(Received for publication, November 27, 1995; and in revised form, January 2, 1996)
From the
2-Nor-2-formylpyridoxal (NFPLP) has been synthesized and coupled
to bovine Hb according to the procedure developed by Benesch and
Benesch(1) . The reaction of bovine Hb with NFPLP leads to a
cross-linkage between the subunits, which greatly stabilizes the
low affinity T state of the molecule and simultaneously abolishes the
tendency of the tetramer to dissociate into
dimers. The
functional properties, examined from both the equilibrium and kinetic
points of view, indicate that the chemical modification affects the
O
affinity, abolishes cooperativity, and induces a slight
decrease of the Bohr effect. From modeling studies we are confronted
with two different structural alternatives; the cross-link of
chains may be formed between lysine 82 of
and the N
terminus of methionine 2 of
or between the two lysine
82 residues of both
chains. Digestion of modified
globin chains and isolation of the cross-linked peptide have
showed that NFPLP cross-links Met-
2 and Lys-
82. This allowed
discussion in some detail of the molecular basis of the Bohr effect of
the modified bovine hemoglobin. On the whole, NFPLP-modified bovine Hb
could be considered as a first step toward the synthesis of a potential
blood substitute.
Over the last few decades, studies on structure-function
relationships in hemoglobin have been greatly helped by naturally
occurring mutants, artificially induced point mutations and specific
chemical modifications at the level of some key amino acid residues. As
far as the latter are concerned, the reaction of human hemoglobin with
NFPLP ()(1) led to a covalent modification of great
interest, as it yielded a hemoglobin with lowered oxygen affinity and
almost unchanged cooperativity, thereby offering a unique opportunity
to study the effect of organic phosphates without the complexity
arising from dissociation of the effector. Moreover, hemoglobin
modified by NFPLP offers additional biotechnological advantages since
it shows some promise as an oxygen-carrying resuscitation fluid. In
fact, this chemical modification overcomes two main disadvantages,
which critically restrict the usefulness of free human Hb as an
oxygen-carrying substitute for red blood cells. The first of these is
inherent to the oxygen dissociation curve of free Hb whose P
(partial pressure of oxygen at which 50%
saturation is achieved) indicates a far greater affinity for O
than normal human blood and therefore a significantly lower
oxygen tension at the level of tissues; the second is related to the
split of free Hb tetramers into
dimers, which are rapidly
eliminated through the renal glomeruli and characterized by the absence
of cooperativity and a very high oxygen
affinity(2, 3) . As previously reported by Benesch and
Benesch(1) , NFPLP, by reacting with valine
1 and lysine
82 of human Hb and forming a covalent bridge located in the
2,3-DPG crevice in between the two
chains, eliminates at a single
blow both the problems reported above. In this perspective, since
bovine Hb does not appear to cause rapid antibody formation when
injected into non-bovine species(4) , many authors have
investigated the possibility of using bovine Hb as a substitute for
human blood(5, 25) . Along the same lines we used
NFPLP to modify bovine Hb, with the aim of obtaining further
information on the structure-function relationships of this hemoglobin,
known to display peculiar functional characteristics. In fact, bovine
Hb belongs to a group of mammalian hemoglobins that, in the presence of
physiological concentrations of chloride, have an oxygen affinity lower
than that of human HbA being also insensitive to the presence of
2,3-DPG. The failure of bovine Hb to respond to 2,3-DPG was explained
by the peculiar N termini of
subunits, i.e. the deletion
of the residue at position
1 and the presence of a hydrophobic
residue, usually Met, at position
2. According to Perutz and
Imai(6) , these structural differences are at the basis of the
decreased influence of organic phosphates. On this basis, bovine Hb
appears to be modulated in vivo essentially by chloride ions,
which may thus be considered its physiologically relevant effector. We
were interested in investigating the possibility of improving the
functional characteristics that render this protein a potential blood
substitute through a specific chemical modification at the level of the
anion binding site between the two
chains. To this end we were
helped by a recent elegant work on the structural features of bovine
hemoglobin(7) , which allowed us to go further into the
structure-function relationships of the NFPLP-modified bovine
hemoglobin.
Unless otherwise stated, all chemicals employed were obtained in the highest grade commercially available from Aldrich. Bovine blood was provided from the local slaughterhouse.
Amino acid analysis was carried out on the purified peptide hydrolyzed in the vapor phase with 6 M HCl at 110 °C for 24 h. The amino acid composition of the hydrolyzed peptide was determined using a Pharmacia 4151 Alpha Plus instrument.
Figure 1:
a, 300-MHz H NMR spectrum of NFPLP solution in D
O at 20
°C; resonances: C8-H: 8.29; C9-H: 6.49; C8-H: 6.35;
C7-H
: 5.14 and 5.12; HOD: 4.80 ppm; the dialdehyde groups
react with water to give an equilibrium concentration of the hydrate, a gem-diol. b, 75.5-MHz
C NMR spectrum for
the same sample.
Figure 2:
Semipreparative reverse-phase HPLC
profiles of the and
subunits of bovine Hb before (straight line) and after (dashed line) reaction with
NFPLP. Brownlee C8 column (200
10 mm) was used following the
procedure described by Shelton et al.; the chromatogram was
developed with a linear gradient between 25% and 89% acetonitrile over
80 min and then 89% for 5 min;
= 278
nm.
Figure 3:
121.5-MHz P NMR spectra in
D
O for free NFPLP (a) and cross-linked Hb-NFPLP (b). The
P resonances of each phosphate groups
give complementary information on this linkage.The NFPLP spectrum was
the result of averaging 800 transients at a repetition rate of 0.4
s
; Hb-NFPLP spectrum was the result of 140,000
transients.
Figure 4:
Dependence on pH of the oxygen affinity of
bovine Hb in the presence of chloride anions () and in absence
of chlorides (
); same results for cross-linked bovine Hb in the
presence (&cjs0800;) and in the absence of chloride anions (
).
Temperature 20 °C.
Moreover, the Bohr effect is largely maintained in
Hb-NFPLP, although somewhat reduced with respect to the unmodified
protein due to the decrease of oxygen affinity observed at pH values
higher than 7.2. However, cooperativity is almost completely abolished,
as indicated by the Hill coefficient (n), which
is very close to unity (1.1 ± 0.1) (Fig. 5).
Figure 5:
Hill plots of oxygen binding to
cross-linked bovine hemoglobin at 20 °C in 0.1 M Hepes,
chloride-free, pH = 7.5 () and pH = 7.8 (
);
plots at the same pH values for bovine Hb:
, pH = 7.5;
, pH = 7.8. For pH = 7.5, the asymptote intercepts
the reference-state axis (log pO
= 0) at intrinsic
constant K
= 0.118
torr
.
In both cases the kinetics of the reaction is strongly biphasic (fast phase: 75%) as the relative proportions of the two processes are essentially pH-independent; the rate of deoxygenation measured for the two kinetic phases appears strongly dependent on pH, although this dependence is more evident in normal bovine Hb. The value of the velocity constant of both phases is independent of hemoglobin and dithionite concentrations, and therefore the reaction may be described in terms of two independent first order processes.
For both the fast and slow phase, the plot of the value
of the rate constant versus pH is a symmetrical curve
corresponding to a simple titration. However, the cross-linked Hb
presented, with or without chloride, a deoxygenation rate constant k slightly slower than the corresponding
constant of bovine Hb; in contrast, k
was
always higher. The titration of Hb-NFPLP showed that both the
pK
values shifted to more acidic pH in relation to
bovine Hb (Table 1).
In principle, and
similarly to human Hb, the cross-link could be established between
lysine 82 of and the N-terminal methionine 2 of
or between the two lysine 82 residues of both
chains(1) .
As reported in Fig. 6, a cross-link
between methionine 2 and lysine 82 can be established by moving the
amino group of methionine and the
amino group
of
lysine 82 closer to the central cavity by
approximately 3.5 and 1.5 Å, respectively. Only a slight movement
was necessary for
histidine 143 and the side chain of
lysine 82 to form electrostatic interactions with the
phenolate anion and the phosphate group, respectively.
Figure 6:
Sketch of the model of the central cavity
between the chains of the cross-linked bovine hemoglobin. The
cross-link was established between Met-2 and Lys-82. The phosphate
group of NFPLP interacts with the
amino group of
Lys-
82 and the phenolate anion interacts with the
imidazole ring of His-
143.
Cross-linking
lysine 82 of and lysine 82 of
did
not necessitate a significant movement of the
lysines; however,
both the amino groups of methionine 2 and histidine 143 were moved by
approximately 3-4 Å in order to form electrostatic
interactions with the phosphate of NFPLP.
Figure 7:
HPLC purification of the peptides
derived from the tryptic digestion of NFPLP-cross-linked globins
of bovine Hb. The arrow indicates the peptide corresponding to
the sum of the fragments 1-7 and 77-95 of the primary
structure of the bovine
globin. The inset shows the
chromatogram of the tryptic digestion of the normal bovine
globin.
According to our modeling studies, it was not possible to
discriminate between two different structural alternatives
(Met-2-Lys-82 or Lys-82-Lys-82) for the cross-link; this
problem has been solved by digestion of globin chains and
following isolation of the cross-linked peptide. Sequence analysis of
this peptide has shown that the reaction with NFPLP occurs between the
Met-2 of one
chain and Lys-82 of the other
subunit of the
bovine Hb molecule. This suggested that the tetrameric structure was no
more able to dissociate into
dimers. This is clearly shown
by electrophoresis on cellulose acetate of the globins, which shows, in
addition to the band relative to the
chains, the presence of a
band relative to the cross-linked
chains.
Regarding the
functional properties of bovine Hb-NFPLP, these indicate that the
chemical modification affects its affinity for oxygen and abolishes
homotropic interactions and the effect of chloride, inducing a decrease
(35%) of the Bohr effect. The decrease in oxygen affinity observed
in the case of bovine Hb-NFPLP in the absence of chloride, when
compared to normal bovine Hb, is of peculiar significance since it
indicates that the negative charges of NFPLP may substitute chloride
ions in regulating the oxygen affinity acting in much the some way as
2,3-DPG does in human HbA. Given the situation, the oxygen affinity of
Hb-NFPLP is no longer affected by Cl
and it is very
similar to and even lower than (depending on the pH) the corresponding
affinity of the native bovine Hb in the presence of these ions.
The
loss of cooperativity and the low oxygen affinity suggest that the
reaction with NFPLP, performed under anaerobic conditions,
preferentially stabilize the T conformational state of the new
molecule. This is also indicated by the oxygen binding curve which
corresponds, at all the pH values examined, to the lower asymptote of
the untreated bovine Hb (Fig. 4). In this respect, it may be
worthwhile recalling that the same chemical modification performed on
human HbA results in a considerable drop in the oxygen affinity but has
only a small effect on the degree of homotropic interactions, being the
Hill coefficient n
1.9, versus 2.7
for the native molecule(18) . In the case of human HbA,
therefore, NFPLP is not able to freeze the Hb molecule in the low
affinity conformational state. The difference seen between human HbA
and bovine Hb in their reaction with NFPLP must be ascribed to specific
structural characteristics belonging to the bovine molecule. This may
well be related to the shift of the A-helix toward the dyad axis of the
tetrameric structure observed in the crystal structure of bovine Hb,
which has been recently interpreted as the main characteristic
responsible for the low intrinsic oxygen affinity of this
Hb(7) . In this perspective, spectroscopic observations on the
nitric oxide derivative may be relevant, as these have clearly
demonstrated that the Hb of ruminants populates its T conformational
state much more than other mammalian hemoglobins, even in the liganded
derivatives(6, 19) . As a matter of fact, a similar
shift, although less pronounced, has been observed in human HbA upon
binding of organic phosphate(20) .
Moreover, previous
studies on the structure of human HbA modified by NFPLP have shown that
the cross-linking reaction induces the amino group of Val-1 to
move about 3 Å into the central cavity. As a result the
N-terminal peptide and the beginning of the A-helix are pulled closer
to the EF corner and the E-helix. All these findings, relative to human
HbA, support the hypothesis that the relative position of A-helix does
play a significant role in the modulation of the oxygen affinity.
In
addition, it would be important to outline that on the basis of
stereochemical characteristics of the cavity between the two
chains Arnone et al.(21) would have expected the
reaction with NFPLP to ``freeze'' the tetramer in the T
state, thereby producing a low affinity non-cooperative tetramer. As
already mentioned, contrary to this expectation HbA-NFPLP modified
appeared to be still cooperative (n
1.9).
The forecast of Arnone et al. is completely fulfilled by
bovine Hb, whose NFPLP derivative appears first to be frozen in the T
conformational state and second to have fully abolished the cooperative
effect.
This differences in behavior may find an explanation on the basis of the following considerations.
(a) In bovine Hb the A-helix is closer to the dyad axis with respect to HbA even in the absence of any organic phosphate effector (7) . In some way this intrinsic difference of the molecule may facilitate the molecule to fully populate the T conformational state.
(b) Our
modeling studies show that the cross-link between Met-2 and
Lys-
82 can be established by moving the amino group of methionine
1 and the
2
-amino group of lysine closer to the central
cavity by approximately 3.5 and 1.5 Å, respectively. Upon NFPLP
reaction, therefore, the A-helix should be further shifted toward the
interior of the molecule thereby strongly stabilizing the T state.
Other interesting observations are related to the Bohr effect of
both the native and the NFPLP-reacted protein. The cross-linked Hb
displays identical Bohr effects both in the presence and in the absence
of Cl. Moreover, this Bohr effect is decreased by 35%
with respect to that of the untreated molecule. It is therefore very
likely that this 35% of the Bohr effect, which in the native protein
appears to be Cl
-linked, has to be ascribed to one
(or both) of the residues that are involved in the cross-linking of the
chains or to some other residues that, to come into operation,
need the switch from the T to the R state of the molecule. As far as
the enhancement of the alkaline Bohr effect by chloride ions is
concerned, point mutations and chemical modification experiments (22, 23) have indicated that, within the central
cavity, the replacement of either two pairs of cationic groups by
neutral ones, or of a single cationic group by an anionic one, is
sufficient to inhibit the Cl
effect no matter which
residues are altered.
On the basis of these findings and of
structural results, it has been suggested (7) the absence of
any specific chloride binding sites. Hence, it has been proposed that
the effect of Cl could be due to the widening of the
central cavity on transition from the R to the T structure. This would
allow Cl
to diffuse in and neutralize the excess of
positive charges, lining the central cavity, without being bound to any
one of them. Consequently, the enhancement of the Bohr effect by
Cl
is due to a rise in pK values (i.e. increased binding of protons) of the cationic groups within the
central cavity.
Our results are in agreement with this view, since
the cross-linkage abolishes the positive charge of Lys-82 and
introduces in the cavity between the
chains the three potential
negative charges of the NFPLP molecule. According to the above reported
considerations, the Cl
effect on the O
affinity and the chloride-induced Bohr effect are vanished.
In
addition, it is necessary to consider that the strong impairment of T
R conformation transition also goes in the same direction. Thus
the O
-linked widening of the central cavity and the
following diffusion of Cl
can no more occur in the
NFPLP-modified bovine Hb; therefore, the differential
``binding'' of these ions cannot show up.
However, it
should not be forgotten that a significant part of the Bohr effect (i.e. the chloride-independent part) appears to be displayed
by a molecule that is frozen in the low affinity T state. This may be
taken as an indication that, at least in NFPLP-modified bovine Hb, a
significant part of the pH dependence of the O affinity
finds its molecular basis at the level of the tertiary structure. The
details of this effect remain to be seen.
In any case, concerning a potential blood substitute, the functional properties of the NFPLP-modified bovine Hb are not completely satisfactory. The main disadvantage is the absence of cooperativity, which may not be fully compensated by the very low oxygen affinity of the protein. However, an additional step could be the synthesis of an intertetramer supermolecule in which several modified bovine Hb tetramers are linked together by means of bifunctional reagents such as dialdehydes. Since most of these modifications are known to result in a strong destabilization of the T structure in favor of the high affinity R state, we may have the chance of partially restoring heme-heme interactions through the formation of intermolecular cross-links and supermolecular structures.
Finally, since NFPLP-bovine Hb has lost all its cooperative interactions, according to allosteric models(24) , the kinetic parameters obtained may be thought to belong to the low affinity T state of the molecule.
The kinetics of
the dissociation of oxygen from NFPLP-Hb was followed by mixing the
oxygenated derivative of both the native and the cross-linked protein
with sodium dithionite using a stopped-flow apparatus. When the
reaction of dithionite with free oxygen is not rate-limiting, the time
course of the observed process obeys first order kinetics, and the
measured rate constant represents an overall dissociation of the ligand
from saturated Hb. The deoxygenation process is characterized by two
phases, one being faster with a relative amplitude corresponding to
nearly 75% of the total change; Table 1reports the k constants for oxygen measured by the
dithionite method. Moreover, the rate of dissociation is significantly
affected by pH (Fig. 8). The titration curve is symmetrical and
corresponds to simple process whose pK values are, for k
, 7.30 ± 0.20 and 6.91 ± 0.52,
and for k
, 7.40 ± 0.31 and 5.93 ±
0.45, respectively, for the native and the modified protein. From these
values it appears that in the cross-linked Hb the Bohr effect does not
receive any significant contribution from the dissociation velocity
constant; in fact, the pK of the transition is only in a
region of pH in which the Bohr effect is completely over. In contrast,
in the case of native protein it appears very clearly that the pH
dependence of the oxygen affinity may find part of its kinetic
expression in the rate constant of oxygen dissociation on the basis of
the pK values of both the Bohr effect and the transition of
the velocity constant. Hence, this difference in behavior seems to be
related to the inhibition of the ligand-linked allosteric transition
brought about by the chemical modification.
Figure 8:
Variation of the first-order rate constant k with pH for the deoxygenation of NFPLP-oxyHb by
Na
S
O
, in presence of chloride ions
0.1 M. The curve is a simple titration with
pK
= 6.91 ± 0.52; k
for acid form = (66.5 ± 8.7)
s
, and k
for basic form
= (32.9 ± 7.6). Temperature = 20 °C. NFPLP-Hb
concentration = 100 mM (in
heme).
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
All ASBMB Journals | Molecular and Cellular Proteomics |
Journal of Lipid Research | Biochemistry and Molecular Biology Education |