Pathway and Mechanism of pH Dependent Human Hemoglobin Tetramer-Dimer-Monomer Dissociations
Luo M (2013) Pathway and Mechanism of pH Dependent Human Hemoglobin Tetramer-Dimer-Monomer
Dissociations. PLoS ONE 8(11): e81708. doi:10.1371/journal.pone.0081708
Pathway and Mechanism of pH Dependent Human Hemoglobin Tetramer-Dimer-Monomer Dissociations
Yao-Xiong Huang 0 1
Zheng-Jie Wu 0 1
Bao-Tian Huang 0 1
Man Luo 0 1
Joseph P. R. O. Orgel, Illinois Institute of Technology, United States of America
0 Funding: This work was supported partly by the Chinese National Natural Science Foundation (Nos. 30940019 and 60377043) and Guang Dong Provincial Science and Technology Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
1 Department of Biomedical Engineering, Ji Nan University , Guang Zhou , China
Hemoglobin dissociation is of great interest in protein process and clinical medicine as well as in artificial blood research. However, the pathway and mechanisms of pH-dependent human Hb dissociation are not clear, whether Hb would really dissociate into monomers is still a question. Therefore, we have conducted a multi-technique investigation on the structure and function of human Hb versus pH. Here we demonstrate that tetramer hemoglobin can easily dissociate into dimer in abnormal pH and the tetramer dimer dissociation is reversible if pH returns to normal physiological value. When the environmental pH becomes more acidic (<6.5) or alkaline (>8.0), Hb can further dissociate from dimer to monomer. The proportion of monomers increases while the fraction of dimers decreases as pH declines from 6.2 to 5.4. The dimer monomer dissociation is accompanied with series changes of protein structure thus it is an irreversible process. The structural changes in the dissociated Hbs result in some loss of their functions. Both the Hb dimer and monomer cannot adequately carry and release oxygen to the tissues in circulation. These findings provide a comprehensive understanding on the pH-dependent protein transitions of human Hb, give guideline to explain complex protein processes and the means to control protein dissociation or reassociation reaction. They are also of practical value in clinical medicine, blood preservation and blood substitute development.
In certain abnormal metabolism and respiration conditions,
especially in some pathological situations such as acidosis or
alkalosis, the pH value of human blood would fall outside of the
normal physiological range of 7.35-7.45. Some preserved
blood specimens in blood bank even have pH values lower
than 6.5 owing to various factors[1,2]. It was reported that
under abnormal pH condition, human tetramer hemoglobin
tetrameric hemoglobin is a very important issue in clinic and
blood substitute research because dissociated Hb in body
circulation would induce some side effects such as renal
tubular damage and toxicity, and be cleared rapidly from
circulation . However, up till now, the mechanism of the
dissociation is not completely clear. Whether the Hb dimer
would further dissociate into monomers is also a question for
lack of solid evidence, although several hypotheses have been
Only Shaeffer and Mrabet reported that they had
found experimentally some free 3H-labeled chain monomers
were incorporated into carbonmonoxy-hemoglobin A dimers by
exchanging with pre-existing unlabeled chain subunits in a
pH range of 6.5-8.4. They believed that it indicated dissociation
of Hb dimers into monomers. Regardless of whether such an
indirect evidence is convincing or not, the dimermonomer
dissociation constant K2 calculated from their experiment was
just 4.710-13 M, suggesting that the possibility of the
dissociation was very low in the circumstance. In other words,
the dissociation was mainly a random behavior, and did not
involve any specific factor which induced change in the
hemoglobin structure thus causing the dissociation.
To our knowledge, besides no direct evidence has been
reported to indicate whether normal human hemoglobin (Hb A)
would dissociate to monomer in solutions of abnormal pH, no
one knows if dissociated hemoglobin can re-associate to a
normal tetramer structure when pH returns to physiological
level. Obviously, clarification of these questions is significant.
Since it can not only lead to a comprehensive understanding
on human hemoglobin dissociations, but also provide methods
of controlling the protein dissociation or re-association reaction,
and give guidelines to clinical medicine and blood preservation,
as well as blood substitute development.
Therefore, we performed a multi-technique systematic study
on the issue. Our objective was to obtain direct evidences on
(1) the size distribution of Hb molecules in the solutions with
various pH values; (2) the pathways of human hemoglobin
dissociation; (3) the protein structural changes under different
dissociation conditions; and (4) the structural changes of the
proteins globin and heme vs. pH. Thereby we can reveal the
mechanism of various Hb dissociations, and disclose whether
the dissociation processes are reversible when the solution pH
returns to normal physiological value.
Materials and Methods
The protocol of this Study was approved by Ji Nan University
Animal Care and Use Committee conforming to the Chinese
Public Health Service Police on Human Care and Use of
Laboratory Animals. Informed written consent was obtained
from the healthy non-smoking adult volunteers providing
All reagents and solutions were prepared from analytical
grade materials. 20 mM phosphate buffer was prepared in
distilled water; its pH was adjusted to the desired values
ranging from pH 5.4 to 9.0 by adjusting different proportion of
Na2HPO4 and NaH2PO4.
Preparation of hemoglobin
Hemoglobin was prepared according to Geraci et al.
Normal blood was obtained from healthy non-smoking adult
volunteers (providing informed written consent) by
venipuncture and poured into heparinized tubes. The blood
was centrifuged at 1500 rpm for 10 min, during which the
plasma and buffy coat were removed by aspiration. Thereafter
the erythrocytes were washed three times with 0.9% NaCl, and
then centrifuged for 10 min at 2000 rpm to remove the
supernatant. The erythrocytes were later kept cold at 4 C for
15min and lysed in 10 vol of ice-cold water, then churned to be
disrupted. The sample was kept at 4 C again for 20min,
afterwards, the hemolysate was obtained by centrifugation for
30min at 12000rpm (4 C), followed by filtration through a
0.22m disposable filter holder. The obtained samples were passed
over a Sephadex G-25 for purification. Later, the purified
hemoglobin was stored in a refrigerator at 4 C and ready to be
The concentrations of hemoglobin in each of the sample
solutions were determined using the hemiglobincyanide (HiCN)
method and detected by an absorption spectrophotometry at
540nm. The purified hemoglobin was diluted by the
phosphate buffers of different pH values (5.4, 5.9, 6.2, 7.4, 8.0
and 9.0) to be 100 M (heme) in concentration. The
concentration was sufficiently low so that inter-particle
interactions could be neglected.
Dynamic light scattering
Considering none of the conventional techniques such as gel
chromatography, gel filtration, sedimentation etc. can
perform non-disturbance, real time, in situ measurement on the
natural situation about the dissociation of Hb in solutions, we
improved the technique of dynamic light scattering (DLS) to
obtain detailed information about the size distribution of Hb
molecules in solution. In the measurements, the scattered light
intensity and its autocorrelation function were measured by a
Zeta PALS instrument (Brookhaven, USA) using a laser with
wavelength of = 678 nm. Detailed procedures of the DLS
measurements were the same as that we described
previously. The diameters of the hemoglobin in different pH
values were detected by analyzing the autocorrelation function
of the scattered light. The size distribution of Hb molecules in
each pH was derived from a NNLS(Non-Negatively constrained
Least Squares algorithm) analysis using number weighting by
which each particle (no mater it is small or large) has equal
weighting once the final distribution is calculated. By this
means, the proportion of each kind of Hb molecules
(monomers, dimers and tetramers) in the solutions can be
calculated accurately. All the reported size distribution data are
the averages of the results from at least five measurements.
The measurements were performed at temperature of 37 C.
To verify the information obtained by DLS, we employed gel
chromatography to determine if the solutions really coexisted
hemoglobin molecules of different molecular weights. Large
zone elution volumes were determined as a function of pH for
human hemoglobin. A Sephadex G-50 column (1.640cm) was
employed with the eluent buffer: 20mM phosphate buffer
(pH7.0). Cytochrome C (Sigma, MW=13KDa) and bovine
serum albumin (Sigma, MW = 67KDa) were used as the
references to determine the elution volumes of human
hemoglobin in different solutions.
UV-visible absorption spectrophotometry
We also performed UV-Visible absorption spectroscopy to
measure the structure of heme and its combination with globin
chain as functions of pH. The UV-visible absorption
spectrophotometry was taken with a Unicam 500(Thermal,
USA) in the wavelength ranging from 350 to 700 nm at room
temperature. The heme peaks of hemoglobin as functions of
pH values were obtained by analyzing the UV-visible spectra
measured in different pH suspensions.
To perform non-disturbance, real-time measurements on the
molecular structure and oxygen carrying capacity of
hemoglobin in various pH solutions, we also developed a
series of novel methods of Raman confocal
microspectroscopy[19,20]. The Raman spectra measurement was
performed using a RAM INV (Horiba JY, France) system with
an inverted Olympus optical microscope. The 514 nm light from
an Ar+ iron laser was chosen as the excitation light for it has the
advantage in obtaining resonant signal at the characteristic
Figure 1. The hydrodynamic diameters and size distributions of human hemoglobin in solutions with different pH
levels. (a) The size distribution of Hb at pH 7.4; (b) the size distribution of Hb at pH 5.4; (c) the averaged diameters of human
hemoglobin vs. pH level; (d) the averaged hemoglobin diameters in the solutions after their pH levels had been returned to normal
condition. The pH values indicated in the horizontal coordinate are the original pH values of the sample solutions.
oxidation marker 4 band (1358cm-1) of hemoglobin and
providing direct and detailed information about the structure of
heme. The spectra were recorded between 1800 and
600 cm-1 with a resolution of 1 cm-1. For each measurement, a
laser exposure time of 10 s was selected and ten scans were
accumulated. Before the measurements, a silicon wafer with a
character band at 520.7cm-1 was used to calibrate the
instrument on daily basis. All the spectra were normalized
using the CH2 band at 1448cm-1 as normalization metric. The
measurements were performed at 37 C. For every pH value,
five parallel samples were prepared and each sample was
measured at least five times, so the spectrum of each pH value
was obtained from the average result of at least 25 spectra.
Results and Discussion
Sizes of hemoglobin under different pH values
Figure 1 shows the size distributions of human hemoglobin in
pH 7.4 and pH 5.4 solutions respectively. From Figure 1(a) we
can see that human hemoglobin in the solution of pH 7.4 was
5.6 nm in size and mono-dispersed. This is consistent with the
diameter of a hemoglobin tetramer (5.5 nm) observed by x-ray
Figure 1(b) illustrates the size distribution of hemoglobin in
the solution of pH 5.4. We can see that, besides the 5.62 nm
tetramers, more particles are either 4.2 nm or 3.16 nm in
diameter. According to the formulas about the ratio between
the sizes of a tetramer and a dimer: RRDT = 2 3 , and between a
diameter particles can be distinguished as dimers and
monomers respectively. From the table shown in Figure 1(b)
about the size distribution of Hb in the pH 5.4 solution, we can
see that the majority (~80%) of the hemoglobin molecules were
monomers, about one-sixth were dimers, and only few were
Figure 1(c) shows how the mean diameter of Hb varies with
pH. We can see that the Hb molecules in the physiological
range (pH 7.1-7.4) have a mean size of tetramer about 5.3
5.6 nm, but become smaller and have a mean size of monomer
about 3.4 - 3.7 nm in acidic ( pH 5.4, 5.9, 6.2) and strong
alkaline (pH 9.0) conditions. At pH 8.0, the mean size of Hb is
4.6 nm. The mean diameter of Hb at each pH value shown in
Figure 1(c) was averaged from the size distribution of
hemoglobin in the pH, the size distribution in turn, gives
detailed information about the proportions of monomers,
dimers and tetramers in each of the hemoglobin solutions.
Table 1 lists the proportions of monomers, dimers and
tetramers in the hemoglobin solutions of different pH values.
Each set of the data was averaged from the results of five
Table 1. The proportions of monomers, dimers and
pH Proportion of Hb with different diameters (%)
3.16nm 4.22nm 5.62nm
5.4 81.97 15.57 2.46
80.65 16.95 2.40
82.65 15.70 1.65
79.37 16.67 3.96
77.52 19.398 3.10
5.9 67.11 26.17 6.72
74.07 21.48 4.45
73.53 22.79 3.68
68.49 25.35 6.16
75.76 20.45 3.79
6.2 65.36 29.41 5.23
68.97 25.52 5.53
71.43 23.57 5.00
65.36 28.76 5.88
70.42 23.94 5.64
parallel samples. The proportion of the Hb molecules with a
certain size d was deduced from the values of G(d) (see Figure
1(a) and 1(b)). G(d) is the intensity of the light scattered from
the particles with size d and is proportional to the number of the
particles. Therefore, by having all the G(d) values of the Hb
molecules with different sizes, one can obtain information
about the proportion of each kind of the molecules. We can see
that in the pH 5.4 solution, most of Hb molecules are
monomers. As pH increases, the proportion of monomers
decreases, whereas the fraction of dimers increases. In normal
pH solution, all the Hb molecules are tetramers. While at pH
8.0, most of Hb molecules are dimers, with some coexisting
tetramers but no monomers. When pH value reaches 9.0, once
again most of Hb molecules are monomers (about 60%), less
than 33% are dimers, and only a few are tetramers (7%).
From the Gel chromatography elution profile of a mixture
solution of Borvine serum albumin and cytochrome C, it was
found that the elution peak of Borvine serum albumin is with a
volume of 30.95 ml, while the elution peak of cytochrome C is
with a volume of 49.4 ml. By using them as the references, we
determined the elution volumes of human hemoglobin in
Figure 2 (a) displays the elution profile of the hemoglobin
solution at pH 7.4. According to the Andrews formula, the
calculated molecular weight corresponding to the elution
volume is about 63KD and should be that of Hb tetramers
(64.5KDa). This is consistent with the result of dynamic light
There are two elution peaks for the solution of pH 5.4 (see
Figure 2(b)). The major one has an elution volume of 46.6ml or
a molecular weight of 16.39 KD and should be the one of Hb
monomers (16 KD). The small one could be the mixture of
tetramers (64.5 KD) and dimers (32 KD). The elution profile
also suggests that the major molecular structure in the solution
is monomer, the same as that implied by the results of dynamic
The reversibility of hemoglobin dissociation
To verify whether or not the dissociated Hb can re-associate
to tetramer or dimer, the Hb solutions which had been adjusted
to different acidic or alkaline conditions for 72 hours were
readjusted to pH 7.4 and incubated for 48 hours (at 4 C). After
that, DLS measurements were performed on the samples at 37
C to determine their particle size distribution. Figure 1(d)
displays the average hemoglobin diameters in the solutions
after their pH had been returned to normal condition for 48
hours. The pH values indicated in the horizontal coordinate are
the original pH values of the sample solutions. We can see that
only the hemoglobin originally in the solution of pH 8.0 can
return to a tetramer structure, whereas the hemoglobin
molecules in the other solutions which originally were in acidic
or other alkaline conditions remained as monomers.
Variation of Hb structure in different pH environments
depicted by the UV-Visible spectra
The absorption peak of Hb at 415 nm is the characteristic
peak of heme and called the Soret band. Its shift under
different pH conditions is displayed in Figure 3. We can see
that, when pH value is lower than 7.4, the Soret band has a
blue shift, indicating that the combination of heme and the
heme socket has changed. However, at higher pH values (8,9),
the Soret band doesnt change.
Raman spectra of hemoglobin under different pH
Raman spectroscopy measurement can sensitively and
accurately detect changes in protein structure, especially the
porphyrin moieties within heme and other metalloporphyrin.
Figure 4 displays the Raman spectra (from 600-1800 cm-1) of
hemoglobin in the solutions of different pH values. As
mentioned above that, each of the spectra was obtained by
averaging the results of at least 25 measurements. No
significant spectrum difference was found from different parallel
groups, each Raman peak was always in the same position
Figure 2. The elution profiles of the hemoglobin solutions (a) at pH 7.4 and (b) at pH 5.4.
among spectra (1cm-1) and the standard deviation in amplitude
is relatively small(see Figure 4(a)).
From figure 4(a) we can also see that at pH 7.4, the major
Raman peaks of hemoglobin are at 1358, 1548, 1581 and 1608
cm-1. These peaks correspond to the characteristic bands of
deoxyHb[19,26,27], so it suggests that in normal pH condition
hemoglobin is mainly deoxygenated. This is consistent with the
results of our previous measurement on the Hb in living human
erythrocyte. As described previously, the samples were
incubated at 4 C for 72 hours before Raman spectroscopy
measurement. When erythrocytes are cooled down in such a
circumstance, the concentration of 2,3 DPG in the cells
increases [28,29]. Since 2,3 DPG is a heteroallosteric effector
of hemoglobin, it can lower hemoglobin's affinity for oxygen by
binding preferentially to deoxyhemoglobin. An increased
concentration of DPG in normal red blood cells favors
formation of the T, low-affinity state of hemoglobin. Therefore,
most of the Hb molecules obtained from the erythrocytes in the
condition of 4 C for 72 hours were in deoxygenated state.
Figure 4(b) shows the Raman spectra of hemoglobin at
different pH values. We can see that the peak intensity at
1358cm-1 varies with pH. This peak is the 4 band of heme and
it is the characteristic band of deoxidized hemoglobin. The
increase of the peak intensity with the deviation of pH value
from normal (see Figure 4(c)) indicates that in abnormal pH
conditions, the heme group becomes exposed, the combination
of heme and globin also has changed . Since the ability of
hemoglobin to pick up or release oxygen depends on the
interaction between oxygen and the iron atom of the heme
groups and hemoglobin's quaternary structure, the change in
the heme group suggests that the oxygen carrying ability of Hb
in abnormal pH is influenced.
The Raman doublet at 830 and 850 cm-1 is due to the Fermi
resonance between a ring-breathing vibration and the overtone
of an out-of-plane ring bending vibration of Tyr, the doublet
reflects the status of tyrosine in the globin chain. When the
intensity ratio I850/830 is less than 1, the tyrosine is buried,
whereas if the ratio I850/830 >1, the tyrosine is exposed[31,32].
Figure 5(a) gives an enlarged image of the Tyr bands at
830cm-1 and 850cm-1 in the solutions of pH 5.4, 7.4 and 9.0
respectively. As mentioned above that, each of these spectra
and those shown in Figure 5(b) and (c) was obtained by
averaging the results of at least 25 measurements at every pH
value. Similar to the spectrum and SD shown in Figure 4(a), no
significant spectrum difference was found from different parallel
groups and the standard deviation in amplitude was relatively
small. We can see that in acidic (pH 5.4-6.2) and strong
alkaline (pH 9.0) conditions, the intensity of the band at 850
cm-1 is greater than that at 830cm-1. This indicates that under
these conditions, the tyrosine in the globin chains is exposed.
Whereas at pH 8.0, the ratio of I850/830 is less than 1, which is
the same as the case of pH 7.4, suggesting that the tyrosine is
Figure 5(b) and 5(c) are the enlarged images of the bands
from 1240 to 1300cm-1. The bands in the range of
1240-1260cm-1 are assigned to the random coil vibration mode
of amide III, and the bands in the 1265-1300cm-1 range are
assigned to the helix vibration mode [33,34]. We can see that
at normal pH, the characteristic spectra of the helix vibration
mode are evident, suggesting that the secondary structure of
hemoglobin mostly exists in helix vibration mode. As pH is
away from neutral, the intensities of the characteristic bands for
random coil vibration mode gradually increase, while those for
helix vibration mode decrease. This suggests that in either
acidic or strong alkaline condition, the secondary structure of
hemoglobin would change from ordered ( helix) to disorder
(random coil), so the protein would be damaged. The exception
is at pH 8.0; the secondary structure of Hb mainly remains as
helix, indicating that Hb has not undergone any secondary
structure change in the circumstance.
Without normal tyrosine and heme group, a Hb molecule
cannot bind or release oxygen normally in acid and strong
alkaline conditions. Therefore, the Raman spectra obtained in
pH 5.4-6.2 and pH 9.0 (see Figure 4(a)) are quite complex.
They exhibit not only the characteristic peaks of deoxyHb
(1358, 1548, 1581, 1608cm-1), but also the characteristic peaks
of oxyHb (1568, 1590 and 1640cm-1) and even some of metHb
(1340 and 1628cm-1).
Mechanism of Hb dissociation from tetramer to dimer
Human hemoglobin includes four subunits, two globin
chains and two globin chains, which held together by
noncovalent bonds in a globular tetramer configuration. It was
believed that the stability of a hemoglobin tetramer is relevant
to electrostatic interactions, which also influences the
dissociation of hemoglobin from tetramer into dimer. Taking the
Hb dissociation process in pH 8.0 as an example, the mean
diameter of a Hb particle is 4.52 0.04 nm and the hemoglobin
concentration is 100 M (heme). Hence the electrostatic free
energy calculated with the model derived by Tanford, etc.[9,36]
is: Gel = 3.31 0.20 RT. This indicates that under the
condition the electrostatic free energy of a dimer is lower than
that of a tetramer, so tetramer favors to dissociate into dimer.
The calculated tetramer-dimer equilibrium constant K4 is
1722.43 303.42 M (heme), also suggesting that hemoglobin
in this circumstance is prone to exist in the form of dimmer.
Theoretically, tetramer 22 can dissociate into dimer in three
pathways: symmetric dissociation (222), asymmetric
dissociation (222+2) and nonspecific dissociation
(2222+2+2). But the actual hemoglobin dissociation is
mainly subjected to a highly symmetric mechanism
(222). For the -subunit and -subunit are mainly
associated by non-polar residues to form a stable interface.
The - and -subunits that interacting along the 11 (22)
packing interface form tight contacts and involve about 34
residues, whereas the 12 (21) sliding interface involves only
about 19 residues and is characterized by weaker contacts.
Consequently, dissociation often happens at 12 or 21 sliding
interface. So the hemoglobin dissociation from tetramer to
dimer can only result in the formation of dimer but not 12
or 12 dimer.
Mechanism of Hb dissociation from tetramer to
11 (or 22) is a stable interface of subunit - and usually
difficult to further dissociate, its dissociation to monomer at
acidic and strong alkaline conditions should be of a mechanism
differing from that of tetramer dimer. According to our
experimental results of Raman scattering spectroscopy, the
secondary and tertiary structures of hemoglobin had changed
in the process of the dissociation. The mechanism of the
dissociation is proposed as follows.
As shown in Figure 5(a), the hydrophobic amino acid has
changed from burial to exposure under acidic and strong
alkaline conditions. So the hemoglobin dimer cannot keep its
stability but dissociates into monomer.
Another secondary structure change of the Hb under pH
5.4-6.2 and pH 9.0 conditions is that the -helix has been
replaced by random coil. The -helix structure is the basis for
the formation of a tertiary structure with particular conformation
for a stable Hb, the loss of -helical structure for hemoglobin
strongly suggests local perturbations in globin, such as the
interactions among the subunits are significantly weakened and
some weak salt-bridge linked among each of them are even
broken. So that dimer is facilitated to dissociate into monomer.
The third change is in the structure of heme pocket as shown
in Figure 3. The Raman spectroscopy of the pyrrole ring shown
in Figure 4(a) also suggests that there is a displacement of
heme in the heme pocket which would expose it to the solution
environment. This was further proved by the increase of the
intensity of the characteristic spectral band of heme at
1358cm-1 shown in Figure 4(b). Since the more exposed
heme would absorb more light to increase the intensities of the
characteristic bands, heme should be exposed in the
circumstance. The exposure to the solvent of the hydrophobic
surface in the inter-subunit interfaces is one of the most
important features of protein unfolding, some other proteins
were also found to be dissociated from tetramers to non-native
monomers due to the effect [37,38].
Therefore, these changes in the secondary and tertiary
structures would disrupt the formation of the secondary bond
that maintains the stability of the 11 interface, thus resulting in
the dissociation from dimer to monomer. One may argue that
besides the dissociation from dimer to monomer, it may not get
rid of the possibility that hemoglobin tetramer directly
dissociates into monomer. However, based on our
experimental evidences, this possibility should be quite rare.
Since the dimer structure was always found in all the solutions
with various proportions under different pH conditions. Its
proportion increases as pH value approaching to the
physiological condition. If hemoglobin dissociates directly from
tetramer to monomer, there should be only monomer existing
in acidic and strong alkaline conditions but without dimer.
Therefore, tetramer Hb should dissociate first into dimer, then
Mechanism of the reversibility of hemoglobin
As shown in Figure 1(d), the dissociation of hemoglobin from
tetramer to dimer is reversible, whereas that from dimmer to
monomer is irreversible. The reversibility of Hb dissociation
from tetramer to dimmer can be explained by using Tanfords
electrostatic free energy model [9,36]. Based on the model, the
electrostatic free energy difference between dimer and
tetramer at pH 7.4 is Gel = 0.75 0.97 RT, so hemoglobin
favors to exit in the form of tetramer structure at pH 7.4. The
equilibrium constant K = 25.65 22.48 M (heme) in the
condition also suggests that the 22 2 processes can be
on reversible direction. Both Raman and UV-visible absorption
spectroscopy measurements showed that the re-associated Hb
has the same molecular structures and oxygen carrying
function as that of normal Hb at pH 7.4, indicating that the
reassociated hemoglobin is active again as it returns to the
In contrast, the dissociation from dimer to monomer is
caused by structural changes of the hemoglobin subunits.
These changes in subunit structure are difficult to recover, so
when the pH environment returns to 7.4, the hemoglobin
monomer cannot re-associate to dimer or tetramer.
In summary, we have conducted a multi-technique
systematic study on the effect of pH on the structure and
function of human hemoglobin, and demonstrated with direct
and convincing evidences that when the environmental pH is
away from normal physiological value, the tetramer hemoglobin
would easily dissociate into dimer by having electrostatic free
energy advantage. The tetramer dimer dissociation is a 22
2 process and it is reversible if the environmental pH
returns to neutral value. When pH becomes more acidic and
alkaline, such as in pH 5.4-6.5 and pH 9.0, dimer Hb will further
dissociate into monomer. The dissociation is accompanied with
series changes of protein structure, so that the secondary bond
is unable to form between the subunits to maintain a stable
state of dimer, thus causing the dissociation of dimer and
inducing the ferrous iron transform to ferrate iron by
peroxidization. Since the dissociation process involves
structure changes, even if the environmental pH returns to 7.4,
it is not reversible. The dissociated Hb is not able to adequately
carry and release oxygen to the tissues in circulation.
Therefore, pH dependent Hb dissociation should be avoided in
patients and preserved blood.
Conceived and designed the experiments: YXH. Performed the
experiments: ZJW BTH. Analyzed the data: YXH ML. Wrote the
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