(Received for publication, February 11, 1997, and in revised form, May 2, 1997)
From the Department of Biochemistry and Cell Biology
and W. M. Keck Center for Computational Biology, Rice University,
Houston, Texas 77005-1892 and Somatogen Inc.,
Boulder, Colorado 80301-2857
Rate constants for hemin dissociation from the
and
subunits of native and recombinant human hemoglobins were
measured as a function of protein concentration at pH 7.0, 37 °C,
using H64Y/V68F apomyoglobin as a hemin acceptor reagent. Hemin
dissociation rates were also measured for native isolated
and
chains and for recombinant hemoglobin tetramers stabilized by
subunit fusion. The rate constant for hemin dissociation from
subunits in native hemoglobin increases from 1.5 h
1
in tetramers at high protein concentration to 15 h
1 in
dimers at low concentrations. The rate of hemin dissociation from
subunits in native hemoglobin is significantly smaller (0.3-0.6
h
1) and shows little dependence on protein concentration.
Recombinant hemoglobins containing a fused di-
subunit remain
tetrameric under all concentrations and show rates of hemin loss
similar to those observed for wild-type and native hemoglobin at high protein concentration. Rates of hemin dissociation from monomeric
and
chains are much greater, 12 and 40 h
1,
respectively, at pH 7, 37 °C. Aggregation of monomers to form
1
1 dimers greatly stabilizes bound hemin
in
chains, decreasing its rate of hemin loss ~20-fold. In
contrast, dimer formation has little stabilizing effect on hemin
binding to
subunits. A significant reduction in the rate of hemin
loss from
subunits does occur after formation of the
1
2 interface in tetrameric hemoglobin.
These results suggest that native human hemoglobin may have evolved to
lose heme rapidly after red cell lysis, allowing the prosthetic group
to be removed by serum albumin and apohemopexin.
Human hemoglobin is protected against denaturation by
encapsulation in red blood cells. The iron atoms are kept in the
ferrous state by intracellular methemoglobin reductases, and
O2 generated by spontaneous autooxidation is rapidly
transformed into H2O and O2 by superoxide
dismutase and catalase (1, 2). Heme loss is inhibited by maintenance of
the reduced state, the high concentration of cytoplasmic hemoglobin,
and the presence of a cell membrane which prevents dispersal and
precipitation of any dissociated prosthetic group. When hemoglobin is
released by a small amount of red cell lysis, it is diluted
significantly in plasma. In the case of human hemoglobin, this dilution
leads to formation of noncooperative,
1
1
dimers which display epitopes that are recognized by circulating
haptoglobin molecules (2, 3). Binding of dimers to haptoglobin
facilitates rapid clearance from the blood stream (Fig.
1).
Hemoglobin dimers also autooxidize more rapidly and lose hemin more
readily than hemoglobin tetramers (4-6). Rapid hemin loss from dilute,
extracellular hemoglobin may be advantageous since the resulting free
heme in plasma is readily taken up by serum albumin and apohemopexin
and transported to the liver for recycling. Thus, we felt that it would
be important to measure quantitatively how the state of aggregation
affects the rate of hemin loss from the and
subunits of human
hemoglobin.
Roughly 30 years ago, Bunn and Jandl (7) measured time courses for
59Fe-labeled hemin exchange between human adult and fetal
hemoglobins and between adult hemoglobin and human serum albumin.
Quantitative analysis suggested that the rate of hemin exchange with
subunits was 5-10-fold greater than with
subunits. Benesch and
Kwong (5) showed that partial hemin exchange between methemoglobin and
human serum albumin can be followed spectrophotometrically at pH values
8. They measured rates of hemin dissociation from
subunits in a
variety of native and mutant human hemoglobins. However, even at
extremely high concentrations, human serum albumin is unable to extract
significant amounts of hemin from
subunits within intact
hemoglobin. To overcome this problem, we developed a genetically
engineered apoglobin for use as a colorimetric reagent to measure
complete time courses of hemin dissociation from both myoglobins and
hemoglobins (8).
The reagent is a myoglobin mutant in which the distal histidine
(His-64) was replaced with tyrosine. The phenolate side chain coordinates to the iron atom giving the ferric form of the mutant holoprotein a "green" color and an absorbance spectrum very
different from those of native metmyoglobins and methemoglobins which
appear "brown." In addition, Val-68(E11) was replaced with Phe to
enhance the stability of the apoprotein and to increase its affinity
for hemin. When methemoglobin Ao is mixed with an excess of
the H64Y/V68F apomyoglobin reagent, complete hemin exchange occurs and
the observed time course is markedly biphasic (see Fig. 2). Using
valence and mutant hybrid hemoglobins, we were able to confirm that the
faster phase represents hemin loss from ferric subunits and the
slower phase hemin loss from
subunits (8).
Our rate constants for hemin loss, which were measured at low
hemoglobin concentrations (1-10 µM), were significantly
larger than those reported by Bunn and Jandl (7), which were measured at high protein concentration. We speculated that the differences were
due to more rapid hemin dissociation from methemoglobin dimers that
were present at the low concentrations used in our experiments. Benesch
and Kwong (6) confirmed this idea directly by measuring the dependence
of the rate of hemin dissociation from subunits on hemoglobin
concentration. However, since Benesch and Kwong (6) were using the
human serum albumin assay, they were unable to examine the effects of
dimer formation on hemin dissociation from
subunits.
In this work, we have measured the rate constants for hemin loss from
isolated and
subunits,
1
1 dimers,
native tetramers, and recombinant tetramers stabilized by
gene
fusion. The rate constants for hemin dissociation from native dimers
and tetramers were obtained by analyzing the protein concentration
dependence of the observed time courses. The results provide a
quantitative description of the linkage between quaternary structure
and hemin binding in the
and
subunits. These data also explain
why it is so difficult to prepare the aquomet forms of isolated chains. Finally, the high rates of hemin dissociation observed in dimers and
monomers support the view that human hemoglobin has evolved to fall
apart rapidly in dilute solution.
A description of the properties of
the H64Y/V68F apomyoglobin reagent and its use in measuring complete
time courses of hemin loss from ferric myoglobins and hemoglobins are
given in Hargrove et al. (8). Native human hemoglobin was
prepared as described by Mathews et al. (9), and isolated
and
subunits were purified by the method of Bucci (10).
Recombinant wild-type human hemoglobin containing V1M replacements in
and
subunits (rHb0.0)1 and
genetically stabilized hemoglobins containing a glycine linker (rHb0.1,
rHb1.1) between the C terminus of one
subunit and the N terminus of
another were purified as described by Looker et al. (11,
12). Some preparations of recombinant hemoglobin were purified by a
modified procedure in which deoxygenated crude lysate was first heat
treated at 65 °C for 30 min to precipitate Escherichia
coli proteins. After cooling to 4 °C, polyethyleneimine was
added, and the dense mass of E. coli proteins and nucleic acids was removed by centrifugation. Following centrifugation, hemoglobin was purified by sequential ion exchange chromatography using
(1) Q-column, (2) S-column, and (3) Q-Sepharose Fast Flow resins
(Pharmacia Biotech). These columns were equilibrated and eluted as
described for columns Q2 and S1 in Looker et al. (11). In
the present work, the Q-column chromatographic step was repeated after
the S-column to remove additional methemoglobin. After the second
Q-column procedure, ferrous hemoglobin containing fractions were pooled
as above, concentrated to 50 mg/mL and stored as small aliquots in
liquid nitrogen.
The general procedures for measurement of hemin dissociation are described in Hargrove et al. (8). Unless otherwise indicated, experiments were performed at 37 °C in 0.15 M potassium or sodium phosphate buffer, pH 7.0 and 0.45 M sucrose. In all experiments, the concentration of apomyoglobin (H64Y/V68F) was at least twice the total Hb (heme) concentration. Under these conditions, the observed rate is equal to the first order rate of hemin dissociation from methemoglobin (8).
Below 12 µM Hb, dissociation time courses were monitored
at 410 nm. Between 12 and 35 µM Hb, time courses were
monitored at 600 nm. It was not possible to record continuous time
courses above 35 µM Hb due to turbidity caused by
precipitation of large amounts of apohemoglobin. Consequently, at high
hemoglobin concentrations (200-600 µM heme) small
aliquots were withdrawn at appropriate time points from a stock
reaction mixture and centrifuged briefly (~30 s) at 14,000 rpm in a
refrigerated bench top microcentrifuge. A measured volume of
supernatant was then diluted into 10 mM sodium phosphate
buffer, pH 8, and the visible spectrum recorded quickly. The changes in
absorbance at 410 and 600 nm were used to monitor the formation of
H64Y/V68F holomyoglobin and the concomitant disappearance of
methemoglobin (see Fig. 3A).
Analysis of Hemin Dissociation Time Courses
Absorbance traces at 410 and 600 nm were exported into data analysis software and analyzed as the sum of two independent exponential decay processes. In the latter stages of the reactions (>4 h), the absorbance traces were frequently distorted for samples at initial Hb concentrations greater than 20 µM due to precipitation of apohemoglobin. In these cases the most useful method of analysis was to truncate the absorbance trace to avoid regions showing absorbance increases due to apoprotein aggregation.
Time courses for complete
hemin loss from hemoglobin Ao are biphasic under all
conditions. As shown in Fig. 2, addition of inositol
hexaphosphate at low protein concentrations preferentially decreases
the rate of the fast or subunit phase of the reaction from ~16
h
1 to ~3 h
1. The rate constant for the
slow phase was unaffected and remained at ~0.5 h
1. This
result suggests that tetramer formation stabilizes hemin in
subunits since inositol hexaphosphate promotes tetramer formation by
binding to the positively charge cleft between the
subunits of
tetrameric hemoglobin (13, 14).
Time courses for hemin loss from native human hemoglobin at high and
low protein concentrations are shown in Fig.
3A. As observed by Benesch and Kwong (6), the
rate of hemin loss from subunits (fast phase) is reduced almost
10-fold when hemoglobin concentration is increased from 1 to 600 µM. Time courses analogous to those in Fig. 3A
were collected at 12 different protein concentrations, and the
dependence of the fitted rate constants on heme concentration is shown
in Fig. 4.
Time courses for hemin dissociation from native hemoglobin were
biphasic under all conditions. The rate constant for the faster or phase decreased markedly with increasing protein concentration whereas
the rate of the slow or
phase showed little change (Fig. 4).
Analysis of these data is simplified by the fact that the rate of
formation and dissociation of hemoglobin tetramers is very rapid
compared with that for hemin dissociation. The rate constants for
tetramer dissociation and dimer aggregation are 1-10 s
1
and 1-5 × 105 M
1
s
1, respectively, for R-state forms of hemoglobin (3, 15,
16), whereas the rate constants for hemin dissociation are
0.005
s
1 (Table I). Thus, the observed rate of
hemin loss from an
or
subunit is a weighted sum of the rate
constants that apply for the subunit in dimers and in tetramers.
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
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The solid line in Fig. 4A for subunits within
native hemoglobin represents a fit in which the values of
k
Hdimer and
K4,2 were varied, and
k
Htetramer was fixed at 1.5 h
1, the value observed for
subunits in genetically
stabilized rHb0.1. The fitted parameters are listed in Table I. The
value obtained for K4,2 was 1.5 µM, which is very close to that determined for native
hemoglobin by Edelstein et al. (17) using flash photolysis and ultracentrifugation techniques. Convergence was more difficult when
all three parameters were varied. Similar values of
k
Hdimer and
k
Htetramer were obtained when
K4,2 was fixed to 1 µM (17).
Regardless of the exact analysis, the results in Fig. 4A
show that k
H for
subunits decreases
greater than 10-fold when
1
1 dimers aggregate to form tetramers in agreement with the previous work of
Benesch and Kwong (6).
In contrast, the rate of hemin dissociation from subunits shows
little dependence on hemoglobin concentration. If
K4,2 is fixed at 1.5 µM, the
fitted values of k
H for
subunits are 0.6 h
1 in dimers and 0.3 h
1 in tetramers. Fits
of similar quality were obtained by assuming no dependence and an
average value of 0.4 h
1. Regardless of the exact
analysis, the results show that dissociation into dimers has little
effect on hemin affinity in
subunits.
Fig.
3B shows time courses for hemin dissociation from
recombinant, wild-type human hemoglobin (rHb0.0) and a recombinant human hemoglobin stabilized against dimer formation by fusion of two
subunit genes into a single gene (rHb0.1). In both genes the codons
for the N-terminal valines in both subunits were replaced with
methionine codons to initiate translation in E. coli. Since the initiator methionine is retained, each subunit has effectively a
V1M mutation (11). In rHb0.1, a glycine residue connects the C terminus
of one
chain with the N terminus of a second
chain. This
subunit fusion prevents dissociation of the (
-
)
2
tetramer into dimers (11).
Although both proteins were at low concentrations (5-10
µM), the rate of hemin dissociation from subunits in
the di-
containing hemoglobin was 10-fold slower than that from
subunits in the wild-type control (Fig. 3B). The absolute
value (~1.5 h
1) is roughly equal to that observed for
subunits within native human hemoglobin at very high heme
concentrations (600 µM trace in Fig. 3A). As
shown in Fig. 4, the rate of hemin loss from
subunits in
genetically stabilized hemoglobin (rHb0.1) shows no dependence on
protein concentration and serves to fix the value of
k
H for these subunits in hemoglobin tetramers.
In contrast, the rate of hemin loss from
subunits in the wild-type
control (rHb0.0) shows a dependence on protein concentration which is similar to that observed for native HbA0 (Fig. 4A). The
fitted values of
k
Hdimer
and K4,2 for (rHb0.0) were 33 h
1
and 2.6 µM, respectively, when
k
Htetramer was fixed at 1.5 h
1. Thus, the V1M mutations in the recombinant protein
appear to increase the rate of hemin loss from
subunits in dimers
approximately 2-fold. As in the case of native hemoglobin, the rate of
hemin loss from
subunits appears to be independent of protein
concentration and is unaffected by genetic fusion (Fig. 4, Table
I).
We examined rates of hemin dissociation from rHb1.1 which, in addition
to the V1M mutations and fused subunits, contains the Presbyterian
mutation (
N108K). The latter substitution was added to enhance the
O2 transport properties of the recombinant protein (11). As
with rHb0.1, no dependence on protein concentration was observed, and
the only difference was a small increase in k
H
for
subunits which is presumably due to the N108K mutation in this
subunit.
Time courses for
hemin dissociation from native isolated and
chains were
measured in a stopped flow apparatus because of their high rates of
hemin loss and the difficulty of sample preparation. A slight excess of
potassium ferricyanide was added to a syringe containing 20 µM of the oxygenated forms of either
or
chains to
generate the corresponding ferric forms. Immediately after formation of
the ferric subunit, the contents of this syringe were reacted with 40 µM H64Y/V68F apomyoglobin. Time courses for hemin
dissociation were measured at 600 nm to avoid background absorbance by
excess ferricyanide (
max = 400 nm). Slow absorbance increases were observed at the end of each reaction due to
precipitation of the newly generated apoglobin chains. Hemin
dissociation rate constants were estimated from the initial portions of
the time courses by fitting to one or two exponential expressions with an offset (Fig. 5). Hemin dissociation from
chains
appears to be monophasic with a rate constant equal to ~12
h
1 at 37 °C, pH 7. Time courses for hemin dissociation
from isolated
subunits were biphasic, with observed rate constants
equal to ~40 h
1 and ~2 h
1 for the fast
and slow phases. McGovern et al. (18) estimated that the
equilibrium constant for dissociation of
tetramers into monomers is
1.25 × 10
12 M3 at pH 7. This value of K4,1 predicts a significant amount
of
tetramers at the concentrations used in our experiments. Thus, the simplest interpretation of the isolated
chain time course is
that hemin dissociation from monomeric
chains is very rapid (k
H
40 h
1), whereas hemin
loss from
tetramers is slow (k
H
2 h
1) and comparable to that from
subunits within
tetrameric hemoglobin.
The quaternary structure of methemoglobin has a profound effect on
the rate of hemin dissociation (Table I). The monomeric forms of the
isolated and
chains lose hemin 30-40 times more rapidly than
the corresponding subunits in a tetramer. Hargrove et al.
(19) have shown that the association rate constant for the binding of
monomeric heme to apoglobins is always ~1 × 108
M
1 s
1 regardless of the exact
protein structure. Thus, the equilibrium constant for hemin
dissociation can be computed as K
H = k
H/(1 × 108
M
1 s
1) where
k
H is converted from units of h
1
to s
1. Equilibrium dissociation constants for hemin
binding to monomeric, dimeric, and tetrameric native hemoglobin are
listed in Table II and compared with the value for sperm
whale myoglobin under the same conditions.
|
The results in Tables I and II provide a quantitative explanation for
why the aquomet forms of isolated and
chains are so unstable.
The half-times for hemin dissociation are 1-3 min at 37 °C, and
although the equilibrium constants are still ~10
10
M, rebinding has to compete with irreversible hemin
aggregation and precipitation. In addition, the apoprotein forms of the
isolated subunits are very unstable at 37 °C and precipitate almost
immediately after heme removal.
The instability of apoglobin monomers and their low affinity for hemin
may explain why it is difficult to express the and
subunits of
human hemoglobin separately as soluble holoproteins in E. coli. In contrast, co-expression of the subunits yields high
levels of soluble hemoglobin (11, 12, 20). Dimer and tetramer formation
are required to stabilize the apoproteins and to increase hemin
affinity. In contrast apomyoglobin is much more stable and has an
affinity for hemin which is 1,000-3,000-fold greater than that of the
isolated subunits of hemoglobin. This result accounts for the ease of
expression of sperm whale holomyoglobin in bacteria (21-23).
Formation of 1
1 dimers from monomers
causes a 30-fold increase in the affinity of
subunits for hemin,
whereas only a 2-fold increase is observed for
subunits. The
structural cause of this selectivity is not clear since the
1
1 interface involves mostly hydrophobic
contacts between the B, G, and H helices of the two subunits, regions
which are far removed from the heme pocket in both proteins.
Presumably, formation of these contacts stabilizes the overall,
tertiary structure of
but not
subunits. Association of dimers
into tetramers is driven primarily by formation of the
1
2 interface which involves more polar
contacts between the C and N termini and the C-helices and FG corners
of both subunits. The C-helix is near the heme group and the FG corner
serves to position the proximal His(F8) for direct coordination to the
iron atom. These interactions are required for strong hemin binding to
subunits. A more detailed structural interpretation will require
systematic mutagenesis studies analogous to those carried out for sperm
whale myoglobin (24).
We thank Dr. Douglas Lemon and Dr. Carol Cech at Somatogen, Inc. for reading the manuscript critically and making several helpful suggestions.