The a1b1 contact of human hemoglobin plays a key role in stabilizing
the bound dioxygen
Further evidence from the iron valency hybrids
Jun pei Yasuda
1
, Takayuki Ichikawa
1
, Mie Tsuruga
1
, Ariki Matsuoka
2
, Yoshiaki Sugawara
3
and Keiji
Shikama
1,4
1
Biological Institute, Graduate School of Science, Tohoku University, Sendai, Japan;
2
Fukushima Medical University, Fukushima,
Japan;
3
Hiroshima Prefectural Women's University, Hiroshima, Japan;
4
PHP Laboratory for Molecular Biology, Sendai, Japan
When the a and b chains were separated from human
oxyhemoglobin (HbO
2
), each individual chain was o xidized
easily to the ferric form, their rates being a lmost the same
with a very strong acid-catalysis. In the HbO

(b
3+
)
2
, and demonstrated that the
autoxidation rate of either the a or b chains (when O
2
-
ligated) is independent of the valency state of the corre-
sponding counterpart chains. From these results, we have
concluded that the formation of the a1b1ora2b2 contact
suppresses remarkably the au toxidation rate o f the b chain
and thus plays a key role in stabilizing t he HbO
2
tetramer. Its
mechanistic details were also given in terms of a nucleophilic
displacement of O
2
±
from the FeO
2
center, and the emphasis
was placed on the proton-catalyzed process p erformed by
the distal histidine residue.
Keywords: Hb oxidation; chain nonequivalence; valency
hybrids; a1b1 contact; a cid-catalysis.
The reversible and stable binding of molecular oxygen to the
heme iron(II) is the b asis of hemoglobin function. However,
the oxygenated form of hemoglobin, as well as of myoglo-
bin, is known to be oxidized easily to the ferric met-form,

remarkably d elayed oxidation rate in the HbO
2
tetramer,
and this is the origin of such chain heterogeneity found in
the hemoglobin autoxidation at acidic pH [8].
To further characterize the nature of the a1b1ora2b2
interface in stabilizing the heme-bound dioxygen, we have
constructed iron valency hybrid hemoglobins, and studied
their autoxidation behavior at several different pH values as
compared with the native or reconstructed HbO
2
.Such
examinations seem to be of primary importance, not only
for a full understanding of the molecular mechanism of
hemoglobin autoxidation, but also for planning new
molecular designs for synthetic oxygen carriers that are
highly resistant against t he heme oxidation under physio-
logical conditions. Finally, we will revisit the hemoglobin
function as seen from the two different types of the ab
contact, and try to reconcile the cooperative oxygen binding
with the stabilization of the bound dioxygen. With respect
to this, we will also give possible implications for the
unstable hemoglobin mutants leading to the formation of
Heinz bodies in red blood cells, resulting in hemolytic
anemia.
MATERIALS AND METHODS
Chemicals
Sodium p-hydroxymercuribenzoate (p-MB) was from Sig-
ma. Mes, Mops, Hepes, Tris and Caps for buffer systems,
2-mercaptoethanol, and all other chemicals were of reagent

value was obtained on the basis of t he pyridine hemo-
chromogen method [10].
Isolation of mercuribenzoated a and b chains
All separations were carried out with fresh HbO
2
solutions
at low temperature (0±4 °C) by a two-column method. The
procedure was essentially the same as described by Geraci
et al . [11] and by Turci & McDonald [12], with our previous
speci®cations [8]. Each time, p-MB (100 mg) was dissolved
in 2 mL of 0.1
M
NaOH and neutralized with 1
M
CH
3
COOH. This was react ed with 10 mL of HbO
2
solution
(4±7 m
M
as heme) in 50 m
M
phosphate buffer, pH 6.0, and
in the presence of 0.1
M
NaCl. After passing through a
Sephadex G -25 column ( 2.5 ´ 40 cm), the mercurated
HbO
2

regenerat ed a or b chains were eluted out complete ly as the
oxy-form by changing the buffer, and kept stably in liquid
nitrogen until use. The concentration of each separated
chain was determined, after conversion into cyanomet-
form, using the following absorption coef®cients at 540 nm:
10.5 m
M
)1
ácm
)1
for the a chain and 11.2 m
M
)1
ácm
)1
for the
b chain. These values were obtained on the basis of the
pyridine hemochromogen method [10].
Titration of SH groups
According to the method of Boyer [13], free sulfhydryl
groups of the regenerated a or b chains were tit rated
spectrophotometrically at 250 nm with p-hydroxy-
mercuribenzoate in 0.1
M
Mops buffer, pH 7.0. The result-
ing contents were 1.0 (1.05  0.08) for the a chain and 2.0
(2.01  0.08) for the b chain, respectively, as might be
expected from the number of cysteines located at positions
a104(G11), b93 (F9) and b112(G14) for HbA.
Preparation of valency hybrid hemoglobins

Hepes ( pH 7.9) to completely elute out the major
peak of the reconstructed HbO
2
. Under this condition, a
small quantity of unassociated aO
2
chains remained on the
top of the column .
Valency hybrids (
a
3+
)
2
(
b
O
2
)
2
and (
a
O
2
)
2
(
b
3+
)
2

360 lLofbO
2
solution (% 750 l
M
). The resultant mixture
was then applied to a CM-cellulose column (2.5 ´ 3cm)
equilibrated with 1 0 m
M
phosphate buffer, pH 6.8. After a
small quantity of unassociated bO
2
chains passed through
the column, the buffer was changed to 50 m
M
Hepes
(pH 7.9) to elute out the major peak of the hybrid tetramer
(a
3+
)
2
(bO
2
)
2
. Under this condition, unassociated a
3+
chains had r emained on the top of the column. E ssentially
the same p rocedure can be used for the preparation of
another hybrid (aO
2

containing 300 l
M
heme. For spectrophotometry, the
reaction mixture was quickly transferred to a quartz cell
held at 35  0.1 °C, and changes in the absorption
spectrum from 450 to 700 nm were recorded on the same
chart at measured intervals of time. For separated a and
b chains, the rate measurement w as usually carried out with
10 l
M
protein (as heme) and in the presence of 20% (v/v)
glycerol. As the ®nal state of each run, the hemoglobin was
completely converted to t he ferric met-form by the addition
Ó FEBS 2002 The a1b1 contact in HbO
2
autoxidation (Eur. J. Biochem. 269) 203
of potassium ferricyanide. The buffers used were Mes,
maleate, Mops, and Caps. The pH of the reaction mixture
was carefully checked, before and after the run, with a
Hitachi±Horiba pH meter (Model F-22).
Spectrophotometric measurements
Absorption spectra w ere recorded in a Hitachi two-wave-
length doub le-beam spectrophotometer (model 557, U-3210
or U-3300) or in a B eckman spectrophotometer (model
DU-650), each being equipped with a thermostatically
controlled (within  0.1 °C) cell holder.
Curve ®ttings
Biphasic autoxidation curves were analyzed by an iterative
least-squares method on a computer (NEC PC-9821 V12)
with graphic display, according to our previous speci®ca-

ferric met-form, with a s et of isosbestic points. Consequent-
ly, the process was followed by a plot of experimental data
as ±ln([HbO
2
]
t
/[HbO
2
]
0
)vs.timet, where the ratio of HbO
2
concentration a fter time t to that at time t  0canbe
obtained by the absorbance changes at 576 nm for the
a-peak of human HbO
2
.
Figure 1 shows such examples of the ®rst-order plot for
the autoxidation reaction of human HbO
2
at two different
pH values. At pH 6.2, HbA showed a biphasic curve that
can be described completely by the ®rst-order kinetics
containing two rate constants as follows:
HbO
2

t
HbO
2

these computations, the initial value for each of the rate
constants was taken f rom the corresponding slo pe of a
biphasic curve (as delineated in Fig. 1 by two dotted lines),
and was re®ned by the step sizes of 0.01±0.001 h
)1
to ®nd
out the best values of k
f
and k
s
, according to our previous
speci®cations [5]. The value of P was a lso allowed to vary a
large range (from 0.40 to 0.60) in all cases. In this way, the
following param eters were established a t pH 6.2;
k
f
 0.82  0.03 ´ 10
)1
h
)1
, k
s
 0.13  0.01 ´ 10
)1
h
)1
,
and P  0.52  0.04 i n 0.1
M
Mes buffer at 35 °C. At

as
heme [5]. When the HbO
2
sample is diluted
2
, the tetrameric
species is known to dissociate into ab dimers along the a1b2
or a2b1 interface, so that the dimers formed are of the a1b1
or a2b2 type [14,15]. From these results, we can unequiv-
ocally conclude that the remarkable stability of the b chain
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
-
ln { [

HbO
2
]
t
/ [HbO
2
]
0

EDTA. E ach
curve (±±) was obtained by a least-squares ®tting to the experimental
points (s), based on Eqn (2). At pH 6.2, HbA showed a b iphasic
autoxidation curve containing two rate constants, k
f
and k
s
, respec-
tively. At pH 9.2, however, the reaction was monophasic. The buer
used was Mes for pH 6.2 and Caps for pH 9.2.
204 J. p. Yasuda et al. (Eur. J. Biochem. 269) Ó FEBS 2002
against the acidic autoxidation must have been produced by
the formation of the a1b1ora2b2 c ontact. To see more
quantitatively the effect of the a1b1ora2b2 contact on the
autoxidation reaction, our next step was to construct the
iron valency hybrid tetramers containing either the a or
b chains in the ferric state, and to examine for their stability
properties as compared with the native H bO
2
and its
separated chains.
Preparation of the valency hybrid hemoglobins
and their autoxidation behavior
By mixing equivalent amounts of the separated a and b
chains whose sulfhydryl groups were completely recovered,
we have prepared the reconstructed HbO
2
and its valency
hybrid tetramers. Figure 2 shows such an example for the
chromatographic separation of the hybrid tetramer

2
)
2
in 0.1
M
Mes
buffer pH 6.2, and in the presence of 1 m
M
EDTA at 35 °C.
In this tetramer, even if freshly prepared, the a-peak (at
577 nm) was always lower than the b-peak (at 541 nm) with
an absorbance ratio of a/b  0.90, this being in contrast to
a value of 1.06 for the native or reconstructed HbO
2
.The
Table 1. Comparison of the two rate constants involved in the autoxidation reaction of human HbO
2
at various pH values and 35 °C.
pH
k
obs
(h
)1
)
k
f
/k
s
Concentration
(l

0.99 ´ 10
)2
1.0 300
9.6 0.25 ´ 10
)1
0.25 ´ 10
)1
1.0 50
0
2
4
6
8
10
12
0102030
Fraction Number (4 ml / tube)
0
2
4
6
8
10
12
A
280
( )
CM-cellulose
Valency hybrid
22

phosphate buer, pH 6.8. A small band of
unassociated bO
2
chains passed through the column with the same
buer. To elute out the major peak o f the hybrid tetramer, the b uer
was changed to 50 m
M
Hepes (pH 7.9) at the point indicated by the
®rst arrow. The unassociated a
3+
chains coul d be remov ed by the
addition of 1
M
NaCl as indicated by the second arrow. The protein
and the heme p rotein leve ls were mon itored by th e abso rbances at
280 nm (s) and 415 nm (d), respectively.
0
0.1
0.2
0.3
0.4
0.5
Absorbance
450 500 550 600 650 700
Wavelength (nm)
22
pH 6.2
Valency hybrid
Start
Finish

seemed to be produced by a spectral overlapping of ferric
a
3+
chains. Furthermore, the reaction spectra evolved to the
®nal state of a run, which was identi®ed as the usual acidic
(or aquo) metHb.
If the contribution o f ferric a
3+
chains could be
subtracted from the oxidation spectra o f the (a
3+
)
2
(bO
2
)
2
tetramer on a computer, we may have the spectral changes
that can b e ascribed to the autoxidation of the b chains
alone. Such computations have disclosed that the reaction
started from the fully oxygenated b chains with an absor-
bance ratio of a/b  1.05, and that the oxidation proceeded
to the usual acidic met-form with a set of isosbestic points at
526 and 592 nm, as depicted in Fig. 4. This process was
therefore followed by absorbance changes at 578 nm for the
a-peak o f the b chain, an d could b e described completely by
a single ®rst-order rate constant of k
obs
 0.19 ´ 10
)1

2
chains in 0.1
M
maleate buffer, pH 6.2, and
inthepresenceof1m
M
EDTA plus 20% (v/v) glycerol at
35 °C. The oxidation began with an absorbance ratio of a/
b  1.04, and proceeded very rapidly with a ®rst-order rate
constant of k
obs
 0.10 h
)1
. T his rate is several times
higher than the c orresponding k
s
value for the b chains
either in the hybrid tetramer (a
3+
)
2
(bO
2
)
2
or reconstructed
HbO
2
. Moreover, the ®nal state of the run was not for the
usual acidic met-form but for an admixture with hemi-

450 500 550 600 650 700
Wavelength (nm)
2
in
pH 6.2
Start
Finish
2 2
(β
O
2
) (α
3+
)
2
(β
O
2
)
Fig. 4. Spectral changes w ith time for the autoxidation of the bch ains of
valencyhybridHb(a
3+
)
2
(bO
2
)
2
in 0.1
M

maleate buer at pH 6.2 and 35 °C. Sc ans wer e made at
70-min intervals in the presence of 1 m
M
EDTA and 20% (v/v) glyc-
erol. The ®nal spectrum was not for the acidic met-form , b ut an ad-
mixture with hemichrome having a peak at 53 0 nm an d a s houlde r
near 560 nm. Heme concen tration: 25 l
M
.
206 J. p. Yasuda et al. (Eur. J. Biochem. 269) Ó FEBS 2002
complex [18]. As shown in Fig. 5, the molar fraction of t he
hemichrome (complex B) was estimated to be 75% at
pH 6.2. Furthermore, Borgstahl et al.[19]reportedthe
1.8 A
Ê
structure of carbonmonoxy-b
4
(COb
4
)tetramerof
human hemoglobin, and compared subunit±subunit con-
tacts between three t ypes of interfaces (a1b1, a1b2and
a1a2) of HbO
2
and the corresponding COb
4
interfaces. As a
result, they found that, in contrast to the stable b1b4
interface, the b1b2 interface of the COb
4

of 20% (v/v) glycerol was most effective in preventing
occasional precipitations.
Kinetic analysis of the autoxidation reaction
of valency hybrid hemoglobins
Figure 6 represents ®rst-order plots to show wide differences
in the autoxidation rate of the b chain , when it exists as the
separa ted (bO
2
)
4
, valency hybrid (a
3+
)
2
(bO
2
)
2
, and reco n-
structed HbO
2
tetramers in 0.1
M
Mes buffer at pH 6 .2 and
35 °C. In this way, all the spectrophotometric data were
subjected to ® rst-order kinetics using Eqn (2). The resulting
rate constants f or the native, separated, reconstructed, and
valency hybrid hemoglobins are summarized in Tables 2±4
at three different values of pH. At pH 6.2, for example, the
HbO

this inherent rate was dramatically suppressed i n the
reconstructed as w ell as the native HbO
2
. More importantly,
such a retarded k
s
value could be kept almost completely in
the valency hybrid (a
3+
)
2
(bO
2
)
2
tetramer, too. All these
features were essentially the same at other pH values as seen
in Tables 3 a nd 4. Certainly, the biphasic nature o f the
autoxidation rate became much less steep at pH 7.5, and
even disappeared at pH 9.0. Nevertheless, the r ate of
oxidation of the separated b chain was markedly reduced
by up to 15-fold at pH 7.5 and up to 23-fold at pH 9.0 in the
tetrameric hemoglobin, either it is native or reconstructed or
valency hybrid species.
The similar situation was also found in the a chain, but its
effect on the HbO
2
tetramer was much less crucial than the
b chain. At pH 9.0, the rate of oxidation of the separated
a chain was reduced by up to 16-fold in the HbO

tetramer, because k
f
³ k
s
at any
physiological pH.
DISCUSSION
In hemoglobin research, the central problem is understand-
ing the cooperative binding of m olecular oxygen to the a
2
b
2
tetramer. For human HbA, the a and b chains contain 141
and 146 amino-acid residues, respectively, and a r epresen-
tative set of the s uccessive oxygen-binding constants is g iven
in terms of mmáHg
)1
as follows: K
1
 0.0188, K
2
 0.0566,
0
0.2
0.4
0.6
0.8
1.0
1.2
-

(β
O
2
)
2
(α
O
2
)
2
(β
O
2
)
Reconstructed
k
f
k
s
Fig. 6. First-order plots to show dierent autoxidation rates of the
b chain between three dierent hemoglobin derivatives in 0.1
M
maleate
buer at pH 6.2 an d 35 °C. Each curve (±±) was obtained by a least-
squares ®tting to the experimental points, based on Eqn (2). The
oxidation of separated b chains could be described by a single rate
constant of k
obs
 0.10 h
)1

 4.28 in 0.1
M
Bis/Tris buffer con-
taining 0.1
M
KCl at p H 7.4 an d 25 °C [20]. In this reaction,
major d ifferences have been de®ned between deoxyhemo-
globin and o xyhemoglobin by c omparing their X -ray crystal
structures. These include a movement of the iron atom into
the heme plane with a simultaneous change in the orienta-
tion of the proximal (F8) histidine, a rotation of the a1b1
dimer relative to the other a2b2 dimer about an axis P by
12±15 degrees, and a translation of one dimer relative to th e
other along the P axis by % 1A
Ê
. The latter two changes are
accompanied with sequential breaking of the so-called salt
bridges by C -terminal residues [21±25]. Therefore, the two
types of the ab contact a re de®ned in the molecule. One is
the a1b1(ora2b2) contact involving B, G, and H helices
and the GH corner, and other is the a1b2(ora2b1) contact
involving m ainly helices C and G and the FG corner [19,24].
When HbA goes from the deoxy to the oxy con®guration,
the a1b2anda2b1 contacts undergo the principal changes
associated with the cooperative oxygen binding, so that
these are named the sliding contacts. At the a1b1anda2b2
interfaces, on the other hand, negligible changes are found
insofar as the crystal structure was examined. Consequently,
these are called simply the packing contacts, and their role in
hemoglobin function was not clear for a very long period of

Table 2. Comparison of the autoxidation rate constants between the whole, separated, reconstructed, and hybrid hemoglobins in 0.1
M
buer at pH 6 .2
and 35 °C.
Hb Sample
k
obs
(h
)1
)
Concentration
(l
M
as heme)
k
f
k
s
Whole HbO
2
0.82 ( 0.03) ´ 10
)1
0.13 ( 0.01) ´ 10
)1
300
Separated chains (aO
2
)
1
0.89 ( 0.03) ´ 10

50
(aO
2
)
2
(b
3+
)
2
0.77 ( 0.03) ´ 10
)1
±50
Table 4. Comparison of t he autoxidation rate constants between the whole, separated, reconstructed, and hybrid hemoglobins in 0.1
M
buer at pH 9.0
and 35 °C.
Hb Sample
k
obs
(h
)1
)
Concentration
(l
M
as heme)
k
f
k
s

)2
50
Hybrid (a
3+
)
2
(bO
2
)
2
± 0.62 ´ 10
)2
50
(aO
2
)
2
(b
3+
)
2
0.61 ´ 10
)2
±50
Table 3. Comparison of t he autoxidation rate constants between the whole, separated, reconstructed, and hybrid hemoglobins in 0.1
M
buer at pH 7.5
and 35 °C.
Hb Sample
k

± 0.75 ´ 10
)1
10
Reconstructed (aO
2
)
2
(bO
2
)
2
0.23 ´ 10
)1
0.50 ´ 10
)2
50
Hybrid (a
3+
)
2
(bO
2
)
2
± 0.63 ´ 10
)2
50
(aO
2
)

H
2
OH

3
k
H
MbIIIOH
2
HO
2
4
MbIIO
2
OH
À
3
k
OH
MbIIIOH
À
O
À
2
5
In these e quations, k
0
is the rate c onstant for the basal
displacement by H
2

than 10
6
mol
)1
, as formulated by Eqn (4). In this proton
catalysis, the distal histidine, which forms a hydrogen bond
to the bound dioxyge n [29], appears t o facilitate the effec tive
movement of a c atalytic proton from the solvent to the
bound, polarized dioxygen via its imidazole ring and by a
proton-relay mechanism [6,7].
In our previous paper [8], such a nucleophilic displace-
ment mechanism w as successfully applied to d etailed
pH-dependence studies of the k
f
and k
s
values, both for
the HbO
2
tetramer and its separated chains, at more than
70 different values o f pH from 5 to 11 in 0.1
M
buffer at
35 °C. When the a and b chains were separated from the
HbO
2
tetramer, e ach individual chain was oxidized much
more rapidly than in the p arent HbO
2
, exhibiting a proton-

b63, and described in terms of a Ôtwo -state modelÕ without
any proton catalysis. Such a unique stability of the HbO
2
tetramer was found to remain even in the low concentra-
tions of hemoglobin corresponding to appreciable dissoci-
ation into a1b1ora2b2dimers[5].
We have recently proposed that the distal histidine
residue can play a dual role in the nucleophilic displace-
ment of O
2
±
from MbO
2
or HbO
2
[30]. One is in a
proton-relay mechanism via its imidazole ring, as random
and undirected access of a proton to the bound dioxygen
cannot yield such an enzyme-like, catalytic e ffect on the
autoxidation rate of MbO
2
or HbO
2
. Insofar as we have
examined for more than a dozen of myoglobins, such a
proton-catalyzed process could never be observed in t he
autoxidation of myoglobins lacking the usual distal
histidine r esidue, no matter what the protein i s, the
naturally occurring or the distal His mutant as well [30].
The other role is in the maximum protection of the FeO

acquired this stability by blocking out the proton catalysis
performed by the distal histidine residue (Eqn 4).
Similarly, Shaanan [31] reported the stereochemistry of
the iron-dioxygen bond in human HbO
2
by single-crystal
X-ray analysis. In the a chain, the distance between N
e
of
His (E7) and the terminal oxygen atom (O-2) is found to be
2.7 A
Ê
, and the geometry favors a similar hydrogen bond as
in oxymyoglobin [29]. In the b chain, however, N
e
of His
(E7) is located further away from both O-2 and O-1 (3.4 and
3.2 A
Ê
, respectively), i ndicating that the hydrogen bond, even
if formed, must be very weak. Recently, Lukin et al.[32]
claimed t hat a hydrogen bond is formed between O
2
and t he
distal histidine in both a and b chains of human HbO
2
,as
revealed by heteronuclear NMR spectra of the chain-
selectively labeled samples. In 0.1
M

respect to the heme-bound dioxygen. Such m arked differ-
ences between the two distal heme pockets may also b e
responsible for our kinetic results of the a and b chains in the
HbO
2
tetramer. I n this context, NMR spectra of the
separa ted bO
2
chain must be most informative if available,
because the autoxidation reaction of the b chain contains a
very strong proton-catalysis in the isolated form but not in
the HbO
2
tetramer.
Ó FEBS 2002 The a1b1 contact in HbO
2
autoxidation (Eur. J. Biochem. 269) 209
As for the dimer, as well as the tetramer, effect on the
oxidation rate, our explanations are as follows. At b asic pH,
both isolated a and b chains are q uite susceptible to
autoxidation. Each heme pocket seems to be suf®ciently
open to allow e asier attack of the solvent hydroxyl ion on
the FeO
2
center. As a result, there occurs a very rapid
formation of hydroxide-metHb, the rate being dependent
directly on the concentrations of OH
±
ion. In a1b1 dimers,
conformational constraints would greatly suppress accessi-

2
tetramer is diluted into ab
dimers. Indeed, this is the most characteristic feature of
hemoglobin autoxidation.
In relevance to a clinical aspect, it should be noted that a
quite large number of unstable hemoglobins have been
reported so far [24,33]. M any of the mutants which occur at
the a1b2 interface have altered oxygen af®nity, but bulk of
evidence suggests that the a1b1 i nterface is much more
important in maintaining normal hemoglobin stability than
is the a1b2 interface. As a matter of fact, hemolytic anemia
is known to result from substitutions affecting the a1b1
interface or the heme pocket. If such mutations occur, the
heme iron will be more easily oxidized, and a sequence of
events leads to the globin precipitation or Heinz body
formation in r ed blood cells that causes hemolytic anemia.
Typical examples of such variants are: E [b26(B8)Glu ®
Lys], Volga [ b27(B9)Ala ® Asp], Genova [b28(B10)-
Leu ® Pro], St Louis [b28(B10)L eu ® Gln], Tacoma
[b30(B12)Arg ® Ser], Abraham Lincoln [b32( B14)Leu ®
Pro], Castilla [b32 (B14 )Leu ® Arg], Philly [b35(C1)Tyr ®
Phe], Rush [b101(G3)Glu ® Gln], Peterborough
[b111(G13)Val ® Phe], Madrid [b115(G17)Ala ® Pro],
Khartoum [b124(H2)Pro ® Arg],J.Guantanamo[b128-
(H6)Ala ® Asp], Wien [b130(H8)Tyr ® Asp], Leslie
[b131(H9)Gln ® deleted], Torino [a43(CD1)Phe ® Val],
L.Ferrara [a47(CD5)Asp ® Gly], Setif [a94(G1)Asp ®
Tyr], St. Lukes [a95(G2)Pro ® Arg]. Surprisingly, almost
all of these pathological mutations are f ound on the b chain,
especially in the a1b1 contact regions. It follows from

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