(Received for publication, February 15, 1995; and in revised form, May 25, 1995)
From the
As in other fish, the cathodic hemoglobin of the eel Anguilla anguilla is considered to play an important role in
oxygen transport under hypoxic and acidotic conditions. In the absence
of phosphates this hemoglobin shows a reverse Bohr effect and high
oxygen affinity, which is strongly modulated over a wide pH range by
GTP (whose concentration in the red blood cells varies with ambient
oxygen availability). GTP obliterates the reverse Bohr effect in the
cathodic hemoglobin. The molecular basis for the reverse Bohr effect in
fish hemoglobins has remained obscure due to the lack of structural
data. We have determined the complete amino acid sequence of the
and
chains of the cathodic hemoglobin of A. anguilla and
relate it to the oxygen equilibrium characteristics. Several
substitutions in crucial positions are observed compared with other
hemoglobins, such as the replacement of the C-terminal His of the
chain by Phe (that suppresses the alkaline Bohr effect) and of residues
at the switch region between
and
subunits (that may alter
the allosteric equilibrium, thus causing the high intrinsic oxygen
affinity and low cooperativity). The residues binding organic phosphate
in the
cleft of fish hemoglobins are conserved, which explains
the strong effect of GTP on oxygen affinity and suggests that these
residues contribute to the reverse Bohr effect in the absence of
alkaline Bohr groups. Moreover, His
that is
considered to be responsible for the reverse Bohr effect in human and
tadpole Hbs is replaced by Lys.
Fish hemoglobin (Hb) systems commonly exhibit functional
heterogeneity, which appears to be adaptive to oxygen (O)
transport under varying environmental and physiological conditions
(Weber, 1990). In eel such differentiation between individual Hb
components may favor an efficient O
transport during
exercise stress and migration between fresh and sea water or overland,
when severe respiratory and metabolic acidosis occurs (Powers and
Edmundson, 1972; Hyde et al., 1987). Eel Hb thus is well
suited for studying molecular and functional adaptations of fish Hbs to
environmental conditions, physiological requirements, and mode of life.
The hemolysate from the European eel Anguilla anguilla resolves into two major types of Hb components, an
electrophoretically ``cathodic'' Hb (HbC) with high
isoelectric point and an ``anodic'' fraction with low
isoelectric point. As in the trout Hb system that has been intensively
characterized in terms of molecular structure and oxygenation
properties (e.g. Binotti et al. (1971) and Weber et al. (1976b)), the cathodic Hb fraction of eels displays
high O affinity and low pH sensitivity (a small Bohr
effect), which appear to safeguard O
loading in the gills
under hypoxic and acidotic conditions, whereas the anodic fraction
displays a low affinity and a pronounced Bohr effect (Weber et
al., 1976a). In contrast to the cathodic Hb of trout (HbI), which
is insensitive to organic phosphates, that of the eel shows greater
sensitivity to the endogenous red cell phosphates GTP and ATP than does
the anodic Hb (Weber et al., 1976a).
Another peculiarity of eel HbC is a reverse Bohr effect. A reverse Bohr effect has been demonstrated in few other cases, namely in cathodic Hbs from the the American eel, Anguilla rostrata (Gillen and Riggs, 1973) and the Amazonian catfishes Pterygoplichthys pardalis (Brunori et al., 1979; Weber and Wood, 1979), Hoplosternum littorale (Garlick et al., 1979) and Mylossoma sp. (Martin et al., 1979), where the first mentioned two species are facultative air breathers and H. littorale is an obligate air breather. A reverse Bohr effect has also been observed in some amphibians, as in Hbs from bullfrog (Rana catesbeiana) tadpoles (Atha et al., 1979), the primitive salamander, Amphiuma means (Bonaventura et al., 1977), and the newts Triton crisatus (Morpurgo et al., 1970) and Triturus crisatus carnifex (Condòet al., 1981). The occurrence of reverse Bohr effect Hbs in air-breathing fishes and in amphibians that still possess gills, such as tadpoles and salamanders, suggests an implication of these Hbs in the transition from water to air breathing, given that air breathing in fishes is induced by water hypoxia and associated with episodes of internal acidosis.
An increase in the Hb O affinity at
low pH (reverse Bohr effect) indicates that oxygenation is associated
with proton binding rather than proton release as occurs in Hbs with a
normal alkaline Bohr effect. This suggests that reverse Bohr effect Hbs
have alternative ionizable groups or that their alkaline Bohr groups
are radically modified. In deoxy human HbA, proton binding responsible
for the alkaline Bohr effect occurs primarily at the N terminus of the
chain (Val
(NA1)) and at the C-terminal histidine
of the
chain (His
(HC3)) (Perutz, 1970; Riggs,
1988; Shih et al., 1993), whereas the endogenous phosphate
(DPG) binds at the N terminus of the
chain
(Val
(NA1)), Lys
(EF6), and
His
(H21) that face the central cavity of the Hb
molecule (Perutz, 1970; Arnone, 1972). The last mentioned residue is
considered to be responsible for the reverse, ``acid'' Bohr
effect, which in human HbA occurs at pH lower than 6.5 (Perutz et
al., 1980). The basic molecular mechanism of the reverse Bohr
effect in fish and amphibian Hbs is still obscure because of the lack
of structural data on these Hbs. Apart from the amino acid sequences of
the
and
chains of the Hb from the tadpole of the bullfrog R. catesbeiana (Maruyama et al., 1980; Watt et
al., 1980), sequences for Hbs with reverse Bohr effects are not
available, and only the C-terminal residues in A. rostrata (Gillen and Riggs, 1973) and A. means (Bonaventura et
al., 1977) Hbs have been identified.
In this paper we report
the complete amino acid sequence of the and
chains of A. anguilla cathodic Hb and new information on oxygenation
properties of this Hb. The elucidation of its primary structure
provides a structural basis for comprehending the special functional
properties of this Hb and provides insight into the mechanism of the
reverse Bohr effect.
Figure 1:
FPLC separation and
simultaneous stripping of the cathodic (HbC) and anodic (HbA) Hb
fractions on a HiLoad 16/10 Q Sepharose Hi Performance anion exchange
column. Absorbance was monitored at 254 nm (continuousline) and at 540 nm (discontinuousline). The inset shows isoelectrofocusing in the
pH range 3.5-10.0 on ultrathin polyacrylamide gel of the
separated HbC (C), HbA (A), unfractionated hemolysate (T), and isoelectric point standard proteins (S),
including, from top to bottom, -lactoglobulin A,
bovine carbonic anhydrase B, myoglobin acidic band, acidic, middle and
basic bands of lentil lectin (with pI values of 5.2, 5.85, 6.85, 8.15,
8.45 and 8.65, respectively) and marker protein near the cathode wick.
The cathode is at the bottom.
Figure 2:
RP-HPLC of the and
chains of
HbC. Absorbance was monitored at 280 nm. Details are given under
``Materials and Methods.''
Tryptic peptides of S-pyridylethylated HbC and
chains were purified by
RP-HPLC. All peptides were sequenced and aligned by homology with the
corresponding sequences of other fish globin chains. The amino acid
compositions deduced from the sequence of the individual tryptic
peptides matched those found by amino acid analyses. The primary
structure of the
and
chains of the cathodic Hb from A.
anguilla is reported in Fig.3, where the sequence portions
elucidated by automated Edman degradation of the intact chain, of the
Asp-Pro cleaved chain and of the peptides (obtained after CNBr and
trypsin cleavage) are indicated. As in other fish Hbs, the N terminus
of the
chain was blocked and thus unavailable to Edman
degradation.
Figure 3:
Amino acid sequence of the and
chains of the cathodic Hb of A. anguilla. T1 to T17 indicate the tryptic peptides; in the
chain, CB indicates the peptide obtained after subdigestion of T13 with cyanogen bromide; in the
chain, C indicates
fragment recovered in the ``core.'' The arrows denote the sequenced fragments of the intact
chain and of
the Asp-Pro cleaved chains. Dottedlines indicate the
sequence portion of the tryptic peptides not resolved by automated
Edman degradation. Discontinuouslines indicate the
sequence portions elucidated after trypsin digestion of the Asp-Pro
cleaved
chain. Helix notation as established for human HbA is
also reported.
In the chain an incomplete trypsin cleavage after
Lys
and Arg
yielded also the uncleaved T2-T3
and T5-T6 fragments. Again, the peptide bond
Lys
-Lys
was only partially digested, and
therefore the peptides T7 and T8 were eluted as double peaks, i.e. one containing and the other lacking an additional Lys residue.
The sequences of the tryptic peptides T13 and T6 of the
chain
that coeluted in the RP-HPLC purification step were unequivocally
established, taking advantage of their different yields (77 and 23%,
respectively). Some difficulties were encountered in the elucidation of
the amino acid sequence of the penultimate tryptic peptide T16 of the
chain and of the corresponding region of the
chain. In the
chain the peptide T16 was not recovered in sufficient amounts to
allow its complete sequencing, and the expected tryptic peptide from
Phe
to Lys
of the
chain was not
recovered at all after RP-HPLC purification. After trypsin digestion of
the
chain the peptide from Thr
to Arg
was obtained as an uncleaved fragment, despite the presence of
Lys
and Lys
. Its sequencing proceeded up to
Lys
. To obtain the sequence of the final segment this
fragment was subjected to further subdigestion with CNBr, and the
resulting peptide from Asp
to the C-terminal Arg was
sequenced, after a purification step on RP-HPLC. In the
chain the
last two residues of the T16 peptide were identified by cleavage of the
chain at the Asp-Pro bond with formic acid, purification of the
C-terminal fragment (from Pro
to Phe
) by
RP-HPLC and its subsequent digestion with trypsin. The peptide mixture
was then subjected directly to automated Edman degradation without
purification, and the complete amino acid sequence of the T16 peptide
was elucidated by difference. In another preparation the sequence of
the T16 peptide was determined after RP-HPLC purification of the core,
where the fragment Leu
-Lys
coeluted
with the (presumably) undigested
globin. The sequence of this
fragment (containing the T16 peptide) was determined after reaction of
the mixture with OPA at the 6th step of the Edman degradation to
eliminate undesired (i.e. non-Pro N-terminal) sequences.
The sequence-deduced molecular weights of the S-pyridylethylated and
chains were 15,338
(including acetylation at the N terminus) and 15,999, respectively.
These values are in agreement with the mass of the two globins
determined by electrospray mass spectrometry of 15,341 Da for the
chain and 15,997 Da for the
chain (Fig.4), which is
consistent with the presence of an acetyl group at the N terminus of
the
chain. Molecular weights of 15,100 and 15,300 obtained for
the
and
chains, in SDS-polyacrylamide gel electrophoresis
experiments are in accordance with these measurements.
Figure 4:
Transformed electrospray mass spectra of S-pyridylethylated (A) and
chains (B).
Figure 5: Bohr effect of HbC measured in 0.1 M HEPES buffer at 20 °C, in the presence and absence of 0.1 M KCl and at different GTP:Hb tetramer ratios. Heme concentration was 0.1 mM. Circles, freshly prepared Hb; othersymbols, samples that had been frozen at -80 °C.
O affinity of eel blood adapts rapidly to ambient
O
depletion primarily through a decrease in the
concentration of organic phosphates, particularly GTP, in the red
cells, whereas Hb heterogeneity (number and relative concentration of
the individual Hb components) remains the same after hypoxic and
normoxic acclimation (Wood and Johansen, 1972; Weber et al.,
1976a). Related to its higher sensitivity to nucleotide triphosphates,
the cathodic Hb from eel plays a major role in the adaptation of the
O
transport function to environmental hypoxia. The
associated increase in blood O
affinity improves O
loading and the arteriovenous O
content difference
under hypoxia (Wood and Johansen, 1973). As shown here, phosphate
modulation occurs even at high pH, such as results from
hyperventilation, which is a primary response in fish subjected to
hypoxic conditions (cf. Weber, 1981). In response to variable
O
tensions, O
affinity of HbC can thus be
modulated over a wide pH range by modification of the GTP:Hb ratio. As
a result of the high O
affinity and low pH sensitivity
cathodic Hbs will predictably secure O
loading under
internal acidosis induced by exercise stress. Although the
physiological significance of eel HbC is clear, the molecular
mechanisms governing the regulation of intrinsic O
affinity
of this Hb are not yet understood. The knowledge of the primary
structure reported here provides a basis for insight.
The inhibition of the alkaline Bohr effect that is
due to the C-terminal histidine of the chain seems to be a common
feature of several reverse Bohr effect Hbs; His
(HC3)
is replaced by Phe also in the American eel A. rostrata (Gillen and Riggs, 1973) and is deleted in A. means (Bonaventura et al., 1977). Although the C-terminal His
residue is conserved in R. catesbeiana tadpole Hb (Watt et
al., 1980), the alkaline Bohr effect here is inhibited by the
presence of an Asn (instead of Asp or Glu) residue at position 94(FG1)
of the
chain, which cannot form the salt bridge with the
C-terminal His in the T state. His(HC3)
is preserved in T.
crisatus carnifex Hb (Condòet al.,
1981), but no further sequence data are available for this Hb. Sequence
data for Amazonian fish Hbs that show a reverse Bohr effect are
similarly lacking.
His(H21) is considered to be
largely responsible for the reverse Bohr effect in human and tadpole
Hbs. In the deoxy human HbA this residue is located between
Lys
(EF6) and Lys
(HC1), so that its
imidazole group has a lower affinity for protons (i.e. a lower
pK
) than in the oxygenated form (Perutz et al., 1980). In tadpole Hb, position 144 of the
chain
is occupied by Ser, which is expected to raise the
pK
of His
, explaining the
occurrence of reverse Bohr effects in tadpole Hb in the pH range
8.5-6.0 and in human HbA below pH 6.5 (Watt et al.,
1980). The replacement His
Lys in eel cathodic Hb
thus indicates a different allosteric mechanism in this Hb.
Another element which could affect O binding at the
subunit is the presence of an Asn residue in
position 93(F9), replacing Cys in human HbA or Ser in other fish Hbs.
In human HbA in the R state the SH group of Cys
(F9)
can be either internal between the F and H helices, sharing the pocket
with Tyr(HC2), or external, depending on the spin state of the iron,
whereas in the T state it is external and in contact with the C
terminus (Perutz and Brunori, 1982). The side chain of Asn in the
cathodic eel Hb may therefore produce perturbations in the F and H
helix packing and in the C-terminal region because of steric hindrance
factors and the ability of Asn to form hydrogen bonds.
The presence of both Cl and GTP causes a
lesser decrease in the O
affinity than that induced by GTP
alone. This indicates that the binding sites for the two anions
overlap, at least in part, and suggests the absence of additional sites
for allosteric Cl
binding in this Hb. It has been
proposed that the excess of positive charges in the
cleft of
human Hb can cause a destabilization of the T state relative to the R
state in the absence of anions (Bonaventura and Bonaventura, 1978). The
lowering of the O
affinity in the presence of
Cl
has recently been explained in terms of
neutralization of the positive charges in the cavity without binding at
any specific residue (Perutz et al., 1993, 1994) or,
alternatively, to binding at the N-terminal group of the
chain
(Fronticelli et al., 1994). On the basis of our results it is
not possible to discriminate between these two mechanisms because the
chain N terminus also participates in the binding of phosphates.
However, the far smaller effect of Cl
than of GTP on
the O
affinity of HbC indicates that in the low affinity
conformation electrostatic repulsions in the central cavity are not a
major factor destabilizing the T state and increasing O
affinity. However, the close packing of positive side chains at
this site in the deoxy form could conceivably induce abnormally low
pK
values in the N-terminal group of the
chain or possibly even in the Lys residues, thus making them acid
Bohr groups. The residues Val(NA1), Glu(NA2), Lys(EF6), and Arg(H21)
(or Lys) of the
chain have so far only been found in Root effect
fish Hbs, such as those from carp (Grujic-Injac et al., 1980),
goldfish (Rodewald and Braunitzer, 1984), and red gurnard (Fago et
al., 1993), where the contribution of these residues to the pH
effect may be masked by the presence of strong alkaline Bohr groups. In
this regard it is suggestive that a small reverse Bohr effect
(+0.13 in the pH range 7.8-9.0, where the alkaline Bohr
groups are inoperative), which was inhibited by ATP, also is seen in
carp Hb (Gillen and Riggs, 1972).
In conclusion, the elucidation of
the primary structure of the cathodic Hb of eel provides new insight
into the the molecular basis for O binding regulation. Some
amino acid substitutions are identified that could affect the
and
particularly the
heme ligand affinities and the side chain
packing at the
switch interface that
could alter the T-R allosteric transition. The presence of the GTP
binding residues in this Hb is in agreement with the stereochemical
model proposed for carp Hb and explains the strong effect of organic
phosphates on the O
affinity.
For further investigation
into the structure-function relationships preparation of Hb crystals in
the T and R state suitable for x-ray diffraction analysis is under way.
Moreover preparation of native and
subunits by reaction
with p-mercuribenzoate for O
binding studies,
chemical modification of the N terminus of the
chain, and
cross-linking at the central cavity of the Hb molecule are also in
progress to verify the hypothesis regarding the cause of the reverse
Bohr effect.