From the Departments of Medicine (Division of
Hematology), § Anatomy & Structural Biology, and
Physiology & Biophysics, Albert Einstein College of Medicine,
Bronx, New York 10461
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ABSTRACT |
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Based upon existing crystallographic evidence,
HbS, HbC, and HbA have essentially the same molecular structure.
However, important areas of the molecule are not well defined
crystallographically (e.g. the N-terminal nonhelical
portion of the and
chains), and conformational constraints
differ in solution and in the crystalline state. Over the years, our
laboratory and others have provided evidence of conformational changes
in HbS and, more recently, in HbC.
We now present data based upon allosteric perturbation monitored by
front-face fluorescence, ultraviolet resonance Raman spectroscopy, circular dichroism, and oxygen equilibrium studies that confirm and
significantly expand previous findings suggesting solution-active structural differences in liganded forms of HbS and HbC distal to the
site of mutation and involving the 2,3-diphosphoglycerate binding
pocket. The liganded forms of these hemoglobins are of significant
interest because HbC crystallizes in the erythrocyte in the oxy form,
and oxy HbS exhibits increased mechanical precipitability and a high
propensity to oxidize. Specific findings are as follows: 1) differences
in the intrinsic fluorescence indicate that the Trp microenvironments
are more hydrophobic for HbS > HbC > HbA, 2) ultraviolet
resonance Raman spectroscopy detects alterations in Tyr hydrogen
bonding, in Trp hydrophobicity at the Naturally occurring According to existing crystallographic evidence, HbS, HbC, and HbA have
essentially the same molecular structure. However, this ignores the
following: 1) important areas of the molecule are not well defined
crystallographically (e.g. the N-terminal nonhelical portion
of the Spectroscopic and biochemical findings by our laboratory and others
revealed distal (away from the mutation site) conformational changes:
for HbS, NMR studies revealed alterations in the pK of surface His,
including A recent study using more sensitive spectroscopic instrumentation,
front-face fluorometry, and ultraviolet resonance Raman (UVRR)
spectroscopy revealed differences in the H-bonding between The present study focuses on a comparison of R-state forms of HbC, HbS,
and HbA. The comparison is made using intrinsic fluorescence, extrinsic
fluorescence spectroscopic probing of the central cavity DPG binding
site, UVRR spectroscopy, CD spectroscopy, allosteric perturbation, and
oxygen equilibrium studies. These results confirm and significantly
expand previous findings and demonstrate a differential response
between the R-states of HbC and HbS to central cavity perturbations.
Human Hemoglobin Preparation--
HbC, HbA, and HbS were
purified and separated from hemolysates obtained from AC, SS, or AS
individuals by CM-52 cation exchange chromatography and stripped of
organic phosphates by Sephadex G-25 (equilibrated with bis-Tris and 0.1 M NaCl) column chromatography. The eluted hemoglobin was
concentrated and chromatographed again on a Sephadex G-25 column
equilibrated with a 0.05 M Hepes buffer at the desired pH
of the experimental conditions. No significant met (oxidized)
hemoglobin content was detected as determined by absorption spectrophotometry.
Front-face Fluorescence Spectroscopy--
A SLM 8000C photon
counting spectrophotometer adapted with a front-face cuvette holder was
used to obtain the steady-state fluorescence intrinsic emission spectra
of the hemoglobin solutions as well as the emission from the
fluorescent DPG analog, 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT) (1).
The properties and binding of HPT to hemoglobins have been described
previously in detail (2, 28-30). The addition of effectors to the
hemoglobin solutions did not significantly alter the pH.
UV Resonance Raman Spectroscopy--
The apparatus used to
generate and collect the Raman signal was described previously (1). The
excitation wavelength was 228 nm, and the UV power at the sample was
1.0 milliwatt. The spectrographic slit width was 200 µm. The Raman
signal was collected at 3-min intervals for a total of 30 min. All
samples were cooled under a stream of nitrogen gas to 6 °C. To
ensure that the hemoglobin ligation state remained unchanged during the
course of the experiment, the absorption spectra of each sample were
taken before and after UV exposure; no absorption band changes were
seen during this experiment. For all hemoglobin variants, the heme
concentration was 0.27 mM in 0.1 M Hepes
buffer, pH 7.35. The internal calibration standard utilized was a Raman
band at 1045 cm Circular Dichroism Spectroscopy--
A Jasco J-720
spectropolarimeter was used to compare the CD spectra of purified and
stripped HbC, HbS, and HbA (0.1 mM heme) in 0.1 M Hepes, pH 6.85, with and without an excess of inositol hexaphosphate (1 Hb:5 inositol hexaphosphate).
Functional Studies--
Oxygen equilibrium measurements in Hepes
buffer (pH 7.35) were made with the use of the HEM-O-SCAN at 37 °C
as described previously (2).
Front-face Fluorescence Spectroscopy--
It is well established
that the Trp microenvironments of proteins may be probed and defined by
fluorescence spectroscopy, including hemoglobin tryptophans and other
tetramer microdomains. Non-tryptophan residues have been studied by the
binding of extrinsic fluorescent probes (for a review of Hb
fluorescence, see Ref. 31).
One approach to understanding the mechanisms of oxy HbC crystallization
is to determine the solution-active structural differences among the
R-state Ultraviolet Resonance Raman Spectroscopy--
UVRR spectroscopy of
hemoglobins site selectively probes the aromatic residues of the globin
domains (35-37). The UVRR spectra for human carbonmonoxyhemoglobin A
(COHbA), COHbC, and COHbS in 0.1 M Hepes buffer (no
chloride), pH 7.35, are shown in Fig. 2. Within the given UVRR frequency window, tryptophan bands are located at
1558 cm Probing the Central Cavity DPG Binding Site with a Fluorescent DPG
Analog--
The fluorescent DPG analog, HPT, has been used previously
to show differences in the central cavity of an altered R-state of HbC
compared with a similarly altered R-state of HbA (2). These comparative
studies are now extended to HbS. Front-face fluorometry permits a
direct spectroscopic view of HPT bound to Hb as well as the ability to
view HPT in the presence of concentrations of R-state hemoglobins
greater than 0.5 g% where dissociation to dimers becomes negligible.
COHbA at pH 6.35 has been shown to be in an altered R-state capable of
binding IHP (38, 39). It was previously shown that IHP displaces HPT at
a 1:1 molar ratio, and that IHP has a higher affinity for the DPG
binding site than HPT (28-30). Released HPT is monitored at 385 nm, a
region of little or no Hb interference under the conditions of our
experiment with COHbA (pH 6.35).
As IHP is added to each of the CO hemoglobins, HbA, HbC, and HbS at pH
6.35, an increase in the HPT fluorescence intensity is observed at the
385 nm emission maximum upon excitation at 280 nm (Fig.
3), corresponding to a release in HPT
(30). These results indicate a differential affinity of IHP for
HbA > HbS > HbC.
Probing the Heme Environment by CD Spectroscopy--
Small
differences in the ellipticity of the oxy liganded state of HbS > HbC > HbA in 0.1 M Hepes buffer, pH 6.85, are
enhanced with the addition of excess IHP (Fig.
4). The addition of excess IHP results in
a small but ~2-fold increase in ellipticity exhibited by intensity
changes and a shift from 420.5 nm to longer wavelengths (HbA, ~424
nm; HbC, ~423; and HbS, ~422 nm).
Oxygen Equilibrium Measurements--
Oxygen affinity differences
between the
Stripped HbA compared with HbC and HbS under conditions similar to that
of the fluorescence studies (0.05 M Hepes buffer, pH 7.35)
shows a small but statistically significant difference in oxygen
affinity (P50). The difference in oxygen affinity between HbC and HbS is not significant (Table
I).
To date, there is no detailed mechanistic explanation as to why
HbC forms crystals in the oxy state and HbS forms polymers in the deoxy
state or why oxy HbS exhibits mechanical instability, greater unfolding
at an air-water interface, and a high propensity to oxidize. The
ligand-specific induction of these distinct processes weakens the
argument that the aggregation phenomena arise exclusively from
electrostatic or hydrophobic differences at Spectroscopic and functional (oxygen equilibria) studies presented here
strongly suggest that global, solution-active conformational alterations, particularly those involving the A-helix and the DPG
binding pocket, are different in HbS and HbC. The intrinsic fluorescence studies show a small but reproducible shift of the emission maxima to shorter wavelengths for these R-state UVRR difference spectra of hemoglobins typically exhibit two categories
of change (37, 40, 41, 42). The first can readily be attributed to
quaternary structure-based differences in the
The second category of change observed in UVRR difference spectra is a
series of intensity changes in several of the tyrosine and tryptophan
bands that occur without any sizable shifts in the peak frequencies
(35, 42-44). A decrease in intensity is observed when comparing either
deoxy T or liganded R to deoxy R. The decrease is attributed to a
generalized loosening of the global structure including a weakening of
the hydrogen bond between the A-helix tryptophans and their respective
bonding partners on the E-helix. A decrease in the intensity of the
tyrosine bands is attributed to a weakening of the hydrogen bond
between the penultimate tyrosines and the carbonyls of the FG5 valines.
An increase in the intensity of these tyrosine and tryptophan bands is
similarly ascribed to a strengthening of these hydrogen bonding interactions due to tighter packing between the A- and E-helices and
the H- and F-helices. A UVRR study of C termini-modified hemoglobins indicates that there is a coupling between the packing of the H-helix
and the packing of the A- and E-helices (43). An explanation for this
effect was given based on the H-helix acting as a scaffold that
supports the A-helix. A disruption of the H-helix as occurs upon
deletion of the The UVRR differences observed in the present study tend to fall into
the second category. At physiological pH 7.35, COHbC and COHbS show a
turn toward greater hydrophobicity in the microenvironment for all
three of the Trp residues, At physiological pH 7.35, the ~1558 cm In general, with respect to the UVRR results, in going from HbA to HbC
to HbS, there are intensity increases in most of the tyrosine and
tryptophan bands. Most significantly, the Y8a band at 1615 cm Similar conclusions regarding the increase in A-E-helix packing in
fluromet HbS at pH 6.5 were arrived at by Sokolov and Mukerji (48).
They also found that the fluorescence change between HbA and HbS was
essentially independent of the quaternary state. This finding, in
conjunction with the observed R-T and HbA-HbS UVRR differences, led to
the conclusion that It should be noted that in our earlier report (1), solution conditions
using pH 6.85 for the spectroscopic studies had been chosen because
hemoglobin exhibits a maximal fluorescence emission difference upon R
A direct confirmation of a perturbed central cavity of HbC and HbS is
seen with the use of the extrinsic fluorescent DPG analog HPT, which
directly probes the DPG binding site (28-30). The results with COHb at
pH 6.35, previously shown to form an altered R-state (38, 39), indicate
that the affinity of IHP for the hemoglobins discussed earlier follow
the order HbA > HbS > HbC. These results, combined with the
UVRR findings, indicate an alteration of the DPG pocket at
physiological pH. A possible source for this alteration is a
The effects of IHP upon the P50 of HbC and HbS compared
with HbA provide further support that there is an A-helix perturbation in the Structural alterations corresponding to the heme environment are
correlated with the change in oxygen affinity upon addition of IHP, as
reflected by the CD Soret spectral changes (~420 nm). The addition of
IHP under the conditions used in this study alters the Soret band in
ellipticity and wavelength for oxy HbA > oxy HbC > oxy HbS
(Fig. 4). Above 300 nm, the optical activity of the heme results from
short and long distance interactions of the heme with the protein
matrix, specifically attributed by theoretical calculations to a
coupled oscillator interaction between the heme transitions and those
of the surrounding aromatic side chains (53, 54). This interpretation
is consistent with the altered microenvironments of the aromatic amino
acids of these For decades, functional studies in which HbS and/or HbC were compared
with HbA (conducted under very different conditions (e.g.
bis-Tris buffer) than those used in the present study) gave rise to the
concept that functional and structural differences distal to the site
of the Padlan and Love (57, 58) found that the deoxy HbS crystal structure
exhibits a narrowing of the DPG pocket compared with HbA by a
comparison of the eight deoxy hemoglobin S subunits with the
symmetry-averaged subunits of deoxy HbA. The biggest structural change
is a shift in a hinge-like motion of the A-helices of 1 In general, the advent of state of the art spectroscopic techniques
with site-specific signal assignments permits superior signal
resolution and, consequently, highly detailed structural information
for many proteins. For hemoglobins, this has resulted in the uncovering
of solution-active structural differences at the tetrameric molecular
level and, as exemplified here, distal to the site of the 1
2
interface (
37), and in the A-helix (
14/
15) of both chains, 3)
displacement by inositol hexaphosphate of the Hb-bound
8-hydroxy-1,3,6-pyrenetrisulfonate (the fluorescent
2,3-diphosphoglycerate analog) follows the order HbA > HbS > HbC, and 4) oxygen equilibria measurements indicate a differential
allosteric effect by inositol hexaphosphate for HbC ~ HbS > HbA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6 hemoglobin mutants aggregate into defined
structures in the erythrocyte. Sickle cell hemoglobin (HbS,
6 Glu
Val) forms polymers in the deoxy state, whereas HbC (
6 Glu
Lys) forms crystals in the oxy liganded state. A complete understanding
of the mechanisms giving rise to deoxy HbS polymers and oxy HbC
crystals remains to be elucidated. In the last few years, our
laboratory has pursued questions related to mechanisms involved in the
ligand-specific, induced crystallization of HbC, starting with
site-specific probing of the R-state tetrameric structure of HbC (1,
2). Here, we extend the studies to include a comparison of the R-state
of HbS. The R-state of HbS is of particular relevance because oxy HbS
exhibits unusual properties compared with HbA, such as mechanical
precipitability (3-5), greater unfolding at an air-water interface (6,
7), and increased autooxidation (8-10).
and
chains), and 2) crystals might constrain
conformation compared with that in solution.
2 His affecting the
DPG1 binding site (11, 12).
Optical spectroscopies and
SH group reactivity demonstrate a
long-range tertiary effect of the
6 Val substitution around the heme
pocket (13-16). Organic phosphates decrease the
93 sulfhydryl
reactivity more for oxy HbA than for oxy HbS, which is indicative of
weaker organic phosphate binding to HbS compared with HbA (16).
Polymerization-independent functional differences were shown
for HbS compared with HbA (17, 18). Hemoglobins HbC > HbS > HbA bind to the cytosolic side of the erythrocyte membrane (19-21). It
is noteworthy that the membrane binding site for hemoglobin is the
11-amino acid N-terminal of the cytosolic side of band 3 that fits into
the hemoglobin DPG pocket (22). A close examination of subunit
dissociation studies indicates a slight but reproducible difference
between the intersubunit bonds for HbA and HbS and the AS hybrid (23).
This is in contrast to functional and ligand binding studies presented
by others (e.g. Refs. 24 and 25). Despite all of the
reported differences (5-21), it is generally concluded that the
similarities between HbA and HbS are emphasized and viewed as more
significant. The "small differences" are typically regarded as
having no structural or functional relevance (e.g. Refs. 26
and 27).
15 Trp
and
72 Ser of HbC versus HbA under conditions of 0.3 M perchlorate (1), suggesting a displacement of the A-helix away from the E-helix with a weakening of the H-bond between
15 Trp
and
72 Ser. Crystal growth kinetics and spectroscopic studies of HbC
in the presence of hemoglobin central cavity-binding proteins and
effectors point to differences in the DPG pocket (2).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 from 0.36 mM potassium
nitrate. Because the Raman cross-section of the 1045-cm
1
band diminishes upon prolonged UV
exposure,2 sample-to-sample
total UV exposure was tightly controlled by keeping sample irradiation
times constant and operating the pump laser in light-controlled mode.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6 mutant hemoglobins and HbA. Differences in the Trp
microenvironment(s) of R-state HbS (sickle cell hemoglobin), HbC, and
HbA are revealed by comparing the intrinsic fluorescence spectra (Fig.
1), which show a decrease in intensity
for the
6 mutants (HbC < HbS < HbA) and a blue-shift of
the emission maximum for HbC and HbS. Tryptophan fluorescence emission
maximum shifts are a function of hydrophobicity, whereas tryptophan
fluorescence intensity changes are not easily interpretable, arising
from any of several contributing factors including nonradiative
processes (32-34).
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Fig. 1.
Differences in the Trp microenvironment(s) of
R-state HbS, HbC, and HbA are revealed by intrinsic fluorescence
differences. Excitation at 280 nm (0.05 M Hepes
buffer, pH 7.35, 25 °C).
1 (W3), 1458 cm
1 (W5), 1358 cm
1 (W7), and 1012 cm
1 (W16), whereas
tyrosine bands are found at 1615/1595 cm
1 (Y8a/b), 1205 cm
1 (Y7a), and 1175 cm
1 (Y9a) (35-37).
When the spectra are normalized at the nitrate internal standard as
shown (Fig. 2), both tyrosine and tryptophan bands for COHbS exhibit
the greatest cross-section, followed by those for COHbC. Bands for
COHbA have the smallest cross-section. The W3 band for each hemoglobin
species may be fit to two bands, one of which is centered at
1558.6 ± 0.2 cm
1 (data not shown). The position of
the lower wavenumber band was centered at 1547.5 cm
1 for
COHbA and COHbC. In the COHbS W3 band, however, this lower frequency
band is shifted to 1545 cm
1. This frequency shift is
illustrated by the 1542 cm
1 difference peak for
COHbS-COHbA (Fig 2). The fractional cross-sections for both W3 bands
were essentially identical for all three hemoglobins. It should also be
noted that the 1547 cm
1 band for COHbC exhibits a small
difference just above the level of noise when compared with COHbA. A
comparison of the non-normalized UVRR spectra of the CO forms of the
three hemoglobins (data not shown) demonstrates that the peak
intensities of the W16 band and the NO3 internal standard
bands are inversely ordered, indicating that the W16 band contribution
to the NO3 standard peak does not affect the global
spectral intensity ordering (i.e. HbS > HbC > HbA).
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Fig. 2.
Ultraviolet resonance Raman spectroscopy
shows differences in the Tyr and Trp microenvironments of the 6
mutants. 228 nm excited UVRR spectra of COHb in 0.1 M
Hepes, pH 7.35, at 6 °C. The spectra are normalized to the 1045 cm
1 nitrate internal standard. COHbA, solid
line; COHbC, long and short dashed line;
COHbS, dashed line. The W3 band from 1530 to 1580 cm
1 is expanded and inset in the upper
right. The UVRR difference spectra between COHbC and COHbA
(long and short dashed lines) and between COHbS
and COHbA (dashed line) are shown at the
bottom.
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Fig. 3.
Displacement by IHP of the hemoglobin-bound
HPT, the fluorescent DPG analog (28, 29), indicates a differential
affinity of IHP for HbA > HbS > HbC. 280 nm excitation
of the hemoglobin solutions. Released HPT is monitored at 385 nm, a
region of little or no Hb fluorescence interference under the
conditions of our experiment with COHbA (pH 6.35, 0.05 M
Hepes).
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Fig. 4.
Soret CD spectroscopy of oxy HbA, HbS, and
HbC reveals changes in ellipticity in the heme environment upon the
addition of excess IHP. (0.1 M Hepes, pH 6.85, 0.1 mM heme). IHP is added in a 5-fold excess of
hemoglobin.
6 mutants and HbA (0.05 M Hepes buffer, pH
7.35) as a function of IHP titration are demonstrated by
oxygen-equilibrium measurements (Fig. 5).
The differences approach a plateau at a 1.5:1 IHP:Hb tetramer molar ratio. This is consistent with the known binding of IHP to the hemoglobin tetramer (1:1 at the DPG binding site) and reports of a
secondary lower-affinity binding site.
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Fig. 5.
Oxygen equilibria measurements indicate an
altered allosteric effect by IHP for HbC ~ HbS > HbA.
The measurements were made using a HEMO-SCAN instrument calibrated to
37 °C as described previously (2). IHP was added to hemoglobin
solutions at pH 7.35 in 0.05 M Hepes buffer. The
P50 was determined and is plotted here as a function of the
IHP:Hb tetrameric ratio.
The oxygen affinity (P50) of stripped HbA, HbS, and HbC in 0.05 M Hepes buffer, pH 7.35 (37 °C) (Student's t
test)
6
mutants under these conditions is significant.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6. Rather, we hypothesize that the ligand-specific characteristics of each of these
6 mutant hemoglobins arise from an intramolecular long-range communication of the conformational change at the
6 site. The alteration need not be the same for HbS and HbC.
6 mutants. Because it is well established that tryptophan emission wavelength shifts are a function of hydrophobicity (32-34), the findings indicate that the Trp environment(s) of HbC and HbS compared with HbA are altered in the direction of increased hydrophobicity (HbS > HbC > HbA) (Fig. 1). UVRR comparative spectroscopy helps to
dissect the specifics of the Trp alterations.
1
2 interface. There are two features that
have been especially characterized. The first is a substantial
derivative shaped feature associated with Y8a at ~1615
cm
1 that has been attributed to the T- to R-state loss of
the
42-
99 hydrogen bond in the switch region of the interface.
The second is a T- to R-state decrease in the intensity of the low
frequency shoulder of the W3 band at ~1548 cm
1 that
originates from
37 Trp in the hinge region of the
1
2 interface.
145 Tyr and
146 His weakens the support of the
A-helix of the
chain, resulting in a tighter packing of the A-helix
and E-helix. This tighter packing is reflected in an increased
intensity of the main peak of W3 at 1560 cm
1 due to the
increase in hydrogen bonding of
15 Trp (43).
14,
15, and
37, as reflected in a
greater W3 band cross-section for both mutants relative to COHbA. In
support of this, at pH 6.35, these effects are shown to be further
exaggerated (45). The position of the W3 UVRR band is a function of the
dihedral angle,
2,1
(C2-C3-C
-C
,), which is the angle between the indole residue and the backbone C
(Ref. 46). An equation relating the W3 frequency to
the dihedral angle has been proposed (47). Applying this equation, we
find that the 1547.5 cm
1 W3 band for
37 Trp of COHbA
and COHbC (Fig. 2) corresponds to a
2,1 dihedral angle
of |87o|, whereas the 1545 cm
1 band of
COHbS maps to a dihedral angle of |81o|. The change in
intensity at the 1558 cm
1 band assigned to differences in
the H-bonding between
15 Trp and
72 Ser of the A- and E-helices
may be predictive of alterations in the DPG pocket. Similar UVRR
findings for the T-state fluromet HbS were reported recently (48).
1 UVRR signal
from HbS, HbC, and HbA (Fig. 2) indicates a tightening of the H-bond in the order of HbS > HbC > HbA. Our earlier studies (1),
which were conducted at pH 6.85, indicated an outward displacement of the A-helix in HbC compared with HbA. UVRR studies comparing the use of
nitrate and perchlorate as the internal standard reveal that the
different observations in our earlier study (1) result from the
presence of 0.3 M perchlorate, which we speculate may function as an allosteric effector. Studies of the effects of chloride
at low pH (45) and other allosteric effectors are underway.
1 does not shift (no derivative signal), and the
differences in W3 are largely but not entirely due to the
14,
15
component at 1560 cm
1. Thus the differences among the
three liganded hemoglobins can be largely attributed to global packing
changes among several of the helices. The wavelength shifts in the
intrinsic fluorescence suggest that the increase in packing between
helices is accompanied by or is induced by an increase in the degree of
hydrophobicity within the globin.
37 Trp is responsible for the R-T fluorescence
difference (31), whereas the A-E-helix packing changes are responsible
for the R-T independent HbA-HbS fluorescence differences, as
also shown earlier for R-state HbC (1).The present results are
consistent with these results and conclusions.
T switching at this pH. This maximal difference in fluorescence was
attributed to alterations at the
1
2
interface, because it appeared to be obliterated when
43 Glu was
covalently modified (49-52). Studies pursuing the structural effects
imposed by this pH difference are ongoing.
6-induced tightening of the
15 Trp-
72 Ser hydrogen bond, with
the consequence that the A-helix moves closer to the E-helix for the
6 mutants HbC and HbS.
6 mutants that translates to an altered DPG pocket and consequently affects oxygen binding properties (Fig. 5). HbC and HbS
stripped of organic phosphates show a small but statistically significant difference in their oxygen affinities when compared with
HbA (Table I). These differences in oxygen affinities are consistent
with the suggested A-helix alterations and/or subtle differences in the
microenvironment of
37 Trp at the
1
2
interface for the
6 mutants, both of which are indicated by the
spectroscopic data. The differences are enhanced by the addition of the
DPG analog IHP.
6 mutants, as indicated by the fluorescence and UVRR
spectroscopic results. The implication of differences in the heme
environment initially arises from the observation that for HbS, the
deoxy form gives rise to polymerization, whereas for HbC, the oxy form
promotes intraerythrocytic crystallization. Hence, the CD Soret
differences in ellipticity may serve as a direct measurement of
conformational changes distally communicated to the heme
microenvironment. Differences in the oxy hemoglobin CD spectra of HbA,
HbS, and HbC were reported earlier by Melki (55) and of COHbS were
reported by Fronticelli et al. (13-15).
6 mutations did not exist on a significant level (24, 25).
Early spectroscopic findings and binding studies questioned these
conclusions (11-21, 55, 56), but the dogma prevailed, especially in
light of the crystal structure showing no differences distal to the
site of mutation, albeit at 3 Å resolution (57, 58).
2 and 2
2 subunits, resulting in a 5 Å displacement of the
carbons of the N-terminal valine and a narrowing of the DPG binding
pocket. This finding is consistent with the conclusion presented here. However, Eaton and Hofrichter (27) reason that because no change in the
A-helices is observed in the 1
1 and 2
1
subunits, the displacement observed in the 1
2 and
2
2 subunits presumably results from the formation of the
intermolecular contacts in the crystal (the E- and F-helices of
subunits in neighboring molecules) and is not a direct result of the
substitution of valine at
6. On the contrary, this difference may
very well be intrinsic to the mutation as indicated by: 1) the findings
presented in this investigation, and 2) the recently published 2.05 Å resolution crystal structure of deoxy HbS by Harrington et
al. (59), reporting movement of the A-helices and subtle changes
in the positions of the residues in the acceptor pocket that
demonstrate that plasticity in both the donor and acceptor chains of
the lateral contact facilitates binding.
6
mutations when compared with HbA. Our results point to A-helix
alterations that may be the likely candidate in the primary mechanism
driving oxy HbC to crystallize and causing destabilization of oxy HbS,
which, under deoxy conditions, may give rise to the polymerization
process. Similar A-helix perturbations have also been reported by
others for both T- and R-state forms of fluoro-met HbS (48). Extensive
comparative investigations of the deoxy forms of these mutants are planned.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL 38655, HL 58247, P01HL51804, HLBI R0132793, and HL 07556.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Medicine (Ullmann 925), Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-3604; Fax: 718-824-3153; E-mail: rhirsch{at}aecom.yu.edu.
2 T. Spiro, private communication.
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ABBREVIATIONS |
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The abbreviations used are: DPG, 2,3-diphosphoglycerate; UVRR, ultraviolet resonance Raman; HPT, 8-hydroxy-1,3,6-pyrenetrisulfonate; COHb, carbonmonoxyhemoglobin.
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