(Received for publication, November 27, 1996, and in revised form, December 18, 1996)
From the Program in Molecular Medicine and the Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605
From the Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06260
The assembly of Scapharca dimeric
hemoglobin as a function of ligation has been explored by analytical
gel chromatography, sedimentation equilibrium, and oxygen binding
experiments to test the proposal that its cooperativity is based on
quaternary enhancement. This hypothesis predicts that the liganded form
would be assembled more tightly into a dimer than the unliganded form
and that dissociation would lead to lower oxygen affinity. Our
experiments demonstrate that although the dimeric interface is quite
tight in this hemoglobin, dissociation can be clearly detected in the
liganded states with monomer to dimer association constants in the
range of 108 M1 for the
CO-liganded state and lower association constants measured in the
oxygenated state. In contrast, the deoxy dimer shows no detectable
dissociation by analytical ultracentrifugation. Thus, the more highly
hydrated deoxy interface of this dimer is also the more tightly
assembled. Equilibrium oxygen binding experiments reveal an
increase in oxygen affinity and decrease in cooperativity as the
concentration is lowered (in the µM range). These
experiments unambiguously refute the hypothesis of quaternary
enhancement and indicate that, as in the case of human hemoglobin and
other allosteric proteins, quaternary constraint underlies
cooperativity in Scapharca dimeric hemoglobin.
To perform biological activities efficiently, protein molecules have often evolved mechanisms to couple functionally independent subunits. Such cooperative activity is known to be involved in the regulation of many protein functions including enzyme activity (1, 2), gene expression (3, 4), and oxygen transport (5, 6). Much of our understanding of this process has come from studies of mammalian hemoglobins (5, 6), for which cooperativity manifests itself as a stepwise increase in oxygen affinity as oxygen binding proceeds.
A particularly simple system for exploring cooperative protein function is the dimeric hemoglobin found in the blood clam Scapharca inaequivalvis, which binds oxygen with a Hill coefficient of 1.5 and shows no change in oxygen affinity or cooperativity as pH varies from 5.5 to 9.0 (7). Although the tertiary structure of the subunits is similar to those of mammalian hemoglobins, the assembly into a cooperative complex is radically different (8). High resolution crystal structure analysis of Scapharca dimeric hemoglobin (HbI)1 has shown that ligand binding is coupled with significant tertiary rearrangements but very small quaternary changes in the relative subunit dispositions (9, 10).
Intersubunit communication depends upon coupling ligand binding with interactions between subunits. As Wyman (11) recognized nearly 50 years ago, cooperative interaction energy may be present as stabilizing energy between unliganded subunits (quaternary constraint; Ref. 12) or liganded subunits (quaternary enhancement; Ref. 13). These alternate conditions can be distinguished either by measuring the strength of the subunit interface as a function of ligation or by following ligand affinity as subunit dissociation occurs. In the case of quaternary constraint (12), the deoxygenated oligomer would be more tightly assembled than the liganded form, and, since the deoxy assemblage acts to lower oxygen affinity, subunit dissociation would result in increased oxygen affinity. In the case of quaternary enhancement (13), the opposite situation exists: the liganded complex would be more tightly assembled, and lower affinity would result from dissociation into subunits.
In human hemoglobin, cooperativity results primarily from quaternary
constraints in which the tight deoxy assemblage lowers the intrinsic
subunit oxygen affinity, and binding of subsequent ligands leads to a
stepwise reduction of these constraints (14). Analysis of equilibrium
binding data has suggested that the fourth ligand is bound with higher
affinity than isolated chains, an effect termed quaternary
enhancement (13). This interpretation has, however, been
questioned on the basis of kinetic experiments that appear in conflict
with the equilibrium results (see Refs. 15-18). In the case of a
mutant human hemoglobin, Hb Ypsilanti (99 Asp
Tyr), assembly of
dimers into noncooperative tetramers results in an 85-fold
increase in oxygen affinity (19), a clear example of quaternary
enhancement.
A key structural change upon ligand binding to Scapharca HbI
is the extrusion of a phenylalanine (97) from the heme pocket into the
subunit interface where it displaces a number of interfacial water
molecules (Fig. 1). In deoxy-HbI packing of the Phe-97
side chain in the heme pocket appears to be largely responsible for the
low oxygen affinity of this state, and its disposition is central to
the proposed cooperative mechanism (9). The ligand-linked movement of
Phe-97 and disruption of interface water molecules led to the
hypothesis that cooperativity in Scapharca HbI could result
from quaternary enhancement rather than quaternary constraint. Binding
of a ligand to an isolated subunit would require extrusion of Phe-97
into bulk water rather than the intersubunit interface. Since in the
dimeric interface Phe-97 can interact with the methyl group of Thr-72
(from the second subunit), Royer et al. (8) proposed that
extrusion of Phe-97 would be more favorable in the dimer than in
isolated subunits. This suggests that isolated subunits would bind
oxygen with lower affinity than would subunits assembled in a dimer.
Then, by the linkage between ligand binding and subunit association, a
liganded dimer would be more tightly associated than an unliganded
dimer. This appeared reasonable given the additional water molecules in
the deoxy interface whose ordering is expected to be entropically
unfavorable in contrast with the liganded interface in which additional
hydrophobic interactions are present involving Phe-97.
The present study was undertaken to test the hypothesis that quaternary enhancement underlies cooperativity in Scapharca dimeric hemoglobin. If this hypothesis were true, the liganded hemoglobin would be more tightly assembled than the unliganded hemoglobin, and oxygen affinity would decrease upon subunit dissociation. Our results demonstrate that the reverse is true: Scapharca dimeric hemoglobin, like human hemoglobin, is more tightly assembled in the unliganded state than in the liganded state, and subunit dissociation leads to an increase in oxygen affinity.
Experiments were performed both with Scapharca HbI derived from the clam, kindly provided by Dr. E. Chiancone, University of Rome, and with bacterially expressed recombinant protein (20). No differences in assembly were noted for samples from these two different sources. The standard buffer consisted of 0.1 M Tris with 1 mM Na2EDTA, titrated to pH 7.2 with concentrated HCl. Titration and pH measurement were carried out at 20 °C for chromatography experiments and at room temperature (23 °C) for centrifugation and oxygen binding experiments. Tris was purchased from Sigma; EDTA was obtained from Fisher Scientific.
Gel ChromatographyAnalytical gel chromatography experiments were carried out with CO-saturated HbI and buffers using a Sephadex G-75 column (0.9 × 40 cm) thermostatted at 20 °C. For these experiments, the CO-liganded derivative was chosen to minimize the effects of heme oxidation. The void and internal volumes of the column were determined with blue dextran and glycyl-glycine, respectively, and the quality of the column packing was determined from experiments on a variety of nonassociating proteins.
Large zone chromatography experiments (21) were carried out at a
variety of HbI concentrations. For each experiment, ~25 ml of
hemoglobin solution was prepared in standard buffer, filtered (Gelman
0.45 µm) and loaded onto the Sephadex G-75 column. Concentration measurements were carried out spectrophotometrically, using an extinction coefficient of 0.202 (µM heme)1
for CO-liganded HbI (7). The flow rate of the column was controlled accurately at ~15 ml/h using a peristaltic pump. Continuous
absorbance measurements in the Soret region were made using a Shimadzu
UV 160U spectrophotometer and recorded on an associated analog
recorder.
From experiments at each concentration, the elution volume was
determined from the centroid position of the leading boundary of the
column profile. The elution volume (V) was used to calculate the weight-average partition coefficient (w) by the
relation
w = (V
V0)/Vi, where
V0 and Vi are the void
and internal volumes of the column, respectively.
For HbI, which undergoes monomer-dimer assembly
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
Data analysis was carried out using nonlinear least squares methods
(22). The analysis program uses a modified Gauss-Newton algorithm to
obtain parameter values that correspond to a minimum in variance. For
experiments on HbI, an independent estimate of the monomer partition
coefficient (1) was obtained by experiments using
myoglobin (horse heart). This value was fixed to the experimental value
of 0.4204 in all data analysis. An estimate of
2 (dimer partition coefficient) of 0.2828 ± 0.0047 was obtained from the data analysis.
Samples of HbI-O2 or HbI-CO at approximately 0.4 mg/ml were dialyzed overnight against the standard buffer. In the case of HbI-CO, all solutions were saturated with carbon monoxide.
For a typical experiment, samples were diluted 10-fold, 20-fold, and 67-fold with buffer prior to loading in ultracentrifuge cells. For each dilution, 100 µl of hemoglobin sample was loaded into one of the three solution channels of a 12-mm six-channel exterior loading analytical ultracentrifuge cell (23) equipped with Quartz windows. The solution channels had previously been loaded with 10 µl of a fluorocarbon oil (Minnesota Mining and Manufacturing Co., FC-43) to make the bottom of the solution visible. 120 µl of dialyzed solvent was loaded into each of the three solvent channels. In some cases additional (higher or lower) loading concentrations were used. The cells were centrifuged at 30 krpm and 1 °C overnight in a Beckman XLA analytical ultracentrifuge.
Absorption data were acquired using the XLA scanning system (at 417 nm for HbI-O2 and 422 nm for HbI-CO), which records absorption as a function of distance from the center of rotation. The typical error in these readings is approximately 0.005 AU which, under these conditions, corresponds to a concentration error of about 0.5-1.5 µg/ml. Absorption data were spaced at 10-µm intervals down the cell and recorded every 4 h to test for equilibrium. When no change in the gradient was observed after subtracting successive scans, equilibrium was judged to be achieved. Equilibrium required about 35 h of centrifugation after which the final absorption measurements were recorded. Following the 30 krpm run, the centrifuge was accelerated to 40 krpm and maintained at this speed until equilibrium was obtained (about 38 h), permitting a second set of equilibrium measurements to be made.
A second series of experiments was performed on other samples of HbI-O2 using the photoelectric scanner of the model E analytical ultracentrifuge. Absorption measurements at 416 and 432 nm were used to monitor the protein gradient. Samples at concentrations of 5, 10, and 20 µg/ml were loaded in 30-mm optical path cells. The cells were centrifuged at 1 and 25 °C at 30 and 40 krpm.
Ultracentrifugation of Deoxy-HbISamples of deoxy-HbI were loaded into centrifuge cells in an anaerobic chamber (Anaerobe Systems, Santa Clara, CA) at concentrations similar to those used for liganded HbI as described above. A significant difficulty resulted from residual oxygen in cell material. Various strategies were pursued to eliminate oxygen from the sample such as using different cell materials (polystyrene and Kel-F), soaking cells in dithionite (sodium hydrosulfite, Sigma), and including dithionite in the deoxy-HbI samples. In the absence of dithionite, the deoxy absorption spectrum was lost within a few hours of loading. In the presence of dithionite, the samples, although maintaining a deoxy spectra, showed varying high values of Z-average molecular mass (Mz) indicating polymerization of HbI. In addition, subsequent exposure to oxygen in the presence of dithionite led to a nearly complete loss of absorbance. Finally, a satisfactory procedure was obtained by loading both solute and solvent channels of Kel-F cells with deoxy-HbI at about 0.4 mg/ml and a small quantity of dithionite and leaving them in the anaerobic chamber for several weeks. The cell solvent channels were then rinsed with several quantities of deoxygenated buffer and then loaded with buffer. The solution channels were rinsed with several volumes of the appropriately diluted deoxy-HbI solution before loading. The hemoglobin remained deoxy, as monitored by the ratio of absorbance at 432 nm to that at 416 nm, for several days of sedimentation at 1 °C. The cells were centrifuged at 24 krpm until equilibrium was attained followed by centrifugation at 32 krpm. The lower speeds of the deoxy-HbI centrifugation, compared with those used for liganded HbI, were chosen because of the higher values of Mz observed for all deoxy-HbI samples. Absorption data were measured at 416 and 432 nm in the XLA scanning system.
An additional deoxy experiment was performed with freshly dialyzed HbI several weeks later. After centrifugation of deoxygenated material, the cells were opened and exposed to air for several hours to permit oxygen binding. The cells were then centrifuged at 32 krpm until equilibrium was achieved.
Analysis of Centrifugation DataThe concentration distribution at equilibrium was analyzed using a nonlinear least squares program (24) testing various models. A model assuming an ideal single species (ISS) was first fitted to the combined sets of data from the different sample loading concentrations and speeds of rotation. This provides the value of the molecular mass if an ideal species is present and an Mz if the material is nonideal or a mixture of species. Many other models were then examined: most notable for this experiment is the monomer-dimer model, which yields the molecular mass (M1) of the associating monomer and its association constant. Criteria for goodness of fit is that the root mean square error be small (usually less than 0.01 AU, or about 1 µg/ml) and that it be random (small systematic error in the residuals).
Calculation of molecular mass and equilibrium constant from the pattern
of concentration with radius in the cell requires the knowledge of the
partial specific volume of the protein () in its solvent and the
density of the solvent (
). Based on the amino acid sequence (25) the
value of
was calculated as 0.745 (cm3/g). This does not
include the heme group, which would be expected to increase its value.
Values of
for human oxy- and deoxyhemoglobin have been reported to
be 0.746 and 0.749, respectively, at 20 °C and 0.09 M
NaCl (28). For the results reported here, a value of 0.75 was used. The
density of the solvent was measured to be 1.004 g/cm3 with
a Paar DMA 602 density meter at 20 °C. (This was corrected to 1.006 and 1.003 g/cm3 at 1 and 25 °C, respectively.)
Oxygen binding to HbI at low concentrations (0.2-8 µM) was followed by tonometric measurements (26) in the Soret region. Samples were deoxygenated at 4 °C by flushing with nitrogen and were then equilibrated at 23 °C. Air was introduced with the use of a 2.5-ml gas-tight Hamilton syringe (Fisher Scientific) through a rubber septum and allowed to equilibrate at 23 °C for 10 min. Absorption readings were recorded at 416, 422, 424, and 434 nm. Significant loss of material was observed with samples at low concentration apparently due to adsorption to the glass tonometer. This problem was eliminated by coating the glass with bovine serum albumin (3 mg/ml) for 30 min prior to oxygen binding measurements. To minimize heme oxidation, the tonometer was also coated for 10 min with a solution containing an enzymatic reduction system (27). Following rinsing, residual remaining components were sufficient to eliminate problems with oxidation. This method was used to minimize the strong absorption in the Soret region by catalase, which would obscure the hemoglobin contribution at very low concentrations.
Large zone gel chromatography experiments were performed on HbI-CO
with solutions ranging in concentration from approximately 5 to 0.05 µM heme. Although the dimeric assemblage is quite stable, a clear increase in elution volume results as concentration is lowered,
indicating detectable dissociation in this concentration range. Fig.
2 shows the weight average partition coefficients plotted as a function of HbI concentration along with a theoretical curve based on a best fit of the equilibrium constant and end points.
This fit yielded a monomer-dimer association constant (2Ka) of 7.6 × 107 M1 (Table
I).
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Sedimentation equilibrium experiments were performed in several runs of liganded (O2 or CO) HbI and two runs with unliganded HbI. For each run samples were loaded at three different protein concentrations and centrifuged at two different speeds, providing a total of six data sets/run. The centrifugal depleting effect at the meniscus and concentrating effect at the bottom of the cell result in a range of concentrations studied from lower than 0.06 µM heme to as high as 10 µM heme.
Results of the sedimentation equilibrium experiments are presented in
Table I. Values are included for the Mz based on the ISS model and for
association constants based on a monomer-dimer equilibrium. Experiments
were performed measuring absorbance at two wavelengths to observe any
wavelength dependence of molecular mass which could indicate the
presence of oxidized hemoglobin (28). The values at the two wavelengths
were found to be within the confidence interval of the measurements,
therefore averages of the values at both wavelengths are reported in
Table I. For liganded species, fits obtained with the monomer-dimer
model were significantly better than the fits obtained with the ISS
model and far better than any of the other models considered. Although this can be seen from the lower degree of systematic error shown in
Fig. 3, a more demanding measure of the superiority of
the monomer-dimer model fit to the data is that the ratio of the
variance of the two models favored the monomer-dimer fit over the ISS
model to greater than a 99.7% confidence limit. In contrast to the
liganded species, ultracentrifugation of the deoxygenated samples
showed no measurable dissociation and indicated Mz values close to the molecular mass (33.1 kDa) of the dimer. In one experiment (labeled reoxy) following a run of deoxy sample, the cell was opened
and allowed to equilibrate with oxygen, then resealed and centrifuged again. This experiment is in good agreement with the other
oxyhemoglobin runs (Table I).
As is clear from Table I, there is good agreement for the monomer-dimer
association constant obtained by gel chromatography and sedimentation
equilibrium for HbI-CO. However, lower association constants are
observed for the oxygenated samples compared with the HbI-CO. This may
reflect a real difference between HbI-CO and HbI-O2,
despite their very similar structures (10), or it could result from a
slight oxidation of the heme iron which was not detected. (Oxidation of
HbI results in a hemichrome species that shows extensive dissociation,
with an association constant of about 3 × 103
M1 at pH 7.0 (29).)
Thermodynamic coupling between ligation and subunit dissociation (14)
predicts that given a tighter assemblage in the deoxy state compared
with the oxy state, oxygen affinity of HbI should increase upon subunit
dissociation. To test this prediction, we carried out oxygen binding
experiments on solutions with hemoglobin concentrations between 0.2 and
8 µM heme. In this concentration range, significant
dissociation of liganded species is expected, based on the
chromatography and sedimentation results discussed above. As
concentration is lowered, a clear increase in oxygen affinity is
observed as indicated by the decrease in p50
(Fig. 4). The observed increase in affinity is coupled
with a decrease in cooperativity as evidenced by the Hill coefficient
declining from 1.45 at 8 µM heme to 1.25 at 0.2 µM heme, indicative of partial dimer dissociation.
Our experiments demonstrate that Scapharca HbI is more
tightly assembled into a dimer in the absence of a heme ligand than it
is once oxygen or carbon monoxide is bound. Consequently, the energetic
coupling of subunit assembly and ligand binding, depicted in Fig.
5, requires that monomers will bind oxygen with higher affinity than dimers. This has been verified by oxygen binding measurements at low hemoglobin concentration which show increased oxygen affinity as a result of partial subunit dissociation. Thus, as
in the case of human hemoglobin and other allosteric proteins, quaternary constraint underlies the cooperative mechanism.
An important ramification of these experiments is that the more hydrated interface of deoxy-HbI is also the form with the greater subunit interface stability. It is relevant to note that no large quaternary structural changes occur upon ligand binding to HbI. Thus, differences in the stability of the dimer can be interpreted in light of the rather localized ligand-linked structural changes at the subunit interface. As shown in Fig. 1, the high resolution crystal structures reveal the presence of 17 well ordered water molecules arranged in a cluster in the core of the deoxy interface compared with 11 water molecules in HbI-CO or HbI-O2. This crystallographic observation has recently been extended by measurements of oxygen binding as a function of osmotic pressure which indicate that oxygen binding is coupled with the release of approximately six water molecules/hemoglobin dimer (30). Significantly, the water molecules in the deoxy interface are more well ordered and provide more favorable hydrogen bonding than those in the liganded interfaces (9, 10). Our present results suggest that these water molecules could contribute to interface stability, despite the entropic cost of their ordering. Removal of just two hydrogen bonds from the protein to the deoxy water cluster by mutation of Thr-72 to Val results in the loss of two water molecules and a 40-fold increase in oxygen affinity (30). This suggests a direct link between the integrity of the interface water cluster and maintenance of the low affinity deoxy conformation, further supporting a contribution by these water molecules to interface stability.
Our experiments also reveal that isolated subunits of HbI bind oxygen
with higher affinity than those in the dimeric assemblage. The high
resolution crystal structures reveal a number of interactions between
deoxy subunits which could stabilize their low affinity conformations.
A key determinant of oxygen affinity appears to be the conformation of
Phe-97 whose side chain packs tightly in the heme pocket in the deoxy
state but is extruded into the subunit interface upon ligation (9).
When packed in the heme pocket, Phe-97 is thought to reduce oxygen
affinity by restricting movement of the iron into the heme plane and by
lengthening a hydrogen bond involving the proximal histidine (9).
Coupled with the deoxy disposition of Phe-97 is a sharp bend of the F
helix and a displacement of the heme group toward the subunit
interface. The dimeric assemblage of deoxy-HbI appears to contribute to
the stability of these conformations by providing hydrogen bond
partners for main chain atoms of the bent F helix (from water
molecules) and the heme propionate groups (from Lys-96, Asn-100
, and
water molecules). Thus, the interactions present in the subunit
interface appear important for the stability of the low affinity
conformation, which is consistent with our present results
demonstrating increased oxygen affinity as partial subunit dissociation
occurs.
The assembly of subunits of Scapharca HbI into a highly stable dimer makes complete elucidation of the thermodynamic linkage between assembly and ligand binding difficult. The results presented here provide estimates for the dissociation constants of liganded forms, but the lack of any observable dissociation of the deoxygenated form precludes an estimate of its stability. As well, the strength of the dimeric interface prevents an accurate estimate of the oxygen affinity of dissociated monomers. To address these issues, we intend to use site-directed mutagenesis of HbI (20) to probe the assembly reaction.
We thank Drs. Emilia Chiancone and Gianni Colotti for providing purified Scapharca HbI used in our initial experiments and Animesh Pardanani for several helpful discussions.