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Characterization of Two Types of Fetal Hemoglobin: cy2‘y2 and cy2*y2
By Kazuhiko Adachi, Jungyop Kim, Toshio Asakura, and Elias Schwartz
The effect of differences in ‘y and ‘y fractions of fetal
hemoglobin (HbF) on the kinetics of polymerization of
HbS-HbF mixtures was studied. We also examined their
effect on oxygen affinity, surface hydrophobicity, mechanical stability, and solubility of HbF. Differences in ‘y:*y ratio
did not affect the polymerization of mixtures of HbF and
HbS, suggesting that the inhibitory effect of HbF on the
polymerization of HbS is independent of the ‘y:’y ratio of
HbF and is totally dependent on the fraction of HbF in the
mixture. The oxygen equilibrium curve of HbF was not
affected by differences in the ratios of ‘7 and ‘7 in HbF. In
contrast, surface hydrophobicity, mechanical stability, and
solubility of HbF were affected by differences in the ‘y:’y
ratio. The higher the ‘y:’y ratio, the smaller the elution
volume on a TSK Gel SW hydrophobic column in high
phosphate buffer. The mechanical stability of HbF was also
dependent on the ratio of ‘y:’?: stability was greater at
higher fractions of ’7. Differences in the ‘ 7 : ’ ~ratio also
affected solubility of HbF: HbF containing the higher fraction of ‘y was the more soluble. These data indicate that
although alanine at the 136th position of the y chains has a
stronger surface hydrophobicity than does glycine, this
difference does not affect either the polymerization of HbS
or the oxygen affinity of HbF.
0 1990 by The American Society of Hematology.
F
or Bantu haplotypes usually have a ‘7 fraction less than 0.5
and a more severe clinical course.” While the differences in
Gy/Aybetween the haplotypes have been well documented,
there is considerable overlap in H b F levels, and the extent of
influence of haplotypes or differences in H b F levels has not
been firmly e~tablished.’~
Although elevation of HbF increases the solubility, minimum gelling concentration, and the delay time before polymerization of HbS,’6-22the role of ‘y and Ayof HbF on the
polymerization of HbS and molecular and functional properties of HbF has not yet been determined. Using blood from a
patient with HbS@+hereditary persistence of fetal hemoglobin (HPFH) who has a H b F level of 28% and only ‘7
present” and hemolysate from sickle cell patients with fetal
hemoglobins having various ‘y fractions, we studied the
effect of the glycine and alanine residues at 7136 on surface
hydrophobicity, oxygen affinity, and stability of the HbF
molecule. Furthermore, we studied the effect of ‘ y and Ayon
the kinetics of polymerization of sickle hemoglobin.
ETAL HEMOGLOBIN (HbF) contains two types of y
chain that differ in the amino acid residue at the 136th
position: one type has alanine (Ay) as its residue and the
other, glycine (‘y).’ There are usually two y-chain genes per
chromosome, and the ‘ y and Ay chains are the products of
nonallelic tandem gene^.^.^ HbF isolated from red blood cells
of newborns contains about 70% ‘y chain and 30% Aychain,
a proportion that is constant throughout fetal de~elopment.~.’
In contrast, the small amount of H b F found in adult red
blood cells contains about 40% ‘7 chain and 60% chain.5
The transition from the fetal ‘yAy ratio to the “adult” ratio
takes place during the first 10 months of
Clinical severity in sickle cell disease is quite varied. The
concept that elevation of H b F should favorably influence
morbidity in persons with sickle cell anemia has been
supported by both biochemical and clinical studies. Modification of clinical expression may also relate in part to the
chromosomal background of the sickle mutation, in particular to four major haplotypes: Benin, Senegal, Central African Republic (Bantu), and Arab-Indian.’.l2 The clinical
course may depend not only on HbF level but perhaps also on
the fraction of ‘7 present in HbF.I2 For instance, ArabIndian haplotypes associated with HbF levels ranging from
18% to 25% have a ‘y:totaI y ratio of 0.60. Sickle cell
patients with the Senegal haplotype who have 12.3% * 5.3%
HbF have a ‘7 fraction of 0.67. Both of these haplotypes are
associated with a milder clinical course, less severe anemia,
and fewer dense irreversibly-sickled cells than are other
hap lo type^.^'"''^ In contrast, patients homozygous for Benin
From the Division of Hematology. The Children’s Hospital of
Philadelphia, University of Pennsylvania School of Medicine.
Philadelphia, PA.
Submitted June 23,1989; accepted January 23.1990.
Supported by Grants No. HL32908, HL28157, and HL38632
from the National Institutes of Health. Bethesda. MD.
Address reprint requests to Kazuhiko Adachi. PhD, Division of
Hematology. The Children’s Hospital of Philadelphia, 34th St &
Civic Center Blvd, Philadelphia, PA 19104.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7510-0004$3.00/0
2070
EXPERIMENTAL PROCEDURES
Hemoglobin S was purified from sickle cell trait hemolysates
(HbA and HbS) by CM Sephadex column chromatography (Sigma,
or a mixture of
St Louis, MO).24Hemoglobin F with only a2y:36Giy,
a2$36G1y
2
and a2y:36Aia,
was purified by fast protein liquid chromatography (FPLC) using a preparative Mono Q column.” HbF is
separated from acetelyated HbF under the same conditions. The
concentration of hemoglobin was determined spectrophotometrically
by the use of a millimolar extinction coefficient of mE,,, = 12.5 and
mEJ6, = 13.4 for monomeric deoxy- and carboxyhemoglobin,
Fractions of ‘7 and A-y of HbF were analyzed by highperformance liquid chromatography (HPLC) using a C,* Waters
Model Bondapak column (Milford, MA) equilibrated with an
acetonitrile-trifluoroacetic acid (TFA) solution?’ The wavelength of
the detector (Waters Model 250) was set at 280 nm. Flow rate was
1.0 mL/min, and the chromatogram was determined at room
temperature using a Waters 720 system controller.
Kinetic studies on the polymerization of hemoglobin were carried
out in 1.8 mol/L phosphate buffers at 3OoCz4Samples of mixtures of
HbS and HbF were prepared by mixing these two hemoglobins with
20 mmol/L phosphate buffer, pH 7.0, under aerobic conditions, and
by keeping the mixture in this solution for at least 12 hours. An
aliquot of the mixture was injected into an anaerobic cuvette
containing deoxygenated 1.8 mol/L phosphate buffer and Na2S204.
Blood, Vol 75. No 10 (May 15).1990:pp 2070-2075
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207 1
W O TYPES OF HBF azGy2AND sty,
Under these conditions, the mixture contains HbF, HbS, and hybrid
hemoglobins.2s
Solubility was determined by measuring the concentration of
dissolved hemoglobin after completion of polymerization by filtration through a 0.45 p m membrane filter (Millipore Co, Bedford,
MA).24The polymer phase was collected by filtration. The fractions
of HbS and HbF in solution were analyzed by ion exchange HPLC.”
Oxygen equilibrium curves of HbF were determined in 0.1 mol/L
phosphate buffer, pH 7.0, at 2OoC with an automatic recording
apparatus in the presence of an antifoaming agent (Silicon Antifoam
Emulsion SAG, Union Carbide Corp, Somerset, NJ) and hemoglobin stabilizer (hexamethylphosphoramide), while removing oxygen
by bubbling with nitrogen gas.29
Mechanical shaking experiments were done according to a method
described previ~usly.’~~’’
Two milliliters of carbonmonoxyhemoglobin (CO-Hb) solution (-50 pmol/L per heme) in 0.1 mol/L
phosphate buffer, pH 8.0, were placed in a 10 x 45 mm cuvette
equipped with a screw cap and were shaken with a TCS-shaker,
Model 250 (Southampton, PA), at 60 Hz for various time intervals
at room temperature. After shaking, the cuvette was centrifuged at
4,000 rpm for 10 minutes, and the absorption spectra of the
supernatant hemoglobin solution were recorded between 500 and
700 nm. The percent of remaining CO-Hb was calculated by the
change of absorbance at 569 nm.
Surface hydrophobicity was determined by the elution volume of a
hemoglobin from a TSK-G-2000 SW column at room temperature
(22°C).32,33
A Pharmacia FPLC system composed of a P-500 pump,
an LCC-500 controller, a UV-1 monitor, and an MV-7 valve
(Uppsala, Sweden) was used. The flow rate was 0.75 mL/min.
RESULTS AND DISCUSSION
Effect of glycine and alanine at the 7136 position of HbF
on HbSpolymerization. The kinetics of the polymerization
of FS mixture in low and high phosphate buffers showed that
the delay time before polymerization of deoxy HbS increases
with increases in the amount of HbF.I9v2’This may be caused
by the exclusion of FS hybrid hemoglobin and H b F from the
p~lymerization,”.’~
suggesting that the initiation of polymerization of FS mixture is controlled only by amounts of HbS
present in the FS mixture.
The effect of glycyl and alanyl residues of the H b F
molecule on the kinetics of HbS polymerization was studied
using equimolar mixtures of HbS and H b F that contained
various mixtures of a27k36G’Y
(‘7) and a2yi36Ala (Fig I).
When hemoglobins F and S are mixed in the oxy-state, the
tetramers rapidly dissociate into dimers, which reassociate
randomly to form FS hybrid hemoglobin.** The amount of
FS hybrid hemoglobin in the FS mixture in high phosphate
buffers also follows the binomial expansion.34The kinetics of
the polymerization of three equimolar FS mixtures under
hybrid-forming conditions was measured in 1.8 mol/L phosphate buffer, pH 7.4, a t 3OoC and compared with that of
HbS. FS mixtures with three different percentages of ‘7
were prepared: loo%, 73%, and 45%.The kinetic curves of
the polymerization of all FS mixtures revealed a delay time
before polymerization, as did the curves for deoxy-HbS
alone.24The length of the delay time depended on the initial
hemoglobin concentration: the higher the total hemoglobin
concentration, the shorter the delay time. Logarithmic plots
of delay time versus hemoglobin concentration of FS mixtures with various ratios of ‘7 and Ayare shown in Fig 1. The
-1.0
”0
%
-1.5
7
m
0
-1
-2.0
-0.5
-1.0
Log
ci
Fig 1. Relationshipbetween the delay time before polymerization and hemoglobin concentration of a 1:l FS mixture. Kinetic
studies of polymerization of the deoxy-form of a 1 : l FS mixture
with various ratios of ‘y:total y were done in 1.8 mol/L phosphate
buffer, pH 7.4, at 30°C using a temperature jump method. A
indicates deoxy-HbS alone; A, 0. and 0 are 1 : l FS mixtures with
100% ‘7.73% ‘7, and 45% ‘y, respectively.
line for all equimolar FS mixtures with different ratios of
‘yAy is shifted toward the right of the line of deoxy-HbS
alone by about 0.6, indicating that one fourth of the total
hemoglobin concentration in the FS mixture, the pure HbS,
determines the length of the delay time before polymerization, as suggested from results of kinetic studies of polymerization of FS mixtures in low and high phosphate
The slight difference among the lines for the three FS
mixtures with different ‘7 percentages must be attributed to
slight differences in the fraction of HbS in the 1:l FS
mixture, rather than to differences in the ‘yAy ratio. The
fractions of HbS in these mixtures were 52.1% 1.25%,
49.1% 1.18%, and 48.5% 1.27%for mixtures with 100%
‘7, 73% ‘7, and 45% ‘7, respectively. The difference
between 52.1% and 48.5% of HbS in the FS mixture can
account for the 0.06 shift, assuming that the length of the
delay time is determined by HbS concentration and that both
the FS hybrid hemoglobins (aFyp, and a/y&) are excluded
from the nucleation step, as well as H b F (a:y2 and a / y z ) .
Multiple types of polymers form in high phosphate buffer.
Among these are the types of polymers formed under low salt
condition^.^^ The nature of HbS polymers in high phosphate
buffers (less than 1.8 mol/L) parallels those formed in
gelling experiments under near physiologic condition^.*^*^^
With increasing salt conditions, the hydrophobic interaction
of proteins increases, and resulting proteins can associate
easily and possibly form multiple types of HbS polymers.
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2072
ADACHI ET AL
Since hybrid HbF and FS hybrid hemoglobin are excluded
from all these multipolymers of HbS formed in 1.8 mol/L
phosphate buffer, the exclusion of these hemoglobins from
HbS polymers is even more likely under near physiologic
conditions.
The solubilities of these three FS mixtures were measured
at the plateau of the polymerization curve. As shown in Fig 2,
although the solubility of HbS was independent of the initial
hemoglobin concentration, the solubility of equimolar FS
mixtures depends on the initial hemoglobin concentration.
This also indicates that HbF and FS hybrid hemoglobin are
excluded from polymers, and that the solubility of the FS
mixture is controlled by HbS. The supernatant of FS mixture
(solubility) is greater when initial hemoglobin concentration
is increased because of increases in the H b F and FS hybrid
hemoglobin that are excluded from polymerization. This
result is similar to that in studies using AS mixtures.36 The
solubility of the FS mixture in 1.8 mol/L phosphate buffer
was independent of the ‘yAy ratio. It is known that the
logarithmic plots of solubility of hemoglobins are linearly
related to phosphate c o n c e n t r a t i ~ nIt. ~is~more
~ ~ ~ likely that
the solubility of FS mixture is independent of ‘yAy ratio
under near physiologic conditions, although direct measurements are required to prove this.
Turbidity increased linearly with increases in initial hemoglobin concentration and was independent of the ‘yAy ratio
(Fig 3). These results indicate that polymerization of FS
mixture is not affected by the ‘yAy ratio in HbF, and that
the inhibitory effect of H b F on the polymerization of HbS
depends solely on the fraction of HbF.
Oxygen equilibrium curves of HbF with various fractions
of ‘7. Oxygen equilibrium curves of HbF with two different fractions of ‘y were determined in 0.1 mol/L phosphate buffer, pH 7.0, at 2OoC by an automatic Hemox
analyzer in the presence and absence of organic phosphate.
As shown in Table 1, the P,, values for HbF containing 100%
‘y and 73% ‘y are the same, both in the presence and
absence of organic phosphates, suggesting that glycyl and
alanyl residues at the y136 position do not affect the
functional properties of HbF.
Eflect of Gly and Ala at y136 on surface hydrophobicity.
HPLC using so-called reversed phase (RP) column packing
I
=I
D
0.30
I
I
t
2.0 I
I
I
I
/
db
I
a 0.5
I
A
A
v
0
0.10
0.20
0.30
Initial C o n c e n t r a t i o n ( g / dl)
Fig 3. Relationship between turbidity after polymerization and
hemoglobin concentration. Turbidity at the plateau of polymerization of 1:l FS mixtures with various hemoglobin concentrations
was measured spectrophotometrically at 700 nm. Experimental
conditions and symbols are as described for Fig 1.
has become a powerful tool for the separation of ‘7 and Ay,as
well as CY and chains.’.27In R P column chromatography,
proteins separate according to their degree of hydrophobicity
by interaction between exposed nonpolar amino acid residues
on the protein and hydrophobic groups on the column matrix.
In addition, in RP-HPLC, very densely distributed nonpolar
groups produce strong hydrophobic interaction, necessitating
the use of organic solvents for elution. Using this method, ‘7
can clearly separate from
and the retention time for Gy is
found to be shorter than that for Ay, suggesting that the
alanyl residue at the 7136 position is more hydrophobic than
the glycyl residue at the same position. However, this finding
can not be applied directly to native tetrameric spy, and
azAy2,since the solvent used for the separation causes
proteins to denature.
Recently, hydrophobic interaction column chromatography that does not use organic solvents has been de~eloped’~-~’
and can be used to quantify the surface hydrophobicity of
proteins in the native state. We found that a highperformance size-exclusion column equilibrated with high
Table 1. P, Values of Oxygen EquilibriumCurves
of HbF With Different G’y and ‘7 Ratios
-A-k-A
I
HbF (% ‘4
P,, (mm Ha)
+ DPG (2 mmol/L)
100
73
8.63
8.65
11.5
11.7
1
I
I
Initial C o n c e n t r a t i o n ( g l d l )
Fig 2. Solubility after polymerization of the deoxy-form of HbS
and 1:1 FS mixtures. Supernatant hemoglobin concentration
(solubility) was determined at the plateau of polymerization.
Experimental conditions and symbols are as described for Fig 1.
~
Measurement of oxygen equilibrium curves of HbF (- 100 wmol/L) was
performed in 0.1 mol/L phosphate buffer, pH 7.0, at 20.5OC to 20.8”C.
The values are the mean value of duplicate. Under the same conditions,
the P,, values of HbA with and without DPG (2 mmol/L) were 8 . 3 0 and
13.7 mm Hg, respectively
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TWO TYPES OF HBF aZGy,AND
2073
sty,
salt concentration behaves similarly to hydrophobic interaction chromatography, making it possible to characterize the
surface hydrophobicity of native proteins with the same
molecular size.32
Using this method, we studied the effect of Gly and Ala at
the y l 3 6 position on surface hydrophobicity of the H b F
molecule. The elution volume of normal CO-HbF with 73%
‘7 was 28.5 mL using TSK Gel-2000 S W (7.5 x 300 mm) in
1.65 mol/L phosphate buffer, pH 7.4, at room temperature.
Under these conditions, CO-HbA and CO-HbS elute much
more slowly than CO-HbF.” The elution volumes were 44
mL and 73 mL for CO-HbA and CO-HbS, respectively. The
elution volume for H b F was dependent on the fraction of ‘y
and Ay:the higher the fraction of Ay,the larger the elution
volume (Fig 4). The elution curves of the mixture of a F y ,
and aZAy,broadened with higher fractions of azAy2.The
elution volume for H b F with 100% ‘y was 27.75 mL; for
H b F containing 45% Ay,30.75 mL. However, the tetrameric
aZGy2
and a2Ay2did not separate from each other, perhaps
indicating the formation of hybrid hemoglobin between these
two hemoglobins, From this result, the difference in the
elution volume between H b F with ‘y and H b F with Aywas
calculated to be about 5 mL, under these conditions. These
has a stronger
data indicate that the native form of a2y:36A1a
as suggested by the
surface hydrophobicity than a2y:36G1Y,
result using denatured ‘y and Aychains.’
Efect of Gly and Ala at y136 on mechanical stability of
HbF. The oxy-form of HbS denatures at a rate approximately 10 times faster than that of HbA during mechanical
agitati~n.~’
We have also found that oxy-HbF is much more
stable than oxy-HbA during mechanical agitation. Our
studies on the stability of hemoglobins during mechanical
agitation have shown that the rate of denaturation of
abnormal hemoglobins depends on the type and site of the
mutation.3334’The rate of precipitation of hemoglobin during
mechanical agitation reflects the surface hydrophobicity of
35
c
01
0
-I
I
I
I
I
20
40
60
80
I
100
0 0
I
I
10
I
20
I
I
I
I
\
30
I
4I0
5,-‘\;\.B
0
60
1
70
Time (min)
Fig 5. Denaturation curve of oxy-HbF with various fractions of
‘7. Oxy-HbF with differing ratios of ‘y/’’y in 0.1 mol/L phosphate
buffer (2 mL), pH 8.0, was shaken for the time intervals shown in
comparison with oxy-HbA. The percentagesof denatured hemoglobin were determined spectrophotometrically. 0,W, 0 , and
indicate HbA, HbF with 100% ‘7, HbF with 73% ‘ y , and HbF with
45% ‘ y , respectively.
tetrameric and dimeric hemoglobin molecules, in addition to
protein conformation and surface a ~ t i v i t y . ~ ~Inv this
~ l *study,
~~
the results indicate that the rate of denaturation of H b F
during mechanical agitation varies with the fraction of ‘y
(Fig 5). The denaturation rates for HbF solutions containing
73% ‘y and 35% ‘y are 1 . 1 and 1.4 times faster, respectively,
than that of H b F that does not contain ‘y (100% Ay).From
these results, it can be calculated that the denaturation rate
of H b F with 100% Ayis 1.6 times faster than that of HbF
with 100%‘y.
The denaturation rate of hemoglobin molecules is also
related to the surface hydrophobicity and the amount of
dimeric and tetrameric hemoglobin present.42 Since the
oxygen affinities of a F y 2 and a2Ay2are similar, the dissociation constants of these two fetal hemoglobins should also be
similar. Our data indicate that the surface hydrophobicity of
dimeric aAy is greater than that of a‘y.
Effect of Gly136 and Ala136 of the y chain on the
solubility of HbF. The solubility of H b F is significantly
higher than that of HbA. This appears to be due to a
weakening of one of the electrostatic contacts between
neighboring molecules in HbF crystals.43 Solubility depends
on solvent conditions; for instance, the higher the phosphate
concentration, the lower the s o l ~ b i l i t y . ~The
~ , ~ ’solubility of
HbF with various fractions of ‘7 and Ay was studied in 2.5
mol/L phosphate buffer and was found to be dependent on
the fraction of ‘y and Ay (Table 2). Solubility of H b F was
higher with increased fractions of ‘y.
100
% GV
Fig 4. Relationshipbetween the elution volume on a TSK-GEL
SW column and the fraction of ‘y of HbF. CO-HbF solution (200 NL)
with various ratios of
in 1.65 mol/L phosphate buffer, pH 7.4,
was applied to a TSK-G-2000 SW column equilibrated with 1.65
mol/L phosphate, pH 7.4, at room temperature and was eluted
with the same buffer. The elution volume of ‘7 was determined by
the extrapolation of the line to 0% ‘7 (dashed line).
Table 2. Solubility of HbF With Various Fractions of ‘y and ‘7 in
2.5 mol/L Phosphate Buffer, pH 7.4, at 22°C
Fraction of ‘7 (%)
100
73
50
Solubility (ma/&)
28.5 + 6.4
27.4 + 2.8
20.5 k 1.4
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2074
ADACHI ET AL
The solubility of protein depends on the protein-solvent
and protein-protein interactions. The 136th position of the H
helix of the HbF molecule does not appear to be a contact
point when HbF aggregates to form crystals.43The difference
in the solubility of a;yz and a / y 2 may be related to
differences in the protein-solvent interaction caused by their
surface hydrophobicity. This depends on the difference in the
hydrophobicity of Gly and Ala at the 136th position of the y
chain.
Our results indicate that the chain composition of fetal
hemoglobin in respect to ‘y and Ay content does not affect
two major properties of sickle hemoglobin: delay time from
deoxygenation to aggregation and oxygen equilibrium. It is,
therefore, very unlikely that the differences in ‘ y and Ay
content found with various p” haplotype backgrounds account for the range of clinical expression of sickle cell
disease. We recognize that the experiments done in this
study, especially those on the kinetics of polymerization in a
high phosphate buffer, were not done under physiologic
conditions. Further experiments on the kinetics of the polymerization of FS mixtures under near physiologic conditions
should be done to confirm our results.
ACKNOWLEDGMENT
We thank Janet Fithian for editorial assistance and Emi Asakura
for secretarial help.
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1990 75: 2070-2075
Characterization of two types of fetal hemoglobin: alpha 2G gamma 2
and alpha 2A gamma 2
K Adachi, J Kim, T Asakura and E Schwartz
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