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J Biol Chem, Vol. 274, Issue 4, 2583-2591, January 22, 1999


Glutaraldehyde Modification of Recombinant Human Hemoglobin Alters Its Hemodynamic Properties*

Michael P. Doyle, Izydor Apostol, and Bruce A. KerwinDagger

From Baxter Healthcare Corporation, Boulder, Colorado 80301

    ABSTRACT
Top
Abstract
Introduction
References

Many cell-free hemoglobin solutions designed as oxygen-carrying therapeutics produce a hypertensive effect in animals. The response is likely due to oxidation of nitric oxide by hemoglobin. Since the site of oxidation may lie outside the vascular compartment, we tested the hypothesis that polymerization of hemoglobin, rHb1.1, by glutaraldehyde would attenuate the hypertensive response. Two products of the cross-linking reaction were isolated, a glutaraldehyde-derivatized monomer (mono-glxrHb) and a glutaraldehyde cross-linked polymer (poly-glxrHb), and evaluated for their effects on systemic hemodynamics in conscious rats. Administration of rHb1.1 caused a mean arterial pressure elevation of approximately 20 mm Hg and an increase in total peripheral resistance of approximately 30%. Administration of mono-glxrHb induced changes in mean arterial pressure and vascular resistance that were significantly diminished relative to those observed with rHb1.1. Poly-glxrHb elicited a mean arterial pressure response that was further reduced compared with that obtained with mono-glxrHb and a change in vascular resistance that was the same as the response to mono-glxrHb. These results suggest that rHb peripheral vasoconstriction elicited by rHb1.1 is significantly attenuated by glutaraldehyde modification of the hemoglobin monomer and that the effect of glutaraldehyde polymerization is likely due to surface modification and/or intramolecular cross-linking, rather than an increase in molecular size.

    INTRODUCTION
Top
Abstract
Introduction
References

As reviewed elsewhere (1-3), the search for a safe and efficacious oxygen-delivering therapeutic has been ongoing for many years. Limitations to the use of hemoglobin solutions as therapeutics have included renal toxicities due to the presence of stromal elements (4, 5), dissociation into alpha beta dimers (6), high oxygen affinities (7), and short intravascular retention times (1). Multiple approaches have been taken to modify hemoglobins to address these limitations (1, 3), including chemical cross-linking of purified human or bovine hemoglobin to prevent dissociation and chemical modification to increase the P50 to a physiologically suitable level (approximately 25-35 mm Hg, (7)). Recombinant technology was employed to genetically cross-link the alpha -globins to form a di-alpha -globin and thereby prevent dissociation. In addition, the P50 was increased by substitution of Lys for Asn at the beta 108 position. The resultant hemoglobin molecule, rHb1.1,1 is a stabilized pseudotetramer of 64 kDa with a P50 of 32 mm Hg (8).

Solutions of rHb1.1 and other hemoglobins have been shown to have vasoactive properties (9-12). Nitric oxide (NO), an important mediator of vasodilation and other physiological processes (13), is known to react rapidly with the oxyHb (14) forming metHb(Fe3+) and NO3- or to bind to deoxyHb, essentially scavenging the NO and causing vasoconstriction. Scavenging of NO by hemoglobin is thought to contribute to the hypertensive response observed with many cell-free hemoglobin solutions in intact animals (15, 16), perfused organs (17, 18), and isolated vascular preparations (19, 20). We have recently shown that genetic modification of the distal heme pocket to reduce the rate of NO scavenging, measured in vitro, can essentially eliminate the hemoglobin-induced pressor response (21).

Although an interaction between cell-free hemoglobin and NO seems clear, the site at which NO scavenging occurs (e.g. intravascular or interstitial) is unknown. Several researchers have suggested that hemoglobin must leave the vascular space to cause constriction through an NO-mediated mechanism (22-24). Direct evidence supporting this hypothesis is scant. However, larger polymerized hemoglobins are known to have longer vascular retention times (25-27), suggesting that they are less likely to leave the vascular space. Such findings indicate that it might be possible to decrease the vasoactive properties of hemoglobin by making the hemoglobin molecule larger, thereby preventing extravasation into the interstitium and blocking NO-induced oxidation.

Glutaraldehyde polymerization of hemoglobin is well known and produces a multitude of cross-linked species, e.g. dimers, trimers, tetramers, and larger species (28). Mixtures of these polymerized hemoglobins have been reported to exhibit minimal vascular responses (29), but none of the glutaraldehyde-treated monomeric species have been purified to investigate their associated vasoactive properties. We tested the hypothesis that the size of rHb1.1 can have a direct impact on its hemodynamic properties. We report here results of a series of experiments in which we compared the hemodynamic responses of underivatized recombinant hemoglobin (rHb1.1) and glutaraldehyde-polymerized rHb1.1 (poly-glxrHb). To account for possible effects of intramolecular cross-linking and/or surface modification by glutaraldehyde without a change in size, we also examined the responses to glutaraldehyde-derivatized monomeric rHb1.1 (mono-glxrHb).

    EXPERIMENTAL PROCEDURES

Hemoglobin Preparation

Preparation and Purification of Mono-glxrHb-- Recombinant hemoglobin was produced at Somatogen as described by Looker et al. (8). The hemoglobin was polymerized with glutaraldehyde (Sigma, grade I) according to the following methods. rHb1.1 (150 mg/ml in 5 mM sodium phosphate, pH 7.4, and 150 mM sodium chloride) was deoxygenated in a 1-liter round-bottom flask by passing humidified nitrogen gas over the hemoglobin solution while mixing in a Rotovap (Brinkman Instruments) for 6 h at 10 °C. Following deoxygenation, the hemoglobin was capped with a white rubber septum, and a 10% solution of deoxygenated glutaraldehyde in deionized water was added to a final molar ratio of 8 mol of glutaraldehyde per mol of hemoglobin. After incubation overnight at 4 °C, deoxygenated sodium cyanoborohydride (126 mg/ml) in 0.1 M sodium hydroxide was added to a final molar ratio of 10 mol of sodium cyanoborohydride per mol of glutaraldehyde and incubated at room temperature for 4 h. The solution was diluted to 2 liters with buffer (5 mM sodium phosphate, pH 7.4 and 150 mM sodium chloride) and diafiltered with a Pellicon diafiltration system (Millipore) against 10 volumes of the same buffer. The solution was stored at -80 °C until purified by size exclusion chromatography.

Polymerized hemoglobin was fractionated by size exclusion chromatography through a 7.5-liter Sephacryl S200 column followed by a 7.5-liter Sephacryl S300 column each equilibrated with 150 mM sodium chloride, 5 mM sodium phosphate, pH 7.4. The isolated monomeric rHb fractions from multiple chromatography runs were pooled and concentrated to a final volume of 200 ml using the Pellicon diafiltration apparatus. The oxidized hemoglobin was reduced by addition of sodium dithionite to deoxygenated mono-glxrHb until the methemoglobin concentration was <2%. Reaction by-products were removed by rechromatographing the solutions on the 7.5-liter Sephacryl S200 column equilibrated in 20 mM Tris, pH 8.9, at 4 °C. Endotoxin was removed by rechromatographing the hemoglobin on a 500-ml Superose-Q ion-exchange column (Amersham Pharmacia Biotech) equilibrated in 20 mM Tris, pH 8.9, at 4 °C. After loading, the column was washed with 2 volumes of loading buffer, and the hemoglobin was eluted with 20 mM Tris, pH 6.5, at 4 °C. The purified mono-glxrHb and poly-glxrHb were each diafiltered against 10 volumes of 150 mM sodium chloride, 5 mM sodium phosphate, pH 7.4, and concentrated to 50 mg/ml. Aliquots were filtered through pyrogen free 0.2-µm filters (Gelman Sciences) and stored at -80 °C in 15-ml polypropylene tubes. The purified hemoglobins were analyzed for methemoglobin and endotoxin as described below. Methemoglobin was less than 10% and endotoxin was less than 1 e.u./ml.

Preparation and Purification of Poly1-glxrHb-- Deoxygenated rHb1.1 (145 mg/ml in 150 mM sodium chloride, 5 mM sodium phosphate, pH 7.4 at 23 °C) was reacted with glutaraldehyde (preparation described previously) at a 7:1 molar ratio of glutaraldehyde:hemoglobin for 5 min at room temperature. The reaction was quenched by addition of sodium borohydride (152 mg/ml) in 0.1 M sodium hydroxide, at a 4:1 molar ratio borohydride:glutaraldehyde. The solution was incubated an additional 15 min at room temperature and then diafiltered against 15 volumes of deoxygenated 20 mM Tris, pH 9.0, at 8 °C. The glutaraldehyde-treated hemoglobin was applied to a Superose-Q ion-exchange column (Amersham Pharmacia Biotech) at a ratio of 50 g of hemoglobin per 1 liter of resin. The column was washed with 2 volumes of 20 mM Tris, pH 9, at 8 °C followed by 12 volumes of 20 mM Tris, pH 7.6, at 8 °C. The poly1-glxrHb was eluted with 20 mM Tris, pH 7.4, at 8 °C, diafiltered against 150 mM sodium chloride, 5 mM sodium phosphate, pH 7.4, and concentrated to 94 mg/ml. Aliquots were filtered through pyrogen-free 0.2-µm filters (Gelman Sciences) and stored at -80 °C in 15-ml polypropylene tubes. The purified hemoglobins were analyzed for methemoglobin and endotoxin as described below. Methemoglobin was less than 10% and endotoxin was less than 1 e.u./ml.

Preparation and Purification of Poly2-glxrHb-- Glutaraldehyde-polymerized hemoglobin with a molecular mass range between 200 and 1000 kDa was isolated from the reaction mixture used for preparing mono-glxrHb. The desired molecular mass range polymerized hemoglobin was isolated by size exclusion chromatography, reduced with dithionite, and endotoxin removed as described previously for the mono-glxrHb.

Hemoglobin Characterization

Methemoglobin Analysis-- Methemoglobin content in hemoglobin samples was determined using the method described by Evelyn and Malloy (30).

Endotoxin Analysis-- Bacterial endotoxin content in hemoglobin samples was determined using the Limulus Amebocyte Lysate assay (Pyrotell Inc.).

Oxygen Equilibrium Binding Measurements-- P50 and nmax were determined using a hemox analyzer as described by Hoffman et al. (31) at 37 °C and pH 7.40 in 50 mM HEPES (free acid) and 150 mM NaCl.

Autooxidation Rates-- Autooxidation rates of the hemoglobins were determined at 37 °C by the methods described by Brantley et al. (32) with the following modifications. The buffer consisted of 50 mM Tris·hydrochloride (pH 8.3 at 37 °C), 10 µM diethylenetriaminepentaacetic acid, 150 nM superoxide dismutase, 150 nM catalase, and equilibrated with 1 atm of oxygen. The hemoglobin concentration was 50 µM in heme.

NO-induced Oxidation-- The rates of NO-induced oxidation were determined at 20 °C using stopped-flow rapid mixing techniques as described by Eich et al. (33). Briefly, hemoglobin (0.2-1 µm) in 0.1 M sodium phosphate, pH 7.4, was mixed with a solution containing dissolved NO and the formation of metHb monitored at 402 nm.

Tryptic Mapping-- Tryptic mapping was performed as described by Lippincott et al. (34). The percent change from rHb1.1 referred to in Table II was derived from the following equation.
100 · [1−(<UP>peak height glxrHb peptide/peak height glxrHb &bgr;4 peptide</UP>) · ((<UP>peak height rHb1.1 peptide/peak height rHb1.1 &bgr;4 peptide</UP>)<SUP><UP>−</UP>1</SUP>)] (Eq. 1)

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis-- SDS-PAGE was performed based on the method of Laemmli (35). Aliquots (5 µg) were diluted with 2 volumes of SDS sample buffer (Novex Corp.) containing 0.1 M dithiothreitol and heated at 65 °C for 5 min. Samples were electrophoresed on an 8-16% polyacrylamide gradient Tris·glycine gel for 2.5 h at 120 V. The gel was stained in Coomassie Blue and then destained with a solution of 40% methanol, 10% glacial acetic acid, and 50% water. Destained gels were digitized using an IS-1000 digital imaging system (Alpha Innotech Corp.).

High Performance Size Exclusion Chromatography-- The molecular mass distribution of the glutaraldehyde cross-linked hemoglobin was monitored using high performance size exclusion chromatography on a Superose 12 column (1 × 30 cm, Amersham Pharmacia Biotech) connected in tandem with a Superose 6 column (1 × 30 cm, Amersham Pharmacia Biotech) mounted on a HP1090 HPLC system (Hewlett-Packard) and equilibrated with 150 mM NaCl, 5 mM sodium phosphate, pH 7.8. The absorbance was monitored at 280 nm. SEC molecular weight standards were obtained from Sigma.

Reverse Phase HPLC-- Samples were prepared by precipitation with ice-cold acid/acetone as described by Witkowska et al. (36) and Lippincott et al. (34). Pellets were solubilized in 0.1% trifluoroacetic acid, 20% acetonitrile at a final concentration of 1 mg/ml. Reverse phase HPLC analyses were performed using a Zorbax C3 analytical column (0.46 × 25 cm) mounted on an HP1090 HPLC system (Hewlett-Packard). The oven temperature was maintained at 40 °C. Solvent A is 0.1% trifluoroacetic acid in water and solvent B is 0.1% trifluoroacetic acid in acetonitrile. The flow rate was 1 ml/min. The column was equilibrated in 65% solvent A, 35% solvent B. Following sample injection the column was maintained at the starting conditions for 5 min and then ramped to 51% solvent A, 49% solvent B over 45 min.

LC-MS-- Mass spectrometry was performed as described by Lippincott et al. (34) using a Finnigan Mat LCQ with an HP1090 HPLC on the front end to run reverse phase separations prior to analysis.

Dynamic Light Scattering-- Dynamic light scattering was performed using a Nicomp 370 Submicron Particle Sizer equipped with a 40-milliwatt HeNe laser. Data were collected and averaged over 10 min and analyzed using the C370 version 12 software program provided by Particle Sizing Systems (Santa Barbara, CA). Samples were passed through 0.2-µm filters prior to analysis.

Oncotic Pressure of Hemoglobins-- The oncotic pressures of hemoglobins used in this study were measured in vitro directly in a colloid osmometer (model 4420, Wescor, Logan, UT). The oncotic pressures were measured at the final hemoglobin concentrations used for injection into the animals.

Evaluation of Hemodynamic Responses

All surgical and experimental procedures were approved by the Somatogen Animal Care and Use Committee. Male Sprague-Dawley rats (Charles River, 250-350 g) were used for all experiments. The animals were chronically instrumented with pulsed Doppler flow probes on the ascending aorta for cardiac output measurement and with indwelling arterial and venous catheters as described previously (37) for blood pressure measurement and hemoglobin infusion.

For all experiments, the animals were studied in a conscious, resting state. On the day of the experiment, each rat was placed in a Plexiglas experimental chamber that was of sufficient size (25 × 15 × 12.5 cm) to allow free movement. The chamber was flushed continuously with fresh air, and fresh bedding covered the chamber floor. The catheters and Doppler flow probe leads were fed through the top of the chamber, and both catheters were opened and flushed with sterile, heparinized saline. The arterial catheter was connected to a pressure transducer for arterial pressure measurement, and the venous catheter was connected to a syringe containing hemoglobin or human serum albumin (HSA, Baxter Healthcare Corp.). HSA was used as a negative control with the assumption that it does not consume NO. Since the molecular mass and concentration of HSA (66.5 kDa, 50 mg/ml) were virtually the same as those of rHb1.1 and mono-glxrHb, it served as a comparator for the mass and volume of protein solution administered. The flow probe leads were connected to a modified high velocity module (HVPD, Crystal Biotech, Northborough, MA) that was used in the autotracking mode at a pulse repetition frequency of 125 kHz to avoid detection of spurious, aliasing signals (38). Arterial pressure, heart rate, and cardiac output were continuously recorded at sampling frequency of 50 Hz using a Windaq data acquisition system (Dataq Instruments, Columbus, OH) and a 160-MHz Pentium computer (Compaq).

After sufficient time for acclimatization to the experimental surroundings and recording of base-line data (generally 30-60 min), hemoglobin or HSA was infused at a rate of 0.5 ml/min until a dose of 350 mg/kg was administered (less than 3 min). Hemodynamic data were collected continuously for 90 min following completion of the infusion. At the end of the 90-min data collection period, phenylephrine (3 µg/ml) was infused at a rate of 6 µg/kg/min for 2 min to verify proper catheter placement and provide a qualitative indication of the vascular responsiveness of each animal. Data were collected for an additional 15 min. Animals that did not exhibit a brisk response to phenylephrine were not included in subsequent analysis (<5% occurrence). Each animal received only a single dose of hemoglobin or HSA.

In order to determine the hemoglobin concentrations in plasma following administration and during the period of hemodynamic measurement, another set of rats was instrumented with venous catheters using the methods described above. After 2-3 days of recovery, the rHb1.1, mono-glxrHb, and poly-glxrHb were again administered to conscious rats by intravenous infusion (350 mg/kg, 0.5 ml/min). At time points of 0, 30, 60, 120 min post-administration, the animals were physically immobilized, and blood samples (0.3-0.4 ml) were obtained by tail vein transection. The blood was collected in heparinized tubes, and centrifuged at 5,000 × g for 5 min to obtain plasma samples. Hemoglobin concentration in the plasma was determined using the analytical technique of Tentori and Salvati (39).

Data and Statistical Analyses

Custom-designed software was used to process the raw hemodynamic data. Mean arterial pressure, heart rate, and cardiac output values were determined by averaging data over 30-s intervals every 5 min prior to and for 30 min following hemoglobin administration. Thereafter, 30-s averages were obtained every 10 min until the end of the experiment. All data shown are mean ± S.E.

Both mean arterial pressure and heart rate are expressed as the change from base line. Base-line values were calculated as the average of the data collected for 30 min prior to hemoglobin or HSA administration. Cardiac output is expressed as percent change from base line. Total peripheral resistance was calculated from mean arterial pressure and cardiac output and is also expressed as percent change from base line. To facilitate statistical comparisons of the hemodynamic responses to several molecules, the data from 10- to 90-min post-administration were averaged for each animal to obtain an "overall" response. The infusion generally required up to 3 min to complete, and the animals were frequently active for another 2-3 min. By 10 min post-infusion, the animals had returned to their control, resting state. Therefore, data from the 5-min time point were excluded from the overall response calculation to avoid potential inclusion of hemodynamic changes due to activity. The overall response data were analyzed by one-way analysis of variance and Newman-Keuls post hoc tests.

    RESULTS

Hemoglobin Preparation

A monomeric and two polymeric glutaraldehyde-modified recombinant hemoglobins were prepared for testing in rat hemodynamic studies. The polymerization reactions were quenched with either cyanoborohydride or borohydride. Cyanoborohydride is a Schiff base-specific reducing agent (40), and borohydride can reduce both Schiff bases and aldehydes (41). Both reducing agents were equally effective in quenching the reactions as analyzed by SEC (data not shown). The mono-glxrHb was purified from the polymer mixture by preparative size exclusion chromatography (SEC) to 91% purity with the remainder being dimeric hemoglobin as assessed by analytical SEC (Fig. 1B). The molecular mass of the mono-glxrHb fraction (Table I) based on comparison with globular protein standards corresponded to an apparent molecular mass of 51 kDa and a diameter of 8.6 nm as determined by dynamic light scattering. Purified rHb1.1 (molecular mass = 64.4 kDa) also showed an apparent mass of 51 kDa (Fig. 1A) although its diameter was 7.3 nm, less than that observed for mono-glxrHb.


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  		Fig. 1.   Size exclusion chromatographic analysis of the purified glutaraldehyde cross-linked rHb fractions mono-glxrHb, poly1-glxrHb, and poly2-glxrHb. Recombinant hemoglobin was reacted with glutaraldehyde and size-fractionated as described under "Experimental Procedures." A, purified rHb1.1; B, purified mono-glxrHb; C, purified poly1-glxrHb; D, purified poly2-glxrHb. The molecular weight reference guide was generated from a linear regression of retention time versus log molecular weight for a series of protein standards.

                              
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Table I
Summary of size and functional characterization for the purified fractions of glutaraldehyde-modified rHb1.1
The data shown here were from single determinations of each assay. Analysis of purified hemoglobins from other batches demonstrated similar results (data not shown).

Large scale purification of polymeric hemoglobin was accomplished using ion-exchange chromatography to fractionate the hemoglobin polymers. The majority of cross-linked hemoglobins greater than 1000 kDa flowed through the column at pH 9.0 and were discarded. Lowering the buffer pH to 7.6 caused elution of the monomeric hemoglobin along with some dimeric and trimeric hemoglobins which eluted over a number of column volumes (data not shown). An additional decrease in the buffer pH to 7.4 caused elution of the polymeric hemoglobin fraction of interest. The SEC profile of poly1-glxrHb is shown in Fig. 1C. This fraction contained less than 1% as monomeric rHb, ~7% dimeric rHb, and ~15% trimeric rHb with the majority ranging from 200 to 1000 kDa (Table I). Approximately 14% of the polymers were greater than 1000 kDa. The apparent diameter was 15.8 nm as measured by dynamic light scattering (Table I). Due to the presence of lower molecular weight hemoglobins in the poly1-glxrHb fraction, a more highly purified subfraction was prepared to discern if these species had any effect on hemodynamic responses. This hemoglobin polymer (poly2-glxrHb) was purified by multiple SEC fractionations to exhaustively remove the monomeric, dimeric, and trimeric hemoglobins along with large polymers >1000 kDa. The poly2-glxrHb had a range of 200-1000 kDa with a peak molecular mass of 439 kDa (see Fig. 1D and Table I) and an apparent diameter of 16.2 nm. The material contained approximately 2% as dimers and trimers and approximately 14% >1000 kDa.

Hemoglobin Characterization

Functional Characterization-- The mono- and poly-glxrHb fractions were examined for changes in the oxygen affinity (P50), cooperativity (nmax), autooxidation rates, and NO oxidation rates of oxyHb (Table I). The P50 for unmodified rHb1.1 was 32 mm Hg with a maximum Hill coefficient of 2.2. Glutaraldehyde modification of the hemoglobin resulted in a slightly decreased P50 for mono- and poly1-glxrHb (30mm Hg) and a slightly increased P50 for poly2-glxrHb (36 mm Hg). The Hill coefficients of the modified hemoglobins were all similar and demonstrated significantly decreased cooperativity when compared with unmodified rHb1.1 (nmax = 1.4 for mono-glxrHb, 1.5 for poly1-glxrHb, and 1.3 for poly2-glxrHb). The autooxidation rates were 0.7 and 1.0 h-1 for the mono- and poly-glxrHb, respectively. Unmodified rHb1.1 has an intrinsic autooxidation rate of 0.7 h-1 under identical conditions. Rates of NO-induced oxidation of oxyhemoglobin for the mono- and poly2-glxrHb fractions were all essentially identical to rHb1.1 which has a rate of 57 µmol-1·s-1.

Characterization of Isolated Fractions-- Mono-glxrHb and both poly-glxrHb preparations were analyzed by SDS-PAGE (Fig. 2). Native rHb1.1 (Fig. 2, lane 1) showed a characteristic banding pattern with the expected bands at approximately 15 and 32 kDa corresponding to the beta -globin and di-alpha -globin, respectively. Mono-glxrHb (Fig. 2, lane 2) contained the expected beta - and di-alpha -globin bands as well as three additional bands with molecular masses corresponding to 44, 46, and 62 kDa. The bands at 44 and 46 kDa may correspond to the cross-link of the beta -globin to the first or second half of the di-alpha -globin resulting in differentiation by SDS-PAGE. The band at 62 kDa is consistent with two molecules of the beta -globin chain cross-linked to the one di-alpha -globin chain. Similar banding patterns, albeit to a reduced degree, were observed for the poly1-glxrHb and poly2-glxrHb fractions. However, the band at 62 kDa was not visible in either of the poly1- or poly2-glxrHb. Additionally, diffuse banding patterns of cross-linked polypeptides with molecular masses greater than 64 kDa were observed in the poly1- and poly2-glxrHb.


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  		Fig. 2.   Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified glutaraldehyde cross-linked rHb fractions. Aliquots of the indicated samples were analyzed on an 8-16% SDS-PAGE as described under "Experimental Procedures." Positions of molecular mass markers are indicated to the right. Lane 1, rHb1.1 control; lane 2, mono-glxrHb; lane 3, poly1-glxrHb; lane 4, poly2-glxrHb.

Glutaraldehyde modification of the purified hemoglobins was further examined using reverse phase HPLC and in-line mass spectrometry (Fig. 3). Unmodified beta - and di-alpha -globin elute at 31.0 and 42.8 min, respectively, with molecular masses of 15,913 and 30,342 atomic mass units (Fig. 3A), and they are in good agreement with the expected values (42). LC-MS of the mono-glxrHb (Fig. 3B) demonstrated the presence of unmodified beta -globin eluting at 31.5 min along with a number of other peaks near the unmodified beta -globin eluting at 31.8, 32.7, and 33.9 min with masses of 15,981, 15,979, and 15,979 atomic mass units. The majority of the globins eluted between 40 and 43 min. It was not possible to define the masses of the polypeptides due to the heterogeneity of the sample giving rise to a noisy m/z spectrum. SDS-PAGE analysis of the polypeptides in the peak eluting between 40 and 43 min demonstrated that it was composed of the 32- (di-alpha ), 44-, 46-, and 62-kDa species. Similar chromatographic profiles to that observed for the mono-glxrHb were observed for the poly1- and poly2-glxrHb samples, although the modified beta -globins eluting between 31.5 and 33.9 min were present to a lesser degree than that observed for mono-glxrHb. The elution time for the majority of the globins shifted to between 41 and 46 min. Again, it was not possible to deconvolute the spectrum due to the heterogeneity of the derivatized globins.


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  		Fig. 3.   Reverse phase HPLC and LC-MS analysis of the purified mono-glxrHb, poly1-glxrHb, and poly2-glxrHb. Hemoglobins were analyzed by reverse phase-HPLC and LC-MS as described under "Experimental Procedures." A, rHb1.1; B, mono-glxrHb; C, poly1-glxrHb; D, poly2-glxrHb. The masses for the peaks are expressed in atomic mass units.

The sites of glutaraldehyde modification/cross-linking were examined using tryptic mapping. The notation of Lippincott et al. (34) is used here to denote tryptic fragments. The tryptic profiles of the mono- and poly-glxrHb fractions were similar but demonstrated significant differences when compared with unmodified rHb1.1 (Fig. 4). Peak height ratios to the beta 4 peptide were used to compare maps. The beta 4 peptide of the beta -globin is flanked by Arg residues that are not modified by glutaraldehyde. Therefore, the degree of trypsin digestion of the beta 4 peptide was used as an internal standard, and the ratio of the peak height of each peptide to the beta 4 peptide was used to elucidate differences between the control and glutaraldehyde-modified samples (Table II). The majority of peptides that decreased by >35% on the beta -globin were beta 1, beta 8/9-1, beta 8/9-2, beta 9, beta 10/11, beta 11, beta 12, beta 13- and beta 14, indicating modification or involvement of cross-linking by the following residues: beta  N terminus, Lys66, Lys82, Lys95, Lys108, Lys120, and Lys132. The modified peptides of the di-alpha -globin included alpha 1, alpha 8,9-1, alpha 8,9-2, alpha 10/11, alpha 11, alpha 12, and alpha 13 corresponding to the modification of the following residues: di-alpha N terminus, Lys61, Lys99, Lys127, and Lys139 from the first half of the di-alpha -globin chain and the corresponding residues in the second half Lys203, Lys241, Lys269, and Lys281. Two difference peaks at 44.8 (1) and 45.2 (2) min were detected in the mono-glxrHb and poly-glxrHb tryptic maps. LC-MS analysis of peak 1 indicated a mass of 1052 atomic mass units which is consistent with a mass of the N-terminal beta  peptide (beta 1, 984 atomic mass units) plus an additional 68 atomic mass units. Peak 2 had a mass of 828 atomic mass units which is consistent with the mass of the di-alpha N-terminal peptide (761 atomic mass units) plus an additional 68 atomic mass units. We were unable to detect any other difference peptides with masses corresponding to either glutaraldehyde decoration or cross-linking.


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  		Fig. 4.   Trypsin maps of purified glutaraldehyde cross-linked rHb fractions. Aliquots were analyzed by trypsin mapping as described under "Experimental Procedures." A, rHb1.1; B, mono-glxrHb; C, poly1-glxrHb; D, poly2-glxrHb. The peptides denotations in A are the same as those used by Lippincott et al. (34). The peptides with asterisks demonstrated decreases in peak heights of at least 35% from rHb1.1 occurring in the mono-, poly1-, and poly2-glxrHb fractions. The same peptides all decreased in C and D. Difference peptides are denoted 1 and 2 in B and were observed in C and D.

                              
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Table II
Quantitation of peak height ratios for tryptic maps of mono-, poly1, and poly2-glxrHb

Evaluation of Hemodynamic Responses

Administration of rHb1.1 caused a rise in mean arterial pressure to levels approaching 20 mm Hg above control (Fig. 5A) and a concomitant fall in heart rate and cardiac output (Fig. 5, B and C). Consequently, total peripheral resistance rose significantly and reached a plateau of approximately 30% above base-line values. In contrast to the responses elicited by rHb1.1, administration of the same volume of a 5% protein solution (HSA) caused little change in mean arterial pressure or vascular resistance (Fig. 5, A and D). Heart rate and cardiac output rose slightly following HSA administration (Fig. 5, B and C). Surprisingly, the changes in mean arterial pressure and total peripheral resistance elicited by mono-glxrHb were depressed compared with those induced by rHb1.1 (Fig. 5, A and D), despite the similarities in molecular weight. The mean arterial pressure response induced by poly1-glxrHb was significantly reduced compared with those obtained with rHb1.1 and mono-glxrHb (Fig. 5A). However, the vascular resistance response was identical to that observed with mono-glxrHb (Fig. 5D). Changes in heart rate and cardiac output were not different for the three hemoglobin preparations (Fig. 5, B and C). Base-line mean arterial pressure and heart rate values were not different in any of the experimental groups (Table III).


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  		Fig. 5.   Overall mean arterial pressure (A), heart rate (B), cardiac output (C), and total peripheral resistance (D) responses determined as the average of the data obtained from 10- to 90-min post-administration. Bars represent mean ± S.E. *, p < 0.05 versus rHb1.1; down-triangle, p < 0.05 versus mono-glxrHb; #, p < 0.05 versus poly1-glxrHb.

                              
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Table III
Base-line hemodynamic values obtained prior to hemoglobin or HSA administration
Data are mean ± S.E. obtained from at least 30 min of base-line recording. n, number of animals; MAP, mean arterial pressure; HR, heart rate; bpm, beats per minute.

From these results, it appears that glutaraldehyde decoration (i.e. mono-glxrHb) significantly attenuates the hemodynamic response induced by rHb1.1 and that an increase in molecular size by glutaraldehyde cross-linking (i.e. poly-glxrHb) further decreases the mean arterial pressure response. Since poly1-glxrHb contained small amounts of dimeric and trimeric rHb species (Fig. 1), we tested the contributions of those lower molecular weight fractions by examining the responses to a more highly purified fraction (poly2-glxrHb) in a second set of experiments. These animals were instrumented with arterial and venous catheters as described under "Experimental Procedures," but pulsed Doppler flow probes were not implanted. Responses to rHb1.1, mono-glxrHb, and HSA were also obtained in animals prepared in the same manner. As with poly1-glxrHb, the change in mean arterial pressure in response to poly2-glxrHb was significantly lower than those obtained with rHb1.1 and mono-glxrHb (Fig. 6A). The heart rate responses to rHb1.1, mono-glxrHb, and poly2-glxrHb were all greater than the response to HSA but were not different from each other (Fig. 6B).


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  		Fig. 6.   Overall mean arterial pressure (A) and heart rate (B) responses determined as the average of the data obtained from 10- to 90-min post-administration. Bars represent mean ± S.E. *, p < 0.05 versus rHb1.1; down-triangle, p < 0.05 versus mono-glxrHb; #, p < 0.05 versus poly2-glxrHb.

To determine if the observed differences in hemodynamic responses could be accounted for by differences in plasma hemoglobin concentration, plasma samples were obtained at several time points during the first 120 min following administration. As shown in Table IV, the concentrations of rHb1.1 and mono-glxrHb were identical and were both significantly less than the concentration of poly2-glxrHb throughout the time course of hemodynamic measurement. Therefore, the reduced pressor responses elicited by mono- and poly-glxrHb cannot be attributed to lower plasma concentrations or more rapid clearance from the vascular space.

                              
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Table IV
Plasma hemoglobin concentrations (mg/ml) obtained at several time points corresponding to the period of hemodynamic measurement
0 min refers to samples taken immediately after hemoglobin administration, and the other columns refer to samples taken at the specified times post-administration. Data are shown as mean ± S.E.


    DISCUSSION

The prevailing hypothesis for the mechanism of the hemoglobin-induced rise in arterial pressure postulates that hemoglobin extravasates into the interstitium and causes vasoconstriction due to scavenging of NO. Sharma and colleagues (16) reported that co-infusion of L-arginine diminishes the hemodynamic responses to diaspirin cross-linked hemoglobin, a monomeric hemoglobin. Similar results with L-arginine were reported by Katsuyama et al. (43) who also showed that inhibition of NO synthesis by Nomega -nitro-L-arginine attenuates the hypertensive response to diaspirin cross-linked hemoglobin. Studies performed in vitro have similarly indicated a dependence on an interaction between hemoglobin and NO (20). We have recently reported that administration of recombinant hemoglobin in which the distal heme pocket was genetically modified to reduce NO scavenging rates by 20-fold demonstrated negligible hemodynamic responses in rats (21). Mono- and poly-glxrHb had NO oxidation rates that were identical to that of rHb1.1, indicating that differences in NO scavenging rates cannot account for the observed reductions in the hypertensive response. One possible explanation for our data is that glutaraldehyde treatment reduced the reactive heme concentration at the site of NO scavenging. Whereas the specific site of NO scavenging important for vasoconstriction is unknown, several investigators have suggested that it lies outside of the vascular compartment (22-24). Although there is little or no direct evidence available to support the hypothesis that extravasation is required for the hemoglobin-induced hypertensive response, our results and those of others (22, 27, 44) with polymerized hemoglobin are consistent with an obligatory role for hemoglobin extravasation. If polymerized hemoglobin such as poly-glxrHb is less able to leave the vasculature, the interstitial concentrations will be lower than those for smaller hemoglobin molecules (e.g. rHb1.1), and the amount of interstitial NO scavenging will be correspondingly diminished. In this context, the reduced hypertensive response elicited by mono-glxrHb is puzzling. If extravasation is required for the hypertensive effect, our results would suggest that molecular size is not the sole determinant of the rate of extravasation and that glutaraldehyde modification of the mono-glxrHb prevents its extravasation to the sites of NO scavenging.

The routes whereby proteins cross- the endothelium are still controversial but likely include a paracellular pathway and one utilizing plasmalemmal vesicles (45, 46). Since plasmalemmal vesicles apparently provide the primary mode of transport for molecules greater than 4 nm in diameter (46), endocytotic transport of rHb1.1 and mono-glxrHb (diameters of 7.3 and 8.5 nm, respectively) is the likely route whereby these hemoglobins extravasate. In support of this possibility, Milici et al. (47) have reported that albumin (64 kDa) movement across the myocardial endothelium occurs via plasmalemmal vesicles. Velky et al. (48) have provided additional evidence for endocytotic transport of hemoglobin in their observation that inhibition of endocytosis by m-dansylcadaverine decreased leakage of stroma-free hemoglobin into the peritoneal cavity. Initiation of the albumin transport process appears to require binding of albumin to either SPARC, gp60, gp30, or gp18 receptors (49). The additional finding that modification of the albumin with either colloidal gold particles or maleic anhydride, which nonspecifically modify primary amines, inhibits its binding to both SPARC and gp60 (49) suggests that similar surface modification of hemoglobin could interfere with its transendothelial movement.

We have ample evidence that the surface of the hemoglobin is altered by glutaraldehyde treatment. Glutaraldehyde is known to modify primary amines (50) which on proteins are available as N-terminal amines and epsilon -amines of the Lys residues. In addition, the x-ray crystallographic structure of the hemoglobin (51) shows that the N termini of the beta - and di-alpha -globins and the majority of the Lys residues are on the surface of the hemoglobin and readily available to the solvent. The Lysbeta 108 and Lysdi-alpha 99 are buried within the diphosphoglycerate-binding pocket of the hemoglobin. Indeed, our tryptic mapping data suggest that the beta  Lys residues 66, 82, 95, 108, 120, and 132 and di-alpha Lys residues 61, 99, 127, 139, 203, 241, 269, and 281 were modified on average to greater than 35% by glutaraldehyde. Other Lys residues were also modified, albeit to varying degrees. The similarities between the tryptic maps of mono-, poly1-, and poly2-glxrHb (Fig. 4 and Table II) suggest that these hemoglobins are all modified comparably and may account for the similar hemodynamic responses observed for the glutaraldehyde-modified monomeric and polymeric hemoglobins. The nature of the modifications on the Lys residues is not clear. We observed only two identifiable difference peaks in the tryptic map of the mono-glxrHb (Fig. 4B), whereas a number of peptides were clearly modified. Both difference peaks, based on MS/MS fragmentation (data not shown), were assigned as 68-Da modifications of the N-terminal peptide from the beta - and di-alpha -globins. The mass increase expected for a single molecule of glutaraldehdye forming Schiff bases between two amines followed by reduction with borohydride to imines is 68 Da. Reaction of the N terminus with one of the aldehyde groups followed by reduction with borohydride should result in a mass increase of 88 Da. Therefore, the exact nature of the modification is unclear and under further investigation.

Cross-linking of the beta - and di-alpha subunits may also be important in masking potential binding sites buried within the rHb1.1 molecule or in further stabilization by internal cross-linking. For example, haptoglobin binds to hemoglobin following its dissociation into alpha beta dimers exposing a previously buried binding site (52, 53). The SDS-PAGE (Fig. 2) and LC-MS analysis (Fig. 3) both demonstrated the presence of beta - to di-alpha -globin cross-linked species in both the mono- and poly-glxrHbs. A single beta  cross-linked to di-alpha -globin was detected by the SDS-PAGE as two distinct bands with either 44- or 46-kDa molecular masses. A beta /di-alpha cross-linked globin would be expected to have an approximate molecular mass of 46.3 kDa eluting near the 46-kDa band on the gel. The lower band may be due to additional intraglobin cross-linking changing its electrophoretic mobility or due to the presence of glutaraldehyde on the globin. Several modifications/decorations to the beta -globin were observed during LC-MS analysis (Fig. 3). The differences in mobility observed by SDS-PAGE were not seen following cross-linking of a second beta -globin to produce the beta 2/di-alpha cross-linked species. Disappearance of that band in the poly1- and poly2-glxrHb fractions may indicate further polymerization of the hemoglobin.

We found that glutaraldehyde polymerization decreased the mean arterial pressure response to a greater extent than did glutaraldehyde decoration (Fig. 5A). However, the vascular resistance responses were the same for both treatments (Fig. 5D). The reason for this apparent discrepancy in unknown but could stem from differences in oncotic or colloid osmotic pressures. The oncotic pressure of rHb1.1 measured in vitro at the concentration used (100 mg/ml) is 42 mm Hg, whereas the oncotic pressure of poly-glxrHb at the same concentration is 11 mm Hg. The oncotic pressure of HSA measured under the same conditions is 58 mm Hg. Although the oncotic pressure of mono-glxrHb was not determined, it would not be expected to differ significantly from the value obtained for rHb1.1. Administration of a highly hypo-oncotic solution such as poly-glxrHb may cause fluid to leave the vascular space, leading to contraction of intravascular volume and reductions in cardiac output and mean arterial pressure. This mechanism, in addition to the observed attenuation of peripheral vasoconstriction, could thus account for the additional attenuation in the mean arterial pressure response compared with that seen for the mono-glxrHb. Although blood volume was not determined in these studies, the observation that the concentration of poly-glxrHb was higher than that of either rHb1.1 or mono-glxrHb supports the hypothesis that fluid moved out of the vascular space after administration of poly-glxrHb.

Vandegriff and Winslow (54) have suggested that the vasoconstriction elicited by cell-free hemoglobin is due to an autoregulatory response to accelerated O2 delivery to arterioles. According to this hypothesis, the O2 affinity of many hemoglobin solutions is sufficiently low that O2 is released in the arterioles, and because of the O2 sensitivity of these vessels, vasoconstriction occurs. The P50 of hemoglobin in whole blood is 26 mm Hg (55). The P50 values of the hemoglobins used here were all similar with P50 values ranging from 30 to 36 mm Hg. Furthermore, the P50 value of rHb1.1 (32 mm Hg) was between those of mono- and poly-glxrHb (30 and 36 mm Hg, respectively), making it highly unlikely that the observed attenuation of the hypertensive response is due to differences in oxygen affinity.

To our knowledge, this study provides the first evidence that modification of hemoglobin with glutaraldehyde and without polymerization can directly impact the peripheral vasoconstriction induced by hemoglobin. The results presented here suggest that the effects of glutaraldehyde treatment on the hemodynamic responses to hemoglobin are by and large due to surface modification and/or internal stabilization. The mechanism(s) by which the modifications alter the hypertensive response remain to be elucidated.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the excellent technical expertise of Jon Vincelette, Anne Armstrong, and Michael Suniga in carrying out the hemodynamic experiments and Dominic Madril, Maria Pagratis, Tim Fattor, and Rita Vali for help with purification of the glutaraldehyde-treated hemoglobins. We also thank Dr. Douglas Lemon for measuring the NO-induced oxidation rates and Tim Fattor for measuring autooxidation rates. This work would not have been possible without the support of the Somatogen technical staff in manufacturing, pilot operations, and assay services. Finally, we thank Drs. Gillian Olins, David Foster, and Richard Gorczynski for critical review of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Amgen, Inc., One Amgen Center Drive, MS 8-1-C, Thousand Oaks, CA 91320. Tel.: 805-447-0712; Fax: 805-498-8674; E-mail: bKerwin{at}amgen.com.

The abbreviations used are: rHb1.1, recombinant human hemoglobin; oxyHb, oxyhemoglobin; deoxyHb, deoxyhemoglobin; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; LC-MS, liquid chromatographic mass spectrometry; HSA, human serum albumin; SEC, size exclusion chromatography; mono-glxrHb, glutaraldehyde-derivatized monomer; poly-glxrHb, glutaraldehyde cross-linked polymer.
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Abstract
Introduction
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