© 1996 American Heart Association, Inc.
Articles |
Removal of Arg141 From the 
Chain of Human Hemoglobin by Carboxypeptidases N and M
From the Departments of Pharmacology (B.M., P.A.D., E.G.E.) and Anesthesiology (V.L., E.G.E.), University of Illinois College of Medicine, and Cook County Hospital Department of Anesthesiology (R.I.), Chicago, Ill.
Correspondence to Ervin G. Erdös, MD, University of Illinois College of Medicine, Department of Pharmacology (M/C 868), 835 S Wolcott Ave, Chicago, IL 60612. E-mail egerdos@uicvm.uic.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and Methods Results Discussion References |
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Abstract Both human plasma carboxypeptidase N (CPN) and membrane-bound carboxypeptidase M (CPM) released the C-terminal arginine (
-Arg141) of the 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 -Arg141 accelerated the dissociation
rate of the tetramer deoxy-des--Arg141 hemoglobin to
dimers 2500-fold over that of deoxyhemoglobin, as measured by
haptoglobin binding. Moreover, the dissociation of the
deoxy-des--Arg141 hemoglobin tetramer to dimers was
not affected by 2,3-diphosphoglyceric acid. Des--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--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 -Arg141 residue
to sustaining the tetrameric structure of hemoglobin and its normal
oxygen affinity and vasoactivity.
Key Words: blood substitutes • carboxypeptidases • cross-linked hemoglobin • oxygen affinity • coronary vasoconstriction
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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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
ß 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
and ß subunits, which have the following COOH-terminal sequences:
chain,
-Thr137-Ser138-Lys139-Tyr140-Arg141
and ß 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 subunit. The importance of
Arg141 in the chains (-Arg141) rests on
forming salt bridges between the chains that stabilize the
deoxyhemoglobin and consequently lower the oxygen affinity of
hemoglobin.5 6 Fasan et al4 attributed
the
lack of -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 -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
-Arg141 on the oxygen-binding and vasoconstrictor
properties of the resulting des--Arg141 hemoglobin and
the dissociation of the hemoglobin tetramer to dimers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Materials
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--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--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--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 (4x10-5 mol/L). After 24 hours' incubation at 37°C, the P50 and Hill coefficient of the sample were determined.
Heart Perfusion
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.
Cross-Linked Hemoglobin
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
ß chains, in contrast to a commercial preparation in which hemoglobin
was cross-linked at the 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 ß
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--Arg141 Hemoglobin
For the
studies in vitro and in the isolated heart,
des--Arg141 hemoglobin was prepared by incubating
oxyhemoglobin (3.2x10-4 mol/L) at 37°C
with CPB3 as control, with CPM
(2.4x10-7 mol/L), or with the active
subunit of CPN (2.2x10-6 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
Statistical Analysis
All data are presented as
mean±SEM. Data were
analyzed by paired or unpaired t tests.
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Results |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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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
-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 chain is arginine, whereas it is
histidine3 in the ß chain. Consequently, CPB, CPM, and
CPN released only -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.
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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 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.
Cross-Linked Hemoglobin
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 ß monomers are primarily involved in the
cross-linking of oxyhemoglobin,21 any deoxyhemoglobin
contaminating our preparation would be cross-linked between the
monomers.24 The low-molecular-weight (15-kD) bands
in lanes 1 and 2 are dissociated and ß monomers of quite similar
molecular mass, and the single band in lane 3 represents
primarily monomers.
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To assess the amount of free ß 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 ß
chain.
Hydrolysis of Hemoglobins
Fig 2 shows the time
course of hydrolysis of the
bond of the C-terminal -Arg141 by CPM. As illustrated,
the oxygenated form of hemoglobin was cleaved more than
twice as fast as the deoxygenated form.
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),
deoxyhemoglobin (
), and cross-linked hemoglobin (
). CPM
(15 nmol/L) was incubated with oxygenated and
deoxygenated hemoglobin
(10-4 mol/L) or with cross-linked
hemoglobin (1.4x10-4 mol/L) in a
reaction volume of 50 µL. Abscissa: time in minutes. Ordinate: amount
of Cross-linked hemoglobin (1.4x10-4 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--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--Arg141 hemoglobin. For deoxyhemoglobin,
the reaction was very slow; t50, 13.9 hours. With
des--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 ß 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--Arg141 hemoglobin. In control experiments
without added haptoglobin, the absorbance did not change.
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The
fluorescence of haptoglobin was quenched similarly, up to
70% to 75%, by increasing amounts of oxyhemoglobin and
des--Arg141 oxyhemoglobin, yielding parallel curves when
the concentrations (up to 400 µg) are plotted against percent
quenching (not shown).
Oxygen Equilibrium
The P50 value of
des--Arg141 hemoglobin
(Table 3) was lower than that obtained with hemoglobin.
Des--Arg141 hemoglobin also had a marked decrease of
cooperativity as indicated by the value of the Hill coefficient.
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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 (4x10-5 mol/L), which blocked the liberation of Arg141 by plasma CPN.8 14
Effect on Coronary Arteries
Injections of hemoglobin or
des--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--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--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--Arg141 hemoglobin was significantly
more vasoconstrictive than hemoglobin. The perfusion pressure increased
by 14.61±1.53 mm Hg (des--Arg141 hemoglobin) versus
7.78 mm Hg (hemoglobin); P<.001. The maximally effective
concentrations of hemoglobin and des--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.
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Very likely, the
dissociation of hemoglobin or
des--Arg141 hemoglobin tetramers to ß dimers
contributed to the vasoconstriction. To test whether the
vasoconstrictor responses of hemoglobin and des--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.
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Mediators
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--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--Arg141 hemoglobin solutions were tested one to
three times).
Neither the basal coronary pressure nor the
vasoconstriction by
hemoglobin and des--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--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--Arg141 hemoglobin by 156±12.7%
(P<.05, paired t test) for hemoglobin, and
147.7±21.7% for des--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--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--Arg141 hemoglobin–induced
coronary vasoconstriction.
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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As shown in this report, the two human enzymes CPM and CPN hydrolyze hemoglobin, and CPM is more effective in removing
-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 -Arg141, by forming salt bridges, is a major
cross-linking factor in the deoxyhemoglobin.5 6 The
carbonyl group of the Arg141 of the 1 chain is
linked to the amino group of the N-terminal valine of the other
chain (2). The guanidine group of Arg141 in the 1
chain is directed to the -carboxyl group of Asp126
in the 2 chain. In contrast, in the oxyhemoglobin,
-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
ß 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--Arg141 hemoglobin very
rapidly dissociated to dimers, as measured by haptoglobin binding. The
dissociation constant for des--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--Arg141 deoxyhemoglobin is
unstable.6 17
The oxygen affinity of des--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
-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--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--Arg141 hemoglobin is more
vasoconstrictive on the coronaries of the rat than
hemoglobin itself. This phenomenon may be due to the dissociation of
des--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 ß 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
Endothelin-1 may contribute to the pressor response to hemoglobin,32 35 36 37 but in our experiments, two endothelin receptor blockers25 did not affect the vasoconstriction.
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--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--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 -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 |
|---|
|
-Me-Leu-D-Trp(COOCH3)-D-Nle
-nitro-L-arginine methyl
ester
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Acknowledgments |
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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.
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References |
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