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INTRODUCTION |
The ability of fish to colonize a large variation of biotopes is
integrally related with the striking molecular and functional differentiation encountered in their hemoglobin
(Hb)1 systems. Variations in
the functional properties of Hb result partly from variations in
molecular structure that determine the intrinsic O2 binding
properties (1) and partly from regulatory changes in the
physicochemical conditions under which they operate in vivo,
such as red cell pH (that varies with ventilation rate and
catecholamine stimulation) and in the type and concentration of
heterotropic effectors like organic phosphates that decrease Hb-O2 affinity (2-6).
In addition to "anodic" Hbs (HbAn) that migrate
anodically under normal electrophoretic conditions (pH ~8.6) and have
relatively low O2 affinities and marked Bohr effects
(decreased O2 affinity that enhances O2 release
in the acid tissues) and Root effects (reduction in O2
binding capacity upon acidification that induces O2
unloading in the swim bladder and retina), many fish species express
"cathodic Hbs" (HbCa) that have high isoelectric points
and lack significant pH effects suggesting that they safeguard
O2 transport to tissues under hypoxic and acidotic
conditions (7-9). Previous studies on the physiological and molecular
implications of Hb multiplicity in fish have been concentrated on only
a few species, such as rainbow trout, Onchorhynchus mykiss,
and the eel Anguilla anguilla that exhibit radical
differences, indicating the existence of diverse molecular strategies
among teleosts. Thus, whereas cathodic HbI of trout lacks a Bohr effect and is insensitive to phosphate effectors (10, 11), cathodic eel
HbCa shows a reverse Bohr effect in the absence of
phosphates and greater phosphate sensitivity than anodic eel
HbAn (12-14). Also, whereas the NTP pool of trout
erythrocytes almost entirely consists of ATP, GTP is the main effector
in eels, where it shows a greater effect on Hb-O2 affinity
and greater decreases in concentration following hypoxic exposure than
ATP (12).
Deoxygenated Hb may also bind the cytoplasmic domain of erythrocytic
band 3 proteins (cd-B3) in competition with glycolytic enzymes, as
demonstrated for the human proteins (15, 16). The absence of effects of
peptides corresponding to N-terminal fragments of trout cd-B3 on
O2 affinity of anodic trout HbIV, despite pronounced
effects on human Hb (17), calls for closer study of Hb-band 3 interaction in fish.
Hoplosternum littorale, a small, heavily armored
catfish from the Amazon basin, is an ideal model for investigating
molecular adaptations in Hb function to extreme environmental
conditions, bimodal breathing and modes of life. While using gills for
gas exchange in well aerated water, it surfaces to swallow air in O2-deficient waters and has a thin-walled part of the
intestine that is kept devoid of food and appears to be a site for
aerial gas exchange (18). The fish constructs floating nests of dead weed that expose the developing embryos to higher O2
tensions than those prevailing in the water (18). A further peculiarity is that its red cells contain the "mammalian" cofactor DPG as well
as "piscine" effectors ATP and GTP in approximately equal concentrations and that the DPG levels vary with environmental temperature (19, 20). It has single anodic and cathodic Hbs (that
exhibits and lacks a Root effect, respectively) and shows no evidence
for polymorphism in Hb multiplicity (21).
We report on the interactive effects of pH, the naturally occurring
effectors ATP, GTP, DPG, and Cl
and of IHP on
O2 binding of Hoplosternum HbAn and
HbCa, and on the oxygenation-linked interaction with a
synthetic peptide corresponding to the N terminus of trout cd-B3. In
order to understand the structural and allosteric basis for its
distinctive functional characteristics, we also analyzed the
O2 equilibria of HbCa in terms of the two-state
model for allosteric transitions (22), and we determined the primary
structures of its globin chains.
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EXPERIMENTAL PROCEDURES |
Adult H. littorale (65) (14-16 cm, 45-66 × g)
locally known as tamoata were collected by throw net in the Solimoes
river near Marchantaria, Brazil. Blood was taken in heparinized
syringes from the caudal blood vessels. Saline-washed red cells were
frozen at 
80 °C until use.
Hb was prepared as described previously (23) and dialyzed against 0.02 M Tris-HCl buffer, pH 8.4 (at 5 °C). Electrophoresis on
cellulose acetate strips revealed only two Hb components that were
separated by anion exchange chromatography on a 27 × 2 cm DEAE-Sephacel column equilibrated with the dialysis buffer and eluted
in a 0-0.1 M NaCl gradient. Separated fractions were
dialyzed for 24 h against three changes of CO-equilibrated 0.01 M HEPES, pH 7.7, containing 5·104
M EDTA. All preparative steps were carried out at
0-5 °C. The Hb was frozen at 80 °C in 90-150-µl aliquots
that were individually thawed immediately before experimentation.
Stripped human Hb for control measurements was prepared as described
previously (24) from blood of a non-smoking adult.
O2 Binding--
O2 binding equilibria
were measured using a modified diffusion chamber, where ultrathin
layers of Hb solution were equilibrated with pure (>99.998%)
N2 or O2 or stepped mixes of these and air prepared with Wösthoff pumps to ensure full equilibration at each
step (23, 25). The pH of Hb solutions was adjusted using HEPES buffers
for pH ~6.5-8.2, MES buffers for lower, and glycine buffer for
higher pH values (final buffer concentration, 0.10 M). The
pH was measured in oxygenated (air-equilibrated) Hb samples using a
BMS2 Mk2 Blood Micro system and PHM 64 Research pH meter (Radiometer,
Copenhagen, Denmark). Chloride was added as KCl and measured using a
Radiometer CMT10 chloride titrator. ATP, GTP, and DPG concentrations in
stock samples were assayed using Sigma test chemicals. The effects of
anions on O2 equilibria were measured at pH near 7.5 and
7.0, whereafter the P50 (half-saturation
O2 tension) and n50 (Hill
cooperativity coefficient at P50) values at
these exact pH values were interpolated from linear regressions. The
overall heat of oxygenation 
H', which includes the heat
of solution of O2 (13 kJ·mole1) and the
heats of processes linked to O2 binding such as proton and
anion dissociation, was evaluated as
R·(lnP50)/(1/T),
where R is the gas constant. The effects of synthetic
peptides corresponding to the first 10 and 20 amino acid residues of
trout cd-B3 on Hb-O2 affinity was examined as earlier
described (17). The sequence of the 20-mer peptide is
Met-Glu-Asn-Asp-Leu-Ser-Phe-Gly-Glu-Asp-Val-Met-Ser-Tyr-Glu-Glu-Glu-Ser-Asp-Ser (the 10-mer comprises the first 10 residues) (26).
To analyze the allosteric interactions, precise O2
equilibria measured with focus on extreme (low and high) saturation
values were analyzed in terms of the MWC model (22), evaluating
KT and KR, the allosteric
constant (L), and derived parameters, including q, G, Pm, and
nmax (where q is the number of
interacting O2-binding sites; G is the free
energy of cooperativity; Pm is the median O2 tension; and nmax is the maximum
cooperativity) (see Table I) as described by Weber et al.
(27).
Reverse-phase Chromatography--
Heme was removed from purified
HbCa by acid-acetone precipitation (28). Globin chains were
reduced for 5 min at 100 °C in 50 mM Tris-HCl, pH 7.0, 6 M guanidinium chloride, 1% 2-mercaptoethanol and modified
with 4-vinylpyridine and maleic anhydride as described previously (29).
Samples were acidified with trifluoroacetic acid and applied to a
Prosphere RP C4 5-µm column (300 Å; 4.6 × 250 mm;
Alltech Associates, Inc.) equilibrated with 5% acetonitrile in 0.1%
aqueous trifluoroacetic acid. The samples were eluted with a linear
gradient of 5-75% acetonitrile in 0.1% trifluoroacetic acid over 45 min at a flow rate of 1 ml/min. Absorbance of the eluate was monitored
at 280 nm.
Enzymatic Digestion and Peptide Isolation--
Globin chains
were digested with trypsin at an enzyme:substrate ratio of 1:50 in 200 mM NH4HCO3, pH 8.3, at 37 °C for
6 h. The digested products were isolated by RP-HPLC as described
for the globin chain isolation. The amino acid sequence of peptides was
determined with an automated protein sequencer ABI 471 B (Applied Biosystems), according to the manufacturer's recommendations.
Primer Design and cDNA Sequencing--
By using the amino
acid sequence of the 
chain, the degenerated primer HOPLO F1,
TGGGGNAARATHCAYATHGA, a 20-mer with 144 redundancies, was designed
corresponding to the sense strand predicted by the peptide fragment
WGKIHID (Fig. 10). Total RNA was isolated from intact erythrocytes with
a micro RNA isolation kit (Stratagene). First strand cDNA was
synthesized with MMLV-RT (Promega) using an oligo(dT) primer. PCR
reactions were carried out using HOPLO F1 and oligo(dT). The PCRs were
carried out for 30 cycles of 94 °C for 30 s, 50 °C for 1.0 min, and 72 °C for 1.5 min with Taq polymerase on a
GeneAmp PCR system 9600 (Perkin-Elmer). Sequencing was then performed
with HOPLO F1 as primer on an ABI 377 automatic sequencer (Applied
Biosystems, Inc.) according to the manufacturer's recommendations.
Electrospray Ionization Mass Spectrometry--
Electrospray data
were acquired on a Quattro II triple quadrupole mass spectrometer
(Micromass Ltd.) as described elsewhere (30).
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RESULTS |
Oxygenation Studies--
Anion exchange chromatography resolves
the Hb into two distinct fractions, HbCa and
HbAn, occurring in a ratio of approximately 38:62 (Fig.
1). The oxygenation characteristics of
HbAn and HbCa are radically different. At pH
7.2, the approximate intracellular value, the affinity of stripped
HbCa markedly exceeds that of
HbAn (P50 = 2.4 and 8.7 mm Hg,
respectively, at 25 °C) (Figs. 2 and 3). In contrast to the pronounced normal
Bohr effect in HbAn (
= log
P50/pH = 0.56 at pH 7.2),
HbCa exhibits a marked, reverse Bohr effect ( = +0.38). Due to opposite pH effects the functional differentiation
between the two isoHbs increases with falling pH.
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Fig. 1.
Separation of cathodic HbCa and
anodic HbAn of Hoplosternum littorale by
DEAE-ion exchange chromatography. , absorption at 540 nm;  ,
chloride concentration; rectangles, fractions pooled for
functional and structural characterization.
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Fig. 2.
O2-binding curves of
Hoplosternum HbAn and HbCa,
measured in 0.1 M HEPES buffer at 25 °C, showing
opposite Bohr effects. Heme concentration, 0.15 mM
(HbAn) and 0.14 mM (HbCa).
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Fig. 3.
P50 and
n50 values of Hoplosternum
HbAn and HbCa at 25 °C and their pH
dependence in the absence (diamonds) and presence
(circles) of chloride and the absence (open
symbols) and presence (solid symbols) of
saturating ATP concentrations (ATP:Hb4 ratio
 100). As shown ATP and chloride enhance the
normal Bohr effect in HbAn and reverse the Bohr effect in
HbCa. Histograms show log P50
values induced at pH 7.2 by 0.10 M chloride (solid
columns), ATP (open columns), and 0.10 M
chloride and ATP (obliquely hatched columns). Other
conditions as in Fig. 2.
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HbCa exhibits much greater sensitivity to ATP than
HbAn. The phosphate effects increase with falling pH,
whereby the presence of ATP induces a slight normal Bohr effect in
HbCa ( = 0.14 at pH 7.2) and almost obliterates
the affinity difference between the two Hb components (Fig. 3).
Significantly, ATP alone decreases O2 affinity of both
components more than ATP in the presence of 100 mM
Cl (as illustrated for pH 7.2 by the log
P50 columns in Fig. 3). The Hill coefficient
n50 approximates 2.0 in both Hbs at pH 6.5-8.0, decreases at low and high pH to 1.5 in HbAn, and at low pH
to 1.7 in HbCa (Fig. 3) but increases to 2.4 in
HbCa in the presence of ATP.
The difference between the Bohr effect curves at 10 and 25 °C (Fig.
4) illustrates a large overall
temperature effect (H' about 85 kJ·mol-1)
in HbAn at high pH (8.7) where the Bohr effect and
phosphate binding disappear (cf. Fig. 3). At lower pH, where
the Bohr effect is operative, the enthalpy of oxygenation decreases to
approximately 45 kJ·mol1 at pH 6.8 reflecting
endothermic proton release. Given that the Bohr factor (0.65) gives the
moles of protons dissociated per mol of O2 bound, the
enthalpy difference (+40 kJ·mol of heme) indicates an apparent heat
of proton dissociation of 62 kJ·mol-1. Analogously
the increase in enthalpy for HbCa (by approximately 18 kJ·mol1 as pH decreases from pH 9, Fig. 4) reflects
proton association upon O2 binding, in accordance with the
reverse Bohr effect. Related to the Bohr factor (+0.38) this increase
indicates an apparent ionization enthalpy of approximately 47 kJ per
mol of protons bound. These values may, however, be biased by
thermodynamic contributions from other oxygenation linked processes,
such as Cl binding, that may account for the lower
H value found in HbAn at pH 6.0 than at pH
8.5 (where oxygen-linked proton binding approximates zero).
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Fig. 4.
Bohr effects (log
P50/pH) of
Hoplosternum HbAn ( ,  ) and
HbCa ( ,  ) at 10 (, ) and 25 °C (, )
(upper panel), and the pH dependence of the overall
heat of oxygenation (H') (lower
panel) measured in the presence of 0.10 M
KCl. Heme concentration, 0.14 mM.
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Chloride ions reduce O2 affinity of both HbAn
and HbCa, except for HbA at pH >7.7 where 0.1 M chloride increased affinity (Fig. 3). Below pH 7.7 chloride and saturating ATP concentration raise the Bohr effect of
HbAn to 0.65 and 1.1, respectively. The chloride
sensitivity of Hoplosternum Hbs is low compared with human
Hb. At pH 7.0, 100 mM chloride increases log
P50 of HbAn and HbCa by
only 0.07 units, compared with 0.45 units in human Hb (Figs. 3 and
5).
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Fig. 5.
Effects of chloride concentration on
P50 of Hoplosternum
HbAn ( ) and HbCa ( ) at pH 7.0 (left panel) and pH 7.5 (right
panel), compared with effects on human Hb (, after Ref.
24) at pH 7.0 (left panel) and 7.4 (right
panel). Heme concentration, 0.6 mM.
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Dose-response curves for the effects of anions (A) on
O2 affinity (Figs. 5 and 6)
can be interpreted in terms of the basic linkage equation: log
Pm/log[A] = X, where X is the amount of anion bound per
(de-)oxygenated heme. Provided close agreement exists between
P50 and Pm values (see
below) and between the concentration and activity of the effector, the
slopes of log P50 versus
log[A] plots at midpoint (designated by 
) approach a
limiting value that cannot be smaller than the number of oxygen-linked
binding sites per heme (24, 31).
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Fig. 6.
Effects of DPG, ATP, GTP, and IHP
concentrations on P50 values of Hoplosternum
HbAn (left panels) and
HbCa (right panels) at pH 7.0 (upper
panels) and pH 7.5 (lower panels), measured
at 25 °C in the presence of 0.10 M KCl. Heme
concentrations, 0.14 (HbCa) and 0.15 (HbAn).
 indicates zero phosphate concentration.
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The log P50 versus
log[Cl] curves indicate values of approximately
0.22 for Hoplosternum HbAn and HbCa
compared with 0.48 for human Hb at pH 7.0 and lower values at higher pH
( = 0.13-0.14 for Hoplosternum Hbs at pH 7.5 and
0.45 for human Hb at pH 7.4) (Fig. 5).
Dose-response curves for the phosphate effectors (Fig. 6) reveal the
order of allosteric effectivity as DPG < ATP < GTP < IHP, greater sensitivities of HbCa than HbAn to
all effectors and greater effects at pH 7.0 than at pH 7.5, where the
cationic phosphate-binding sites are less charged. Curiously, DPG and
low concentrations of the other effectors increased O2 affinity of HbAn at pH 7.5 (Fig. 6D).
For HbCa the curves at pH 7.0 and 7.5 (Fig. 6, A
and B) indicate lower maximum P50
values induced by DPG than by IHP, ATP, and GTP, indicating formation
of additional bonds (cf. Ref. 32) with the latter effectors
at saturating phosphate:Hb ratios. Whereas the slope for DPG and
HbCa ( = 0.22) tallies with the release of one
phosphate molecule per oxygenated tetramer, higher values (>0.25)
obtained for ATP, GTP, and IHP suggest the existence of additional
sites of phosphate interaction.
Interpolated on the basis of the P50 maximum
induced by IHP (Fig. 6A), the data indicate apparent
dissociation equilibrium constants, Ka (estimated as
the effector concentration that induces half of the maximum change in
log P50) for the reactions of HbCa
with ATP, GTP, and IHP at pH 7.0 of approximately 11 × 104, 5.4 × 104, and 2.2 × 104 M, respectively. Interpolated in terms of
the P50 maximum induced by DPG, the constant for
DPG approximates 13.2 × 104 M. Compared
with values for the reaction of DPG with human and Eskimo dog Hbs
(3.2 × 104 M at pH 7.5 and ~1 × 104 M at pH 7.2, respectively, at 20 °C
and in the presence of 100 mM Cl) (33, 34),
this illustrates relatively low DPG sensitivity in
Hoplosternum HbCa.
In contrast to the pronounced effects of the 10- and 20-mer synthetic
trout band 3 peptides on the O2 affinity of human Hb (Fig.
7; see also Ref. 17), the peptides had no
effect on Hoplosternum HbAn at pH 7.2 and only
marginally decreased the O2 affinity at lower pH (6.4)
(Fig. 7). This aligns with the absence of effects in trout Hbs I-IV
(17),2 despite the large
effects of these peptides in human Hb (17). Significantly, the peptide
exerts a distinct effect on Hoplosternum HbCa at
pH 7.2 and an even greater effect at lower pH (6.58) (Fig. 7). The
effect on human Hb (17) and the marked pH-dependent effects
in Hoplosternum HbCa (Fig. 7) attest to the
functionality of the peptides and the presence of a putative band
3-binding site in both Hbs.
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Fig. 7.
Semilogarithmic plots of O2
equilibria of Hoplosternum and human Hbs in the
absence (open symbols) and presence (closed
symbols) of synthetic peptides corresponding to the
N-terminal segment of the cytoplasmic fragment of band 3 protein
(cd-B3) of rainbow trout at the indicated pH values. Effects of
20-mer peptide on human Hb and Hoplosternum HbAn
(left panel) and of 10-mer peptide on
Hoplosternum HbCa (right panel). Heme
concentration, 0.30 mM (Hoplosternum Hbs) and
0.63 mM (human Hb). Peptide:tetrameric Hb ratio, 5.
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The allosteric and derived MWC model parameters are given in Table
I. The agreement between
n50 and nmax and between
P50 and Pm values
reflects highly symmetrical O2 equilibrium curves that
permit rigorous analysis of P50 plots. Moreover,
the mean value for the number of interacting O2-binding
sites per molecule (q = 4.03 ± 0.74), obtained
when q was fit along with the other parameters to obtain the
best possible fit in the 13 condition sets described in Table I,
tallies neatly with a tetrameric structure. The derived parameters
summarized in Table I were thus obtained with q fixed at
4.
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Table I
MWC and derived parameters for Hoplosternum HbCa and their pH
and cofactor sensitivities, derived for q = 4
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Extended Hill plots for the effects of pH and organic phosphates in
HbCa are shown (Figs. 8 and
9). In contrast to anodic vertebrate Hbs where the normal alkaline Bohr effect primarily results from a decrease
in KT with increasing proton concentrations (23,
35, 36), the control mechanism of the reverse Bohr effect of
Hoplosternum HbCa is an increase in
KT with falling pH (Fig. 8, Table I), indicating a more constrained T state with increasing pH. The Bohr factor of the
deoxygenated (T state) Hb markedly exceeds that at median saturation
( = +0.35 versus +0.25, respectively, at pH 7-8;
Fig. 8, inset). Increased pH accordingly raises the free
energy of cooperativity (G increases from approximately
6.5 to 8.5 kJ·mol between pH 7 and 8, see Table I) as illustrated by
the greater distance between the upper and lower linear asymptotes of
the extended Hill plots at high pH (see Fig. 8). The allosteric
constant L of Hoplosternum HbCa
increases at high pH (Table I), compared with the opposite effect in
human and anodic fish Hbs (23, 35).
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Fig. 8.
Extended Hill plots of stripped
Hoplosternum HbCa at 25 °C and the
indicated pH values. As shown, KT and
KR values are evident from intersections of
upper and lower asymptotes of slope unity with x axis at log
Y/(1 Y) = 0. Inset, pH
dependence of KT, 1/Pm,
and KR values. Heme concentration, 0.64 mM.
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Fig. 9.
Extended Hill plots of stripped
Hoplosternum HbCa at 25 °C and pH 7.5 in the absence of phosphates (str) and in the presence
of ATP and GTP (left panel) and DPG and IHP
(right panel) at the indicated phosphate to
Hb(tetramer) ratios. Insets, dependence of
log(KT, 1/Pm, and
KR (mm Hg1)) on log[phosphate].
Heme concentration, 0.64 mM.
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In contrast to protons, ATP, GTP, DPG, and IHP modulate O2
affinity of Hoplosternum HbCa by decreasing
KT (Fig. 9) as in anodic mammalian and fish Hbs (23, 35, 37). The values for KT,
Pm, and KR (deduced from the slopes of data sets in the inset of Fig. 9) reflect T
state, median, and R state "DPG factors" of 0.33, 0.25, and 0.0. The lack of pronounced effects on KR values in
Hoplosternum HbCa (Fig. 9, inset)
differs from the reductions in KR observed in the presence of high NTP:Hb4 ratios in anodic tench Hb
(23).
Structural Characterization--
Separation of the 
and chains of the Hoplosternum HbCa by RP-HPLC and
their molecular masses determined by electrospray ionization mass
spectrometry as 15,542.0 and 15,978.0, respectively, are shown in Fig.
10.
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Fig. 10.
Separation of globin chains of
Hoplosternum HbCa by RP-HPLC
(inset) and the electrospray mass spectrum obtained
for the chain (left)
and chain (right).
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N-terminal sequencing showed that the chain was blocked, whereas
the chain was directly accessible for Edman degradation, as
commonly observed in teleost Hbs. The chain was unblocked by
heating at 55 °C in 30% trifluoroacetic acid for 3 h. The
S-pyridylethylated and
S-pyridylethylated/maleilated globin chains were digested with trypsin, and the resulting peptides were separated by RP-HPLC. All
peaks were sequenced. Some peaks contained 2 or 3 peptides, but their
sequences were deduced unambiguously by subtraction of peptides
sequenced in other peaks.
The and chains of Hoplosternum HbCa
consist of 142 and 146 amino acid residues, respectively. Alignment of
the globin chains with those for eel A. anguilla (13) Hb and
rainbow trout O. mykiss (38) Hb is presented in Fig.
11. The sequences align well and
without any ambiguity with other fish globin chains. In order to
confirm the sequence showed in Fig. 11, we performed partial cDNA
sequencing as described under "Experimental Procedures." The amino
acid sequence thus deduced confirms the sequence obtained at the
protein level.
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Fig. 11.
Alignment of the amino acid sequences
of and chains of
Hoplosternum HbCa (Swiss-Prot data base
accession numbers P82315 and P82316, respectively) with those of
eel (A. anguilla) cathodic HbCa
(accession numbers P80726 and P80727, respectively) and trout
(O. mykiss) anodic HbIV (accession numbers P14527 and
P02141) and cathodic HbI (accession numbers P02019 and
P02142).
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The sequence-deduced molecular weights are 15,544.1 and 15,976.3 for
the and chains, respectively. These values are in excellent
agreement with the experimentally determined mass data (15,542.0 ± 2.0 and 15,978.0 ± 2.0 for the and chains,
respectively) where the mass for the chain is corrected for the
N-terminal acetylation.
Remarkably, position NA2(2) is occupied by His, as in mammals, in
contrast to other teleosts that have Glu or Asp (exceptionally Lys)
(39) at this phosphate-binding site. Similarly notable is the presence
of Ser at H-21(143), compared with His in mammals and Lys or Arg in
other fish (except trout HbI that has Ser and lacks phosphate sensitivity).
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DISCUSSION |
The marked functional differentiation between
Hoplosternum HbAn and HbCa agrees
with earlier findings of Garlick et al. (21). In contrast to
their study carried out in the presence of ionic (Tris/BisTris) buffers
that may perturb the Bohr and phosphate effects due to higher chloride
levels at low pH values (24), the present work carried out using
zwitterionic HEPES buffer shows much lower Bohr factors ( = 0.56 compared with 0.98 for HbAn).
The Reverse Bohr Effect--
What may be the significance of a
reverse Bohr effect in HbCa that is obliterated by ATP? In
view of the greater reduction of O2 affinity by phosphates
at low pH, we propose that a reverse Bohr effect in phosphate-free
solution is a precondition for small in vivo pH effects
associated with pronounced phosphate sensitivity.
Apart from Hoplosternum, Hbs with pronounced reverse Bohr
effects occur in the facultative air-breathing teleost
Pterygoplichthys pardalis (21, 40), the surface skimmer
Mylossoma sp. (41), frog tadpoles, and aquatic salamanders
(cf. Ref. 13) suggesting implication in the utilization of
alternative sources of O2. The reverse Bohr effect and
strong phosphate sensitivity in Hoplosternum HbCa contrast with lack of Bohr and NTP effects in cathodic
trout HbI but accord with data for eel Anguilla (12-14),
Mylossoma (41), and Pterygoplichtys (40),
indicating that the intensively studied trout HbI is an exceptional
rather than prototype cathodic Hb.
In human Hb, the main Bohr groups are N-terminal Val-NA1(1) and the
C-terminal His-HC3(146) that account for about 30 and 50-65%,
respectively, of the normal Bohr effect, whereas His-H21(143) is
considered to be involved in the expression of the reverse ("acid")
Bohr effect that reflects the uptake of protons upon oxygenation at low
pH (<6.5) (42-44). With Val-NA1 acetylated in fish Hbs, the absence
of a normal Bohr effect in stripped Hoplosternum HbCa correlates with the His 
Phe-HC3(146)
replacement, as found in cathodic Hbs of trout, eel, and catfish (7,
13, 38). The reverse Bohr effect becomes apparent only when the major
alkaline Bohr groups are replaced (as in cathodic Hbs) or inoperative
(as in anodic Hbs that exhibit reverse Bohr effects at high pH) (13, 45). Apart from the HC3(146) substitution, Hoplosternum
HbCa shows a His Asn-FG4(94) replacement that also
is encountered in eel HbCa and other reverse Bohr effect
Hbs, providing further evidence for involvement of His-FG4(94) in
the alkaline Bohr effect of fish Hbs (45). Interestingly,
Ser-F9(93), which typically is conserved in fish Hbs with normal
Bohr and Root effects and which has been considered to donate a
hydrogen bond to His-HC3(146) (46), is substituted by Cys in
Hoplosternum HbCa and by Asn in eel
HbCa. Cys at F9(93) is another mammalian trait and
highly exceptional in fish Hbs.
The molecular mechanism proposed for the reverse Bohr effect in eel
HbCa (13) visualizes the implication of the residues at the
phosphate-binding site that in fish Hbs include Val-NA1(1),
Glu-NA2(2), Lys-EF6(82), and Arg-H21(143). In the T state the
proximity of positively charged amino acid residues in the central
cavity reduces their affinity for protons (whereas their
pKa values are normal in the R state), whereby the
groups implicated in organic phosphate binding become reverse Bohr
groups in the absence of phosphates. In other words, protons
destabilize the T state, as is evident from the increase of
KT with pH decrease, whereas the O2
affinity of the R state is practically unaffected (Fig. 8; Ref. 14). Accordingly, the reverse Bohr effect in Hoplosternum
HbCa having His at NA2(2) is almost twice as large as
that in eel HbCa having Glu-NA2(2) ( = +0.38 and
+0.2, respectively), indicating that more positively charged groups in
the central cavity contribute to this effect in
Hoplosternum.
To our knowledge the increase in overall oxygenation enthalpy of
HbCa (increased temperature sensitivity) with falling pH
(Fig. 4) provides the first demonstration of the thermodynamic
consequences of O2-linked proton binding associated with a
reverse Bohr effect. The opposite pH dependence of the temperature
effects in HbAn and HbCa (Fig. 4) would tend to
keep a constant and pH-independent in vivo heat of oxygenation.
Organic Phosphate Interaction--
Most fish Hbs have Glu at
NA2(2), which accepts hydrogen bonds from strain-free ATP and GTP
molecules (47). The presence of His at NA2(2) in
Hoplosternum HbCa is exceptional for teleosts
and other ectothermic vertebrates, where its distribution suggests a
correlation with air breathing or the presence of alternative red cell
phosphates. As listed (48) it occurs in the Hbs of the lungfish
Lepidosiren paradoxa, where 6-8% of its erythrocytic
phosphates is inositol diphosphate (49), the sharks Squalus
acanthias and Heterodontus portusjacksoni, where high
erythrocytic urea levels antagonize the modulator effectivity of ATP
(50), and tadpoles of the frog Rana catesbeiana and the toad
Xenopus laevis. Alternatively, the episodic occurrence of His-NA2 in elasmobranchs, lungfish, and developmental stages of higher
vertebrates suggests that it may be a phylogenetically primitive
character that was deleted in most non-mammalian vertebrates.
The occurrence of high levels of DPG in Hoplosternum
erythrocytes together with the "mammalian DPG-binding" residue
His-NA2(2) in HbCa appears to impart no selective
advantage for DPG binding, given that HbCa, as does
HbAn, exhibits markedly lower sensitivities to DPG than to
ATP and GTP (Fig. 6).
In view of the large phosphate effects in Hoplosternum
HbCa, the presence of uncharged Ser at H21(143),
compared with His in human Hb and Arg or Lys in other fish Hbs, is
unexpected and calls for reconsideration of the importance of
individual phosphate-binding sites. Moreover, Ser-H21(143) also
occurs in trout HbI and human fetal Hb that have no and small phosphate
effects, respectively. These findings suggest minor significance
of H21(143) for phosphate interaction or that the
GluHis-NA2(2) exchange in HbCa compensates for
absence of phosphate binding at this site. A recent NMR study of mutant
recombinant Hbs (44) similarly indicates that H21(143) is not
essential for DPG binding in the neutral pH range.
The progressively increasing effects of DPG, ATP, and GTP on
O2 affinity of Hoplosternum Hbs (Fig. 6)
contrast with human Hb that exhibits similar sensitivities to these
effectors (51) and similar binding constants for ATP and DPG (35). In
life, however, NTP effects may be drastically reduced as a result of complex formation with divalent cations, since the ATP-Mg2+
stability constant exceeds the DPG-Mg2+ constants by an
order of magnitude (52, 53).
The maximal slope of log P50 versus
log[DPG] curve (Fig. 6) is consistent with a 1:1 (DPG/Hb tetramer)
stoichiometry found in human and other mammalian Hbs (34). The values exceeding 0.25 observed with IHP and NTP (Fig. 6) could result
from binding of these effectors at additional sites. In dromedary Hb,
the pattern of Cl and phosphate binding similarly
indicates the presence of two polyanion sites per tetramer in deoxy and
oxygenated Hb, one of which becomes stronger and the other weaker, in
terms of affinity, as a result of oxygenation of the molecule (31).
The greater effects of phosphates on O2 affinity of
HbCa than HbAn in Hoplosternum
indicate a dominant role of HbCa in adapting blood
O2 affinity to variations under the environmental conditions. In the armored catfish Hypostomus and
Pterygoplichthys hypoxic exposure induces (gut) air
breathing and lowers ATP and GTP levels that may increase blood
O2 affinity and exploitation of the O2 reserves
during submersion (54, 55).
Chloride Effects--
In human Hb, Cl may act either
by neutralizing the positive charges in the central cavity without
binding to specific residues (56) or bind at specific sites (57). Two
major sites generally considered to be implicated in chloride binding
in human Hb are Val-NA1(l) that interacts with Ser-H14(131) and
Lys-EF6(82) that interacts with Val-NA1(l) (cf. Ref.
43). The values for Hoplosternum Hbs A and C (0.22) and
human Hb (0.48) (Fig. 5) indicate oxygenation-linked binding of 1 and 2 chloride ions, respectively, per tetramer, which accords with
acetylation of Val-NA1(l) in Hoplosternum
HbCa (Fig. 11).
The unexpected increase in O2 affinity induced in
Hoplosternum HbAn by 0.1 M
Cl at pH >7.5 (Fig. 3) may result from Cl
binding to the R state. It agrees with the observation that in the
presence of Cl, low DPG and ATP levels raise
O2 affinity of HbAn at pH 7.5 (Fig.
6D). Human Hb similarly provides evidence for Cl binding in the oxygenated state (58). The lesser
effects of ATP + Cl than of ATP in both Hb components
(Fig. 3) suggest that Cl ions block binding of the
phosphate effector at common binding sites.
Band 3 Peptide Effects--
The effect of the synthetic trout
cd-B3-peptide on the O2 affinity of Hoplosternum
HbCa provides the first evidence for functionally
significant interaction between fish Hb and band 3 proteins, suggesting
a possible transducer role for Hoplosternum HbCa
in regulating cellular processes in an oxygen-dependent
manner. Band 3 proteins are responsible for
HCO3/Cl exchange across
the red cell membranes, and Hb and glycolytic enzymes (such as
aldolase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase) compete for binding to their
cytoplasmic domains (16, 59, 60). Thus high O2 availability
releases Hb from the cd-B3 protein that thus become available for
inhibiting glycolytic activity and controlling red cell volume via
cAMP-dependent NaCl uptake (61).
Why does trout cd-B3 undergo oxygenation-linked binding with
Hoplosternum HbC and human Hb but not with trout Hbs? We
suggest that this is due to the presence of positively charged His at NA2(2) in Hoplosternum HbCa and human Hb,
given that the lack of effect on trout HbIV may result from repulsion
between the peptide and Asp at NA2(2) (17).
Allosteric Transitions--
The allosteric mechanisms controlling
O2 affinity and its dependence on allosteric effectors in
HbCa are illustrated by the parameters of the MWC model
(Table I). At pH 7.5 the
KT:KR ratio for
HbCa indicates a 22-fold increase in O2
affinity between fully deoxy and fully oxygenated Hb, compared with a
35-fold augmentation in human Hb at pH 7.4 (62). Phosphates decrease
KT without significantly changing
KR, thereby increasing G (Fig. 6,
Table I). As in eel (14), the reverse Bohr effect in
Hoplosternum HbCa is associated with an increase
in KT and resultant decrease in G
with falling pH (Table I). These effects are opposite those in human
and other anodic Hbs with normal Bohr effects and suggest that
increased bond energies constrain the molecules in the deoxy conformation as pH increases, in contrast to human Hb where additional bonds are formed at low pH (14, 63). The increase in deoxy state bond
energies between pH 7.0 and 8.0 (calculated from
GT = RT·ln
(KT7/KT8))
is 1.9 kJ·mol1. Analogously, increases in deoxy bond
energies imparted in the presence of DPG, ATP, GTP, and IHP at pH 7.5 (estimated as
RT·ln(KTP/KTstr))
are 1.46, 1.35, 2.21, and 2.91 kJ·mol1, respectively,
at phosphate/Hb 2.5, 2.19, 3.16, 3.98, and 4.19 kJ·mol1, respectively, at phosphate/Hb ~28. These
values are low compared with the stabilization energy of internal
hydrogen bonds (12 kJ·mol1) (64) illustrating that
small bond energy differences may account for large differences in the
effects of individual heterotropic phosphate effectors.
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