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Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1438-1445
Effects of Increased Anionic Charge in the  -Globin Chain on
Assembly of Hemoglobin In Vitro
By
Kazuhiko Adachi,
Takamasa Yamaguchi,
Jian Pang, and
Saul Surrey
From the Division of Hematology, The Children's Hospital of
Philadelphia, and Department of Pediatrics, University of Pennsylvania
School of Medicine, Philadelphia, PA; the Departments of Research and
Pediatrics, duPont Hospital for Children, Wilmington, DE; and the
Department of Pediatrics, Jefferson Medical College, Philadelphia, PA.
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ABSTRACT |
Studies on assembly in vitro of  -globin chains with recombinant
 16 Gly Asp, 95 LysGlu, 120 LysGlu
and 16 GlyAsp, 120 LysGlu human -globin chain
variants in addition to human A- and
S-globin chains were performed to evaluate effects of
increased anionic charge in the chain on hemoglobin assembly using
soluble recombinant -globin chains expressed in bacteria. A 112
CysAsp change was also engineered to monitor effects on
assembly of increased negative charge at 11 interaction sites.
Order of tetramer formation in vitro under limiting -globin chain
conditions showed Hb G16D, K120E = Hb K120E = Hb K95E > Hb G16D > Hb A > Hb S >>> Hb C112D. In addition, 112
CysAsp chains exist as monomers rather than 4
tetramers in the absence of chains, and the chain in Hb C112D tetramers was readily exchanged by addition of
s. These results suggest that affinity between and
chains is promoted by negatively-charged chains up to a maximum
of two additional net negative charges and is independent of location on the surface except at the 11 interaction site. In addition, our findings show that 112 Cys on the G helix is critical for facilitating formation of stable dimers, which then form
functional hemoglobin tetramers, and that 112 CysAsp
inhibits formation of stable 11 and 12 interactions in
22 and 4 tetramers, respectively.
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INTRODUCTION |
HEMOGLOBIN IS COMPOSED of two - and
two non--polypeptide chains and has served as a model macromolecule
to study various aspects of structure, synthesis, and assembly of
multisubunit proteins.1,2 The human - and -like
globin genes are located on different chromosomes and give rise to the
two different chains involved in hemoglobin biosynthesis.2
A number of factors can influence relative levels of human hemoglobin
variants, which are produced in vivo.3-8 Formation of
hemoglobin requires balanced production of -and -polypeptide
chains. The - and -globin mRNAs are first translated into their
respective polypeptide chains, and the two hemoglobin chains diffuse
into the cytoplasm and assemble into dimers, which then form
stable, functional 22 tetramers.
The rate-limiting step in hemoglobin assembly is the bimolecular
reaction involving + , which is thought to be
governed by electrostatic attraction between monomeric partner
subunits.2,3 The higher proportion of Hb A than Hb S in AS
heterozygotes has been explained by assembly rate differences with chains of A and S chains, which have Glu
and Val at the 6 position, respectively.2,3 In addition,
extensive work by Bunn et al3,6,7 showed using naturally-occurring hemoglobin variants that an additional negative charge in the chain promotes assembly of hemoglobin.
Although the additional negatively-charged chains promote
electrostatic attraction between partner subunits, the maximum
enhancement by increased negative charge, as well as the role of direct
interaction sites in promoting stable assembly, is not clear.
Recent studies using site-directed mutagenesis should provide further
elucidation of the mechanism of subunit assembly of hemoglobin.
Studies of hemoglobin assembly in vitro require isolation of large
amounts of individual - and -chain variants from their tetramers,
and the isolated chains are then reconstituted in vitro to form
hemoglobin tetramers. To facilitate assembly studies and further our
understanding of this process, production of soluble - and -chain
variants is critical. We recently succeeded in producing authentic
human, soluble -globin chains in bacteria using an expression vector
containing cDNAs for methionine aminopeptidase and human globin.9 Of interest, the -globin chain fraction contained monomers and disulfide cross-linked dimers. The dimers, which
are formed by oxidation of cysteine residues, could be reduced to
monomers by addition of dithiothreitol (DTT). Furthermore, dimers were
unable to form tetramers in vitro on addition of exogenous chains,
while monomeric chains, which are in the reduced form as a result
of DTT addition, were able to form tetramers.9 Our results
indicate that - and -globin chains fold independently and that
conditions for efficient dimer-tetramer assembly are now available. We
are now able to produce soluble -chain variants to systematically
evaluate factors affecting hemoglobin assembly by engineering various
mutations at predetermined sites. In this report, we expressed five
variant -globin chains to confirm the role of subunit surface charge
using soluble -globin chains expressed in bacteria and to assess the
role of 11 direct interaction sites on assembly of hemoglobin.
Three of the variants, 16 GlyAsp, 95 LysGlu,
and 120 LysGlu, are found in J-Baltimore, N-Baltimore, and
Hb Hijiyama, respectively, and contain an additional one or two net
negatively-charged amino acid substitutions on the surface of the chain. Heterozygotes express more than 50% of these variant hemoglobins.3 In addition to these variants, we engineered a three net negatively-charged -chain variant, 16
GlyAsp, 120 LysGlu, to assess the maximum effect of
negative charge addition on promotion of assembly. A fifth
variant, 112 CysAsp, was also made, which contains a
negatively-charged amino acid substitution at an 11 interaction
site, to clarify the role of this site in stable assembly.
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MATERIALS AND METHODS |
Expression of soluble recombinant human -globin chain variants in
Escherichia coli.
16 GlyAsp, 95 LysGlu, 120
LysGlu, 16 GlyAsp, 120 LysGlu and
112 CysAsp chains were expressed as described
previously9 using the vector pHE2, which contains cDNAs
coding for the human -chain variant and methionine
aminopeptidase. The basic strategy for generation of these variants by
site-specific mutagenesis of the normal chain involves
recombination/polymerase chain reaction as described
previously.10 Clones were subjected to DNA sequence
analysis of the entire -globin cDNA region using site-specific
primers and fluorescently-tagged terminators in a cycle sequencing
reaction in which extension products were analyzed on an automated DNA
sequencer.11 Plasmids were transfected into E. coli
(JM 109) (Promega Co, Madison, WI), bacteria were grown at
30°C, and soluble -globin chain variants were isolated and purified as described.9
Authentic human chain was purified from tetrameric Hb A isolated
from erythrocyte lysates according to previously described methods.12 Removal of p-mercuribenzoate was
accomplished using 20 mmol/L DTT, and globin chains were isolated by
gel filtration on a fast protein liquid chromatography (FPLC) Superose
12 column (Pharmacia Biotech, Uppsala, Sweden).
Biochemical characterization of purified -globin chains.
Molecular mass and sample purity were assessed by sodium dodecyl
sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) as described.13 Electrospray ionization mass spectrometry
(ESMS) was performed on a VG BioQ triple quadrapole mass spectrometer (Micromass, Altrincham, Cheshire, UK).14 The
multiply-charged ions derived from globin (Mr:
15,126.4) served as internal and external standards for mass scale
calibration. Data analysis used the MassLynx software package
(Micromass, Altrincham, Cheshire, UK).
Purified -globin chains were also analyzed by cellulose acetate
electrophoresis on Titan III membranes at pH 8.6 with Super-Heme buffer
(Helena Laboratories, Beaumont, TX). Isoelectric focusing of purified
-chain variants, A and S was performed
on an Ampholine PAG plate (pH 5.5 to pH 8.5) using a Multiphor II
system (Pharmacia Biotech, Piscataway, NJ). After focusing for 2 hours
at constant 25 W at 4°C, the gel plate was stained with Coomassie
Brilliant Blue R-250 to detect proteins. Isoelectric point of each
-globin variant was estimated from a calibration curve prepared with
isoelectric focusing (IEF) standards (Bio-Rad, Hercules, CA).
Absorption spectra of purified globins in the CO forms were
recorded using a Hitachi U-2000 spectrophotometer (Hitachi Instruments
Inc, Danbury, CT). Circular dichroism (CD) spectra of -globin
variants were recorded using an Aviv model 62 DS instrument (Varian
Analytical Instruments, San Fernand, CA) employing a 0.1-cm light path
cuvette at 10 µmol/L globin concentration. CD spectra of -globin
variants compared with normal A were monitored in the
wavelength range from 190 to 260 nm. Oxygen dissociation curves were
determined in 50 mmol/L Bis-Tris buffer containing 0.1 mol/L NaCl, pH
7.1 at 20°C using a Hemox Analyzer (TCS Med Co, Huntington Valley,
PA).15
Hemoglobin concentration was determined spectrophotometrically on a
Hitachi U-2000 spectrophotometer using a millimolar extinction coefficient of 13.4 at 540 nm for carbon-monoxy
hemoglobin.16 Assembly studies of purified -chain
variants (75 µmol/L) were performed after addition of varying amounts
of -globin chain in the CO form in 0.1 mol/L phosphate buffer, pH
7.0 at 25°C,9 and tetramer formation was assessed by
FPLC (fast protein liquid chromatography) using Mono S and Superose 12 gel-filtration chromatography.
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RESULTS |
Expression and characterization of -chain variants.
After DNA sequence confirmation, the five -chain variant cDNAs were
expressed in bacteria. All five purified variants migrated as single
bands following cellulose acetate electrophoresis at pH 8.6 (Fig 1). As expected, addition of negative
charges in 16 GlyAsp, 95 LysGlu, 120
LysGlu and 16 GlyAsp, 120 LysGlu compared with A chains resulted in increased anodic
mobility on electrophoresis. These results suggest that these
negatively-charged chains, like A chains, exist as
4 tetramers in solution. In contrast, electrophoretic mobility of 112 CysAsp was similar to that of
S chains, indicating that 112 CysAsp chains
exist as monomers (charge of -2) and not 4 tetramers
(charge of -4) like the A chain.17, 18
Isoelectric focusing (IEF) of the purified -chain variants, A and S was also performed on an
Ampholine PAG plate (pH 5.5 to pH 8.5) to assess effects of additional
negative charges on the surface charge and pI (isoelectric point) of
the -chain variants (Table 1). Our
results show that the pIs for four of the five variants were lower than
that of the A chain and that the lowest pI was
associated with the variant with three net additional negative charges
compared with the A chain (16 GlyAsp, 120 LysClu). In addition, the pI of 95 LysGlu was
slightly higher than that of 120 LysGlu (5.84 v 5.73) even though both variants have the same two net negative charge
changes. These findings indicate that 120 is more exposed on the
surface compared with 95. The only variant with a pI > the
A chain was the 112 CysAsp chain,
indicating again that the 112 CysAsp chains migrate as
monomers rather than tetramers like 4A
chains as shown on cellulose acetate electrophoresis.
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| Fig 1.
Electrophoresis of purified -globin chain variants
16 GlyAsp, 95 LysGlu, 120
LysGlu, 16 GlyAsp, 120 LysGlu and
112 CysAsp and in vitro assembled tetramers. Purified
-globin chain variants 16 GlyAsp, 95
LysGlu, 120 LysGlu, 16 GlyAsp, 120 LysGlu and 112 CysAsp (A) and in vitro assembled
tetramers (B) formed by mixing with -globin chains isolated from
human red blood cells and incubating in 10 mmol/L potassium phosphate buffer pH 7.0 at 25°C were analyzed by electrophoresis on cellulose acetate membranes at pH 8.6 using Supre-Heme buffer (Helena Lab, Beaumont, TX). (A) Lane 1, A chain (purified from human
red blood cells); lane 2, 112 CysAsp chain; lane 3, 16
GlyAsp chain; lane 4, 95 LysGlu chain; lane 5, 120 LysGlu chain; lane 6, 16 GlyAsp, 120 LysGlu chain; and lane 7, S chain (purified
from human red blood cells). (B) Lane 1, Hb A (22 purified from human red blood cells);
lane 2, in vitro assembled 22 (112
CysAsp); lane 3, in vitro assembled
22 (16 GlyAsp); lane 4, in
vitro assembled 22 (95
LysGlu); lane 5, in vitro assembled
22 (120 LysGlu); lane 6, in
vitro assembled 22 (16
GlyAsp, 120 LysGlu); and, lane 7, Hb S
(22S purified from human
sickle red blood cells).
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We previously reported that the A-globin chain fraction
isolated after expression in bacteria contained monomers and disulfide cross-linked dimers.9 The dimers formed by oxidation of
cysteine residues and were reduced to monomers by addition of DTT. The purified negatively-charged -chain variants, except for the 112 CysAsp chain, also contained a mixture of monomers and
disulfide-linked dimers like the A-chain
fraction.9 In addition, dimer formation of the variants and
A chain was apparent during purification. In contrast,
112 CysAsp chains migrate as monomers on SDS-PAGE,
suggesting that disulfide bond formation generating - dimers is
caused by cross-linking of 112 Cys residues (Fig 2).
Mass spectral analysis of the five variants using ESMS showed the
expected -globin chain molecular masses (Table 1).
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| Fig 2.
SDS-PAGE of purified -globin variants. -chain
variants (2 to 5 µg) expressed in bacteria were purified and
subjected to SDS-PAGE after treatment with (+) or without (-) 200 mmol/L DTT for 30 minutes at 25°C. After heating for 3 minutes in
the presence of 3% (wt/vol) SDS in a boiling water bath, samples were
electrophoresed on a 12.5% (wt/vol) polyacrylamide gel at a constant
voltage of 100 V. Gels were stained with Coomassie Brilliant Blue R-250
to detect proteins. (A) Molecular weight standards (Amersham Life Science, Arlington Heights, IL); (B) 16 GlyAsp chain; (C)
95 LysGlu chain; (D) 120 LysGlu chain; (E)
16 GlyAsp, 120 LysGlu chain; (F) 112
CysAsp chain; and (G)  chain.
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Characterization of assembled 22
tetramers containing the five -chain variants.
The negatively-charged chains were assembled in vitro with chains to form tetrameric hemoglobins, and tetramers were then purified
by Mono S-FPLC chromatography. Electrophoretic mobility (Fig 1B) and
FPLC elution profile of assembled tetrameric
22 (112 CysAsp)
were similar to Hb A (22). In contrast,
surface charges of assembled tetramers of
22 (16 GlyAsp),
22 (95 LysGlu),
22 (120 LysGlu), and
22 (16 GlyAsp, 120 LysGlu), as assessed by cellulose acetate electrophoresis
were more negative than that of Hb A, and their elution from Mono
S-cation FPLC occurred just before that of Hb A. These results indicate
that 16 GlyAsp, 95 LysGlu, 120
LysGlu and 16 GlyAsp, 120 LysGlu are
exposed on the surface, while 112 CysAsp is located at an
internal position in the 22
tetramer.1 Absorption spectra of the CO forms of these
tetrameric variants containing negatively-charged chains were the
same as those of native tetrameric Hb A.
Naturally-occurring variants J-Baltimore (Hb G16D) and N-Baltimore
(Hb K95E) and reconstituted hemoglobin tetramers containing 16
GlyAsp or 95 LysGlu have the same oxygen
affinity and cooperativity as normal Hb A.19 We performed
functional studies of Hb G16D, K120E, and Hb C112D in 50 mmol/L
Bis Tris buffer, pH 7.1 containing 0.1 mol/L NaCl at 20°C in the
presence and absence of 2,3-biphosphoglycerate (BPG) and compared
results with those of Hb A (Table 2). Reconstituted Hb
G16D, K120E exhibited the same oxygen affinity and cooperativity as
those of normal Hb A tetramers. These results indicate that these
recombinant hemoglobins are correctly folded and assembled, and that
changes in amino acids on the surface, which do not involve direct
11 and 12 interaction sites of hemoglobin do not affect functional properties of dimeric and tetrameric hemoglobins. In contrast, oxygen affinity of Hb C112D, which has a substitution at
an 11 interaction site, was slightly higher (P50 of
2.3 v 3.7 for Hb A); and its cooperativity was slightly lower
than that of normal Hb A tetramers (2.60 v 2.74).
Tetramer formation in vitro.
Previous studies in vitro in the presence of limiting amounts of chains and mixtures of equal amounts of purified normal and mutant
subunits showed that mutant hemoglobin percentages were higher when
using more negatively-charged chains like J-Baltimore (16
GlyAsp) and N-Baltimore (95
LysGlu).3,6,7 These results suggest that more
negatively-charged chains bind positively-charged chains more
readily than A chains.3,6-8 To confirm and
extend those studies, we produced 16 GlyAsp, 95
LysGlu, 120 LysGlu and 16 GlyAsp,
120 LysGlu chains using a bacterial expression system and
then performed assembly studies in vitro with purified chains and
compared results with those using A and S
chains. Results of assembly in vitro with chains and equimolar mixtures of A and 16
GlyAsp or 95 LysGlu chains were the same as
those reported previously.3,6,7 In addition, assembly
results for 120 LysGlu chains were similar to those of
95 LysGlu chains. The ratio of Hb X/Hb A as the -chain
concentration approached zero was 2.7, 2.5, 1.5, and 0.4 for Hb
K120E, Hb K95E, Hb G16D, and Hb S, respectively
(Fig 3A). These results confirm results from earlier
studies3,6-8 showing that subunit surface charge plays a
critical role in relative affinity of chains for chains before
formation of tetrameric hemoglobin.
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| Fig 3.
Effect of -chain surface
charge on relative amounts of in vitro assembled tetramers as a
function of varying amounts of input chains. (A) Equimolar mixtures
of normal (A), sickle (S), 16
GlyAsp, 95 LysGlu or 120 LysGlu
chains (75 µmol/L) were added to varying amounts of -globin chain
in 0.1 mol/L phosphate buffer, pH 7.0 at 25°C, and assembled
tetramers were analyzed by FPLC. The relative ratio (y axis) of Hb S to
Hb A ( ) in (S + A)/ mixtures, Hb
G16D to Hb A ( ) in (16 Asp +A)/ mixtures,
Hb K95E to Hb A ( ) in (95 Glu +A)/
mixtures and Hb K120E to Hb A ( ) in (120 Glu
+A)/ mixtures was calculated as a function of
varying amounts of input -globin chain. (B) Equimolar mixtures of
s with 16 GlyAsp, 95 LysGlu,
120 LysGlu, 16 GlyAsp, 120 LysGlu
or 112 CysAsp chains (75 µmol/L) were added to
increasing amounts of -globin chain in 0.1 mol/L phosphate buffer,
pH 7.0 at 25°C. Tetramer formation was analyzed by FPLC, and the
relative ratio of Hb A to Hb S (), Hb C112D to Hb S ( ), Hb
G16D to Hb S (o), Hb K95E to Hb S (), Hb K120E to Hb S
(), and Hb G16D, K120E to Hb S ( ) was calculated (y axis).
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Previous competition experiments in vitro using mixtures of purified
and chains showed that A dimers form about
twice as readily as s dimers when the concentration
of chains becomes limiting.3,8 This results in assembly
of less Hb S relative to Hb A when equimolar amounts of
A and S chains compete for limiting
amounts of globin. Our results also show that tetramer formation
occurs efficiently in vitro; and, that under limiting -chain
conditions, less Hb S compared with Hb A formed in mixtures containing
equal amounts of S and A chains (Fig 3).
In addition, we performed subunit competition experiments in which
varying amounts of chains were added to equimolar mixtures of
S and either 16 GlyAsp, 95
LysGlu, 120 LysGlu, 16 GlyAsp, 120 LysGlu or A chains to further assess the
effect of 6 GluVal on assembly. Under limiting -chain
conditions, percentages of hemoglobin tetramers containing
negatively-charged chains were much higher in mixtures containing
S instead of A chains (Fig 3B). Total
amounts of Hb K120E and Hb K95E were always more than that of Hb
G16D, while values for the double mutant Hb G16D, K120E were
similar to those of Hb K120E and Hb K95E. The order of tetramer
formation in vitro under limiting -globin chain conditions was Hb
G16D, K120E = Hb K120E =Hb K95E > Hb G16D > Hb A. Ratios of Hb X/Hb S as -chain concentration approached zero were
6.2, 6.3, 6.0, 4.0, and 2.5 for Hb G16D, K120E, Hb K120E, Hb
K95E, Hb G16D, and Hb A, respectively (Fig 3B). These results
also indicate that promotion of assembly by additional negative charges
in the chain is independent of location on the surface, and that
two net additional negative charges compared with A
chains appear to be the maximum charge for facilitating formation of
+ ----- electrostatic intermediates.
Furthermore, the ratios of (Hb K120E/Hb S)/(Hb K120E/Hb A), (Hb
K95E/Hb S)/(Hb K95E/Hb A) and (Hb G16D/Hb S)/(Hb G16D/Hb A)
approached 2.5, which is the same value obtained for the ratio of Hb
A/Hb S under limiting -chain conditions.
Subunit competition studies were also performed in which varying
amounts of chains were added to equimolar mixtures of
S and 112 CysAsp chains to assess the role
of 11 interaction sites on assembly. Competition experiments with
A, S, and the 112 Asp chain variant
were difficult to do, as Hb A tetramers were not readily separated from
Hb C112D tetramers by FPLC. Our results show under limiting
-chain conditions that relatively much less Hb C112D compared
with Hb K95E, Hb G16D, and Hb A tetramers formed in these
mixtures containing equimolar amounts of the S chain
(Fig 3B). Furthermore, Hb C112D levels in mixtures containing S and 112 CysAsp chains were almost zero
when the ratio of chain to total chains was less than 0.5. Tetramer formation for the 112 CysAsp variant after
addition of chains was also monitored by cellulose acetate
electrophoresis (Fig 1) and FPLC (Superose 12 gel-filtration).
Gel-filtration patterns of mixtures containing 112 CysAsp
or the other -chain variants and chains were similar to those of
A and chains (Hb A), indicating that 112
CysAsp chains, just like native A chains, form
hemoglobin tetramers with exogenously-added chains (data not
shown). These results indicate that the relative affinity of for
chains is dependent on direct 11 interaction sites, even
though surface charge of the chains plays a critical role in the
initial stage of assembly. After initial electrostatic interactions of
the two chains, stable 11 interactions may then occur between the
two subunits.
Dissociation of Hb C112D tetramers.
Tetrameric Hb readily dissociates in solution and exists in equilibrium
with dimers.17,20 In contrast, native dimers dissociate slowly to monomers with a first-order dissociation rate
constant of about 3 × 10-3h-1.21 This indicates that over
a 72-hour period, only 33% of the total single chains are exchangeable
from normal tetrameric hemoglobin by a competing chain. In contrast, Hb
C112D tetramers can be readily exchanged by addition of
S chains (Fig 4), as shown
in timed FPLC chromatography experiments of mixtures of Hb C112D
tetramers and S chains. Over a period of only 30 minutes
(Fig 4C), almost all of the S chains were incorporated
into tetrameric Hb S (peak "c" in Fig 4), generating free 112
CysAsp chains (peak "a" in Fig 4). In contrast, Hb
G16D, K120E, Hb K120E, Hb K95E, Hb G16D, or Hb A tetramers
do not exchange with S chains during these same time
intervals.
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| Fig 4.
S-chain exchange as a function of
incubation time with Hb 112 CysAsp tetramers. Tetrameric
Hb C112D was incubated with s chains in 0.1 mol/L
phosphate buffer, pH 7.0 at 25°C, and Hb S tetramer formation, as
well as generation of 112 CysAsp chains, were analyzed by
FPLC. (A), (B), and (C) correspond to chromatographic analyses before
(zero time point) and after 15 and 30 minutes incubations in the
presence of s chains, respectively. Peaks a, b, c, and d
represent 112 CysAsp, Hb C112D, Hb S and
S, respectively. The dotted line is a trace of the
gradient profile for NaCl.
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Gel filtration of 112 Cys chain.
It is known that isolated -globin chains aggregate to form
4 homotetramers.18 Our gel-filtration
results show that A and the four negatively-charged chains, containing 16 GlyAsp, 95 LysGlu,
120 LysGlu and 16 GlyAsp, or 120 LysGlu, exist almost totally as 4 tetramers,
which depends on concentration; while 112 Asp chains exist only as
monomers (Fig 5). Mixtures of
A and 112 Asp chains show high and low molecular
weight forms, indicating that 112 Asp chains exist as monomers and
inhibit formation of 4 tetramers as observed in our
electrophoretic studies. These results suggest that 112 Cys is a key
amino acid in formation of 4 tetramers and that loss of
112 Cys inhibits tetramer formation. It is also important to note
that the circular dichroism spectrum measured from 190 to 260 nm for
112 Asp chains in the CO form was identical to that of
A-globin chains. These results indicate that 112 Asp
chains made in the Escherichia coli expression system are
properly folded like authentic globin. Furthermore, our findings
suggest that altered properties of assembly of the 112 Asp chain
with chain are caused by the Cys to Asp change at 112 rather
than by incorrect folding.
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| Fig 5.
Gel-filtration chromatography of 112 CysAsp
chains. Gel-filtration chromatography of purified ( 70 µmol/L in
200 µL) A chains (A), 112 CysAsp chains
(B), and a mixture of the two chains (C) was performed at a flow rate
of 0.5 mL/minute in 0.1 mol/L phosphate buffer, pH 7.0. Vo and Vt refer
to void and total column volumes, respectively.
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DISCUSSION |
The kinetics of human hemoglobin assembly in vitro were studied
previously using naturally-occurring variants, and three intermediate steps were proposed.3,6-8 The first involves dissociation
of oligomers to monomers; the second, interaction of and monomers to form essentially irreversible dimers; and the third,
aggregation of dimer to form functional
22 tetramers. Formation of monomers and
tetramers of chains depends on experimental conditions. Even though
our experimental conditions for assembly favor formation of
predominantly 4 tetramers rather than monomers due
to the relatively high concentration of chains used (eg, 75 µmol/L in 0.1 mol/L phosphate buffer at 25°C), our results on
effects of charge were similar to those previously
reported.7 In the previous study, conditions favored
formation of predominantly -chain monomers because of lower
hemoglobin concentrations (1.25 to 12.5 µmol/L), low ionic strenth
(10 mmol/L) and low temperature (0°C). These results suggest that
the different amounts of hemoglobin variants and Hb A formed in these
earlier studies and in our studies are caused by differences in
affinities of the individual -chain variants for chains.3,7,8 In addition, our findings demonstrate that
both 95 LysGlu and 120 LysGlu promoted
assembly with chain more than that of 16 GlyAsp, while
addition of another negative charge in 16 GlyAsp, 120 LysGlu chains did not influence assembly compared with 120
LysGlu. These results suggest that surface charge effects of
the chain on assembly are independent of position and are dependent
on total surface net amino acid charge up to a maximum of two
additional net negative charges compared with A chains.
These results also support the previously proposed electrostatic model
of assembly.3,7
Cys 112 is located at the interface of 11 in
22 hemoglobin tetramers, and interacts
with Val 107 (G14) and Ala 110 (G11), which is critical for
stabilization of the interface.1,20 Our present
studies show that Hb C112D levels in equimolar mixtures containing
S and 112 CysAsp chains were almost zero
when the ratio of chain to total chains was less than 0.5. Furthermore, the order of tetramer formation was Hb G16D, K120E = Hb
K120E = Hb K95E > Hb G16D > Hb A > Hb S >>> Hb
C112D, and dissociation of 22 (112
CysAsp) into monomers was much faster than that of Hb A tetramers. In addition, oxygen affinity of Hb C112D was slightly higher than that of Hb A with slightly less cooperativity than Hb A,
which is comparable to results showing lack of cooperativity (n = 1)
and higher oxygen affinity for recombinant Hb R40D (P50 of 1.2 v 5.1 for Hb A).22 This -chain variant
also has a negatively-charged -chain substitution at an 12
interaction site of tetrameric hemoglobin and dissociates into monomers
more readily than A chains; however, complete
dissociation to monomers did not occur under similar hemoglobin
concentrations used for our experiments.22 These results
reinforce the notion that oxygen affinity of tetrameric hemoglobin is
affected mainly by 12 interaction sites.1,2 It is
also interesting to note that recombinant chains containing Gly
instead of Cys at 112 on the 11 interface appear to stabilize 12 interactions and affect the allosteric equilibrium of
hemoglobin.23 Even though assembly of this variant with chains was not studied, the change to Gly at 112 should affect
11 assembly and the 12 interface differently compared with
the Cys to Asp change we engineered at 112. The small differences in
oxygen-binding properties of 22 (112
CysAsp) compared with those of Hb A may be caused by
propagation of changes induced at the 11 site to the 12
interface by this substitution. Further studies are required to
evaluate effects of this change at the 11 site on the 12
interface. These studies should facilitate further understanding of the
allosteric transition of hemoglobin. Furthermore, our results indicate
that relative affinity of for chains is dependent on direct
11 interaction sites, even though surface charge of the chains
affects interactions at the initial stage of assembly.
Analysis of 22 and 4
subunit interfaces by x-ray diffraction showed a high degree of
similarity between the quaternary structures of CO 4 and
CO Hb (22).20 Unlike the
22 tetramer, the 4
tetramer has high oxygen affinity, does not bind oxygen cooperatively,
and is influenced much less by allosteric effectors of native
hemoglobin oxygen affinity.18,20 In addition, the and
subunits of hemoglobin assemble to form tetramer through a stable
dimer intermediate, whereas 4 assembles from
monomeric chains with relatively little dimer
formation.17 Recent x-ray analysis of 4
hemoglobin at 1.8 Å resolution indicated that 112 Cys (G14) is
located at the -chain interface, and the side chains of 112 Cys
at 1 and 2 in the 4 tetramer are very close to the
molecular dyad at the 12 interface.20 These two
residues exist on the surface of the chains and may be involved in
weak interactions with other residues. Our present results also show the absence of disulfide dimer formation for 112 Asp (G14), which normally occurs in negatively-charged chains like the
A chain.9 These results clearly indicate
that 112 Cys residues in the 1 and 2 globin chains are close
together and that disulfide -chain dimer formation is governed by
these two cysteine residues, and not by Cys 93.9 In
addition, 112 Asp chains do not form 4 tetramers and
these chains in 22 (112
CysAsp) tetramers exchange readily with other chains,
probably because of unstable interactions between 112 Asp (G14) and
107 Val (G14) at the 11 interaction sites. This finding
suggests that 112 Cys (G14) is a critical amino acid in formation of
stable 4 tetramers, as well as dimers.
Studies aimed at production of more efficient hemoglobin variants are
now critical for development of gene therapy approaches to sickle cell
disease and thalassemia. There are limitations on expression levels of
Hb A or Hb F with viral vectors, and it is critical to design the most
efficient hemoglobin variants.24 Design and testing of more
efficient Hb A or Hb F variants for gene therapy and the growing
knowledge regarding hematopoietic stem cell biology will facilitate
future efforts to enhance expression levels and gene transfer
efficiency of vectors containing - or  -chain variants. In
addition to changing oxygen affinity of the hemoglobin variant for
potential use in gene therapy,24 we can now apply results
from studies of subunit assembly to increase Hb A or Hb F levels by
engineering more efficient - or -chain variants, which will be
more stable and promote assembly. Modification of surface charge of
hemoglobin variants is now expected to facilitate increases in total
hemoglobin variant levels in red blood cells, as the
assembly rate of and non- chains to form or dimers depends on electrostatic attraction. More negatively-charged or chains in addition to stabilization of 11 or 11 interaction sites would be expected to promote higher affinity of the variant -like chain for rather than A or s
chains. Information gained from these studies can be applied to produce
better hemoglobin variants for gene therapy in the future, as well as
to facilitate our understanding of the mechanism of assembly of a
number of other multisubunit proteins.
| |
FOOTNOTES |
Submitted August 12, 1997;
accepted October 1, 1997.
Supported by Grants No. P60 HL38632 and DK 16691 from the National
Institutes of Health, Bethesda, MD; the March of Dimes Birth Defects
Foundation (FY95); American Heart Association; the Nemours Foundation;
and UNICO National Inc.
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.
| |
ACKNOWLEDGMENT |
We thank Dr Eric Rappaport and members of the Nucleic Acid/Protein Core
at The Children's Hospital of Philadelphia for automated DNA sequence
analysis. We are grateful to Dr H.E. Witkowska for mass spectral
analysis of the -chain variants performed at the Children's
Hospital Mass Spectrometry Facility in Oakland, CA (Dr C. Shackleton,
Director), which is supported in part by National Institutes of Health
Grant No. HL20985 and a Shared Instrumentation Grant No. RR06505.
| |
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Abstract
Introduction
Abstract