From the
The role of Lys-95(
The primary cause of sickle cell anemia is a single DNA base
change encoding for the amino acid substitution Glu
Some of the interactions
between HbS tetramers were identified by Bookchin and Nagel
(8) ,
who measured the sparing effect of other natural hemoglobin mutants
with substitutions at various positions on the insolubility of
deoxy-HbS. More recently, many of the contact sites and interactions
have been identified by x-ray crystallography and electron microscopy
of sickle
hemoglobin
(9, 10, 11, 12, 13, 14) .
However, the strength of most of these interactions is not yet
appreciated in quantitative terms. Using site-directed replacement, we
are attempting to answer this question
(15, 16, 17) for selected amino acids in sickle hemoglobin.
Other
studies on sickle hemoglobin performed before the structure of the
aggregate was known focused on the chemical modification of certain
amino acid side chains of HbS in order to improve its solubility
(18-21). These studies, as well as the results of Bookchin and
Nagel described above, provided information on the participation of
certain amino acids in the polymerization process. However, each
approach had its limitations with respect to the sites that could be
studied. For example, the chemical modification studies could address
only those sites to which a given chemical reagent had some affinity,
and this function could not generally be either predicted or directed.
The experiments with the natural mutants could focus only on those
sites for which natural mutants were available. With the advent of
site-directed mutagenesis, especially in the yeast
system
(22, 23) , the range of sites that can be
evaluated is unlimited, because it is now possible to substitute any
amino acid at any position on either the
In the present study, the
particular site that we substitute is Lys-95(
To prepare the
E6V/K95I(
After purification on a CM-Cellulose 52
column as described earlier (15-17), the double mutant eluted as
a single peak when rechromatographed analytically on the SynchroPak
CM-300 HPLC column. Its purity was verified by isoelectric focusing as
described previously (15-17), and its migration was consistent
with the charge differences at the two mutation sites (i.e. the removal of one negative charge in sickle Hb (Glu
If a therapeutic intervention for sickle cell disease is
directed at the sickle Hb molecule itself, it would be advantageous to
know the strengths of the various side chain interactions between the
deoxyhemoglobin S tetramers in order to choose the most effective
target site. The solution of the structure of the sickle hemoglobin
aggregate
(3) revealed many contact sites in the aggregate but
did not provide an indication of their relative strengths. Our
objective in this communication was to measure such a value for
Lys-95(
Hofmann et al.(34) have reported the considerable formation of a sulfur adduct
with heme, sulfheme, produced in a yeast expression system beginning
about 16 h after the start of the galactose induction period and
increasing for the subsequent 3 days. In our studies, we do not exceed
a 20-h induction period with
galactose
(15, 16, 17, 26) , and we find
the production of only one major hemoglobin with the correct spectral
properties, indicating the absence of sulfheme. The minor
heme-containing proteins produced were less than 10% of the total
hemoglobin and were separated from the main hemoglobin component on the
HPLC-column
(16) . The K95I(
In considering the earlier approach of using
mixtures of different mutant hemoglobins with substitutions at various
positions to assess possible points of interaction in the sickle Hb
aggregate
(8) , the argument could be raised that, because the
hemoglobins had the substitutions on different tetramers, it was not an
accurate representation of the aggregation process in red cells.
However, our conclusions do not support such an argument because our
results on the recombinant double mutant tetramer of HbS with the
substitutions on the same
In general, in
choosing the amino acid to be introduced by site-directed mutagenesis,
it has been our experience that it is useful to first evaluate the
properties of the natural hemoglobin mutants at the same
position
(12, 26) . In this instance the effects of the
natural mutants Hb Detroit (Lys-95(
In addition to the very
large effect of the Ile-95(
The oxygen affinity method used in the
present study measures the overall extent of polymerization but does
not provide information on the details of the process
(32) .
Using this same procedure, it has been reported that HbF participates
very little in the process of polymerization (i.e. an 80%
increase in the gelling concentration)
(32) . Our data indicate
an increase in the gelling concentration for K95I(
The
The value found for Ile was used to normalize
the values for the other amino acids. The mutant peptide was isolated
as described in the legend to Fig. 4. There is some destruction of Ser
during acid hydrolysis.
The underlined Ile shows the target site of
mutagenesis. The numbers correspond to the amount of PTH-derivative in
picomoles found at each cycle of Edman degradation. This peptide was
isolated as described in the legend to Fig. 4.
The Hb concentration was 0.7 mM in 50 mM bis-Tris, pH 7.5.
We are grateful to Adelaide Acquaviva for expert help
with the typescript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
), which is on the exterior of the
hemoglobin (HbS) tetramer, in the aggregation process has been
addressed because there is a lack of agreement on its importance. The
early studies on the aggregation of HbS in the presence of other mutant
hemoglobins are consistent with the subsequent electron microscopic
studies in demonstrating the participation of Lys-95(
) in
gelation; the results of the crystal structure do not agree with these
conclusions. Therefore, with the objective of clarifying its role we
have carried out site-directed substitution of Lys-95(
) to an
isoleucine residue. The mutation was introduced by polymerase chain
reaction recombination methodology, and the absence of other mutations
in the
-globin gene was established by sequencing the gene in its
entirety. The recombinant mutant hemoglobin was expressed in yeast and
characterized by peptide mapping and sequencing, which demonstrated
that the only different tryptic peptide had the Ile substitution at
position 95(
). The recombinant hemoglobin had the correct amino
acid composition and molecular weight by mass spectrometric analysis.
It was also pure as judged by isoelectric focusing. It was fully
functional because it had an average Hill coefficient of 3.1 and
responded normally to the allosteric regulators, chloride,
2,3-diphosphoglycerate, and inositol hexaphosphate. Of particular
interest was the finding that this hemoglobin mutant aggregated at a
concentration of about 40 g/dl, nearly twice that at which HbS itself
aggregated (24 g/dl). Therefore, Lys-95(
) has a very important
role in the aggregation process and is a good candidate site for the
design of a therapeutic agent for sickle cell anemia.
Val at
position 6 of the
-chain of hemoglobin
(1, 2) . This
replacement leads to a strong interaction between Val-6(
) and a
hydrophobic pocket in the region of Phe-85(
)/Leu-88(
) of an
adjacent tetramer in the deoxy conformation
(3) . Subsequent
interactions, some hydrophobic and others hydrophilic in nature
involving both lateral and axial intertetrameric contacts, lead to the
aggregation of deoxy-Hb tetramers and eventual distortion and sickling
of red cells in the venous circulation. The thermodynamics of the
polymerization of sickle hemoglobin have been elucidated by Eaton and
co-workers
(4, 5, 6, 7) , as well as by
other investigators cited in Ref. 4.
- or the
-chain of
the tetramer. The yeast expression system that we employ for HbS
(15) has the human
- and
-globin genes on the same
plasmid and produces a recombinant sickle hemoglobin that is identical
by about a dozen biochemical and physiological criteria with the
natural sickle hemoglobin purified from the red cells of sickle cell
anemia patients
(15, 16, 17) . Most importantly,
the gelling concentration of this recombinant sickle hemoglobin is the
same as that of the hemoglobin S purified from human sickle red cells.
These findings indicate that this system is well suited to produce
sickle Hb double mutants to explore those areas of the hemoglobin S
tetramer whose roles in the gelation process are not yet defined and to
measure quantitatively the strength of such interactions at certain
intertetrameric contact sites in the deoxy-HbS aggregate. In the
initial study with this objective
(17) , a sickle hemoglobin
double mutant was expressed in which Leu-88(
), part of the initial
hydrophobic contact region with Val-6(
), was substituted by an Ala
residue. Surprisingly, this conservative replacement led to a 30%
decrease in gelation concentration of deoxy-HbS, a value that indicated
the strength of this initial interaction. Adachi et al.(24) have also addressed this initial hydrophobic interaction by
making substitutions at both the donor Val-6(
) and the part of the
acceptor region involving Leu-88(
).
), which is on the
exterior of the hemoglobin tetramer, yet near the Phe-85/Leu-88
hydrophobic pocket. The role of this amino acid side chain in the
aggregation process has been implicated in some studies but not in
others so that its role at present is uncertain. When the natural Hb
mutants with Lys-95(
) replaced either by asparagine (Hb Detroit)
or by glutamic acid (HbN Baltimore), neutral polar and charged polar
side chains, respectively, were mixed with HbS, the gelling
concentration of the latter increased from about 29 g/dl for HbS to
31.6 and 37.0 g/dl in the presence of the two mutant hemoglobins,
respectively
(8) . However, the crystal structure of sickle
hemoglobin
(9, 10) did not show the participation of
Lys-95(
) in the aggregate, although it did not specifically
exclude this site. The findings of Edelstein and co-workers
(13, 14) and of Josephs and co-workers
(11) , who used
electron microscopic studies of the sickle HbS fiber, show involvement
of Lys-95(
) as a contact site. Dickerson and Geis
(25) have
pointed out this distinct difference between the crystal and fiber
structures of HbS. Hence, our objective in this communication is to
answer the question of its involvement in the aggregation process and
to establish its role quantitatively.
Reagents
The restriction endonucleases, T4
polynucleotide kinase, alkaline phosphatase, and DNA ligase were from
Boehringer Mannheim. The DNA sequencing kit and the T7 DNA Polymerase
(Sequenase version 2.0) were obtained from U. S. Biochemical Corp. The
GeneAmp PCR(
)
reagent kit and AmpliTaq DNA
polymerase were purchased from Perkin-Elmer. The oligonucleotides were
synthesized by Operon Technologies (Alameda, CA). Universal pUC/M13
Forward and M13 Reverse were obtained from Promega and U. S.
Biochemical Corp., respectively. The Prep-A-Gene DNA purification kit
was from Bio-Rad. All the other reagents were of analytical purity.
Yeast Expression System
The double mutant Hb was
expressed in yeast (Saccharomyces cerevisiae GSY112
cir°) as described previously
(15, 16, 17) after making the desired mutation in the - and
-globin gene-containing plasmid by site-directed mutagenesis.
pGS389 is a yeast/Escherichia coli shuttle plasmid containing
the human
- and
-globin cDNAs and the 2-µm yeast
element
(22) . It can replicate in E. coli, where the
selection is facilitated by the use of its ampicillin resistance gene.
The transcription of the globin genes is induced by growing the cells
in a medium containing galactose. pGS189 is a derivative of Bluescript
II SK(+) plasmid containing the human
- and
-globin
cDNAs and replicates only in E.
coli(15, 16, 17) .
) double mutant, an XhoI fragment of pGS189
containing the sickle
-globin cDNA was first inserted in the
XhoI site of Bluescript SK(+)
(12) . This plasmid
was used as a template in the PCR reactions. Two overlapping PCR
products were synthesized by a PTC-100-60 instrument (MJ Research
Inc., Watertown, MA) as described earlier
(12) using separately
the 5`-ATCCACGTGCAGGATGTCACAGTGCAG and pUC/M13 Forward and the
5`-CTGCACTGTGACATCCTGCACGTGGAT and M13 Reverse primers. The underlined
bases were those used to bring about the desired mutation. These
products were recombined in a separate PCR reaction by using the
pUC/M13 Forward and M13 Reverse primers. The final amplified DNA was
digested by XhoI, and the 1280-base pair fragment was purified
from agarose gel by a Prep-A-Gene DNA purification kit. This fragment
was subcloned to the 4130-base pair XhoI-fragment of pGS189.
The correct insertional direction was confirmed by restriction mapping,
and the sequence was verified by the conventional dideoxy method.
Finally, the
- and
-globin gene cassette was isolated as a
NotI fragment after digesting the newly synthesized pGS189
derivative with NotI and BglI and inserted into
pGS389 previously digested with NotI. The yeast cells were
transformed by this plasmid using a lithium acetate method referred to
in Refs. 15-17, and the transformants were selected on a complete
minimal medium without uracil (15).
Growth of Yeast and Purification of Recombinant
Hb
The yeast was grown in YP medium
(22) for 4 days using
ethanol as the carbon source. The promoter controlling the
transcription of the globin genes was induced for 20 h by the addition
of galactose; collection and breakage of the cells has been described
earlier
(15, 16, 17, 26) . The
purification of the double mutant Hb was accomplished by chromatography
on CM-Cellulose 52 and Synchropak CM-300 HPLC columns
(16) . The
gradient for CM-300 was optimized to fit the elution characteristics of
the newly synthesized double mutant.
Mass Spectrometry Analysis
The hemoglobin K95I
sample was subjected to mass spectrometric analysis on a
matrix-assisted laser desorption time-of-flight mass spectrometer
constructed at Rockefeller University and described
elsewhere
(27, 28) . The mass spectra were acquired by
adding the individual spectra of 200 laser shots. Hemoglobin samples
were prepared for laser desorption mass analysis as follows: the laser
desorption matrix material (4-hydroxy--cyano-cinnamic acid) was
dissolved in 50 mM formic acid/water/isopropanol (1:6:4)
(v/v/v). A 10 mM bis-Tris acetate solution, pH 7.5, containing
the hemoglobin sample was then added to the matrix solution to give a
final hemoglobin concentration of approximately 2 µM. A
small aliquot (0.5 µl) of this mixture was applied to the metal
probe tip and dried at room temperature with forced air. The sample was
then inserted into the mass spectrometer and analyzed. Bovine
cytochrome c was used for internal calibration.
Analytical Methods
Isoelectric focusing, amino
acid analysis, and other procedures were performed as described earlier
(15-17, 26). A Vydac C-4 reversed phase column was used for the
separation of the globin chains. The hemoglobin samples were injected
onto the column previously equilibrated with 38% acetonitrile in 0.1%
trifluoroacetic acid. The isolated -globin chains were digested
with trypsin, and the resulting peptides were separated on a Vydac C-18
column with a linear gradient of acetonitrile from 12 to
80%
(15, 16) . The amino acid sequence of the mutant
tryptic peptide was determined on an Applied Bioscience instrument. For
determination of the concentration of DPG and IHP, inorganic and total
phosphate were determined by the spectrophotometric method of Lowry
et al. (29). Hydrolysis was accomplished by using 11
N sulfuric acid, 4.7% perchloric acid.
Functional Studies
The oxygen dissociation curves
were determined at 37 °C on a modified Hem O Scan instrument
(Aminco) as described
previously
(15, 16, 17, 26) . Before the
measurements, the Hb samples were dialyzed and converted to the oxy
form
(30, 31) . When evaluating the effects of allosteric
modulators, the samples were in 50 mM bis-Tris buffer, pH 7.4.
Prior to determination of the concentration of hemoglobin at the onset
of gelation (C*) as described by Benesch et al. (32),
the samples in 100 mM potassium phosphate buffer, pH 6.8, were
concentrated using CentriPrep, Centricon, and MicroCon ultrafiltration
devices (Amicon; molecular weight cut-off of 10,000). The final protein
concentrations were verified by amino acid analysis on a Beckman 6300
analyzer.
Expression and Purification of the Double
Mutant
The hemoglobin double mutant, Hb E6V/K95I(), was
expressed in yeast from the plasmid pGS389 containing the human
-
and
-globin cDNAs with the desired substitutions introduced by the
two mutagenic oligonucleotides described under ``Materials and
Methods.'' The final product synthesized by PCR was rather large
(about 1,300 base pairs), and the sequence of the entire
-gene was
checked by the dideoxy method. No mutations other than the two desired
(i.e. encoding the amino acid substitutions Glu-6(
)
Val and Lys-95(
)
Ile) were detected. The expression
level of the recombinant hemoglobin, which was 5-10 mg/liter, was
not markedly influenced by increasing the galactose concentration from
2 to 3% during the induction period. In addition to the K95I(
)
recombinant Hb, only small amounts of minor hemoglobins were detected
during the 20-h induction.
Val)
compensated for by the removal of one positive charge in the double
mutant (Lys
Ile)) (Fig. 1). Thus, the double mutant is
near HbA upon isoelectric focusing. Both mutations are on the exterior
of the protein, so that the full effect of these
pK
changes is reflected in its
electrophoretic behavior.
Figure 1:
Isoelectric focusing of the purified
K95I() Hb. A gel from Isolab (pH 6-8) was electrophoresed at
10 W for 45 min. Lane a, natural HbS; lane b, natural
HbA; lane c, K95I(
) Hb.
Mass Spectrometry
The molecular mass of the
purified E6V/K95I() double Hb mutant was determined by
matrix-assisted laser desorption mass
spectrometry
(27, 28, 33) . In this procedure
hemoglobin is dissociated into its constituent
- and
-subunits and their individual molecular masses are measured by a
time-of-flight method. A molecular mass of 15,823.9 mass units was
obtained for the
-subunit (Fig. 2). This value agrees well
with the calculated molecular mass of 15,823.3 mass units. The
difference of 14.4 Da from the mass of sickle Hb (15,838.3 mass units)
is within experimental error of the expected difference of 15 Da
between a Lys (146.2 mass units) and an Ile (131.2 mass units) residue.
The molecular mass obtained for the
-chain (15,128.4 mass units)
is consistent with the calculated value (15,126.4 mass units) within
the error of the measurement.
Figure 2:
Mass spectrometric analysis of the double
mutant sickle hemoglobin with Lys-95() substituted by Ile.
Matrix-assisted laser desorption mass spectrum of HbS K95I(
) is
shown. Peaks corresponding to the protonated
- and
-chains
are designated
and
(K95I), respectively.
The small peak at m/z 15,476 likely arises from a
doubly protonated heterodimer of the
- and
-chains. The
origin of the weak peak at m/z 16,456 has not been
elucidated.
HPLC Analysis of Globin Chains and Amino Acid
Analysis
For further characterization, the - and
-chains were first separated by HPLC. The double mutant
-chain eluted after the
-chain (Fig. 3) with an elution
time of 56.2 min compared with an elution time of 36.6 min for a normal
-chain and 38.7 min for a sickle
-chain. Thus, the double
mutant
-chain showed a dramatic difference in its elution behavior
compared to both sickle Hb and HbA. Indeed, the order of elution of
- and
-globin chains was the reverse of that usually observed
(Fig. 3). Subtle changes in the amino acid composition of
polypeptides can lead to dramatic changes in their behavior on reverse
phase HPLC columns, which is not always strictly according to the
hydrophobicity of the amino acids. The influence of the Ile residue was
probably accentuated due to its location on the outer surface of the
-subunit.
Figure 3:
Separation of the - and
-globin
chains of K95I(
) Hb by HPLC. The purified double mutant was
injected in a Vydac C-4 column previously equilibrated with 38%
acetonitrile in 0.1% trifluoroacetic acid.
The - and
-chains were identified by the
amounts of Ser, Glu, Gly, Ala, and Val, which are the amino acids that
show significant differences between the two chains. The amino acid
composition of the isolated
-chain showed the theoretical value of
1 mol of Ile/mol of Hb chain. The values for the other amino acids were
in reasonable accord with the known composition (). The
choice of Ile as the substitution also facilitated the identification
of the mutation site, because natural HbS does not contain Ile. No Ile
was found in the
-chain, which also had the expected composition.
Peptide Mapping and Sequencing of the Mutant
Peptide
The double mutant -chain isolated as described in
Fig. 3
was subjected to tryptic digestion, and the peptides were
analyzed on a Vydac C-18 column. The mutant peptide eluted in a unique
position (Fig. 4, bottom) compared with the peptide map
shown of this region of HbA (Fig. 4, top). Amino acid
analysis of this peptide was consistent with its assignment as a
peptide fragment comprising amino acids 83-104 and showed 1 mol
of Ile/mol of the peptide (). The correct mutation site
was verified by sequencing the peptide isolated in Fig. 4with a
high yield of Ile at the position 95(
) (I).
Figure 4:
Tryptic peptide maps of the -globin
chains. The
-globin chains of HbA and K95I(
) Hb were isolated
as shown in Fig. 3, carboxymethylated, and digested with trypsin. The
resulting peptides were chromatographed on a Vydac C-18 column using a
linear acetonitrile gradient of
12-80%.
Oxygen Binding
In order to ascertain that the
recombinant Hb double mutant had retained the functional properties of
hemoglobin, its oxygen binding curve was measured. As shown in
Fig. 5
, at high Hb concentration (4.1 mM), a typical
sigmoidal curve was found; the P value at this
concentration of the E6V/K95I Hb was 33.5 mm Hg, and it was fully
cooperative with a Hill coefficient of 3.1 (Fig. 5,
inset).
Figure 5:
Oxygen binding curve of K95I() Hb.
The purified double mutant (4.1 mM in tetramer) in 50
mM bis-Tris buffer, pH 7.4, was converted from the CO to the
oxy form (26, 27), and the oxygen binding was measured at 37 °C
using a modified Hem O Scan instrument. The inset shows the
cooperativity with an n value of 3.1 calculated from the slope
of the line.
Response to Allosteric Effectors
The response of
the E6V/K95I() double mutant to chloride, DPG, and IHP is shown in
. At a concentration of 0.7 mM in tetramer, the
recombinant Hb had a basal P
value of 10 mm Hg.
As with sickle Hb
(20) , NaCl at concentrations of 20 mM
or lower did not significantly change the oxygen binding of the
recombinant Hb E6V/K95I(
). With increasing NaCl concentration, the
P
value hyperbolically increased, reaching a
maximum of 25 mm Hg at a concentration of about 1 M NaCl.
Under these conditions, the Hill coefficient decreased from 3.2 to 2.0,
especially with NaCl concentrations above 0.5 M. DPG, a much
more potent effector of Hb function, showed a marked increase in the
P
value of the double mutant at low
concentration. Above 1.3 mM DPG, no further increase in the
P
value was observed. IHP, at a concentration of
0.7 mM IHP, led to a maximum P
value of
56 mm Hg.
Gelation of the Hemoglobin Double Mutant
To
measure the gelation concentration of the recombinant Hb
E6V/K95I(), the P
value of different
concentrations of the double mutant was determined in 100 mM
potassium phosphate buffer, pH 6.8, as described by Benesch et al. (32). In this procedure physiological concentrations of
hemoglobin, such as those that occur in red cells, are used. The
P
values ranged from 23.5 to 58.5 mm Hg between
the Hb concentrations of 5-48 g/dl (Fig. 6). There was a
rapid decrease in the oxygen affinity once the concentration of the
E6V/K95I(
) double mutant reached a value of 39.5 g/dl. This point
was defined by Benesch et al.(32) as the concentration
of hemoglobin at the onset of gelation (C*). When compared
with a value of 23.7 g/dl for sickle Hb (Fig. 6, dashed
line)
(15, 16) , it is obvious that the substitution
of Lys-95(
) by Ile has had a major effect on the process of
aggregation. The cooperativity was not markedly affected by the
concentration of Hb used, because the Hill coefficients varied between
2.5 and 3.3 throughout the concentration range studied. Our data
indicate that Lys-95(
) is an important site in the aggregation
process in agreement with the conclusions of Bookchin and
Nagel
(8) , of Josephs and co-workers
(11) , and of
Edelstein and co-workers
(13, 14) .
Figure 6:
Gelation of K95I() Hb. The purified
double Hb mutant was dialyzed against 100 mM potassium
phosphate buffer, pH 6.8, and concentrated to 48.6 g/dl. This solution
was diluted to obtain the concentrations shown in the figure. The
measurements were done at 37 °C using the method of Benesch et
al. (32). The dashed line is the aggregation profile of
both natural and recombinant HbS taken from Ref.
11.
), a prominent site on the exterior of the molecule. From
the results in this communication, the contribution of this site to the
aggregation process appears to be even greater than that of the initial
hydrophobic interaction involving Val-6(
) and the
Phe-85/Leu-88(
) region, because the Hb concentration for the
E6V/K95I double mutant at the onset of gelation is increased much more
(39.5 g/dl) than that for the E6V/L88A(
) double mutant (31.2 g/dl)
compared with 23.7 g/dl for HbS (17). Considering that the E6V/K95I
double mutant retains the original Leu-88(
) (I), this
effect on gelation is especially dramatic.
) Hb double mutant described in
this manuscript displayed the normal spectral profile of ferrous Hb and
had the correct functional properties of hemoglobin as shown by a Hill
coefficient of 3.1 and a normal response to the allosteric regulators
DPG, chloride, and IHP.
-chain agree with those of Bookchin and
Nagel
(8) on the importance of Lys-95(
).
)
Asn) and HbN Baltimore
(Lys-95(
)
Glu) on the aggregation of HbS
(8) were
evaluated. The latter hemoglobin impeded the gelation of HbS
substantially more than did the former, consistent with the idea that
an ionic bond between Lys-95(
) and some negative side chain on an
adjacent tetramer was involved as a contact site. Thus, the acidic
substitution in HbN Baltimore would be expected to generate the charge
repulsion at this site and reduce gelation. The Asn substitution in Hb
Detroit would not be expected to have such an effect. It was
anticipated that if this rationale was correct, the Ile side chain in
the recombinant Hb might seek an interaction elsewhere rather than with
the putative negatively charged acceptor and, hence, lower the gelation
concentration. On the other hand, if the Ile substitution were without
effect on gelation, such a movement would not have been necessary
because no such interaction existed at this site. The findings of a
large inhibitory effect for the Ile-95(
) substitution are
consistent with the possibility that an ionic contact involving
Lys-95(
) was prevented by the replacement. Eaton and Hofrichter
(4) point out that Lys-95(
) is near the dimer interface,
and hence its replacement by a nonpolar side chain could have a large
effect on the intratetrameric contacts.
) substitution on the overall process
of aggregation as measured by the oxygen affinity decrease with
increasing hemoglobin S concentration, there are some other interesting
differences with the profile of the aggregation of HbS alone. Hence, at
Hb concentrations lower than that at the onset of gelation, the slope
of the line for the initial increase in P
is less
than that for HbS itself (Fig. 6, dashed line). However,
once aggregation commences (above 39.5 g/dl), the rates of the oxygen
affinity decrease (P
increase) are parallel for
both HbS and the K95I(
) double mutant. This behavior is likely due
to the decreased ability of the double mutant to aggregate during the
initial phase, thus leading to an increased concentration requirement
for the onset of gelation.
) of about 70%
(i.e. of the same order of magnitude as the inhibitory effect
of HbF). It will be interesting to compare the relative strength of the
interaction at Lys-95(
) with those of other sites in the aggregate
on which there is currently no quantitative information. The results of
the present study clearly indicate the importance of Lys-95(
) in
the process of sickle Hb aggregation and reveal this site to be a
likely candidate as a target for the development of a therapeutic agent
for sickle cell anemia directed at the hemoglobin S molecule itself.
Table:
Amino
acid analysis of the - and
-chains of K95I(
) Hb
- and
-chains were isolated as described in the legend to
Fig. 2. The values have been normalized to the values found for Pro,
which was set at the theoretical value of 7.0. The values for Thr and
Ser are uncorrected for their 5-7% destruction during acid
hydrolysis. Under the conditions of acid hydrolysis employed, Cys, Met,
and Trp are destroyed. For the amino acid values that are underlined,
there is a significant difference between their amounts in the
-
and
-chains.
Table:
Amino acid composition of mutant tryptic
peptide of K95I()
Table:
N-terminal sequence of the mutant
peptide of K95I()
Table:
Functional properties of the purified
K95I() Hb
), another name for the
double HbS mutant E6V/K95I(
).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.