Haptoglobin Preferentially Binds β but Not α Subunits Cross-Linked Hemoglobin Tetramers with Minimal Effects on Ligand and Redox Reactions
Alayash AI (2013) Haptoglobin Preferentially Binds b but Not a Subunits Cross-Linked Hemoglobin Tetramers with Minimal
Effects on Ligand and Redox Reactions. PLoS ONE 8(3): e59841. doi:10.1371/journal.pone.0059841
Haptoglobin Preferentially Binds b but Not a Subunits Cross-Linked Hemoglobin Tetramers with Minimal Effects on Ligand and Redox Reactions
Yiping Jia 0
Francine Wood 0
Paul W. Buehler 0
Abdu I. Alayash 0
Jose M. Sanchez-Ruiz, Universidad de Granada, Spain
0 Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration , Bethesda, Maryland , United States of America
Human hemoglobin (Hb) and haptoglobin (Hp) exhibit an extremely high affinity for each other, and the dissociation of Hb tetramers into dimers is generally believed to be a prerequisite for complex formation. We have investigated Hp interactions with native Hb, aa, and bb cross-linked Hb (aaXLHb and bbXLHb, respectively), and rapid kinetics of Hb ligand binding as well as the redox reactivity in the presence of and absence of Hp. The quaternary conformation of bb subunit cross-linking results in a higher binding affinity than that of aa subunit cross-linked Hb. However, bb cross-linked Hb exhibits a four fold slower association rate constant than the reaction rate of unmodified Hb with Hp. The Hp contact regions in the Hb dimer interfaces appear to be more readily exposed in bbXLHb than aaXLHb. In addition, apart from the functional changes caused by chemical modifications, Hp binding does not induce appreciable effects on the ligand binding and redox reactions of bbXLHb. Our findings may therefore be relevant to the design of safer Hb-based oxygen therapeutics by utilizing this preferential binding of bbXLHb to Hp. This may ultimately provide a safe oxidative inactivation and clearance pathway for chemically modified Hbs in circulation.
Funding: This work was supported in part by National Institutes of Health (NIH) grant HL 110900 (AIA), the U.S. Food and Drug Administration (MODSCI 2011)
(AIA), and H.H.S. Medical Counter Measures Grant (PWB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Haptoglobin (Hp) has been extensively studied as an
hemoglobin (Hb) scavenging protein due to its naturally high binding
affinity towards extracellular Hb in plasma . The Hb-Hp
protein complex is removed from circulation through the CD163
scavenger receptor on the surface of peripheral blood and tissue
monocytes and macrophages [2,3,4]. Free Hb is cleared to prevent
cellular and tissue damage caused by oxidative reactions mediated
by its heme iron. There has been renewed interest in Hp as
a potential therapeutic for indications involving acute and chronic
hemolysis, and as an adjuvant for acellular Hb-based oxygen
Hp is an acute phase plasma protein that exists in three primary
phenotypes: Hp 1-1, Hp 2-1, and Hp 2-2 . A Hp monomer
consists of one a subunit and one b subunit linked via disulfide
linkage, and one Hp monomer binds with one Hb ab dimer. The
Hp molecule consists of two types of a subunits, a1 (approximately
9 kDa) and a2 (approximately 16 kDa), and a single type of
b subunit of Hp which weights approximately 45 kDa. The a1
subunit contains a single cysteine residue, while the a2 subunit
contains an extra cysteine residue capable of forming multiple
disulfide bonds. Therefore, Hp 1-1 is a dimer that consists of two
a1 subunits and two b subunits. Hp 2-1 and Hp 2-2 contain a2
subunits to form polymers of different sizes and shapes. The Hb
binding site on Hp has previously been mapped to the large
b subunit .
It has been shown that all types of Hp bind oxygenated Hb
almost irreversibly with the equilibrium dissociation constant (Kd)
reportedly as low as 10212 M or 10215 M [8,9]. Purified Hb
subunits can form much weaker complex with Hp, whereas the
deoxyHb remains mostly as tetramers and appears to display no
binding to Hp. The Hb dimer associates rapidly with Hp under
oxygenated conditions, and Hb dimer formation appears to be
essential to Hb-Hp binding .
In the absence of antioxidant enzymatic system within red blood
cells, acellular Hb undergoes a series of oxidative reactions
generating potentially toxic reactive oxygen species (ROS) such as
superoxide (O2N-), hydrogen peroxide (H2O2). H2O2 is one of the
well-examined Hb pro-oxidants that react with ferrous Hb (Fe2+)
in a two-electron transfer process to produce the oxo-ferryl
(Fe4+ = O22) Hb species. When it reacts with met Hb (Fe3+),
a protein radical (NHbFe4+ = O22) is formed. Both of these higher
oxidation states of Hb can cause redox side reactions, damaging
Hb itself as well as other nearby proteins and lipids. These
reactions have yet to be fully understood, but they are believed to
originate from the highly reactive heme group. Radicals are
thought to migrate via tyrosine residues to other amino acids .
As a consequence, irreversible oxidative modifications of specific
amino acids, e.g., Trp15, Met55, Cys93, Cys112 and Tyr145 in
the b subunits have been consistently observed in the presence of
oxidants such as H2O2. Cross-linkages between Hb globin chains
and heme which can trigger further oxidative reactivity have also
been reported to occur .
Nitrite, another powerful oxidant, reacts with oxyHb in
a complex process of multiple reaction steps influenced by Hb
quaternary state intrinsic oxygen and ligand reactivity, and heme
accessibility . Nitrite also reacts with deoxyHb to generate
metHb and NO. The latter enzyme-like function may have some
physiological implications in the regulation of blood vessel dilation
and blood pressure control. Hp has been demonstrated to enhance
nitrite reductase activity of deoxyHb by approximately ten-fold,
much like that of Hb dimers [14,15]. Moreover recent studies have
shown that Hp impedes radical formation, or stabilizes the
damaging radicals formed in the presence of H2O2, which led us
and others to explore the protective mechanisms of Hp more fully
for possible therapeutic applications .
Hp interactions with several chemically modified Hbs have been
previously evaluated using fluorometric methods and by surface
plasmon resonance (SPR) analysis . This analysis consistently
revealed a strong affinity of Hp for Hbs, but a much lower affinity
for internally cross-linked Hbs. Furthermore, these studies suggest
that the aa subunits cross-linked Hbs may have more reduced
affinity to Hp.
The Hp binding with modified and unmodified Hbs was
examined closely in the present study using SEC-HPLC, native gel
electrophoresis, and stopped-flow kinetic techniques. The
functional effects of Hp binding on oxygen dissociation and ligand
binding to internally cross-linked, non-dissociable tetrameric Hb
were studied in comparison with that of unmodified Hb. The
redox reactions of Hb with typical oxidants such as H2O2 and
nitrite were assessed in order to determine the impact of specific
chemical cross-linking and Hp binding of these reactions. Our
results suggest that bb subunit cross-linked tetrameric Hb retains
substantial Hp binding capacity which does not alter ligand
binding or redox reactions beyond that observed with chemical
modification alone. This can have an important application in the
design of safe cross-linked and/or polymerized hemoglobin-based
blood substitutes. These results are also discussed in the light of
a recently published crystal structure of the porcine Hp-Hb
Figure 3. Rapid kinetics of Hp reaction with Hb measured by fluorescence emission change. A. The time courses of Hp binding with Hb
(15 mM) were fitted to exponential equations to obtain the pseudo-first-order rate constants for Hb, aaXLHb, and bbXLHb. B. The second order rate
constants of Hp binding with native Hb (close circle) and bbXLHb (open circle) were derived from the Hb concentration dependence of the obtained
Human HbA was prepared as previously described via
ammonium sulfate precipitation and anion-exchange FPLC, and
stripped of endogenous organic phosphate cofactors . Both aa
subunits cross-linked Hb (aaXLHb) and bb subunits cross-linked
Hb (bbXLHb) by bis(3,5-dibromosalicyl)fumarate were prepared
as previously described , and provided by Research Institutes
of the United States Army (Washington, DC). The Hp sample
containing primarily dimers (Hp 1-1) and to a lesser extent
polymers (Hp 2-1, and Hp 2-2) was purified , and kindly
provided by Bio Products Laboratory (BPL, Hertfordshire, UK).
All chemicals and reagents were purchased from Sigma Aldrich
(Saint Louis, Missouri) or Fisher Scientific (Pittsburgh,
Pennsylvania) unless indicated otherwise, and gases were purchased from
Roberts Oxygen Company, Inc. (Rockville, Maryland) or
Matheson Tri Gas (Basking Ridge, New Jersey).
Figure 4. Stopped-flow kinetics of oxygen dissociation from Hbs in the presence and absence of Hp. A. The time courses of oxygen
dissociation from HbA (open cirlce), and the Hb and Hp complex (gray triangle) mixed with 1.5 mg/mL sodium dithionite (Na2S2O4) were plotted for
comparison. B. The time courses of oxygen dissociation from bbXLHb (open circle), and the bbXLHb complex with Hp (gray triangle) were illustrated.
HPLC and Gel Electrophoresis Analyses
Hp was mixed in excess with Hb (Hp:Hb, in a 2:1 ratio) to allow
for the complete binding of Hb to Hp. The extent of Hb binding
to Hp was measured by size exclusion chromatography on
a BioSep-SEC-S3000 column ((600 mm67.5 mm), Phenomenex,
Torrence CA) attached to a Waters 2535 quaternary gradient
module and 2948 photodiode array detector (Waters Corporation,
Milford, MA). Each run was normalized using a 50 ml injection
loop. The mobile phase consisted of 50 mM potassium phosphate
buffer, pH 7.4, at 22uC, pumped at a flow rate of 1.0 mL/minute
over a 30 min run time. An injection loop flush step was
performed prior to each run using 500 mL of mobile phase. The
percentage of Hb bound to Hp was determined by dividing the
area of Hb-Hp peaks (1517 min elution time) at 405 nm by the
total area of Hb containing peaks (1521 min elution time)
multiplied by 100.
The molecular weight compositions of the Hb, Hp, and Hb-Hp
samples were assessed by native PAGE using an Invitrogen
NovexH Minigel System and NativeMarkTM Unstained Protein
Standard (Carlsbad, CA). A 416% precast NativePAGETM
Novex Bis-Tris gel was employed, and each lane was loaded with
35 mL of sample solutions (Invitrogen NativePAGETM sample
prep kit) containing about 100 mM Hb and Hp in excess. The gel
was run at 120 V for 90 minutes on a PowerEase 500 Power
Supply. After electrophoresis, the gel was stained overnight with
Coomassie blue G250 stain buffer, and then destained in water.
Stopped-flow Fluorescence Measurement
The rapid reaction of Hb and Hp was monitored in an Applied
Photophysics microvolume stopped-flow spectrophotometer
(Leatherhead, UK) with a dead time of approximately 1.5 ms.
Hp solutions (, 1 mM) were mixed in the stopped-flow with Hb
solutions of various excess concentrations (up to 30 mM) in 50 mM
sodium phosphate buffer, pH 7.4. The fluorescent change of the
reaction was measured with an excitation wavelength at 285 nm,
Figure 5. Stopped-flow kinetics of CO ligand association with HbA in the presence and absence of Hp. A. Representative time course of
CO (500 mM after mixing) binding with HbA was fitted to single exponential equation by non-linear least squares regression analysis. B.
Representative time course of CO (250 mM after mixing) binding with the HbA and Hp complex was biphasic, and fitted to double exponentials to
derive apparent association rate constants.
Figure 7. Nitrite reaction with oxy HbA and bbXLHb in the presence and absence of Hp. The spectral changes over time were measured in
a spectrophotometer for the reaction of oxy HbA (30 mM) and nitrite (6 mM) in the absence (A) or the presence (B) of excess Hp. The kinetics of oxy
HbA (C) and bbXLHb (D) reacting with freshly prepared nitrite as measured by rapid mixing, and the absorbance change was monitored at 577 nm.
The time courses of the complex nitrite-induced Hb oxidation processes in the absence (open circle) and presence (open triangle) of Hp were
illustrated, and the half times of the reaction under the same conditions were derived and listed in Table 2.
and a cutoff filter ,360 nm for emission as a function of time. At
least three time courses of Hp binding with Hbs were fitted to
exponential equations to obtain the averaged pseudo-first-order
rate constant for each reaction. Bimolecular rate constants were
derived from the slope of the linear relationship of the apparent
association rate constants as a function of Hb concentration.
Nitrite oxidation (t1/2) (s)
H2O2 oxidation (M21s21)
SulfHb initial rate (mM/min)
Rapid Kinetics of Ligand Reactions
The kinetics of oxygen dissociation from oxy Hb or the Hb-Hp
complex, and binding of carbon monoxide (CO) to deoxy Hb or
the Hb-Hp complex were measured in an Applied Photophysics
microvolume stopped-flow instrument as previously described
[21,22]. Hb solutions (30 mM in heme) were rapidly mixed with an
equal volume of 1.5 mg/mL sodium dithionite, and the
absorbance changes of the oxygen dissociation process were monitored
at 437.5 nm in 50 mM Bis-Tris buffer at pH 7.4 at room
temperature. The CO binding kinetics were measured at
437.5 nm in the same instrument and buffer containing freshly
made 1.5 mg/mL sodium dithionite. The CO solution was
prepared by saturating the degassed buffer with a flow of
prewashed CO gas. For each reaction, at least three kinetic traces
were averaged and fit to exponential equations using the
Marquardt2Levenberg fitting routines included in the Applied
Nitrite- and H2O2-induced Hb Oxidation
The spectral changes of the Hb reaction with nitrite were
measured in an Agilent 8453 diode array spectrophotometer in the
presence or absence of Hp. Rapid mixing methods using Applied
Photophysics microvolume stopped-flow instrument were used to
measure the kinetics of nitrite oxidation reaction of Hbs with and
without Hp. Hb solutions (30 mM) were mixed with equal volumes
of freshly prepared nitrite solution (6 mM) in potassium phosphate
buffer, pH7.4, to initiate oxidation, and the absorbance changes
were monitored as a function of time. The reaction time courses
were recorded at a single wavelength, 577 nm, and repeated at
least three times for each reaction condition.
The oxidation of met Hbs by excessive amount of H2O2 was
performed under pseudo-first-order conditions using Applied
Photophysics microvolume stopped-flow spectrophotometer with
a diode array detector at 25uC, as described previously . Ferric
(met) Hb solutions (20 mM) with and without excess Hp in
0.5 mM Tris buffer, pH 7.4, were rapidly mixed with H2O2
stabilization measured as sulfHb concentrations in the reaction of metHb and H2O2. The close (N) and open (#) circles in the graph represent the
Figure 8. Hydrogen peroxide-induced Ferryl/sulf bbXLHb formation in the presence and absence of Hp. Ferryl Hb formation and
sulfHb concentrations at reaction times of H2O2-induced metHb oxidation in the absence and presence of excess Hp, respectively. The inset shows
typical spectral changes of resultant sulfHb at approximate 620 nm as a function of time. The approximate initial reaction rates were obtained and
listed in Table 2.
solutions of increasing concentrations up to 100 mM. The
absorbance spectral changes (at least 200 spectra) were recorded
as a function of time. The whole set of spectral data were subjected
to global curve fitting analysis (Applied Photophysics software) to
derive the reaction rate constants of the oxidation reaction of met
Hb to ferryl Hb. The second-order rate constants were obtained
from the dependence of the apparent rate constants on H2O2
The Measurement of Ferryl Hb Intermediate Formation
The intermediate formation of ferryl Hb in the reaction of Hb
or Hb-Hp with H2O2 was detected by its reaction with sodium
sulfide (Na2S), generating sulfonated Hb (sulfHb) that is spectrally
detectable . In a typical experiment, metHb (50 mM) was
mixed with 1:5 molar ratio of H2O2 in 50 mM potassium
phosphate buffer, pH 7.4, to initiate the oxidative reaction in
a cuvette monitored in an Agilent 8453 diode array
spectrophotometer. After a 2 minute incubation time, Na2S (2 mM) was
added to the reaction mixture and spectral changes between
450 nm and 700 nm were recorded for the conversion of ferryl Hb
to sulfHb. The sulfHb concentrations were calculated using the
extinction coefficient of 20.8 mM21 cm21 at 620 nm .
Purified human Hb, aaXLHb and bbXLHb, and complexs of
each protein with Hp were analyzed using both analytical
sizeexclusion chromatography (SEC) and native gel electrophoresis
methods. SEC consistently revealed the major Hb peaks at the
elution time of 20.9 min (monitored at 405 nm) in all Hb samples
(Figure 1A). Three main peaks were observed between elution
times 13 min and 18 min for the HbA sample with Hp, in
agreement with the previously reported results . The peak
eluting at 17.3 minutes represents HbA bound to Hp 1-1, while
Hb binding to polymeric species (Hp 2-2 or Hp 2-1) were observed
with predominant peaks at 15.5 and 16.4 minutes, respectively. A
slightly different elution profile with additional peaks eluting
between 13 min and 16 min was observed for the mixture of
bbXLHb and Hp, indicating further molecular association and the
formation of larger molecular weight complexes. Only small peaks
could be detected in the same elution time frame for the mixture of
aaXLHb and Hp. Since Hp was added in excess, 99.6% 60.4%
purified human Hb sample was bound with Hp and no free Hb
peak was observed. It was determined from peak % area
calculations within the boxed region that bbXLHb preserved
approximately 52.9% 64.1% of the binding capacity with Hp
relative to unmodified Hb, whereas aaXLHb and Hp showed
minimal binding with less than 15.8% 63.5% including
nonspecific bindings and low levels of contaminants (Figure 1B).
The SEC results were verified and confirmed on a native PAGE
(Figure 2). The unmodified Hb was resolved largely into Hb
dimers and some tetramers (Lane 2) as contrasted to the native gel
molecular markers (Lane 1). The samples of aaXLHb (Lane 3)
and bbXLHb (Lane 4) appeared primarily as tetramers, as a result
of the chemical cross-linking and tetramer stabilization processes,
with some minor protein impurities that may not be Hb species,
and were not observed in the SEC-HPLC results. The Hp sample
(Lane 8) was resolved primarily as Hp dimers (Hp 1-1), with some
Hp polymer species, i.e., Hp 2-1 and Hp 2-2. In the presence of
slight excess Hp (Lane 5), the formation of additional protein
bands, and the disappearance of free Hb indicated the binding
between Hp and unmodified Hb. The Hp dimers, and Hp
polymers composed of Hp 2-1 and Hp 2-2 can form multiple
protein complexes with native Hb to give extra bands. Conversely,
relatively few new protein bands were observed in the case of the
aaXLHb and Hp sample (Lane 6), which could represent a merge
of the two samples run separately in Lanes 3 and 8. However,
protein complexes of high molecular weight polymers formed
between Hp and bbXLHb can be seen (Lane 7) with some
bbXLHb tetramer also visible. This observation suggests a
moderate binding capability, consistent with the SEC data. In addition,
Hb dimers bind to Hp with strong monovalent binding
characteristics, and bbXLHb binds to Hp with polyvalent binding
characteristics. Our previous data suggest that bbXLHb
polyvalent binding with Hp leads to a time dependent gelation
phenomenon . These binding characteristics also contribute to
the observation of multiple band patterns observed in lanes 5 and
Hb binding to Hp is accompanied by the quenching of Hp
intrinsic fluorescence as previously reported [10,26]. We measured
the kinetics of Hb and Hp binding by monitoring fluorescence
changes in a rapid mixing stopped-flow spectrophotometer
equipped with a fluorescence detector. Representative time
courses of Hp (1 mM) association with 15 mM Hb, aaXLHb, or
bbXLHb under pseudo first order conditions are shown in
Figure 3A. The interaction of Hb and Hp was fitted to a single
exponential equation. No reaction was observed between
aaXLHb and Hp under the same experimental conditions. In
contrast, bbXLHb and Hp displayed a relatively slower reaction
followed by a much slower second kinetic phase, possibly due to
polymer formation. These results are consistent with the
observations using SEC and native PAGE studies of unmodified and
modified Hbs binding with Hp. The Hb concentration
dependence of the apparent association rate constants as shown in
Figure 3B revealed that bbXLHb and Hb bind to Hp under our
experimental conditions with bimolecular rate constants at about
0.01560.004 mM21s21 and 0.05260.004 mM21s21, respectively.
The latter value is close to that reported previously for human Hb
binding to Hp type 11 .
The effects of Hp binding with unmodified Hb and cross-linked
Hb tetramers on the rapid kinetics of ligand association and
dissociation were evaluated. Figure 4A shows the time courses of
O2 dissociation from Hb and the Hp-Hb complex. The binding of
Hp clearly altered the overall time course from a single
exponential process (34 s21) to a biphasic reaction with apparent
rate constants of 69 s21 and 14 s21 based on the nonlinear least
squares curve fitting. In contrast, identical O2 dissociation time
courses were recorded for bbXLHb bound or not bound to Hp
(Figure 4B), with no appreciable difference in derived rate
constants (Table 1).
Figure 5A and 5B illustrate representative time courses of CO
association with Hb in the absence and presence of Hp,
respectively. The Hb reaction with CO is typically a single
exponential process, whereas Hb-Hp complexes exhibit kinetic
heterogeneity, including an additional fast phase with
approximately 10 times larger second-order rate constant under the same
experimental conditions (Table 1). In contrast, the recorded time
courses of CO reaction with bbXLHb and the complex of
bbXLHb and Hp were identical as depicted in Figure 6, and no
significant differences in the derived second-order rate constants
were observed (Table 1).
The effects of Hp binding on Hb oxidative reactions were
examined using nitrite and H2O2 as oxidants. It is known that
oxygenated Hb reacts with nitrite following a complex process to
form metHb and nitrate as end products [28,29,30]. Figure 7A &
7B present the progressive spectral changes from oxy to met Hb
over the reaction time of nitrite and native Hb with and without
Hp. Fewer intermediate spectra and faster reaction were observed
in the presence of Hp (Figure 7B). Figure 7C shows the typical
auto-accelerating time course by monitoring the absorbance
change of the oxy Hb and nitrite reaction to completion. Hp
binding shifted the curve to the left, enhancing the
autoaccelerating reaction of nitrite with the Hb and the Hb-Hp
complex and changing the reaction half-time from 19.0 s to 8.4 s
under our experimental conditions. Conversely, nitrite-induced
oxidation of bbXLHb was not altered by Hp (Figure 7D), and
both reactions exhibited similar auto-accelerating process with
a half-time of 2223 s21 under the same conditions (Table 2).
Hp binding was previously shown to have no effect on Hb
pseudoperoxidase activity, the conversion of met to ferryl Hb with
H2O2 consumption . The absorbance changes of the formation
of ferryl Hb were measured under pseudo first-order conditions in
a rapid mixing stopped-flow instrument using a photodiode array
detector. The progressive spectral changes in the Soret and visible
regions were subjected to global kinetic analysis to de-convolute
and reconstruct the spectra of met and ferryl Hbs and to derive the
reaction rate constants. The observed rates were a linear function
of the H2O2 concentrations, resulting in the second-order rate
constants for this oxidation reaction from met to ferryl Hb
(Table 2). No appreciable differences of the H2O2 oxidation rates
were obtained for either Hb or bbXLHb in the absence or
presence of Hp.
The formation of ferryl Hb is followed by a process of
autoreduction to generate metHb which completes a pseudoperoxidase
cycle . The transient formation of ferryl Hb can be captured
prior to its decay to metHb by adding sodium sulfide to convert
the ferryl species to a spectrally more distinct and stable sulfHb
species. It was shown in our previous report  that although the
ferryl Hb concentration reached similar initial levels, the decay of
ferryl Hb was attenuated by Hp binding. Figure 8 depicts the
kinetic stabilization of ferryl bbXLHb as result of Hp binding in
comparison with that of unmodified Hb. The levels of the initial
ferryl bbXLHb formation were comparable to that of Hb, and
progressive spectral changes focusing on the sulfHb peak at
620 nm is shown as a function of time in the inset. These Data
demonstrate that the ferryl form of bbXLHb decayed similarly
with initial rates of 0.32 mM/min and 0.36 mM/min in the
absence and presence of Hp (Table 2). Although Hp reduced the
initial rates from 1.01 mM/min to 0.26 mM/min for wild type Hb,
no kinetic effects on ferryl Hb decay were detected in Hp
complexed with bbXLHb.
The interaction between Hb and Hp is extremely strong and
almost irreversible, equivalent to that of antigen-antibody
interactions. Hb dimers have long been regarded as the only Hb
molecular form that binds with Hp. However, non-dissociable
chemically cross-linked Hbs have been observed to bind with Hp
[16,32]. The nature of chemically modified Hb and Hp protein
complex formation may provide valuable insights into the
molecular pathway of Hb clearance under physiological
conditions, and may provide a better understanding of Hp mediated
attenuation of Hb oxidative reactions .
The chemical modifications of Hb used in this study are
sitespecific in which the intramolecular reagent,
bis(3,5-dibromosalicyl)fumarate was used to either cross-link the two Lys99 residues in
a subunits of the deoxy or the two Lys82 residues in b subunits of
the oxy forms of human Hb, resulting in aaXLHb and bbXLHb
respectively [33,34,35,36]. These structural changes produce
stable, low oxygen (T-state) and high oxygen (R-state) affinity
Hbs for transfusion purposes in animals and in humans [37,38].
We reasoned that Hb quaternary conformations of aaXLHb and
bbXLHb differ sufficiently to restrain aaXLHb and Hp
interaction, but allow bbXLHb enough flexibility in its R
conformation to serve as points of contact with Hp.
Our protein binding analyses clearly showed that the b subunit
cross-linked Hb tetramers bind Hp, whereas tetrameric Hb
crosslinked between a subunits has minimal interaction with Hp.
SECHPLC revealed that approximately 50% of bbXLHb formed
complex with Hp compared to 100% complex formation with
unmodified Hb and Hp and that bbXLHb appeared to form
larger sized complexes. Polymer formation is likely a result of
bbXLHb divalent interaction with Hp, in which binding can
occur at dimeric sites on the stabilized Hb tetramer . The
binding patterns of native Hb and bbXLHb with Hp were
resolved by non-denaturing electrophoresis gel analysis which
confirmed the SEC-HPLC results. Our rapid mixing analyses
indicated that bbXLHb reacts with Hp at a rate that is about 4
fold slower than that of the native Hb. This was followed in the
case of bbXLHb by a much slower second kinetic phase, which
can be attributed to polymer formation.
We have characterized the impact of Hp interactions on the
ligand binding reactions of chemically modified Hbs in order to
understand the extent of the structural perturbation introduced by
Hp. We also reported that both oxygen equilibrium binding and
the kinetics of CO association and oxygen dissociation were
altered after complex formation. In addition, we find that chemical
modification of aaXLHb significantly decreases oxygen
equilibrium binding affinity while increasing the oxygen off rate, whereas
bbXLHb exhibits a higher oxygen affinity and CO on rate. Our
equilibrium (data not shown) and kinetic parameters determined
for the bbXLHb and Hp complex are similar to that of the
uncomplexed bbXLHb. Interestingly, it appears that the complex
formation between the R-state bbXLHb and Hp did not perturb
its heme group reaction environment. Therefore, it is possible to
alter Hb structure, promote Hp binding and retain desired ligand
In agreement with previous reports, our results showed that Hp
does not change the intrinsic reactivity of Hb with H2O2, but
largely increased ferryl Hb stability. It was previously shown that
heme accessibility plays a major role in determining the reaction
rate with nitrite . We observed enhanced nitrite oxidation of
native Hb in the presence of Hp due to Hb dimerization and
potentially heme accessibility. Although chemical modification
typically enhances redox reactivity of modified Hb deviations, Hp
association with bbXLHb did not cause any additional H2O2 and
nitrite reactivity changes. This suggests that modified Hb can bind
Hp and retain heme reactivities.
Our results are in agreement with a recent crystal structure
analysis of the porcine Hb and Hp complex . Although
porcine blood was used as source, porcine and human Hb and Hp
exhibit 82% homology by sequence alignment, and the same
dumbbell shape with two serine protease domains connected by
two complement control protein (CCP) domains. The serine
protease domain contains several loops exposed on the surface and
the amino-terminal region that were determined to constitute the
Hb-binding site. Hp interacts with both Hb a and b subunits, and
amino acid residues in helix C, G and FG were determined as the
primary sites, but also as Hb dimer interface in the formation of
tetramer. These structural data also reveal that the conformation
of deoxygenated Hb dimers does not promote Hp binding.
Remarkably, these data also show that Hb-Hp interaction
originates from an initial complex between C terminus of Hb
a subunit and the active serine protease of Hp. This may explain
the preferred binding to Hp we observed, as a subunits are more
surface exposed due to the cross linkage of the b subunits within
In summary, although Hb binds to Hp with a high affinity via
Hb ab dimerization, non-dissociable Hb tetramers may also form
protein complex with Hp. Hb tetramers cross-linked between two
b subunits retain an R-state like conformation and display much
higher Hp affinity than that of a subunit cross-linked Hb
tetramers. Because the binding of bbXLHb to Hp like unmodified
Hb retains its ability to decrease the propagation of damaging
ferryl radicals, site specific cross-linking of the b subunits may
provide a basis for an improved design of Hb-based oxygen
Conceived and designed the experiments: YJ. Performed the experiments:
YJ FW PWB. Analyzed the data: YJ PWB. Wrote the paper: YJ PWB AIA.
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