(Received for publication, August 10, 1995; and in revised form, October 16, 1995)
From the
Bovine erythrocytes do not contain 2,3-diphosphoglycerate, the
principal allosteric effector of human hemoglobin. Bovine hemoglobin
has a lower oxygen affinity than human hemoglobin and is regulated by
physiological concentrations of chloride (Fronticelli, C., Bucci, E.,
and Razynska, A.(1988) J. Mol. Biol. 202, 343-348). It
has been proposed that the chloride regulation in bovine hemoglobin is
introduced by particular amino acid residues located in the
amino-terminal region of the A helix and in the E helix of the
subunits (Fronticelli, C.(1990) Biophys. Chem. 37,
141-146). In accordance with this proposal we have constructed
two mutant human hemoglobins,
(V1M+H2deleted+T4I+P5A) and
(V1M+H2deleted+T4I+P5A+A76K). These are the
residues present at the proposed locations in bovine hemoglobin except
for isoleucine at position 4. Oxygen binding studies demonstrate that
these mutations have introduced into human hemoglobin the low oxygen
affinity and chloride sensitivity of bovine hemoglobin and reveal the
presence of a previously unrecognized allosteric mechanism of oxygen
affinity regulation where all the interactions responsible for the
lowered affinity and chloride binding appear to be confined to
individual
subunits.
Hemoglobin (Hb) ()is present within the red cell at
about 5 mM concentration and functions to transport oxygen
from the lungs to the tissues (Antonini and Brunori, 1970). This
protein is a tetramer composed of two pairs of identical
and
subunits. The four subunits have the same tertiary folding, and
they each contain eight
helices, which are identified as A
through H. The tetrameric molecule can assume two conformations that
are in equilibrium with one another. The predominant conformation when
no oxygen is bound is called T state, and the predominant conformation
when the oxygen is coordinated to the iron is termed R state. The
affinity of the protein for oxygen is regulated by position of the
conformational equilibrium, i.e. the equilibrium between the R
and T states. The position of the conformational equilibrium is also
influenced by pH and the concentration of certain anions, termed
effectors. In the absence of effector anions, the oxygen affinity of
human Hb is too high for efficient release of oxygen at the oxygen
partial pressure maintained in most tissues, approximately 40 torr
(Vandegriff, 1992).
In human Hb, the effector regulating the conformational equilibrium in the red cells is 2,3-diphosphoglycerate (DPG) (Benesch et al., 1968). On the other hand, bovine Hb has an intrinsically lower oxygen affinity than human Hb, and the affinity is lowered further by interaction with physiological concentrations of chloride ions (100-150 mM) (Fronticelli et al., 1984, 1988; Perutz et al., 1993). At this concentration of chloride, the oxygen affinity of bovine Hb is insensitive to polyanions such as DPG and has an oxygen affinity similar to that of human whole blood (Bucci et al., 1988).
In the development of hemoglobins to be used clinically as a cell-free oxygen carrier, an understanding of the molecular mechanism that regulates the functional characteristics of bovine Hb could lead to the design of human Hb variants that are capable of efficient oxygen transport in cell-free solutions within the circulatory system at the physiological chloride concentrations.
Searching for structural differences between the
human and bovine hemoglobins, we examined their hydropathy plots
(Fronticelli, 1990). No differences were detected between the
chains of human and bovine Hb; however, in the
chains two regions
of different hydrophobicities were observed. One comprised a portion of
the A helix, and the other comprised a portion of the E helix. In order
to correlate the different hydrophobicities with relevant differences
in sequence, we compared sequences of several primate and ruminant
subunits. The results, reported in Table 1, show the
presence of consistently different sequences that in ruminant
subunits result in an increase in the hydropathic index of the
amino-terminal end of the protein and a decrease in the index of the E
helix relative to human
subunits. Thus, we hypothesized that
amino acid substitutions of the amino-terminal residues and of the A
and E helices of bovine Hb were responsible for introducing in bovine
Hb a different mechanism of oxygen affinity regulation (Fronticelli,
1990).
To test this hypothesis we have constructed mutant human
hemoglobins containing some of the amino acid substitutions reported in Table 1. Substitution of valine 1 with a methionine and
deletion of histidine
2 produced a stabilization of the T state
but was not sufficient to introduce the low oxygen affinity of bovine
hemoglobin into human hemoglobin (Fronticelli, 1992). However, this
mutation removed the Cl
binding site between
Val
and Lys
as well as the
Cl
-dependent Bohr effect and introduced at the
amino-terminal of the human
subunits the conformation present in
bovine
subunits (Fronticelli et al., 1994; Perutz et
al., 1993). Additional amino acid substitutions have now been
introduced, and in this paper we report the functional properties of
two mutant hemoglobins,
(V1M+H2deleted+T4I+P5A) and
(V1M+H2deleted+T4I+ P5A+A76K).
Our results
confirm the original hypothesis that the functional characteristics of
bovine hemoglobin are principally dependent on the presence of specific
amino acid residues in the A and E helices. Most important, they also
reveal a novel mechanism of oxygen affinity modulation within a single
subunits, regulated by tertiary conformational changes.
Synthesis of the mutant
NS1-FX- globin fusion protein was obtained in Escherichia coli strain AR-120, induced with nalidixic acid as described previously
(Fronticelli et al., 1991). The molecular weight of the fusion
protein and the amount produced were indistinguishable in
SDS-polyacrylamide gel electrophoresis from that produced from pJKO5
under the same conditions. The DNA was sequenced by the dideoxy method
through the region of the mutagenic oligonucleotide confirming the
mutations indicated above. The additional mutation, A76K, was
introduced using the method of Kunkel et al. (1987) and the
Bio-Rad Muta-Gene mutagenesis kit.
Cleavage of the isolated fusion
protein with Factor Xa and reconstitution with cyano hemin and
subunits were carried out as described previously (Fronticelli et
al., 1991). All of the amino acid substitutions were confirmed by
peptide sequencing. The
globin from human Hb and
(PB5) were
purified by reverse phase HPLC (Shelton et al., 1984) on a
Vydac 4 column using a gradient of 0.1% trifluoroacetic acid with an
increasing concentration of acetonitrile. In each case the protein was S-pyridylethylated and digested with trypsin treated with
1-tosylamide-2-phenylethyl chloromethlyl ketone. The tryptic peptides
were separated by HPLC and analyzed as described previously (Bucci et al., 1993). Sequential Edman degradation was carried out on
a Hewlett Packard G1000A sequenator.
Figure 1:
Electrophoretic separation (Paragon
system, Beckman) of human Hb (lane 1), bovine Hb (lane
2), (bovine)
(human)
(lane
3), and
(human)
(bovine)
(lane 4). The dots represent the site of the samples
deposition.
The rationale for the experiment was that if the entire molecule is necessary for expressing the decrease in oxygen affinity, the two hybrids would have similar oxygen affinities. Otherwise it would be found only in the hybrid containing the enabling subunit.
The hybrid containing the bovine chains has a 3-fold
decrease in oxygen affinity (Fronticelli, 1992) with respect to the
hybrid containing the human
chains (Fig. 2). This is the
same oxygen affinity difference observed between bovine and human Hb
(Fronticelli et al., 1984), supporting the proposition that
the reduced oxygen affinity of bovine Hb is expressed via the
subunits.
Figure 2:
Oxygen binding curves of hybrids of
(bovine)
(human)
(circles)
and
(human)
(bovine)
(squares). Buffer, 100 mM Hepes with 100 mM Cl
at pH 7.4. Temperature, 25
°C.
Figure 3:
Tryptic peptide maps of chains of
human Hb (a) and of
(PB5) (b).
Figure 4:
Oxygen binding curves at pH 7.4 and
protein concentration of 30 mg/ml. Temperature, 25 °C. A,
human Hb (circles), (PB4) (triangles),
(PB5) (squares), and bovine Hb (
). Buffer, 50
mM Hepes. B, human hemoglobin (circles),
(PB4) (triangles),
(PB5) (squares), and
bovine hemoglobin (
). Buffer, 50 mM Hepes and 200
mM Cl
. C, human hemoglobin (circles) and
(PB5) (squares) in the absence (open symbols) and in the presence (filled symbols)
of 2 mM DPG. Buffer, 50 mM Hepes and 100 mM
Cl
.
In the presence of 200 mM chloride, the
oxygen affinity of human Hb is decreased; however, the oxygen affinity
of (PB4) remains nearly the same as that observed in the absence
of chloride (Fig. 4B). This result demonstrates that
although the bovine-like sequence of the amino-terminal region lowers
the oxygen affinity, it also greatly decreases the sensitivity to
chloride. This latter property is not unexpected because we have
previously shown that the replacement of Val
Met and the deletion of His
destroys the anion binding
site between Val
and Lys
as well as
the chloride-dependent Bohr effect (Fronticelli et al., 1994).
In contrast to
(PB4), the oxygen affinities of both bovine Hb and
(PB5) are decreased to the same extent in the presence of 200
mM Cl
(Fig. 4B). Clearly,
the additional substitution of Ala
Lys in
(PB5) creates a new chloride-dependent regulatory site. A possible
location of the chloride binding site is between Lys
,
Lys
, and perhaps His
of the same
subunit, as earlier proposed (Fronticelli, 1990). The same
Cl
binding site has been postulated to be present in
pig hemoglobin, where these three residues are also present. (Condo et al., 1992; Katz et al., 1994)
In the presence
of 100 mM Cl, the oxygen affinity of
(PB5) is 3-fold lower than the oxygen affinity of human Hb but
similar to the oxygen affinity of human Hb in the presence of 2 mM DPG (Fig. 4C). Like bovine Hb in presence of 100
mM Cl
, the oxygen affinity of
(PB5) is
virtually unaffected by the presence of 2 mM DPG (Fronticelli et al., 1988; Perutz et al., 1993). This indicates
that the amino acid substitutions in
(PB5) have introduced a
mechanism of oxygen affinity modulation similar to the one present in
bovine Hb.
Fig. 5presents the Cl titration
of
(PB4),
(PB5), human Hb, and bovine Hb, in 50 mM Hepes buffer at pH 7.4. From the slope of the curve, it can be
calculated (Wyman, 1964) that the Cl
exchanged is
2.5/tetramer for human Hb, bovine Hb, and
(PB5). This indicates
that these hemoglobins bind the same number of chloride ions. In
(PB4) the Cl
exchanged is only 0.8/tetramer.
This confirms that the replacement of Val
with Met and
the deletion of His
results in the loss of the
Cl
binding site present at Val
in
human Hb (Fronticelli et al., 1994) and indicates that a new
chloride binding site is introduced in
(PB5) by the mutation
Ala
Lys.
Figure 5:
Chloride titration of human Hb (circles), (PB4) (triangles),
(PB5) (squares), and bovine Hb (
) in 50 mM Hepes at
pH 7.4 at 25 °C.
Fig. 6A shows the
electrostatic potential of the proposed Cl binding
site calculated from the structure of the double mutant
(V1M+H2del) (Fronticelli et al., 1994) using the
program GRASP (Nicholls et al., 1991). The substitution
Ala
Lys was manually introduced, and the side
chain of Lys
was repositioned. A positively charged
cleft is observable between Lys
and
Lys
. These two residues are about 11 Å apart
and can be bridged by a Cl
ion (Van der Waals radius,
2.5 Å). Fig. 6B shows the electrostatic
potential of the same region in deoxy human Hb, where the putative
Cl
binding site is clearly absent. This model is at
variance with the proposition that the mechanism of the chloride linked
cooperative effects is the same in human and bovine hemoglobins (Perutz et al., 1993).
Figure 6:
Electrostatic potential shown from
-8 kT (red) to +8kT (blue), calculated using the program
GRASP (Nicholls et al., 1991). A, proposed
Cl binding site calculated from the coordinates of
the double mutant
(V1M+H2del) (code 2hhe). The substitution
Ala
Lys was manually introduced. B,
electrostatic potential of the same site in deoxy human Hb (code 2hhd),
where an alanine is present at
76.
It should be emphasized that all the
interactions that generate bovine Hb-like properties in (PB5)
appear to be localized in individual
subunits. Thus, these data
present evidence for the presence of an allosteric mechanism of oxygen
affinity modulation regulated solely by tertiary conformational
changes. Notably, although there are 24 amino acid differences between
the human and bovine
globins, this mechanism is introduced into
human Hb by the replacement of only five amino acid residues.
This paper is dedicated to the memory of Professor Jeffries Wyman, beloved teacher and friend.