Water regulates oxygen binding in hagfish (Myxine glutinosa) hemoglobin
Department of Zoophysiology, Institute of Biology, Building 131, University of Aarhus, DK-8000 Aarhus C, Denmark
* Author for correspondence (e-mail: angela.fago{at}biology.au.dk)
Accepted 5 February 2003
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Summary |
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Key words: water effect, allostery, hagfish, Myxine glutinosa, hemoglobin, bicarbonate, osmolality, oxygen affinity, cooperativity, linkage plot
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Introduction |
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Osmotic stress studies have shown opposite effects of water activity on the
O2 affinity of tetrameric vertebrate hemoglobins and dimeric
hemoglobin of the clam Scapharca inaequivalvis. In human hemoglobin,
approximately 60 water molecules are bound per tetramer in the transition from
the low-affinity T to the high-affinity R state, which is in good agreement
with the increase in solvent-accessible area that follows the TR
allosteric transition (Colombo et al.,
1992; Colombo and
Bonilla-Rodriguez, 1996
;
Colombo and Seixas, 1999
;
Arosio et al., 2002
). By
contrast, upon O2 binding the clam hemoglobin releases 6-8 water
molecules that stabilize the hemoglobin in the deoxy state by forming highly
structured water bridges at the dimer interface
(Royer et al., 1996
).
Hagfishes, together with lampreys, belong to the Cyclostomes, and occupy a
crucial phylogenetic position as the most ancient craniates
(Martini, 1998). Their
hemoglobin system is thus considered to represent the transition state between
invertebrate and vertebrate hemoglobins
(Goodman, 1981
). The
hemoglobin system of Myxine glutinosa consists of three major
hemoglobin fractions: HbI, HbII and HbIII
(Paléus et al., 1971
),
occuring in a ratio of approximately 15:50:35
(Fago et al., 2001
), and are
monomeric in the oxygenated state, but reversibly associate into dimers and
tetramers when deoxygenated. This monomerdimertetramer
equilibrium between high-O2-affinity monomers and low-affinity
oligomers replaces the TR equilibrium of vertebrate tetrameric
hemoglobins and is basic to the allosteric properties of hagfish hemoglobins,
such as cooperative O2 binding and the effects of pH and of
bicarbonate, the latter being a major physiological allosteric effector in
M. glutinosa (Fago and Weber,
1995
,
1998
;
Fago et al., 1999
). With the
exception of HbII, which self-aggregates at acidic pH, interactions between
monomers under physiological conditions occur mainly between HbI and HbII and
between HbII and HbIII in the presence of bicarbonate, whereas HbI and HbIII
do not show functional interaction (Fago
et al., 2001
).
The red blood cells of M. glutinosa are unable to recover their
original volume after osmotic swelling or shrinking
(Brill et al., 1992;
Nikinmaa et al., 1993
;
Dohn and Malte, 1998
). This
unusual feature is attributed to the lack of membrane proteins essential to
volume regulatory responses, such as those involved in the
K+/Cl- cotransport and in the taurine efflux pathways
(Nikinmaa et al., 1993
).
Moreover, absence of the anion exchanger band III membrane protein
(Ellory et al., 1987
;
Peters et al., 2000
) has
important consequences for CO2 and O2 transport, whereby
bicarbonate, a major regulator of O2 affinity in M.
glutinosa hemoglobin (Fago et al.,
1999
), is transported within the red blood cells rather than in
plasma. Hagfishes are probably the only marine organisms to have evolved
without entering freshwater (see Nikinmaa
et al., 1993
), and have one of the highest blood electrolyte
concentrations, with an osmolality close to that of seawater, which is almost
entirely due to high levels of inorganic ions
(Robertson, 1963
). They are
osmoconformers and unable to regulate the osmotic concentration in their blood
in response to that of the ambient seawater. While sodium and chloride
concentrations in the blood plasma are the same as in seawater, the
concentrations of the divalent cations calcium and magnesium are regulated
(Robertson, 1963
;
Fänge, 1998
).
These unusual characteristics provide a unique opportunity to study the effects of water activity on hemoglobin function at both the molecular and the cellular (erythrocytic) levels and to gain insight into the evolutionary origin of water effects in hemoglobins. We report here the effect of changes in osmolality on O2 binding in isolated hemoglobins and in intact red blood cells of the hagfish M. glutinosa, using the osmotic stress approach.
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Materials and methods |
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Because of the high degree of polymorphism and hemoglobin multiplicity
(Paléus et al., 1971;
Fago and Weber, 1995
;
Fago et al., 2001
), individual
hemolysates from 91 animals were analysed on a 0.2 mm-thick polyacrylamide
isoelectric focusing gel containing a 1:2 ratio of 3.5-10 and 7-9 Ampholines
using the Multiphor II System (Amersham Pharmacia Biotech, Uppsala, Sweden).
Samples showing identical multiplicity and having three major bands only were
selected and pooled (see Fago et al.,
2001
). Hemoglobin was `stripped' by gel filtration on a Sephadex
G-25 Fine (23x350 mm) column equilibrated with 50 mmol l-1
Tris buffer, pH 7.8, 0.1 mol l-1 NaCl. The hemoglobin solution was
then dialysed against CO-equilibrated 10 mmol l-1 Tris buffer, pH
7.9 and the three individual components were separated by anion exchange
chromatography on a DEAE-Sephacel column (23x140 mm), equilibrated with
10 mmol l-1 Tris buffer, pH 7.9, and eluted in a 0-0.25 mol
l-1 NaCl linear gradient as described
(Fago et al., 2001
). The
isolated hemoglobins and the stripped hemolysate were concentrated under CO
pressure in an Amicon Ultrafiltration Cell or in Ultrafree-4 Centrifugal
Filter Units (Millipore, Billerica, MA, USA) (5000 MW cut-off) and dialysed
against three changes of CO-equilibrated 10 mmol l-1 Hepes buffer,
pH 7.7, 0.5 mmol l-1 EDTA. The samples were then divided into small
portions that were thawed immediately before O2 equilibrium
measurements according to standardized procedures
(Weber, 1992
;
Weber et al., 2000
). All
preparative steps were performed at 0-4°C. Recordings of the visible
spectrum (450-700 nm) during hemoglobin preparation showed no evidence of
oxidation, as judged by absorbance at 630 nm.
O2 equilibria of hemoglobin solutions at various
osmolalities; the osmotic stress method
O2 equilibria of the hemolysate and of solutions containing HbI
and HbII or HbII and HbIII at 1:1 molar ratios were measured at different
osmolalities at 10°C, 0.5 mmol l-1 heme concentration, in 0.1
mol l-1 Hepes buffer at pH 7.3, close to the physiological pH in
hagfish red blood cells (Tufts and
Boutilier, 1990). A modified gas diffusion chamber connected to
cascaded Wösthoff gas pumps, mixing pure N2 (>99.998%),
O2 and air, was used to obtain stepwise increases in O2
saturation, while changes in absorption at 436 nm were continuously recorded
(Weber, 1981
). Bicarbonate was
added to hemoglobin solutions by introducing 4% CO2 in the gas
mixture and adding 1 µmol l-1 carbonic anhydrase to catalyse the
rapid conversion of CO2 into bicarbonate and hydrogen ions.
Oxidation during O2 equilibrium measurements was typically less
than 5%, as judged from changes in absorbance between 0% and 100% oxygenation.
Hill plots, log[S/(1S)] versus
logPO2 (where S is the fractional
saturation), were used to interpolate O2 affinity
(P50, half-saturation O2 tension) and
cooperativity (expressed as Hill coefficient n50, the
slope of the Hill plot at half-saturation).
A BMS 2 MK 2 (Radiometer, Copenhagen, Denmark) thermostatted microelectrode was used to measure pH. In O2 equilibrium experiments made in the presence of CO2, pH measurements were carried out in hemoglobin subsamples placed in microtonometers equilibrated near P50 and in the presence of 4% CO2.
Different water activities (aw) in the samples were
obtained by adding glucose or glycine to final concentrations ranging between
1 and 1000 mmol l-1. These solutes were chosen for their opposite
effects on the dielectric constant of the solution, in order to eliminate
factors related to electrostatic effects that may affect O2
affinity (Colombo and Bonilla-Rodriguez,
1996). Osmolalities (Osm) were measured on a Semi-Micro Osmometer
(Knauer, Kiel, Germany) in 50 µl samples and water activities
(aw) were calculated
(Colombo and Bonilla-Rodriguez,
1996
) as:
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O2 equilibria of red blood cells suspensions at various
osmolalities
Individual blood samples were centrifuged at 40 g for
plasma removal, and the red blood cells were washed in ice-cold 1000 mosmol
kg-1 Ringer consisting of (mmol l-1): 504 NaCl, 8 KCl, 5
CaCl2, 3 MgSO4, 9 MgCl2, 5 glucose and 13.4
NaHCO3. The cells were then resuspended by adding the same volume
of Ringer as the volume of plasma removed and stored on ice overnight. The
cells were then washed twice in several volumes of either 250, 500, 1000,
2000, 3000 or 4000 mosmol kg-1 saline Ringer, centrifuged and
resuspended by adding solutions of the different molalities to obtain the
original blood volumes. The Ringer solution was made hypo-osmotic by adding
distilled water containing 13.4 mmol l-1 NaHCO3 and
hyperosmotic by adding 1 mol l-1 NaCl. The ensuing
shrinkage/swelling of the red blood cells was confirmed by measurement of the
hematocrit and followed the linear relationship Hct/Hct1000=0.71
(mosmol kg-1/1000)+ 0.29, where Hct1000 is the
hematocrit at 1000 mosmol kg-1
(Dohn and Malte, 1998).
Oxygen equilibrium curves were measured by the diffusion chamber method described above. Spectra in the range 410-600 nm (2 nm wavelength interval and a measuring time of 0.4 s at each wavelength) were recorded by a Cary 50 Probe spectrophotometer (Palo Alto, CA, USA) connected to a computer employing the Cary WinUV program for spectra analysis. After baseline recording with the microscope slide in the chamber, a 1 µl sample of red blood cell suspension was spread on the measurement area of the slide and left in the chamber for stabilization for at least 20 min. Spectra were measured after equilibration to 1, 2, 4, 8, 16, 32 and 55% air, as well as in pure N2 (deoxy) and 100% air (oxy). The concentration of CO2 in the gas mixtures entering the chamber was held constant at 0.5% (approx. 3.5 mmHg, 0.47 kPa). All measurements were carried out at 12°C. A spread-sheet programme (developed by H. Malte and S. Frische, Aarhus University) was used to calculate the O2 saturations at each O2 tension by linear least-square fitting of the oxy and deoxy reference spectra to the observed spectra. P50 and n50 values were interpolated from Hill plots as described above.
The number of O2-linked water and solute molecules was calculated from Equations (2) and (3), respectively. The osmolality values employed for the calculations were those of the Ringer solution.
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Results |
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Fig. 3 shows O2
equilibria of red blood cell suspensions at different osmolalities. An
increase in osmolality of the medium from 1000 to 4000 mosmol kg-1
causes shrinkage of the red blood cells and increased O2 affinity
(the O2 equilibrium curve shifts to the left), whereas
O2 affinity decreases upon swelling (the curve shifts to the
right), while cooperativity remains close to unity for all osmolalities, which
agrees with data for M. glutinosa blood
(Perry et al., 1993).
Hemoglobin concentrations in intact red blood cells (before equilibration at
different osmolalities) ranged from 9.3 to 11.7 mmol l-1 heme.
Following equilibration at different osmolalities, protein concentration
linearly increased from 3.3±0.4 mmol l-1 heme (250 mosmol
kg-1) to 22.3±2.5 mmol l-1 heme (4000 mosmol
kg-1). Fig. 4 shows
the relative shifts in P50 values as a function of water
activity for both intact red blood cells and the hemolysate in the absence and
presence of CO2. As shown, the slope of the regression line for red
blood cells is remarkably similar to that of the hemolysate in the range
0.25-2.0 osmol kg-1, but increases at higher osmolalities
(Fig. 4, Table 1), indicating that a
greater number of water molecules is released upon oxygenation. Solutes from
the Ringer solution do not bind directly to hemoglobin in red blood cells,
which is indicated by the dependence of the relative shifts in
P50 as a function of logeOsm
(Table 1).
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Discussion |
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M. glutinosa possess a complex system of interacting and
non-interacting monomeric hemoglobins that reversibly associate when
deoxygenated to form dimers and tetramers
(Fago and Weber, 1995;
Fago et al., 2001
). One would
expect that an increase in water activity would favour dissociation into
monomers as the protein surface area exposed to solvent increases, but in
hagfish hemoglobin the opposite is true. Since water molecules stabilize the
dimeric/tetrameric state, it appears that during the association between
monomers, specific water binding sites become available that bridge individual
monomers. Accordingly, the number of water molecules stabilizing the
associated state neatly correlates with increasing tendency of the hemoglobins
to associate, being highest in HbI+II in the presence of bicarbonate, where
tetramers are formed upon deoxygenation, and lowest in HbII+III in the absence
of bicarbonate, where the hemoglobin remains monomeric when deoxygenated, as
observed by sedimentation velocity experiments
(Fago et al., 2001
).
Bicarbonate increases the tendency to associate and favours formation of
low-affinity oligomers (Fago et al.,
1999
,
2001
), and consequently the
number of water molecules involved in oligomer stabilization increases in the
presence of CO2 (Fig.
1, Table 1). In
addition, bicarbonate and water may have a synergistic effect in stabilizing
the dimeric and tetrameric states of the hemoglobin.
A major finding of this study is that O2 affinity is similarly
affected by changes in osmolalities (and water activities) both in red blood
cells and in the purified hemolysate; in both cases it increases as water
activity decreases. Moreover, around the physiological value of approximately
1000 mosmol kg-1 (logeaw approx.
-0.02) the number of water molecules stabilizing the oligomeric state of the
hemoglobin (that are liberated upon O2 binding) is similar in the
purified hemolysate and in the intact cells
(Fig. 4,
Table 1). This finding, which
takes advantage of the exceptional ability of M. glutinosa red blood
cells to withstand osmolality changes, reflects a true allosteric role of
water in regulating O2 binding at a cellular level. Since the
monomermonomer association constants are not known, it is not possible
to evaluate the extent of association of the hemoglobin complexes formed
either in hemolysate or in intact red blood cells at a specific protein
concentration. However, at much lower protein concentrations (60 µmol
l-1 heme) than those used in this study (500 µmol l-1
heme) and in the presence of bicarbonate, the deoxygenated hemolysate consists
entirely of monomers and tetramers, as shown by gel filtration and
ultracentrifugation experiments (Fago et al.,
1999,
2001
), indicating that
formation of the tetrameric species HbI+II is favoured over the dimeric
HbII+III species, and that water sensitivity in the hemolysate and in the red
blood cells may thus be largely attributed to HbI+II, although this species
represents only approximately 15% of the total hemoglobin
(Fago et al., 2001
). Moreover,
since the dependence of the O2 affinity on changes in water
activity becomes larger in intact red cells than in pure hemolysate at high
osmolalities (logeaw below -0.04), the
formation of other types of monomermonomer associations in shrunken
cells (i.e. where protein concentration is high) than those hitherto
identified in vitro cannot be excluded. Interestingly, the six
monomeric hemoglobins from the sea lamprey Petromyzon marinus create
a complex network of possible associations
(Rumen and Love, 1963
).
Alternatively, the weak binding of solute to oxygenated hemoglobin
(Table 1) may become strong at
higher solute concentration.
Changes in red blood cell volume have opposite effects on intracellular
water activity and protein concentration. Specifically, in swollen red blood
cells, an increase in water activity would shift the allosteric
monomeroligomer equilibrium towards oligomer formation and thus lower
O2 affinity, whereas protein dilution would favour monomer
formation and thus increase O2 affinity. Our results indicate that
the water effect is strong enough to overshadow protein dilution and to be
measurable during osmotic stress experiments. In lampreys, monomeric
hemoglobins similarly associate upon deoxygenation, and erythrocyte shrinking
(with the consequent increase in protein concentration) decreases
O2 affinity (Airaksinen and
Nikinmaa, 1995); this is in contrast to hagfish red blood cells,
suggesting that lamprey hemoglobins have little or no sensitivity to changes
in water activity. Accordingly, no water molecules were detected at the
dimeric interface of the deoxy form of the lamprey Petromyzon marinus
HbV (Heaslet and Royer, 1999
),
although this could be due to the fairly low resolution of the
crystallographic structure (2.7 Å).
Although the O2 affinity of hagfish hemoglobin depends on water
activity, blood O2 binding is unlikely to be affected via
this mechanism in the natural environment where salinity is constant.
Presumably hagfishes have never been exposed to changes in water osmolality
during evolution and they do not show the adrenergic red cell swelling upon
exposure to hypoxia encountered in teleosts, despite the increased production
of noradrenaline (Perry et al.,
1993). The low cooperativity and the small Bohr and anion effects
(except that of bicarbonate) observed in the hemoglobin of hagfish appear to
secure O2 transport under their natural conditions and mode of life
(constant salinity and temperature, slow-moving behaviour;
Wells et al., 1986
).
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Acknowledgments |
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