Removal of Arg141 From the α Chain of Human Hemoglobin by Carboxypeptidases N and M
Abstract Both human plasma carboxypeptidase N (CPN) and membrane-bound carboxypeptidase M (CPM) released the C-terminal arginine (α-Arg141) of the α chain of human adult hemoglobin. An arginase contamination present in the hemoglobin preparation, which converted the released arginine to ornithine, was removed by gel filtration. CPM was about 20 times more efficient than CPN or its active subunit in hydrolyzing oxyhemoglobin and cleaved oxyhemoglobin twice as fast as deoxyhemoglobin. The hydrolysis of the peptide bond of α-Arg141 accelerated the dissociation rate of the tetramer deoxy-des-α-Arg141 hemoglobin to dimers 2500-fold over that of deoxyhemoglobin, as measured by haptoglobin binding. Moreover, the dissociation of the deoxy-des-α-Arg141 hemoglobin tetramer to dimers was not affected by 2,3-diphosphoglyceric acid. Des-α-Arg141 hemoglobin had a higher oxygen affinity (P50, 5.51 mm Hg; control, 19.94 mm Hg [P50 is the partial pressure of oxygen that gives 50% of the saturation of hemoglobin]) and a lower apparent cooperativity (Hill coefficient: n, 1.02; control, 2.24) than unhydrolyzed hemoglobin. After hemoglobin was incubated in human plasma, its oxygen-binding parameters, the P50, and the Hill coefficient decreased drastically due to cleavage by CPN. In the perfused rat heart, des-α-Arg141 hemoglobin was a more effective coronary vasoconstrictor than hemoglobin, possibly because it dissociated to dimers in the coronary vascular bed. A covalently cross-linked hemoglobin was less active than native hemoglobin. The coronary vasoconstriction was caused by multiple factors, including interference with vasodilation by nitric oxide and eicosanoids. Thus, the hydrolysis of hemoglobin by CPM and CPN demonstrated the contribution of the α-Arg141 residue to sustaining the tetrameric structure of hemoglobin and its normal oxygen affinity and vasoactivity.
Interest in the oxygen-binding properties of human adult hemoglobin and the dissociation of its subunit is stimulated by the potential use of various forms of hemoglobin as substitutes for erythrocytes in transfusion,1 but cell-free hemoglobin itself cannot be employed directly as an oxygen carrier. Indeed, in the absence of 2,3-DPG, outside the red blood cells, the affinity of hemoglobin for oxygen is too high to oxygenate tissues. Moreover, the free hemoglobin in the circulation dissociates into αβ dimers that are removed rapidly from the circulation by glomerular filtration, with the possibility of renal damage. This behavior necessitates polymerization or cross-linking of hemoglobin if it is to be used as a blood substitute.1
Hemoglobin is a tetramer composed of two pairs of dissimilar α and β subunits, which have the following COOH-terminal sequences: α chain, -Thr137-Ser138-Lys139-Tyr140-Arg141 and β chain, -Ala142-His143-Lys144-Tyr145-His146.2 3 Recently, Fasan et al4 used stored frozen human placentas as a source for large-scale production of hemoglobin. However, the hemoglobin extracted from this tissue had a very high oxygen affinity, caused by the lack of the C-terminal Arg141 residue in the α subunit. The importance of Arg141 in the α chains (α-Arg141) rests on forming salt bridges between the α chains that stabilize the deoxyhemoglobin and consequently lower the oxygen affinity of hemoglobin.5 6 Fasan et al4 attributed the lack of α-Arg141 to enzymatic hydrolysis by plasma CPN in blood trapped in the placentas. The placental brush-border membrane, however, is a very rich source of another enzyme, CPM.7 8 9 Plasma CPN and the membrane-bound CPM liberate the C-terminal basic amino acids arginine and lysine of proteins and peptides.8 9 10 11 Nevertheless, CPN cleaves the bond of C-terminal lysine preferentially, while membrane-bound CPM has a higher affinity for C-terminal arginine. Although the two enzymes can hydrolyze the same peptide bond in substrates, the human tissue enzyme CPM has less than 50% sequence identity with the active subunit of the blood-borne CPN.
Because of the proven importance of the α-Arg141 in the hemoglobin molecule,3 5 6 we studied its release by carboxypeptidases that are present in the circulation (CPN) or on the plasma membrane of many organs (CPM). We also investigated the consequences of the hydrolysis of the peptide bond of α-Arg141 on the oxygen-binding and vasoconstrictor properties of the resulting des-α-Arg141 hemoglobin and the dissociation of the hemoglobin tetramer to dimers.
Materials and Methods
MGTA was from Calbiochem. BQ610 and BQ788 were from Peptides International. Outdated human plasma was obtained from Rush-Presbyterian St Luke’s Hospital Blood Bank, Chicago, Ill. Sephadex columns were from Pharmacia. Human oxyhemoglobin, human haptoglobin, enzymes, and reagents were from Sigma Chemical Co.
Electrophoresis of oxyhemoglobin on native polyacrylamide gel without added SDS revealed only a single band. The methemoglobin content of the oxyhemoglobin was determined by the method of Evelyn and Malloy12 and kept below 6% by treatment with sodium dithionite, which was subsequently removed from the solution by gel filtration (Sephadex G-25; PD10 column). The hemoglobin solutions were always used on the day of their preparation. In general, if not otherwise indicated, experiments were done in duplicate and repeated.
Purification of Carboxypeptidases M and N
CPM was used either as purified or as recombinant enzyme. Purified CPM was from human placentas purified after solubilization with phospholipase C.7 Recombinant CPM expressed in insect cells (Tan et al, unpublished data, 1995) was also used.
CPN was purified from outdated human plasma by ion exchange and by arginine-Sepharose affinity chromatography.11 13 Purified CPN was treated with 3 mol/L guanidine-HCL to dissociate it to two inactive regulatory 83-kD subunits and two active 50-kD subunits.11 Both the 280-kD tetramer of the enzyme, containing two active sites, and its isolated 50-kD subunit were used in the experiments.
CPM and CPN activities were routinely measured during purification with dansyl-Ala-Arg as a substrate.14 As control, commercial porcine pancreatic CPB was employed.
The protein concentrations were determined according to Bradford,15 with bovine serum albumin as standard.
Hydrolysis of Hemoglobin
The kinetics of hydrolysis of oxyhemoglobin by CPM and CPN were established by measuring the C-terminal release of Arg141 by HPLC, performed after phenylthiocarbamyl derivatization in a Waters automated gradient system following the manufacturer’s protocols. Because of trace contamination by arginase,16 the enzyme that converts arginine into ornithine and urea, the oxyhemoglobin was further purified by gel filtration using an S200 column in 0.1 mol/L phosphate buffer, pH 7.5, and then was concentrated in an Amicon concentrator with a YM-10 membrane. The efficiency of this step was tested by adding free arginine (10−4 mol/L) and incubating for 3 hours with the purified hemoglobin (10−4 mol/L) to see whether arginine was converted to ornithine.
Oxyhemoglobin (in 0.1 mol/L phosphate buffer, pH 7.5) and enzyme (8 nmol/L CPB, 16 nmol/L CPM, 21 nmol/L CPN, or 18 nmol/L active subunit of CPN) were incubated in a final volume of 150 μL at 37°C. The reactions were terminated by adding 150 μL of trifluoroacetic acid (10%). The samples were centrifuged for 1 hour at 100 000g and then stored at −70°C until analysis by HPLC.
The amount of arginine released was calculated by comparing the integrated peak area of product with the peak area of a known amount of authentic standard. Kinetic constants were obtained by initial velocity measurements of product formation at five substrate concentrations, ranging from 4.10−4 to 2.10−5 mol/L. Data were plotted according to Lineweaver-Burk (1/[S] versus 1/v) and the best straight line calculated by linear regression. Correlation coefficients better than .95 were obtained in all cases.
Oxyhemoglobin was deoxygenated by adding solid dithionite (0.4% wt/vol), followed by desalting on a PD10 column. The deoxyhemoglobin (10−4 mol/L) was then divided into two aliquots. One aliquot was kept in an open flask to be fully oxygenated and the other was kept under nitrogen. At zero time, CPM to 15 nmol/L was added to the flasks to start the reaction. With deoxyhemoglobin solution, CPM was introduced through a needle inserted into the cap of the flask. Cross-linked hemoglobin was incubated with CPB, CPM, or CPN in oxygenated solution.
Dissociation of Deoxyhemoglobin Tetramer
To determine the dissociation rate constants of hemoglobin and des-α-Arg141 hemoglobin from tetramer to dimer, human haptoglobin was used to bind the hemoglobin dimer released. The dissociation of deoxyhemoglobin was recorded by measuring the decrease in absorbance at a wavelength of 430 nm.17 Hemoglobin and haptoglobin in 0.05 mol/L Tris/HCl, 0.1 mol/L NaCl, and 1 mmol/L EDTA, pH 7.5, were mixed in a cuvette in the presence of sodium dithionite (0.3% wt/vol). The cuvette was immediately sealed and placed in a DMS100 spectrophotometer (Varian), and the decrease in absorbance at 430 nm was recorded at room temperature. The binding capacity of haptoglobin for hemoglobin was 0.7 mg of hemoglobin per milligram of haptoglobin. The absorbance at 430 nm of the reference deoxygenated hemoglobin and des-α-Arg141 hemoglobin without the haptoglobin was also measured.
For oxyhemoglobin, the binding of dimers to haptoglobin in 0.05 mol/L Tris, 0.1 mol/L NaCl, and 1 mmol/L EDTA at pH 7.5 was followed by measuring the quenching of haptoglobin fluorescence using an excitation wavelength of 282 nm and an emission wavelength of 331 nm in a Perkin-Elmer spectrophotofluorometer.17
P50 and Hill Coefficient
Binding of one oxygen molecule to one subunit of hemoglobin makes the binding of subsequent oxygen molecules to another subunit much more likely. Cooperative oxygen binding to hemoglobin is achieved by the molecule changing its quaternary structure when oxygen binds.
The oxygen-binding parameters P50 and cooperativity (given by the Hill coefficient) were measured in hemoglobin concentrations adjusted to 0.1 g/mL. The P50 and Hill coefficient of hemoglobin and des-α-Arg141 hemoglobin were determined in 0.1 mol/L phosphate buffer and 0.1 mol/L NaCl, pH 7.5, at 37°C after reduction of methemoglobin by 1% (wt/vol) sodium dithionite.18 19 The percentage of residual methemoglobin was below 6%. The pH, Po2, and oxygen saturation were measured on an ABL4 analyzer/OSM2 hemoximeter (Radiometer Copenhagen).
Oxyhemoglobin (10 mg/mL) was incubated in human plasma in the presence or absence of the added carboxypeptidase inhibitor MGTA (4×10−5 mol/L). After 24 hours’ incubation at 37°C, the P50 and Hill coefficient of the sample were determined.
The experiments were carried out with the approval of the Animal Care and Usage Committee of the University of Illinois at Chicago. Male Sprague-Dawley rats (300 to 350 g) were anesthetized with pentobarbital (60 mg/kg IP, N=45). The abdomen was opened and heparin, 500 USP units per 100 g, was injected into the vena cava. The thorax was cut open, a cold modified Krebs-Henseleit solution poured into the thoracic cavity, and the heart quickly removed and placed in cold Krebs-Henseleit solution, then perfused according to Langendorff’s technique at 37°C.20 The perfusion solution contained, in mmol/L, NaCl 118, KCl 4.70, CaCl2 2.52, MgS04 1.66, NaHC03 24.88, KH2PO4 1.18, glucose 5.55, and sodium pyruvate 2.0, and was filtered through a 0.45-μm-membrane filter. The perfusion solution was gassed with 5% CO2, 95% O2. The flow rate was controlled by a variable-speed peristaltic pump (Varioplex II 2120, LKB) and adjusted to establish a starting coronary perfusion pressure of ≈35 mm Hg. The coronary flow, 6 to 7 mL/min, was kept constant so that the changes in perfusion pressure reflected changes in the resistance of coronary blood vessels. Coronary perfusion pressure was measured with transducers connected to a sidearm of the aortic cannula using a Grass recording system model 79 (Grass Instrument Co) and heart performance analyzer (Digi-Med). Electrocardiograms from the perfused heart were recorded with Hewlett-Packard instruments (models 78534B and 78574A) using atraumatic platinum electrodes. Hemoglobins (0.1 to 0.5 mL) were injected (via an in-line injection port placed at the aortic cannula) by an infusion pump (IITC, Inc, model 210), at a flow rate of 1 mL/min. Stock solutions of hemoglobins were kept at 100 mg/mL in 0.1 mol/L phosphate buffer, pH 7.5. The order of administration of the hemoglobin and its derivatives was regularly varied.
Bis(3,5-dibromosalicyl)-fumarate was reacted with oxyhemoglobin in 0.2 mol/L Bis-Tris buffer, pH 7.2, for 3 hours at 37°C.21 This reaction cross-linked hemoglobin at the β chains, in contrast to a commercial preparation in which hemoglobin was cross-linked at the α chains.22 The concentrations of oxyhemoglobin and the cross-linking reagent were 2 mmol/L each. After the reaction mixture was passed through a column of Sephadex G-25 (Pharmacia PD10), the cross-linked and nonreacted derivatives were separated by gel filtration on an S200 column in 1 mol/L MgCl2 in 0.1 mol/L Tris, pH 7.2.21 To check the purity of the cross-linked hemoglobin, the fractions of cross-linked tetramer were rechromatographed on a Superdex 200 HR 10/30 column (Pharmacia) using the same buffer. The extent of cross-linking was quantified by integration of the peaks obtained. The cross-linking between the subunits of the hemoglobin tetramer was also detected in SDS-PAGE. The amount of separate αβ dimers still present in the cross-linked tetrameric hemoglobin preparation was determined by adding haptoglobin and recording the quenching of fluorescence.17
Preparation of Des-α-Arg141 Hemoglobin
For the studies in vitro and in the isolated heart, des-α-Arg141 hemoglobin was prepared by incubating oxyhemoglobin (3.2×10−4 mol/L) at 37°C with CPB3 as control, with CPM (2.4×10−7 mol/L), or with the active subunit of CPN (2.2×10−6 mol/L). Under our conditions, amino acid analysis indicated 100% hydrolysis of oxyhemoglobin after 4 hours (CPB and CPM) or after overnight incubation with the active subunit of CPN. Hemoglobin concentration was routinely determined after conversion to cyanomethemoglobin.22
All data are presented as mean±SEM. Data were analyzed by paired or unpaired t tests.
Kinetics of the Hydrolysis of Oxyhemoglobin
In our preliminary studies, after cleaving oxyhemoglobin by a carboxypeptidase, we found by HPLC analysis that the split product yielded two peaks, arginine and ornithine. Thus, some of the α-Arg141 released was converted to ornithine by a trace contamination of arginase in the hemoglobin solution derived from red cells.16 23 The presence of arginase in the hemoglobin preparation was confirmed by adding free arginine to hemoglobin in the absence of carboxypeptidases; amino acid analysis showed a time-dependent decrease of the amount of free arginine and in parallel the appearance of ornithine (not shown). After 3 hours of incubation at 37°C with hemoglobin (10−4 mol/L), the amount of added arginine (10−4 mol/L) decreased by 31%. The arginase, however, was removed by gel filtration, and after this step of hemoglobin purification, no ornithine was found.
The C-terminal amino acid of the α chain is arginine, whereas it is histidine3 in the β chain. Consequently, CPB, CPM, and CPN released only α-Arg141 and no other amino acids. Table 1⇓ summarizes the kinetic parameters of the hydrolysis of oxyhemoglobin by CPB, CPM, CPN, and the 50-kD active CPN subunit. Turnover numbers were expressed as micromoles of hemoglobin hydrolyzed per minute per active site, assuming that two arginines were released from each molecule of hemoglobin tetramer (65 kD) and that both active sites in the intact 280-kD CPN molecule, but only the single active site in the 50-kD subunit of CPN, functioned.
As expected from the known properties of CPB, CPM, and CPN,7 8 9 10 11 the peptide bond of the C-terminal arginine in the α chain of oxyhemoglobin was hydrolyzed faster by CPB and CPM than by CPN or the 50-kD active subunit of CPN. The specificity constants (kcat/Km) of CPB and CPM were about 20 times higher than that of CPN. The Km for oxyhemoglobin was lower with the tetramer of CPN than with the 50-kD active subunit, but the kcat was also lower with the 280-kD CPN; thus, the specificity constants of CPN and its active subunit were similar.
Both CPM and CPN hydrolyzed the substrate: the carboxypeptidase inhibitor MGTA inhibited the hydrolysis of oxyhemoglobin by CPM or CPN completely, with a Ki of 14.3 nmol/L or 30.3 nmol/L, respectively.
The elution from a Superdex 200 HR 10/30 column of the products of the cross-linking of oxyhemoglobin with an equimolar concentration of bis(3,5-dibromosalicyl)-fumarate21 indicated that under our conditions, about 80% to 85% of the hemoglobin was cross-linked. SDS-PAGE (Fig 1⇓) of non–cross-linked (lanes 1 and 2) and cross-linked (lane 3) hemoglobin, in the presence of dithiothreitol and 2-mercaptoethanol, revealed two molecular masses of about 32 kD in the cross-linked preparation. The two bands in lane 3 may have resulted from the fact that although the β monomers are primarily involved in the cross-linking of oxyhemoglobin,21 any deoxyhemoglobin contaminating our preparation would be cross-linked between the α monomers.24 The low-molecular-weight (15-kD) bands in lanes 1 and 2 are dissociated α and β monomers of quite similar molecular mass, and the single band in lane 3 represents primarily α monomers.
To assess the amount of free αβ dimer remaining in the preparation of cross-linked hemoglobin, the quenching of fluorescence of haptoglobin was measured. Thirty micrograms of the preparation quenched 2% to 3% of the fluorescence of 43 μg of haptoglobin. By comparison, the same amount of native hemoglobin quenched 28% to 30%; thus, by this estimate, about 85% to 95% of the preparation consisted of hemoglobin cross-linked at the β chain.
Hydrolysis of Hemoglobins
Fig 2⇓ shows the time course of hydrolysis of the bond of the C-terminal α-Arg141 by CPM. As illustrated, the oxygenated form of hemoglobin was cleaved more than twice as fast as the deoxygenated form.
Cross-linked hemoglobin (1.4×10−4 mol/L) was resistant to the actions of CPB, CPM, and CPN; even after 6 hours’ incubation with enzymes, only trace amounts of arginine were released by the carboxypeptidases.
Dissociation of the Tetramer
Dissociation rate constants of deoxyhemoglobin and deoxy-des-α-Arg141 hemoglobin were determined by measuring the decrease in absorbance at 430 nm. This is an indicator of the dissociation of hemoglobin tetramers into dimers, which in turn are complexed by the added haptoglobin. The absorbance data fit well in the equation for a first-order reaction.17 The rate constants (k) listed in Table 2⇓ show typical time courses of changes in absorbance for deoxyhemoglobin and deoxy-des-α-Arg141 hemoglobin. For deoxyhemoglobin, the reaction was very slow; t50, 13.9 hours. With des-α-Arg141 hemoglobin, the rate was faster by several orders of magnitude; t50, 19.9 seconds. Added 2,3-DPG stabilizes the deoxy tetrameric structure of hemoglobin against the dissociation into αβ dimers. In the presence of 2 mmol/L 2,3-DPG, the rate of dissociation of deoxyhemoglobin decreased (Table 2⇓). In contrast, 2,3-DPG was ineffective when added to deoxy-des-α-Arg141 hemoglobin. In control experiments without added haptoglobin, the absorbance did not change.
The fluorescence of haptoglobin was quenched similarly, up to 70% to 75%, by increasing amounts of oxyhemoglobin and des-α-Arg141 oxyhemoglobin, yielding parallel curves when the concentrations (up to 400 μg) are plotted against percent quenching (not shown).
The P50 value of des-α-Arg141 hemoglobin (Table 3⇓) was lower than that obtained with hemoglobin. Des-α-Arg141 hemoglobin also had a marked decrease of cooperativity as indicated by the value of the Hill coefficient.
Oxygen-binding parameters of hemoglobin were also determined after incubation (10 mg/mL) in human plasma for 24 hours at 37°C. The consequences were that the P50 decreased to 4.78 mm Hg compared with 15.81 mm Hg for the control, and the Hill coefficient decreased to 1.41 from a 2.14 control value. As described in “Materials and Methods,” the control samples were incubated under identical conditions but in the presence of the inhibitor MGTA (4×10−5 mol/L), which blocked the liberation of Arg141 by plasma CPN.8 14
Effect on Coronary Arteries
Injections of hemoglobin or des-α-Arg141 hemoglobin increased coronary perfusion pressure for 1 to 2 minutes in the isolated perfused rat heart preparation. This period of increase in the perfusion pressure, which was due to the presence of hemoglobin or des-α-Arg141 hemoglobin, was preceded by an initial brief increase followed by a slight immediate decrease in the perfusion pressure. These slight initial changes were reproduced by the injection of perfusion fluid alone.
The dose-response curves of hemoglobin and des-α-Arg141 hemoglobin in causing coronary vasoconstriction are shown in Fig 3⇓. There were no significant differences between the two types of hemoglobin at the two lower doses of 150 and 300 nmol. However, when 450 nmol was administered, des-α-Arg141 hemoglobin was significantly more vasoconstrictive than hemoglobin. The perfusion pressure increased by 14.61±1.53 mm Hg (des-α-Arg141 hemoglobin) versus 7.78 mm Hg (hemoglobin); P<.001. The maximally effective concentrations of hemoglobin and des-α-Arg141 hemoglobin were not tested because they could cause arrhythmias or an irreversible increase in coronary resistance (data not shown). No variations in the heart rate were detected.
Very likely, the dissociation of hemoglobin or des-α-Arg141 hemoglobin tetramers to αβ dimers contributed to the vasoconstriction. To test whether the vasoconstrictor responses of hemoglobin and des-α-Arg141 hemoglobin were partially due to the presence of dimers, tetrameric cross-linked hemoglobin was also tested. The cross-linked hemoglobin (450 nmol) was 50% less effective as a vasoconstrictor than the unmodified hemoglobin (Fig 4⇓). The perfusion pressure in this series of experiments was increased 8.85±1.44 mm Hg by hemoglobin and only 4.44±1.07 mm Hg by cross-linked hemoglobin; P<.001.
To determine the possible involvement of endothelins in the vasoconstriction, 1 μmol/L of two endothelin receptor blockers, BQ610 and BQ788, was added to the perfusion fluid. BQ610 is a specific antagonist of the endothelin A receptor, whereas BQ788 blocks the endothelin B receptor.25 These two antagonists did not affect basal coronary pressure but completely blocked the vasoconstrictor effect of endothelin-1 given as control (0.3 nmol). The receptor blockers did not significantly inhibit the vasoconstriction caused by hemoglobin. The vasoconstrictor effects of an injection of 450 nmol hemoglobin or des-α-Arg141 hemoglobin increased pressure over the control by 23±1% or 40±4%, respectively, in the presence or absence of BQ610 and BQ788 (n=3; with each heart, hemoglobin and des-α-Arg141 hemoglobin solutions were tested one to three times).
Neither the basal coronary pressure nor the vasoconstriction by hemoglobin and des-α-Arg141 hemoglobin was affected by superoxide dismutase or catalase (170 and 300 U/mL, respectively).26
Infusion of L-NAME, 10−4 mol/L, increased the coronary basal pressure by 6.23±2.17 mm Hg (n=3). The vasoconstrictor effects of hemoglobin and des-α-Arg141 hemoglobin were significantly enhanced with this nitric oxide synthesis inhibitor27 (359±66% and 598±244%, P<.05, paired t test; n=3).
Indomethacin (10 μmol/L), an inhibitor of cyclooxygenase, plus NDGA (10 μmol/L), an inhibitor of 5-lipoxygenase, potentiated the vasoconstriction induced by 450 nmol hemoglobin and des-α-Arg141 hemoglobin by 156±12.7% (P<.05, paired t test) for hemoglobin, and 147.7±21.7% for des-α-Arg141 hemoglobin. The simultaneous perfusion of these two blocking compounds also increased the basal pressure by 6.0±3.16 mm Hg (n=3).
Injections of 450 nmol hemoglobin or des-α-Arg141 hemoglobin were as vasoconstrictive in the presence as in the absence of 10−5 mol/L prazosin, an adrenergic receptor blocker, in the perfusion solution.
A control bolus injection of 450 nmol bovine serum albumin had no vasoconstrictor activity. Nifedipine (10−5 mol/L), an L-type calcium channel antagonist; cromakalim (10−5 mol/L), a KATP channel opener; papaverine (10−4 mol/L); and atropine (10−4 mol/L) failed to reverse hemoglobin- or des-α-Arg141 hemoglobin–induced coronary vasoconstriction.
As shown in this report, the two human enzymes CPM and CPN hydrolyze hemoglobin, and CPM is more effective in removing α-Arg141 than is CPN. The release of this amino acid enhances the dissociation of the tetrameric molecule to dimers, the affinity for oxygen, and the coronary vasoconstriction caused by hemoglobin. The higher affinity of hemoglobin for oxygen means that potentially less oxygen could be released to tissues.
The α-Arg141, by forming salt bridges, is a major cross-linking factor in the deoxyhemoglobin.5 6 The α carbonyl group of the Arg141 of the α1 chain is linked to the α amino group of the N-terminal valine of the other α chain (α2). The guanidine group of Arg141 in the α1 chain is directed to the α-carboxyl group of Asp126 in the α2 chain. In contrast, in the oxyhemoglobin, α-Arg141 does not interact with other groups and consequently forms an unblocked C-terminus.5 6 The free C-terminal arginine of oxyhemoglobin is more accessible to CPM than that of deoxyhemoglobin and is released more than twice as fast, as is also shown with pancreatic CPB.3
Under deoxy conditions, the dissociation of hemoglobin tetramers to αβ dimers is limited but still possible;28 29 thus, some dimers will still be substrates of CPN or CPM. In contrast, the covalently cross-linked hemoglobin does not dissociate and was not significantly hydrolyzed by CPN or CPM in our experiments, possibly owing to steric hindrance.
Under deoxy conditions, des-α-Arg141 hemoglobin very rapidly dissociated to dimers, as measured by haptoglobin binding. The dissociation constant for des-α-Arg141 hemoglobin increased >2500-fold compared with that of hemoglobin, in agreement with data obtained by others.17 In the present study, the apparent lack of effect of 2,3-DPG also indicates that the tetrameric structure of des-α-Arg141 deoxyhemoglobin is unstable.6 17
The oxygen affinity of des-α-Arg141 hemoglobin increased in parallel with the disappearance of cooperativity, in agreement with the high degree of oxygen binding by hemoglobin purified from the placenta, which is probably a product of CPM instead of plasma CPN as suggested.4 9 The oxygen-binding parameters found above are also consistent with the data obtained with naturally occurring variants of hemoglobin that lack α-Arg141.2
When 10 mg of hemoglobin was added to 1 mL of human plasma, a level that can be reached in severe intravascular hemolysis,30 the P50 and Hill coefficient drastically decreased in 24 hours, suggesting a nearly complete hydrolysis of hemoglobin by CPN. Pronounced hemolysis may yield enough free hemoglobin to be hydrolyzed by CPN and especially by CPM, enzymes present in the circulation or in tissues.7 8 9 10 11 Indeed, des-α-Arg141 hemoglobin was found in plasma and urine of patients with acute hemolysis of various etiologies.31
Vasoconstriction in the basilar and coronary arteries caused by hemoglobin32 33 may complicate its use as a blood substitute. We found that des-α-Arg141 hemoglobin is more vasoconstrictive on the coronaries of the rat than hemoglobin itself. This phenomenon may be due to the dissociation of des-α-Arg141 hemoglobin to possibly more vasoconstrictive dimers in the coronary circulation. Covalently cross-linked hemoglobin was less vasoconstrictive, supporting this hypothesis.
Modified hemoglobins appear to be effective oxygen-carrying substitutes for blood. The results presented support this development by showing that cross-linking prevents the metabolism of hemoglobin that leads to the dissociation of the tetrameric molecule and increase in renal clearance. In addition, cross-linking reduces the release of αβ dimers that appear to be more coronary vasoconstrictive.
The vasoconstrictive effect of hemoglobin can be secondary to several factors, including the release of vasoactive eicosanoids and endothelin from the vascular wall, blocking of NO, or generation of free radicals,32 but is independent of its oxygen affinity.34
Oxyhemoglobin autooxidizes to methemoglobin and releases superoxide anion radicals, which promote lipid peroxidation.32 Lipid peroxides can be vasoconstrictive and damage arteries in vitro and in vivo,32 and the free radicals can break down endothelium-derived relaxing factor or NO.38 In our experiments, superoxide dismutase and catalase, two scavenging enzymes, did not abolish coronary vasoconstriction; therefore, superoxide anions or hydrogen peroxide are not involved. Vasospasm that follows subarachnoid hemorrhage can be caused by the formation of free radicals from oxyhemoglobin, but it develops slowly, with onset 3 days after the hemorrhage.32
Hemoglobin can also release vasoactive prostaglandins and inhibit the vasodilation by NO.32 The affinity of NO to hemoglobin was established in the 19th century,39 40 but reports on the involvement of NO or prostaglandins in the vasoconstriction to hemoglobins are equivocal.32 40 41 In our experiments, the NO synthase inhibitor L-NAME enhanced vasoconstriction to hemoglobins. The effect of a combination of indomethacin and NDGA was similar, but inhibitors of the arachidonic acid cascade potentiated the vasoconstriction less. The phenomenon is a complex one, owing to blocking the synthesis of both vasodilator prostaglandins and vasoconstrictor thromboxane.
The coronary vasoconstriction by hemoglobin and des-α-Arg141 hemoglobin in the rat can be attributed to multiple factors acting at multiple sites in the vessel wall,40 41 42 but the sympathetic and parasympathetic nervous systems did not contribute in our experiments.
Hemoglobin is potentially an important substrate of CPN and CPM, the kininase I-type enzymes that cleave Arg9 from bradykinin.7 8 9 10 11 16 In addition, CPN is a major inactivator of anaphylatoxins8 43 and cleaves creatine kinases released from damaged myocardium.44 CPM also hydrolyzes epidermal growth factor45 and is a monocyte/macrophage maturation marker (MAX.1,11).46 Although CPN is in circulating blood after extravasation, for example, in cerebral hemorrhage, it can convert hemoglobin to the more potent vasoconstrictor des-α-Arg141 derivative. CPM, widely distributed,8 47 48 can catalyze the same reactions.
In summary, the hydrolysis of hemoglobin by CPN and CPM proves the importance of α-Arg141 in sustaining the tetrameric structure of hemoglobin and maintaining its normal oxygen-carrying capacity and vasoactive properties. The covalently cross-linked hemoglobin is more resistant to hydrolysis by circulating CPN and membrane-bound CPM and less coronary vasoconstrictive than native hemoglobin.
Selected Abbreviations and Acronyms
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|P50||=||partial pressure of oxygen that gives 50% of saturation of hemoglobin|
This study was supported in part by National Institutes of Health National Heart, Lung, and Blood Institute MERIT grant No. HL36473 (to Dr Erdös) and by the French Ministry of Foreign Affairs Lavoisier fellowship to Dr Michel. The authors gratefully acknowledge Claudie Hecquet and Li-Xiu Wang for their cooperation in cross-linking the hemoglobin, Drs Randal Skidgel and Herbert Jackman for their useful advice and discussions, Dr Fulong Tan for the recombinant CPM, and Sara Thorburn for editorial assistance.
- Received September 5, 1995.
- Accepted December 29, 1995.
- © 1996 American Heart Association, Inc.
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