(Received for publication, May 30, 1995; and in revised form, January 2, 1996)
From the
The stability of the heme-globin linkage in dimers
and in the isolated chains of human hemoglobin has been probed by
studying the transfer of heme from the proteins immobilized onto
CNBr-activated Sepharose 4B to human albumin. The kinetic and
equilibrium features of the reaction have been measured
spectrophotometrically given the stability of the heme donors and the
ease with which heme donor and acceptor can be separated. Isolated
and
chains transfer heme to albumin at similar rates
(1-6
10
s
at pH 9.0
and 20 °C) in the ferrous CO-bound and in the ferric state. In
dimers the heme-globin linkage is strengthened considerably,
albeit to a different extent in the ferrous CO-bound and ferric
met-aquo derivatives. Only in the latter heme is lost at a measurable
rate, 0.065 ± 0.011
10
s
for
heme and 2.8 ± 0.6
10
s
for
heme at pH 9.0 and 20 °C,
which is very close to the rate measured with soluble
met-aquo-hemoglobin at micromolar concentrations. These results
indicate that in human hemoglobin the heme-globin linkage in the
chains is stabilized by interactions between unlike chains at the
interface, whereas heme binding to
the
chains is stabilized by interactions at the
interface. These long range factors
have to be taken into account in addition to the local factors at the
heme pocket when evaluating the effect of point mutation and chemical
modification.
The polypeptide chain-heme equilibrium is an important element in the description of all hemoproteins. However, a direct measure of this parameter is difficult due to the high stability of the holoprotein under physiological conditions(1) . The heme-apoprotein linkage, therefore, is usually assessed by studying the heme transfer reaction from a donor to a heme acceptor protein. When the heme acceptor has a very high affinity for heme, like apomyoglobin, the kinetics of the heme transfer process is followed. This is governed by the rate of heme release from the donor protein as the rate of heme association is very high(2, 3) . When the heme donor and acceptor proteins have similar affinities for heme, the heme is partitioned between the two proteins and hence both kinetic and equilibrium features of the heme transfer reaction can be exploited(4, 5) .
In the case of human hemoglobin
(HbA) there are relatively few quantitative estimates of the stability
of the heme-globin linkage despite its relevance in the study of mutant
and chemically modified
hemoglobins(2, 3, 4, 5, 6) .
The interaction between heme and globin is known to be affected by a
number of parameters. Of major importance are the redox state of the
protein and pH(2, 4, 5) . Thus, heme is
released only from oxidized, met-aquo-hemoglobin, but not from the
reduced protein and more so upon departure from neutral pH values. In
met-aquo-hemoglobin, the rate of heme release from chains is
considerably faster than from
chains. Furthermore, the kinetics
of heme dissociation from
dimers is much greater than from
tetramers(5, 6) .
In the isolated
and
chains to our knowledge the heme-globin
affinity has never been measured, presumably due to the very marked
tendency of the respective globins to precipitate. As a matter of fact
in all studies of heme dissociation from hemoglobin or from myoglobin
the formation of free, precipitable globin upon heme depletion of the
ferric protein is a common problem (3, 5, 6) that has been alleviated in part by
adding sucrose to the incubation medium(2) .
We have
addressed the problem of globin precipitation in a different manner and
propose the use of dimers immobilized on Sepharose 4B (7, 8, 9, 10) as heme donor. This
material offers a number of advantages: it is stable after heme
depletion, it does not undergo changes in state of association, it can
be lyophilized, and, most importantly, it can be separated easily from
the acceptor protein. Therefore formation of the heme-acceptor complex
can be followed spectrophotometrically in a facile way under any
experimental condition. As heme acceptor we have used human albumin
which is endowed with two high affinity heme binding sites (11) and whose affinity for heme does not change in the pH
range 5-10(12) . Transfer of heme from hemoglobin to
albumin to form methemalbumin is known to occur in the blood of
patients with plasma hemoglobinemia and ``in vitro''
when albumin is mixed with ferric hemoglobin(4, 5) .
The immobilized
dimers behave like the soluble protein in
that they do not transfer heme to albumin when in the oxy or CO form,
but only when in the oxidized state, an indication that the
immobilization process does not produce significant alterations in the
heme environment. The direct comparison of the rate of heme loss from
soluble dimers has confirmed this contention.
The behavior of
isolated and
chains both in solution and after
immobilization on Sepharose 4B has also been studied. Quite
unexpectedly, soluble
and
chains transfer heme readily to
albumin even when oxygenated or in the CO-bound form, a finding which
points to a major change in the heme pocket induced by the assembly of
unlike chains into a heterodimer.
Human serum albumin, crystallized and lyophilized, was a
commercial product of Sigma and was used without further purification.
Albumin concentration was determined spectrophotometrically at 280 nm,
using the molar absorbance E =
9.8
10
.
Human hemoglobin was prepared
according to Berger et al.(13) and was immobilized
covalently in the oxygenated state on CNBr-activated Sepharose 4B
(Pharmacia Biotech Inc., Uppsala, Sweden). The coupling reaction was
carried out in 0.1 M sodium bicarbonate, pH 8.3, in the
presence of ethanolamine in 10:1 molar excess with respect to heme. The
suspension was stirred at room temperature for 1 h and washed
thereafter on a filter funnel as described in (9) . This
procedure leads to immobilization of hemoglobin as dimers
which are coupled to Sepharose via either chain and maintain the
capacity to interact in a specific and reversible manner with soluble
dimers(7, 8, 9, 10) . The
concentration of immobilized oxyhemoglobin was typically around 8 mg/ml
of packed resin. It was determined on every preparation using a 1-mm
light path cell and a Cary 219 spectrophotometer; the effect of
turbidity was minimized by the use of protein-free gel in the reference
cell(9) . Immobilized oxy-
dimers (Fig. 1A) were oxidized by addition of two to three
equivalents of potassium ferricyanide at neutral pH; excess
ferricyanide and the ferrocyanide produced during the oxidation
reaction were removed by washing extensively the gel on a filter
funnel. The immobilized met-
dimers were then lyophilized in
the presence of 10% sucrose. The optical absorption spectrum of the
immobilized met-
dimers equilibrated with buffers of pH
6.5-9.0 (Fig. 1, B-D) displays the same pH
dependence of the soluble protein ((1) , page 45).
Figure 1:
Optical absorption spectra of HbA
dimers immobilized on CNBr-activated Sepharose 4B.
Derivative: A, oxygenated at pH 7.0; B-D,
oxidized at pH 6.5 (B), 7.5 (C), and 9.0 (D).
Isolated
and
chains were prepared according to Geraci et
al.(14) . Heme transfer from the soluble chains to albumin
was followed in polyacrylamide gel electrophoresis experiments carried
out according to Davis(15) . The chains were mixed with
albumin, and the mixture was incubated at 4 or 20 °C and then
subjected to electrophoresis. The gels were stained for heme with
benzidine and for protein with Coomassie Blue. The immobilization
reaction was carried out on the oxygenated derivative in 0.1 M phosphate buffer at pH 7.4 and yielded a concentration of
immobilized chains between 1 and 5 mg/ml of packed resin. Oxidation of
the immobilized chains was performed as described for hemoglobin.
where is the immobilized dimer, A stands for
albumin and H for heme. In view of the different behavior of
and
chains, the kinetic analysis was carried out by
approximating the overall reaction to the following
equilibria,
where and
are the hemoglobin chains in the
immobilized
dimer. An iterative diagonalization of the rate
constants matrix relative to the processes described by the equilibria () was used to fit the time course
of methemalbumin formation. The second order processes were linearized
by assuming dH/dt = 0(2) . In the fitting procedure: (i)
the rate constants k
and k
were fixed at the values determined in independent experiments, i.e. at 5
10
M
s
and 3.2
10
s
; (ii) the concentrations of heme-depleted
and
chains as well as the dissociation rate constants k
and k
were allowed to float; (iii) the amount of heme associated with
the globin chains and with albumin was fixed to the value determined
experimentally at the end of the reaction (see Fig. 8), thereby
fixing the values of the rates for heme binding, k
and k
, at each iteration.
Figure 8:
Heme transferred from immobilized ferric
dimers to albumin at different pH values plotted as a
function of the heme/albumin molar ratio in the initial mixture. The
experimental points were taken after attainment of an equilibrium
distribution, i.e. after a 24-h incubation at 20 °C at pH
9.0 (
), 7.5 (
), 6.5 (
). The solid lines are
arbitrary guide lines.
where Y is the fractional saturation of albumin with
heme, [H] is the concentration of free hemin, and K and K
are the affinity
constants of the two albumin binding sites. The kinetic parameters of
the reaction were measured at 20 °C on an Applied Photophysics
(Applied Photophysics Ltd., Leatherhead, United Kingdom) stopped flow
apparatus (dead time 3 ms) upon mixing hemin with albumin in 0.1 M Tris-HCl plus 0.1 M NaCl, pH 9.0. All reactant
concentrations are after mixing unless otherwise stated.
All the algorithms used to fit the experimental data were elaborated with the software package Matlab (The Math Works Inc., Natick, MA).
Sedimentation velocity experiments were performed using a Beckman
model XL-A analytical ultracentrifuge at 40,000 rpm and 20 °C over
the concentration range 0.0043-0.5 g/dl (0.27-3.1
10
M heme). The gradient of protein
concentration in the cells was determined by absorption scans along the
centrifugation radius at a single wavelength (410, 540, or 630 nm) with
a step resolution of 0.001 cm. Sedimentation coefficients were
evaluated with the software provided by Beckman and were reduced to s
according to standard procedures.
The weight fraction of tetramers,
, at any given concentration c, was calculated on the basis of the measured value of s
(a weight average property) and of
s
-
s
, where s
and s
correspond to the
sedimentation velocity of tetramers and dimers, respectively, at
concentration c. In turn, s
= s
(1 - 0.07c) and
s
= s
(1 -
0.07c) with c expressed in g/dl(16) . The value of
s
, the sedimentation coefficient for the
tetramer at zero protein concentration, namely 4.7 S, was used to
calculate s
, the corresponding value for the
dimer, assuming that sedimentation coefficient is proportional to (M
)
(16, 17) . The
curves for K
, the dimer-tetramer association
constant, were calculated from the mass law expression according to (17) .
Sedimentation equilibrium experiments were performed
using a Beckman model XL-A analytical ultracentrifuge at 20,000 rpm and
10 °C in 0.1 M Tris-HCl plus 0.1 M NaCl, pH 9.0,
over the concentration range 0.75-5.0 10
M heme. The data (20 averages/scan) were analyzed with
the software ``Multi'' for self-associating systems provided
by Beckman.
Figure 2: Spectrophotometric titration of albumin with hemin. Small amounts of a hemin solution were added to a 1.3 µM albumin solution in 0.1 M Tris-HCl plus 0.1 M NaCl at pH 9.0 and 20 °C. The solid line represents the best fit to a two-site Adair equation ( under ``Materials and Methods'').
The kinetics of
the reaction has been investigated by mixing in a stopped flow
apparatus 3.6 µM hemin with albumin at concentrations
varying between about 20 and 250 µM. Hemin was used at a
low concentration to minimize its tendency to polymerize. The time
course is monophasic and has been analyzed as a first order reaction.
The pseudo first order rate constant (Fig. 3) depends linearly
on albumin concentration up to approximately 25 µM; at
higher protein concentrations a constant value of 1.3 s is reached. This value, which relates to a rate-limiting
monomolecular step, is close to the rate of hemin
depolymerization(18) . From the data in Fig. 3an
association rate constant for hemin binding to albumin of 5
(±0.9)
10
M
s
can be obtained. This value in combination
with the average affinity constant determined independently (see above)
yields an average rate of heme dissociation from albumin of 3.2
(±0.11)
10
s
.
Figure 3: Pseudo first order rate constants of the reaction between hemin and albumin as a function of albumin concentration. Hemin (before mixing): 3.6 µM; buffer: 0.1 M Tris-HCl plus 0.1 M NaCl at pH 9.0. The solid line represents the best linear regression fit to the first five experimental points.
Figure 4:
Heme transfer from soluble CO-bound
and
chains to albumin monitored in polyacrylamide gel
electrophoresis experiments. Buffer: 0.1 M Tris-HCl plus 0.1 M NaCl at pH 9.0. Lanes: 1, HbA-CO standard; 2,
or
chains; 3, equimolar mixture of
CO-bound
or
chains and albumin incubated under a CO
atmosphere for 3 h at room temperature; 4, albumin. Staining: top, Coomassie Blue; bottom,
benzidine.
Immobilized CO-liganded and
chains
exposed to albumin at pH 9.0 and 20 °C behave similarly to the
soluble ones and give rise readily to a significant amount of
methemalbumin (Fig. 5A). It is of interest that
methemalbumin is formed indicating that CO is lost upon or before
binding. An equilibrium distribution is attained after about 180 min;
at equilibrium, when albumin is in 3-fold molar excess (in terms of
heme binding sites), about 50% of the heme is in the form of
methemalbumin. Under similar experimental conditions immobilized
oxidized chains loose all their heme (Fig. 5A).
Figure 5:
Absorption spectra of methemalbumin formed
upon mixing immobilized chains (A) and immobilized
dimers (B) with albumin. After 3-h incubation at 20
°C in 0.1 M Tris-HCl plus 0.1 M NaCl at pH 9.0,
the liquid phase was separated from the immobilized protein (see
``Materials and Methods''), and the spectra were
recorded.
The
heme transfer process displays two clearly separated kinetic phases (Fig. 6). The time course was fitted using the scheme and the
approximations presented under ``Materials and Methods'' by
considering the contribution of only one chain. The rate of the slow
phase was taken as 3.2 10
s
, which corresponds to the rate of heme
dissociation from albumin estimated from the measurements on the
heme-albumin system. The rate of the fast phase, which can be assigned
to heme release from the
or
chains is 4.5-7.5
10
s
and does not depend within
experimental error on the chain type and on the state of heme oxidation (Table 1).
Figure 6:
Kinetics of heme transfer from immobilized
ferric (A) and
chains (B) to albumin at
pH 9.0 in 0.1 M Tris-HCl plus 0.1 M NaCl. A,
immobilized
chains, 1.8
10
M;
albumin, 5
10
M (bottom
trace), 2
10
M (top
trace). B, immobilized
chains, 1.2
10
M; albumin 5
10
M (bottom trace), 2
10
M (top trace). The solid lines represent the fit to the reaction scheme given under
``Materials and Methods.''
In a further set of experiments the observation
that methemalbumin is formed upon transfer of heme from the CO-bound
chains was exploited as it enables measurement of heme transfer from
soluble chains to albumin. Upon mixing chains at 7
10
M with albumin at 3.4
10
M at pH 9 and 20 °C in the presence
of 0.3 M sucrose(2) , a biphasic reaction is observed (Fig. 7). The fast rate, 0.2
10
s
, is roughly 5-fold slower than that measured
with immobilized
chains.
Figure 7:
Kinetics of heme transfer from soluble
ferrous CO-bound chains to albumin at pH 9.0 in 0.1 M Tris-HCl plus 0.1 M NaCl and 0.3 M sucrose.
Concentrations:
chains, 7
10
M; albumin, 3.4
10
M. Temperature: 20 °C. The solid line represents the fit to the reaction scheme given under
``Materials and Methods.''
Thereafter heme transfer experiments were carried out
by mixing different amounts of immobilized met-aquo dimers
with a constant concentration of albumin at pH 9.0 and incubating the
mixture at 20 °C. Under these experimental conditions an
equilibrium distribution is reached within 24 h. The heme transferred
from the immobilized dimers to albumin at equilibrium is around 30%
when heme is in excess over albumin and approaches 100% at the lowest
heme/albumin ratio tested (Fig. 8); conversely, the saturation
of albumin with heme increases with increase in heme/albumin molar
ratio (data not shown).
The time course of methemalbumin formation at different heme/albumin molar ratios is given in Fig. 9A. It can be described by a fast and a slow process whose apparent rate differs by approximately 50-fold, both rates increase slightly upon increasing pH from 6.5 to 9.0 (see Table 1). The amplitude of the fast process predominates at the higher heme/albumin molar ratios (Table 2).
Figure 9:
Kinetics of heme transfer from immobilized
ferric dimers to albumin at pH 9.0 (A) and 7.5 (B) and 20 °C. A, immobilized
dimers,
1.0
10
M; albumin (from bottom to top), 5
10
, 1
10
, 4
10
, 6
10
, 8
10
M. B, immobilized
dimers, 1.0
10
M; albumin, 5
10
, 1
10
, 2
10
, 4
10
, 6
10
, 8
10
M. The arrows show the end
points of the reaction measured after 24 h. The solid lines represent the fit to the reaction scheme given under
``Materials and Methods.''
Bunn and
Jandl(4) , when studying the exchange of hemes between
hemoglobins A and F, observed that the process is biphasic and proposed
that the fast rate reflects the dissociation of heme from non-
chains and the slower one dissociation from the
chains. This
assignment has been confirmed recently by Hargrove et al.(2) with the aid of mutant hybrid methemoglobins and
valence hybrids in which one subunit is oxidized.
The data in Fig. 9can be interpreted in the same way: when hemoglobin is in
excess, rapid dissociation of heme from the chains predominates
as indicated by the greater amplitude of the fast process; when albumin
is in excess, the contribution of heme dissociation from the
chains becomes relevant and the amplitudes of the fast and slow process
are approximately equal. Under all conditions, as the reaction
proceeds, there is an accumulation of the heme-albumin complex, and
thus, a significant contribution of the rate of hemin dissociation from
albumin, in particular to the slow phase. This interpretation was
substantiated by the analysis of the heme transfer reaction carried out
as outlined under ``Materials and Methods.'' Both rate
constants pertaining to albumin, k
and k
, 5
10
M
s
and 3.2
10
s
, respectively, were
calculated using the data of Fig. 2and Fig. 3. Likewise,
the equilibrium partitioning of hemin between albumin and the
immobilized
dimers was fixed at the value determined at the
end of the experiment reported in Fig. 8. The global fit of the
whole set of data yields rate constants of 6.5
10
and 2.8
10
s
,
respectively, for the slow and fast phase; the fitted time courses are
shown in Fig. 9.
Heme transfer experiments similar to those just described were carried out also at pH 7.5 and 6.5. The amount of heme transferred to albumin at equilibrium increases at any given pH with decrease in the heme/albumin molar ratio; at any given ratio of albumin to heme it decreases with decrease in pH. The rate constants which describe the time course of heme exchange are affected only slightly by pH (Table 1). Representative fits at pH 7.5 are included in Fig. 9.
Last a set of heme transfer experiments
from soluble hemoglobin was performed at pH 9.0 and 20 °C. These
measurements were designed to compare under the same conditions of pH
and temperature the rate of heme loss from immobilized dimers with that
from soluble dimers. To this end it appeared necessary to determine the
dimer-tetramer association constant of methemoglobin by
ultracentrifugation. To our knowledge there are no reports in the
literature on this equilibrium. Under the conditions used for the heme
transfer experiments, namely Tris buffer I = 0.1 M + 0.1 M NaCl at pH 9.0 and 20 °C, the
dimer-tetramer association constant was found to lie between 3.2
10
and 8.0
10
M
(dimer basis) by sedimentation
velocity (Fig. 10). Preliminary sedimentation equilibrium
experiments carried out at 10 °C yielded 5
10
M
. In view of the small temperature
dependence of the dimerization reaction at alkaline pH values
(
H°
13.2 ± 2 kcal/mol; (19) ), the K
value corrected to 20
°C, 9
10
M
, is
close to the upper limit obtained by sedimentation velocity. Thus, when
the concentration of soluble met-hemoglobin is 2.5
10
M (heme), the fraction of dimers is
close to 90%. The fit to the heme transfer data at this hemoglobin
concentration, carried out as described under ``Materials and
Methods,'' yields rates of 1.1
10
s
and 0.2
10
s
for heme loss from
chains in dimers
and tetramers, respectively, and 3.8
10
s
for the slow process (Fig. 10). The
rate of heme release from
chains in soluble dimers is in very
good agreement with that from immobilized ones (Table 1).
Likewise the rate of the slow process is fully consistent with the
values obtained for heme loss from chains in the dimer (Table 1)
and from methemalbumin.
Figure 10:
Kinetics of heme transfer from soluble
ferric HbA to albumin at pH 9.0 in the presence of 0.3 M sucrose and 20 °C. Concentrations (after mixing): hemoglobin
2.5 10
M (heme), albumin 2.5
10
M. The solid line represents the fit to the reaction scheme given under
``Materials and Methods.'' The amplitude of the slow phase is
0.008 and the value of the offset 0.261. The inset shows s
as a function of hemoglobin
concentration determined at pH 9.0 and 20 °C in parallel
sedimentation velocity experiments; different symbols refer to
different sets of experiments. The solid lines were calculated
with dimer-tetramer association constants of: 1, 1.6
10
; 2, 3.2
10
, 3, 8.0
10
M
(dimer
basis).
Sedimentation velocity experiments were also
carried out in 0.15 M phosphate buffer, pH 7.0 and 20 °C,
in order to assess the amount of tetramers present in solutions of
met-hemoglobin under conditions similar to those used by Hargrove et al.(2) . Dissociation into dimers is less
pronounced than in Tris buffer at pH 9.0 (K = 1.5
10
M
). This finding is not unexpected
since dissociation into dimers of oxyhemoglobin, which entails cleavage
of the same
interface, is likewise
enhanced at alkaline pH values(20) .
The experiments presented here show that the stability of the
heme-globin linkage in dimers and isolated chains of human
hemoglobin can be probed by studying the heme transfer reaction from
the immobilized proteins to albumin. Soluble and Sepharose-bound
heme-proteins transfer heme to albumin in a similar fashion. From a
qualitative viewpoint, only those derivatives which transfer heme to
albumin in solution do so when immobilized, and conversely, those
derivatives which do not release heme to albumin in solution do not
release heme when bound to Sepharose (Fig. 5, A and B). At a quantitative level the direct comparison carried out
with both
dimers and isolated chains indicates that the
immobilization step does not alter the heme environment significantly
while providing material that is not easily denatured after heme loss.
Thus, the rates of heme transfer from the immobilized proteins are
increased only 3- to 6-fold relative to the soluble ones (Table 1). Immobilized heme donors have other advantages; for
example they do not undergo changes in state of association, which may
complicate analysis of the heme transfer reaction in solution, and can
be separated easily from the heme acceptor. The latter property enables
one to monitor the relevant spectral changes easily even when the
spectral properties of heme donor and acceptor are very similar. In
brief, immobilized heme donors provide a solution to several problems
that have limited the study of the heme transfer reaction in
solution(2, 5, 6) .
In the present work human albumin has been used as the heme acceptor, because its affinity for heme is comparable with that of human hemoglobin(11) . This feature permits determination not only of the kinetic, but also of the equilibrium aspects of the heme transfer reaction. In turn, knowledge of the amount of heme partitioned at transfer equilibrium between the hemoprotein and albumin provides a useful constraint in the fit of the time courses to the reaction scheme.
The most interesting finding of
this study is that the stability of the heme-globin linkage changes
dramatically upon formation of dimers. This change is
especially striking in the ferrous CO-bound state: at pH 9.0 and 20
°C the isolated chains transfer heme significantly to albumin over
a time scale of a few hours, whereas no heme release takes place from
dimers ( Fig. 4and Fig. 5), which behave like
the
tetramer(4) . The
assembly of unlike chains, therefore, brings about a major
rearrangement in the heme pocket of the ferrous protein which results
in considerable strengthening of the heme-globin interaction, such that
in practice heme dissociation cannot be measured.
Heme oxidation
enhances heme release in human hemoglobin (2, 4, 5) and does so also in the
dimers (Fig. 5B), but has a very small effect on the
heme transfer properties of the isolated chains (Table 1). This
difference in behavior can be ascribed to the different nature of the
ferric forms of the proteins. The isolated chains give rise to
six-coordinate low spin hemichromes, in which the iron-proximal
histidine bond is effectively covalent, while HbA and its immobilized
dimers form high spin met-aquo derivatives in which this bond is
weakened. It is of interest that isolated
and
chains loose
their heme at approximately the same rate (1-6
10
s
), whereas in the
dimer heme dissociation from the
chains is some 50 times slower
than from the
chains (e.g. 0.065
10
s
versus 2.8
10
s
at pH 9.0 and 20 °C). In turn release of
heme from the
chains in the
dimer is only
2-3-fold slower than from isolated
chains. The interaction
between unlike chains, therefore, enhances the stability of the
heme-globin linkage significantly in the
chains and very little
in the
chains. It may be envisaged that the constraint on the
flexibility of the
chains imposed by the
chains (21) increases the activation energy for
-heme release and
that this constraint is transmitted to the
-heme pocket through
the
interface, since immobilized
dimers like the soluble ones are of the
type (7, 8, 9, 10) . The rate
of
-heme release increases with decrease in HbA
concentration(6) , indicating that dissociation of the
tetramer, and hence a change at the
interface, affects the stability of
the heme-globin bond in the
chains. Our data (Fig. 10)
suggest that
chains are little affected.
Benesch and Kwong (6) report rates of heme loss from chains in HbA dimers
and tetramers under pH and temperature conditions very similar to those
used in the present work; release from the
chains was not
determined as only 20% of the reaction was followed. The values given
by Benesch and Kwong (6) are 5-fold lower relative to those
reported in Table 1; however, the difference would be less if
heme release from albumin had been taken into account. Hargrove et
al.(2) report rates of 1.7 ± 1.1
10
s
for
-heme dissociation
and 2.2 ± 0.6
10
s
for the
-heme at pH 7.0, 37 °C, 2-4 µM heme, a roughly 2-fold increase in both rates at pH 8.0 and
relatively small temperature effects. These rates are lower than those
reported in Table 1for
and
chains. However, if one
takes the pH dependence into account and the fact that the rate of heme
loss from the
chains is decreased by the presence of tetramers
(10-25% at 2-4 µM heme under the conditions
used by Hargrove et al., 1994), the agreement between the two
sets of data is quite good.
Last, a comment on the dimer-tetramer
association constant of met-HbA is in order, since the value obtained
by sedimentation velocity and equilibrium in the present work is
considerably lower than that reported recently by Benesch and
Kwong(6) . The ultracentrifugation measurements carried out as
a function of met-HbA concentration at pH 9.0 yield values of K in the range 3-9
10
M
, whereas Benesch and Kwong (6) obtained 1.4
10
M
from measurements of the initial rate of heme transfer to serum
albumin at different hemoglobin concentrations. However, the following
considerations render this indirect method questionable. The curve given in Fig. 1, which was used to calculate the
dissociation constant for HbA, is not symmetrical as expected on the
basis of mass law considerations, indicating that factors other than
dissociation into dimers influence the heme loss assay as used by
Benesch and Kwong(6) . Accordingly, in a graph like that
presented in Fig. 2of the same reference, the data points do
not lie all on a straight line (in (6) the data below zero are
not reported). Furthermore, Benesch and Kwong (6) take the
concordance of the K
value assessed by means of
the heme loss assay for met-HbA at pH 9.0 with that obtained by Turner et al.(22) for oxy-HbA at pH 7.4 as a suggestion that
neither pH nor the oxidation state of the heme iron can have an
important effect on the dimer-tetramer equilibrium. This conclusion is
in contrast with current knowledge on the subunit dissociation behavior
of hemoglobin (20, 22) . A wealth of studies carried
out on ferrous HbA have shown subunit assembly to be affected by heme
and non-heme ligands, by buffer composition and pH. For instance,
alkaline pH values favor dissociation, and the sensitivity to heme
ligands is such that dissociation into dimers of oxy- and CO-HbA
differs significantly. Oxidation is therefore expected to affect
subunit dissociation and indeed the present sedimentation data show
that it enhances dissociation. Moreover, dissociation of met-HbA can be
expected to be influenced by the same factors which influence the
stability of the ferrous tetramer, since the same subunit interface is
involved.
In conclusion, the present data bring out that in
hemoglobin the heme-globin linkage in the chains is stabilized by
interactions between unlike chains at the
interface, whereas heme binding to the
chains is stabilized
by interactions at the
interface. In
accordance with this conclusion in mutant hemoglobins with point
mutations at the
interface, the rate
of heme release from the
subunits is increased with respect to
HbA (3) . No data on the effect of mutations at the
interface are available. In addition
to these long range effects, which have to be taken into account in any
systematic analysis of heme dissociation in mutant and chemically
modified hemoglobins, local factors at the heme pocket play an
important role in the stabilization of the heme-globin bond. These
comprise steric contacts of the porphyrin ring and electrostatic
interactions of its propionates with specific side chains in the distal
heme pocket like His-64, Val-68, Ser-92, and Arg 45, as discussed by
Hargrove et al.(2) in their recent study on
myoglobins and hemoglobin hybrids. In the understanding of the complex
interplay between all these factors in determining the stability of the
heme-globin linkage, the use of immobilized heme donors and of high
affinity heme acceptors, which are in preparation, will be of value.
This paper is dedicated to the memory of Jeffries Wyman, beloved and unforgettable master.