(Received for publication, July 17, 1995; and in revised form, October 25, 1995)
From the
Human deoxyhemoglobin cross-linked with trimesyl
tris(3,5-dibromosalicylate) produces the previously reported
cross-linked hemoglobin in which the amino groups of the two
chain 82 lysyl residues are joined by a trimesyl bridge. Further
specific modification of this protein directed to the
subunits
with bis(3,5-dibromosalicyl)fumarate gives a doubly cross-linked
material in which the
-amino groups of the two
chain 99
lysyl residues are now joined by a fumaryl bridge. The singly
cross-linked
chain species binds oxygen cooperatively with a high
oxygen affinity (P
= 4.8 torr at pH 7.4).
The addition of the second cross-linking reduces the oxygen affinity to
15.9 torr, which compares with 13.0 torr for the singly cross-linked
chain species. The doubly cross-linked hemoglobin retains
significant cooperativity with a Hill coefficient of 2.3 compared with
3.0 for unmodified hemoglobin. Because some of the groups responsible
for the Bohr effect are acylated, this doubly cross-linked hemoglobin
exhibits 25% of the normal Bohr effect and less than 20% of the normal
chloride effect. The use of two distinct cross-links within the same
tetramer provides a material for physical and structural analysis as
well as for further modifications for specific applications. The
results indicate that the cross-link introducing the lowest oxygen
affinity in the two singly cross-linked species appears to control the
overall affinity in this doubly cross-linked species.
Specifically cross-linked hemoglobins are useful for the
investigation of structure-function relationships, (Schumacher et
al., 1995) as well as in the development of potential substitutes
for red blood cell transfusion (Snyder et al., 1987; Keipert et al., 1989; Bucci et al., 1989; Vandegriff and
Winslow, 1991; Kluger et al., 1994). By retaining a reactive
site in a cross-linker after modification of hemoglobin, other
modifications can be readily prepared by addition of nucleophiles to
the modified hemoglobin (Kluger and Song, 1994). This makes it possible
to use hemoglobin as a carrier for drugs or other chemicals that may
have pharmacologic value (Seymour, 1992) or provide probes for physical
and structural analysis. However, hemoglobin containing a trimesyl
dibromosalicylate ester as the bisamide of the -amino groups of
both
82 lysine residues (
82-TDBS-82
; (
)schematic structure is shown in Fig. Z1), has a
relatively high affinity for oxygen.
Figure Z1: Structure 1
Due to its high affinity for oxygen, this is unlikely to deliver oxygen efficiently. Since combining oxygen delivery and bioconjugation is potentially of theoretical as well as medicinal significance, we considered the possibility of further modification to cause a reduced oxygen affinity by introducing a second cross-link.
Walder et al.(1994) developed an
efficient hemoglobin-based oxygen carrier that contains a fumaryl
bisamide of the -amino groups of both
99 lysine residues
(
99-F-99
; the Baxter Healthcare red cell
substitute-derived form of this is called DCLHB) (Walder et
al., 1994; Chatterjee et al. 1986; Snyder et
al., 1987). If the fumaryl cross-link at the
99 lysines is
added to
82-TDBS-82
(Kluger et al.,
1992b), the oxygen affinity might be altered to make it suitable as an
oxygen carrier (schematic structure shown in Fig. Z2).
Figure Z2: Structure 2
Whatever the case, the structure and properties of the doubly cross-linked material would provide an interesting basis for functional studies. We have been able to produce these chemical modifications of the same hemoglobin efficiently. The resulting material has the desirable features of both individual modifications. Olsen et al.(1991) have also reported in abstract form the preparation of multilinked hemoglobins.
TTDS was synthesized
and analyzed as previously reported (Kluger et al. 1992b).
Because TTDS is not readily soluble in water, 1,4-dioxane was used to
dissolve the reagent. (We have also added the reagent in water with
sonication.) The solution of TTDS was degassed under an aspirator and
infused with nitrogen to remove oxygen. An amount of this solution was
added to the deoxy-Hb to make the final concentrations of Hb 0.5 mM and TTDS 0.75 or 1.0 mM. The reaction was allowed to
proceed for up to 2 h under a stream of humidified N at 35
°C in the same rotating flask as used for the deoxygenation.
DBSF was either synthesized as described by Walder et al. (1979) or purchased. Although DBSF can be added as a solid to deoxy-Hb (Walder et al., 1994), we added it as a solution in 1,4-dioxane and 0.1 M pH 9.0 borate. (We have also added the reagent in water with sonication). Oxygen was removed in the same way for TTDS solutions. An amount of DBSF was added to the deoxy-Hb that had been reacted with TTDS to equal twice the moles of Hb present, i.e. to make the DBSF about 1.0 mM. This reaction was allowed to proceed for another 2 h under a stream of humidified nitrogen at 35 °C in the same rotating flask as used for the reaction with TTDS. The final reaction mixture was then cooled to 0 °C in an ice bath, and the modified Hb was converted to the CO form by passing a stream of humidified carbon monoxide over the solution for 2-3 min. The COHb mixture was separated from the excess reagents and low molecular weight reaction products by passing through a Sephadex G-25 column equilibrated with 0.005 M pH 8.0 MOPS and concentrated by pressure filtration. Portions of the stripped COHb were stored on ice, while others were flash-frozen with dry ice and stored at -80 °C until purified or studied further.
The reaction of deoxy-Hb with two equivalents of TTDS in
borate buffer at pH 9 (producing 82-TDBS-82
)
followed by reaction with two equivalents of DBSF yields the doubly
cross-linked hemoglobin tetramer containing an intact ester linkage (i.e. two cross-links connecting four lysine side chains with
a 3,5-dibromosalicylate ester on the trimesyl
-cross-link),
99-F-99
82-TDBS-82
. The structure is shown
schematically in Fig. Z3.
Figure Z3: Structure 3
Figure 1:
Globin chain
separation on C-4 reversed phase column after reaction of 0.5 mM deoxy-Hb with 1.0 mM TTDS in 0.05 M borate at pH
9.0 and 35 °C for 30 min. More than 95% of the chains were
chemically modified mostly to
82-TDBS-82
(peak eluting at 55
min).
Figure 2:
Globin
chain separation on C-4 reversed phase column after reaction of 0.5
mM deoxy-Hb in 0.05 M borate at pH 9.0 and 35 °C
with 1.0 mM TTDS for 1 h. followed by 1.0 mM DBBS for
another 1 h. All of the chains were chemically modified mostly to
82-TDBS-82
and its hydrolysis product
82-T-82
. In
this preparation about 85% of the
chains were chemically modified
to
99-F-99
, and 15% remained
unmodified.
Figure 3:
Globin chain separation on C-4 reversed
phase column of the same reaction mixture shown in Fig. 2after
heating to 60 °C for 3 h. The primary change is the conversion of
all but about 20% of the 82-TDBS-82
mainly to
82-T-82
.
Kluger and Song(1994) have reported that
82-TDBS-82
will react with a variety of
nucleophiles to form derivatives with the third carboxyl of the
trimesyl cross-linker. Fig. 4shows the chain separation results
after reacting a sample of stripped reaction mixture containing
99-F-99
82-TDBS-82
with 0.07 M lysyl-lysine
at 35 °C in 0.05 M borate, pH 9.0, for 2 h. Other
nucleophiles including glycine, lysine, and Tris react under the same
conditions to convert the
82-TDBS-82
to
covalently linked derivatives.
Figure 4:
Globin chain separation on C-4 reversed
phase column of a similar reaction mixture shown in Fig. 2after
treating it with 0.07 M lysyl-lysine for 2 h more at 35
°C. The globin peak eluting at about 40 min was the conjugate of
lysyl-lysine with 82-T-82
.
The peaks assigned to the
82-T-82
and
99-F-99
globin chains were isolated by
preparative C-4 reverse phase HPLC for further structural
characterization. The
82-T-82
chains were hydrolyzed to a set
of peptides with trypsin followed by Glu-C endoproteinase. The
99-F-99
chains were oxidized with performic acid and then
hydrolyzed with trypsin in the presence of 2 M urea. Fig. 5, A and B, show the C-18 reverse phase
HPLC peptide pattern of these globin chains. In Fig. 5A, all of the normal tryptic, Glu-C peptides of
the
chain are present except for those that occur from tryptic
cleavage adjacent to Lys
:
T-9 and
T-10a. A
tryptic peptide not found in native Hb elutes at about 94 min and
absorbs at 258 nm, indicative of the presence of trimesyl moiety. Amino
acid analysis indicates it has the aminoacyl components of
T-9,10a. These results are the same as those reported earlier for
82-T-82
obtained from the reaction of COHb
with trimesoyl tris(methylphosphate); Kluger et al., 1992a).
This peptide pattern and the amino acid composition of the new peptide
combined with the molecular mass measurement of the globin chain are
the basis for deducing the structure to be
82-T-82
.
Similarly, Fig. 5B shows all of the normal tryptic
peptides observed for oxidized
chains except for the absence of
T-11 and ox
T-12. In their place is a new peptide that elutes
near the end of the chromatogram that has the amino acid composition of
ox
T-11,12. These results are the same as those found for the
99-F-99
present in a preparation of DCLHB from Baxter
Healthcare Corp. and reported elsewhere (Jones, 1994).
Figure 5:
A, HPLC peptide pattern of a trypsin-Glu-C
hydrolysate of globin corresponding to the material eluting at about 45
min from the C-4 column separations (see Fig. 1Fig. 2Fig. 3Fig. 4). The peptides
were separated on a C-18 reversed-phase column using a
water-acetonitrile gradient in 0.1% trifluoroacetic acid. All of the
normal peptides were present except T-9 and
T-10a. A new
peptide was found eluting at about 94 min, which had the amino acid
composition of
T-9 and
T-10a plus the UV absorption of the
trimesic cross-linker. B, HPLC peptide pattern of a trypsin
hydrolysate of the oxidized globin corresponding to the material
eluting at about 75-77 min from the C-4 column separations (see Fig. 2and Fig. 3). The peptides were separated as
described for panel A. All of the normal peptides were present
except
T-11 and
T-12. A new peptide was found eluting at
about 83 min. which had the amino acid composition of
T-11 and
T-12.
A
trimesyl cross-link between the two Lys residues does
not appear to change P
appreciably from that of
unmodified Hb at this pH. Differences in the Bohr effects do result in
different outcomes at other pH levels. It is likely that there is
little difference in structure of this species compared with native Hb
in the oxy or deoxy forms. However, a fumaryl cross-link between the
two Lys
residues increases P
by
a factor of 2.5, indicating a marked decrease in oxygen affinity
through a higher energy R state, lower energy T state, or a combination
of both. Thus, the two types of cross-links in this double cross-linked
Hb are manifested very differently in their effects on oxygen affinity.
The Hill coefficients at pH 7.4 are somewhat reduced compared with that
of unmodified Hb for both
82-T-82
and
99-F-99
82-T-82
with n
values
of 2.4 and 2.3, respectively, while n
=
2.7 for
99-F-99
82-T-82
at pH 7.0. (
)Both
the Bohr effect and chloride effect are lower than the normal values
for unmodified Hb (in decreasing order:
82-T-82
to
99-F-99
to
99-F-99
82-T-82
).
The measurement and interpretation of the effects of one or
two cross-links on the properties of hemoglobin depends on being able
to produce these species efficiently. The reaction of TTDS with
hemoglobin is highly selective for the -amino groups of the
Lys
residues in the DPG binding site of the central
cavity of hemoglobin (Kluger et al., 1992b). The yield of
modified hemoglobins is over 95%, the products being mainly either
82-TDBS-82
or
82-T-82
, when the
preparation is carried out under nitrogen (pH 9.0 in borate, 35
°C), even with only a 50% molar excess of TTDS over Hb. The
relative amount of
82-T-82
compared with
82-TDBS-82
at the end of the reaction
depends upon the extent of hydrolysis of DBS from the third carboxyl
group of the trimesic cross-linker, which in turn depends upon the
temperature, pH, and duration of the reaction.
By heating the final
reaction mixture used to produce the doubly cross-linked species to 60
°C for 3 h in the presence of CO, most of the DBS will be
hydrolyzed from the trimesic cross-linker, and the main product is
99-F-99
82-T-82. This approach to the preparation of a
modified Hb with an
99-F-99
cross-link may be an efficient
alternative to the use of polyanions to promote formation of this
modification.
As Kluger and Song(1994)
observed for 82-TDBS-82
, we find that
99-F-99
82-TDBS-82
can act as an acylating reagent
toward nucleophiles, forming conjugates of hemoglobin. This presents
opportunities to investigate combining oxygen delivery with
bioconjugation for a transfused or perfused material. It also permits
the attachment of probes and other molecules that may be useful in
physical and structural analyses.
In alkaline solutions, the rate of
hydrolysis of DBS from 82-TDBS-82
increases. Thus, by
controlling the pH of the solution in which the product is kept, the
free acid
99-F-99
82-T-82
or the ester,
99-F-99
82-TDBS-82
, will predominate. The latter is
useful for subsequent reaction with nucleophiles that form stable
derivatives by displacement of the ester retained on the trimesyl
cross-link.
It
is well documented that nearly 45% of the overall alkaline Bohr effect
is due to the releasing of protons from the His residues in human Hb A that occurs with the decrease in pK
associated with oxygenation (Kilmartin et al., 1980; Shih et al., 1993). The remaining 55% of the Bohr effect is
attributed to the release of protons linked to the chloride effect. The
positively charged cluster of amino acid residues in the central
cavity, i.e.
-NH
of Val
,
-NH
of Lys
and
Lys
, and Arg
, has been identified
as important in the structural mechanism of the chloride effect (Perutz et al., 1994). According to Perutz et al.(1994),
modifications of any positively charged residue in the central cavity
should change the chloride effect and thus the Bohr effect. Therefore,
the additive losses of Bohr effect and chloride effect in the order of
82-T-82
,
99-F-99
,
and
99-F-99
82-T-82
would be expected because of the
extent of acylation of the
-NH
groups of
Lys
and Lys
. Our data show that the
cross-linking modification of Lys
reduces the
chloride effect by 55% and the cross-linking of Lys
reduces it by 66%. The accompanied decrease in overall Bohr
effect with each modification is 26 and 48%, respectively. In the case
of the doubly cross-linked
99-F-99
82-T-82
Hb in
which four of the positively charged residues in the central cavity
have been eliminated, the accumulated reduction of chloride effect of
84% and accompanying large reduction of Bohr effect of 73% are
consistent with the concept of chloride binding proposed by Perutz et al.(1994).
As noted above, His is
known to be responsible for the chloride-independent part of the
overall alkaline Bohr effect of Hb A. Although the double cross-linking
modifications do not chemically alter the histidyl residues of
146, the reduction of the Bohr effect of this hemoglobin to 27% of
normal, which is about one-half of the chloride-independent part of the
Bohr effect, is probably due to constraints in oxygen-linked structural
changes in the doubly cross-linked Hb. The high P
and low n
values of the doubly cross-linked
Hb, which indicate a reduction in the shift in the allosteric
equilibrium toward the R state, support this explanation. The extra
structural constraint in the
99-F-99
82-T-82
Hb that
is manifest in its decreased cooperativity is presumably due more to
the cross-linking modification of the two Lys
residues than the Lys
residues because the
modification of the latter two does not influence the n
value of
99-F-99
as shown in Table 3.
These results show that sequentially introduced site-specific cross-links provide practical means for adjusting physical properties of proteins. The use of a reactive ester on one of the cross-links permits the introduction of probes for further analysis, conjugation, or modification.