are introduced into the circulation to enhance oxygen delivery to respiring tissues. Redox reactions of cell-free Hbs with nitrite may also be of significance in vivo, as these reactions can lead to formation of nitrosylated Hb (NO-Hb) along with oxidised Hb (MetHb). To clarify the factors governing these redox reactions we measured the kinetics of nitrite-induced and oxygen-induced heme oxidation and obtained oxygen binding and oxidation curves for unmodified human Hb and four cross-linked Hbs. The four crosslinked Hbs studied were generated by cross-linking Hb with glutaraldehyde, dextran, O-raffinose or bis(3,5-dibromosalicyl)fumarate. Oxygen binding by the cross-linked Hbs occurred with reduced oxygen affinity, reduced cooperativity and reduced responses to organic phosphate effectors. The redox potentials of the cross-linked Hbs were shifted to higher potentials relative to unmodified Hb in the absence of allosteric effectors, indicating a reduced thermodynamic driving force for oxidation. In spite of this, these Hbs showed increased rates in oxidative reactions. Elevated rates of heme oxidation were observed for their oxy derivatives under aerobic conditions, and upon exposure to nitrite under both aerobic and anaerobic conditions. These results show that heme accessibility rather than heme redox potential is the major determinant of the kinetics of redox reactions of Hb with both oxygen and nitrite.

Introduction Haemoglobin (Hb) within red blood cells is subject to homotropic and heterotropic allosteric control mechanisms that regulate oxygen affinity, which provides for efficient oxygen uptake in the lungs and effective oxygen delivery to respiring tissues (Perutz 1970, 1978). In contrast, cell-free Hbs are more readily lost to the circulation by renal filtration, and lack mechanisms for maintenance of reduced heme, and fine-tuning of oxygen uptake and delivery. It has 1

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more recently been shown that packaging Hbs within red blood cells also helps avoid nitric oxide (NO) scavenging and concomitant increases in blood pressure (Lancaster 1994). To substitute for effectors normally found in red blood cells and avoid loss by renal filtration, cross-linkers that stabilise Hb in the low-affinity (T-state) conformation have been used in the preparation of blood substitute candidates (Reiss 2001). Here we report on our studies of oxygen binding and of oxidation kinetics and thermodynamics for adult human Hb (Hb A0) and four cross-linked Hbs that are in varying stages of clinical evaluation as blood substitute candidates. Hb-DBBF, generated by reaction of deoxy Hb A0 with bis(3,5-dibromosalicyl)fumarate, has a single intra-tetrameric cross-link between the ? chains at 99Lys. It is an analogue of the commercially developed DCLHb™ and has been extensively evaluated as a potential blood substitute (D’Agnillo and Alayash 2000). PolyHbBv (Oxyglobin) is a highly purified bovine Hb that is intra- and inter-molecularly cross-linked with glutaraldehyde that has only recently been made commercially available for use as a blood substitute in dogs with anaemia. It contains a heterogeneous mixture of tetramers (~5%) and larger polymeric aggregates (~95%) ranging in size up to 500 kDa (Pearce and Gawryl 1998). HbDex is a human Hb polymer with intra- and inter-tetrameric cross-links formed by reaction with dextran. Dextrans of average molecular weight of approximately 10 kDa were used in the reaction and the resultant conjugate has a molecular weight average of 300 kDa (Prouchayret et al. 1992). O-R-PolyHbA0 (Hemolink™) is a human Hb polymer with intra- and intertetrameric crosslinks formed by reaction of highly purified human Hb A0 with O-raffinose. It has been reported to contain 32-kDa dimers (5%), 64-kDa tetramers (33%) and larger aggregates ranging in size up to 600 kDa (Adamson and Moore 1997). These cross-linked Hbs were compared to unmodified human Hb in the presence and absence of effectors. Our characterisation of these cross-linked Hbs includes determinations of their oxygen-binding curves, redox potentials, rates of MetHb and Hb-NO formation from nitrite, and rates of autoxidation.

Materials and methods Haemoglobins Hb A0 was prepared by ammonium sulphate precipitation, stripped of endogenous organic phosphates and FPLC purified as previously described (Bonaventura et al. 1991). All Hbs used in this study were maintained at –80°C prior to their use in the studies described here. Hb-DBBF was a kind gift from the Walter Reed Army Institute of Research (Washington D.C). PolyHbBv, an FDA approved blood substitute for use in dogs with anaemia, is a glutaraldehyde-polymerised bovine Hb and was purchased from Biopure Inc. (Cambridge, MA). HbDex was a kind gift from Dr. Patrick Menu, Department

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of Hematology and Physiology, School of Pharmacy, University of Henri Poincaré-Nancy, 54001 Nancy, France, and was prepared as previously reported (Prouchayret et al. 1992) by cross-linking human Hb with benzene tetracarboxylate dextran. O-R-PolyHbA0 was a kind gift of Hemosol, Inc. Canada. It was prepared by reaction of purified Hb A0 with O-raffinose, a hexaldehyde obtained by oxidation of the trisaccharide raffinose (Adamson and Moore 1997). The air-equilibrated cross-linked Hbs had high initial percentages of MetHb when received. For use in the studies described here, they were reduced by addition of sodium dithionite (British Drug Houses) and put through a Sephadex G-25 column to remove dithionite byproducts immediately prior to functional studies. The spectra of the dithionite-treated Hbs showed no evidence of irreversible metHb, ferrylHb or hemichromes.

Oxygen Binding Oxygen equilibrium curves were determined tonometrically (Riggs and Wolbach 1956). Deoxygenation of the Hb samples before air addition was achieved within 15–20 min by three cycles of exposure of ~4 ml of Hb in large volume tonometers to N2 and vacuum. A gastight syringe was used to inject measured volumes of room air through the rubber septum of the tonometer containing the Hb sample, ending with an equilibration with 100% oxygen. After each addition the tonometers were rotated in a water bath for 10 min before an absorbance spectrum was measured. At each equilibration step the PO2 was calculated and changes in the visible absorption spectrum were used to calculate the corresponding fractional O2 saturation.

Spectroelectrochemistry Experiments were carried out in an anaerobic OTTLE cell with Ru(NH3)6Cl3 as a cationic electrochemical mediator. This mediator does not bind at Hb’s anion binding sites and therefore allows anion effects on Hb redox potentials to be determined (Taboy, Bonaventura and Crumbliss 2002). Initial and final spectra were recorded from 350 nm to 750 nm. Absorbance changes were monitored at 406 nm (MetHb peak) and 430 nm (deoxy Hb peak) as a function of the applied potential. In a typical experiment, spectra were recorded starting at +250 mV, and proceeding to –100 mV (vs. NHE) in 20-mV increments. At each applied potential, the absorbance was monitored until no change was detected (5–30 min). Nernst plots of Hb’s oxidation state vs. applied potential were derived from the observed changes in absorbance as previously described (Taboy, Bonaventura and Crumbliss 2002). Although most experiments were performed going from fully oxidised to fully reduced protein, the system was shown to be reversible under our experimental conditions (i.e. the Nernst plot can be generated in either the oxidation or reduction direction).

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Nitrite-induced Oxidation Deoxygenated or air-equilibrated Hb samples were exposed to 10–100-fold excess of NaNO2 over heme. Changes in the visible spectrum were recorded to determine the rate of nitrite-induced heme oxidation. The rate and extent of nitrite-induced Hb-NO formation that accompanied formation of oxidised (MetHb) for the deoxygenated Hb samples was determined by spectral analysis.

Results Oxygen Binding We measured oxygen-binding equilibria for the cross-linked Hbs and unmodified Hb A0. Figure 1 shows representative Hill plots for samples at 20°C at pH 7.4. Under these conditions the cross-linked Hbs have lower oxygen affinities and greatly reduced cooperativity in oxygen binding. We purposefully omitted use of a MetHb reducing system so that the intrinsic (anion-free) properties of the samples could be determined. This experimental detail merits mention, as the cross-linked Hbs were found to be very susceptible to oxidation. In spite of

FIG. 1. Oxygen-binding curves for Hb A0 and cross-linked Hbs. Hill plots are shown for Hb A0 (), O-R-PolyHbA0 (), PolyHbBv (), HbDex () and HbDBBF () without anionic effectors and for Hb A0 with 0.15 mM IHP (▲). The x axis represents log of oxygen pressure in mmHg and the y axis represents the log of Y, the oxygen-bound species, divided by (1–Y). Hbs were 0.06 mM (in heme) in 0.05 M bis-Tris/HCl at pH 7.4±0.1

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this tendency, their P50 values at 20°C were still fairly reproducible. Both O-RPolyHbA0 and HbDex contain subpopulations of material with elevated oxygen affinity, as evidenced by left-shifted Hill plots at low levels of oxygen saturation. The heterogeneous binding curves are consistent with the heterogeneous character of these cross-linked Hbs, which are known to contain subpopulations of unpolymerised material. While the presence of appreciable amounts of MetHb can also result in left-shifted Hill plots, the initial levels of MetHb were low (