(Received for publication, November 3, 1995; and in revised form, December 27, 1995)
From the
The binding of four monoclonal immunoglobulins, two with
specificity for (1
6)-linked D-galactopyranans (IgA
X24 and IgA J539) and two with specificity for the chain terminus of
(1
6)-linked D-glucopyranans (IgA W3129 and IgA
16.4.12E), was measured with a number of their homologous
oligosaccharide ligands at different temperatures. The results show a
linear relationship between lnK
and
1/T, where K
is the affinity
constant and T is the absolute temperature. The unitary free
energy of binding,
G
, is virtually
independent of T, and the
S
is small
when compared with
G
. The enthalpy changes
derived from van't Hoff plots are large and negative, indicating
an exothermic binding effect, whereas the entropy changes are small and
negative, indicating minor overall hydrophobic contributions.
Measurements of the free energies of binding, in low and high salt
buffers, of methyl
-D-galactopyranoside and the methyl
glycoside of
(1
6)-D-galactopyranotetraose with
anti-galactan IgA X24 indicate that the monosaccharide has no
hydrophobic interaction with the highest affinity subsite of IgA,
whereas the tetraoside might have a modest hydrophobic interaction with
the three other hapten-binding subsites of IgA. The standard entropy
change of binding of the two groups (galactosyl and glucosyl) of
oligosaccharides to the two respective sets (anti-galactan and
anti-dextran) of antibodies shows a distinct, differing correlation
with the hapten chain length within each set. This correlation agrees
with the type of association previously established between the
antibodies and either the interior determinants of the antigen (in the
case of the anti-galactans) or the chain terminus (in the case of the
anti-dextrans).
We have shown in the past how murine monoclonal antibodies of
the v441 gene-family (Hartman and Rudikoff, 1984) can bind
to the interior stretches of their homologous antigenic linear
(1
6)-linked galactan polysaccharide (Glaudemans, 1987;
Glaudemans and Kovác, 1988;
Ziegler et al., 1992). Each of the two (per monomeric IgA)
antibody combining areas made up of the interface of the N-terminal
ends of the heavy and the light chain is filled by binding maximally to
a stretch of four sequential galactosyl residues in the homologous,
antigenic polysaccharide chain. It must be kept in mind that we showed
(Glaudemans, 1991; Glaudemans and
Kovác, 1988) that for this
family of galactan-specific antibodies, each of the four galactosyl
residues of the determinant has its own subsite on the antibody
surface, and these four antibody subsites have differing affinities for
``their'' galactosyl residue.
In the case of the binding
of murine monoclonal antibodies to (1
6)-linked glucan
polysaccharide (dextrans), other work (Cisar et al., 1974;
Weigert et al., 1974), as well as our own work (Bennett and
Glaudemans, 1979a; Nashed et al., 1990), has shown that there
is a special group of these antibodies that bind only to the terminal,
upstream end of the
(1
6)-linked glucan polysaccharide chain
and that these antibodies are incapable of binding interior segments of
the dextran polysaccharide chain. These latter antibodies also have
combining areas that can optimally accommodate a stretch of some four
sequential glucosyl residues in this case. However, here that stretch
must include the one and only terminal glucosyl residue that occurs at
the nonreducing (upstream) end of the dextran polysaccharide antigen.
Again, we showed that similar to the galactan/anti-galactan system,
each glucosyl residue has its own subsite, this time the terminal sugar
residue having maximal affinity for its subsite and being bound inside
a cavity that is part of the antibody combining area (Nashed, et
al., 1990). Thus, we see that in the case of the anti-galactans,
antibodies can bind all along the galactan polysaccharide chain,
``loading up'' the polysaccharide chain with antibody
molecules. On the other hand, for the group of anti-dextran antibodies
discussed above, they only engage the nonreducing tetrasaccharidic
terminus of the dextran polysaccharide chain, whereas the rest of that
antibody-bound polysaccharide chain exits from the antibody combing
area into the solution, incapable of binding additional antibody
molecules.
Evidence has been presented previously that the key
interactions of both the (1
6)-linked galactans and the
(1
6)-linked dextrans with their respective homologous
antibodies are mediated by hydrogen bonds (Glaudemans, 1987, 1991;
Glaudemans and Kovác, 1988;
Nashed et al., 1990). We wished to investigate if the binding
of these two quite differing anti-carbohydrate antibody systems with
their homologous antigen (galactan binding by two monoclonal intrachain
binding anti-galactans and dextran binding by two monoclonal chain
end-binding anti-dextrans) could also involve significant hydrophobic
contributions. Thus, the enthalpy change (
H°) of
binding of these two sets of immunoglobulins with a series of their
homologous oligosaccharide determinants was measured by using
van't Hoff plots. The interactions were found to be associated
with large enthalpy changes and with a small negative opposing entropy
change.
Three possible modes of noncovalent interaction between proteins and ligands can a priori take place: electrostatic (i.e. charge interaction), hydrophilic (i.e. involving hydrogen bonding), and hydrophobic bonding (driven by the tendency of water to self-associate). It was shown before that interactions due to charge differences between a monoclonal immunoglobulin and several antigens are greatly diminished or even eliminated with increased salt concentration of the medium (Manjula et al., 1982). In order to evaluate this approach for a neutral ligand carrying charge separation, we here report on the affinity of a small zwitterion ligand, phosphorylcholine, for its monoclonal antibody HOPC8 (Bennett and Glaudemans, 1979b) in medium of high salt concentration.
Reaction rates between organic hydrophobic components, when occurring in aqueous solution, have been shown to be accelerated by higher salt concentrations (Breslow, 1991). Recently, it was reported that peptide hydrolysis catalyzed by retroviral proteases is accelerated by high salt concentration of the medium, and the effect is suggested to be due to hydrophobic substrate-protein interaction (Tropea et al., 1992). These observations led us to so evaluate a possible hydrophobic contribution to the binding of anti-galactan IgA X24 with galactosyl saccharides.
In addition, we report herein our correlation of thermodynamic parameters with the differing nature of the binding of the galactan/anti-galactan and the dextran/anti-dextran systems. As pointed out above, we had previously demonstrated that the two monoclonal anti-galactan antibodies are capable of binding any interior segment of the antigenic galactan chain (Glaudemans et al., 1993; Ziegler et al., 1992). On the other hand, the two monoclonal anti-dextran antibodies studied here bind exclusively to the chain terminus of their homologous dextran polysaccharide antigen (Glaudemans et al., 1989; Nashed et al., 1990). This fundamental difference is also reflected in the thermodynamic binding behavior of these two sets of antibodies with their homologous haptens, as we shall here demonstrate.
Affinity constants for the association between ligands and
antibodies were measured by monitoring the ligand-induced tryptophanyl
fluorescence of the antibody as a function of ligand concentration
(Jolley and Glaudemans, 1974) using a Perkin-Elmer LS 50 Luminescence
Spectrophotometer. These were then expressed as the free energy of
binding (-G° =
RTlnK
) and are listed in Table 1, Table 2, III, IV, and V. Affinity measurements, including those
in solutions of varying NaCl concentrations, were conducted in buffered
(pH 7.4-7.6) solutions. When affinity measurements were conducted
at differing temperatures, the temperatures were monitored by
thermocouple probes in the cuvettes during measurement. The isolation
of the various antibodies has been described (Bennett and Glaudemans,
1979a, 1979b; Glaudemans et al., 1989; Nashed et al.,
1990; Potter and Glaudemans, 1972). We are grateful to Drs. Tsukasa
Matsuda and Elvin A. Kabat for a gift of IgA 16.4.12E. We thank Dr.
Pavol Kovác for samples of
the synthetic oligosaccharides used in this work
(Kovác, 1988;
Kovác and Lerner, 1988).
Phosphorylcholine was used as its Na
Cl
salt (Bennett and Glaudemans, 1979b).
In the ensuing discussion, two things must be kept in mind.
The first is that antibody binding of small ligands, be they the
complete determinant part of the antigen or only part thereof, is the
same when measured with whole antibody, Fab fragment, oligomer, or
monomer as we have shown previously (Jolley et al., 1973;
Pavliak et al., 1993). Thus, results obtained here can be
discussed strictly in terms of the combining area of any given
antibody, irrespective of the nature of the monomeric or dimeric nature
of the affinity purified antibody in question. Secondly, it should be
kept in mind that the N-terminal ends of the heavy and light chains
that together make up the general combining area of an antibody
molecule measure some 40 Å across (Suh et al., 1986).
The extended size of a (16)-linked glycosyl group is
approximately 5 Å. The largest ligands here used are hexomeric
glycosides, i.e. their optimal length cannot exceed 30
Å. Therefore, none of the ligands here used in binding
measurements can ever occupy more than one antibody combining area at
any one time.
The free energies of binding of phosphoryl choline,
methyl -D-galactopyranoside (me
Gal
), (
)phenyl
-D-galactopyranoside
(phenyl
Gal), and the methyl
-glycoside of
(1
6)D-galactotetraose (me
Gal
) with
their respective homologous monoclonal antibodies as a function of NaCl
concentration in the buffer medium at near neutral pH are given in Table 1. For IgA HOPC8 (Potter, 1977), it can be seen that
phosphorylcholine shows a reduction in affinity in buffer solutions
having higher NaCl concentrations. Charge interactions between specific
protein amino acid residues and the hapten's zwitterion have been
observed in the crystal structure of phosphorylcholine bound to IgA
McPC603 (Padlan et al., 1985), which belongs to the same
family as does IgA HOPC8. In other binding studies on IgA HOPC8 using
phosphorylcholine and several of its analogs, evidence was presented
for charge interactions in solution between ligand and immunoglobulin
(Bennett and Glaudemans, 1979b). It was previously shown also that the
interaction between charged antigens and an oppositely charged
immunoglobulin were greatly diminished or eliminated in solution by the
presence of high salt concentrations in the medium (Manjula et
al., 1982). Table 1shows that in contrast to the
phosphorylcholine/HOPC8 system, the influence of high NaCl
concentration in the buffer on the affinity of either me
Gal
or phenyl
Gal with IgA X24 is essentially nil. Others have
shown that hydrophobic interactions between either small molecules
(Breslow, 1991) or proteins and small molecules (Tropea et
al., 1992) are emphasized by the presence of high salt
concentration in the medium. Thus, our observation on the binding of
me
Gal
or phenyl
Gal with IgA X24 indicates the
absence of hydrophobic contributions to the binding of a galactosyl
residue in the highest affinity subsite of that antibody, one of the
four subsites that together can bind the tetrasaccharide
immunodeterminant specified by that immunoglobulin (Glaudemans, 1987).
It can also be seen in Table 1that me
Gal
, which
is essentially the complete immunodeterminant, in contrast to
me
Gal
shows a small increase in affinity for IgA X24
when measured in phosphate buffer containing increasing salt
concentration. Because me
Gal
does not show this
increase, this indicates that for me
Gal
, any or all of
the second, third, and fourth residues possibly have some hydrophobic
interaction with their respective subsites on the antibody's
surface. Thus, our observations on the binding of these haptens as a
function of salt concentration in the medium tend to validate this
method to distinguish between charge-, neutral-, and
hydrophobic-related interactions.
The free energies
(-G°=
RTlnK
) of association of the methyl
-glycoside of
(1
6)-D-galacto(oligo)saccharides
(me
Gal
where n = 1, 2, 3, 4,
5, or 6) with the anti-galactans IgA X24 and IgA J539 and of the methyl
-glycoside of
(1
6)-D-gluco(oligo)saccharides
(me
Glc
where n = 1, 2, 3, 4,
5, or 6) with the anti-dextrans IgA 16.4.12E and IgA W3129 measured at
several temperatures are given in Table 2, Table 3, IV, and
V, respectively.
The van't Hoff equation
lnK/
T =
H°/RT(
), where K stands for the
equilibrium constant at constant pressure, can be rearranged to
lnK = (
H°/R)
(1/T), which means that
lnK/
(1/T) =
-
H°/R. Thus the slope of the plot of
lnK measured at constant pressure versus 1/T is equal to -
H°/R for any given
temperature. Because
[
(
H°)/
T]
=
C
°, this means that if the
change in the heat capacity (
C
°) with
temperature is 0, the plot of lnK versus 1/T would be
linear. This is what we observe over the temperature range studied
(shown only for IgAs X24 and 16.4.12E, Fig. 1and 2).
Figure 1:
Van't Hoff plot
for the binding of the methyl -glycoside of
(1
6)-D-galacto-oligosaccharides with anti-galactan
IgA X24.
The unitary entropy (S
) is
independent of concentration units chosen for the standard state and is
defined as
S° =
S
+ R ln
, where R is the
gas constant (8.317 J) and
is the cratic
contribution to entropy (resulting from the entropy of mixing). Because
the
is the reciprocal of the molarity of water, we
obtain
S° =
S
- 33.42. Thus,
G° =
H° - T(
S
- 33.42) or
G° - 33.42T =
H° - T
S
. One
defines
G° - 33.42T =
G
, which is the unitary free energy (Gurney,
1953). We observe that the unitary free energy of binding for each
oligosaccharide appears to be nearly independent of temperature ( Fig. 3and 4).
Figure 3:
Temperature dependence of the unitary free
energy of binding (G
) of the methyl
-glycoside of
(1
6)-D-galacto-oligosaccharides
with anti-galactan IgA X24.
Because (S
/
T)
=
C
°/T, becomes
(
G
)/
T =
(
H°)/
T - T(
C
°/T) -
S
. Because
(
H°)/
T is also equal to
C
°, we have
(
G
)/
T =
-
S
. We find
G
to be nearly invariant with temperature, because
S
is very small compared with
G
(see Fig. 3and Fig. 4).
Figure 4:
Temperature dependence of the unitary free
energy of binding (G
) of the methyl
-glycoside of
(1
6)-D-gluco-oligosaccharides
with anti-dextran IgA 16.4.12E.
Table 6and Table 7list the values for
H°,
S°, and
S
for the various oligosaccharides and their respective homologous
monoclonal antibodies. It can be seen from those data that the observed
ligand-antibody association results in a large enthalpy change. The
standard entropy change is small and negative and thus disfavors the
binding. A distinct difference can be seen on the one hand between the
anti-galactan monoclonal antibodies binding to
-(1
6)-galactosyl ligands and on the other hand the
anti-dextran monoclonal antibodies binding to
-(1
6)-glucosyl
ligands ( Table 6and Table 7and Fig. 5and Fig. 6). It is known that the latter antibodies (W3129 and
16.4.12E) bind only to the chain terminus of their antigenic
polysaccharide (Glaudemans et al., 1989; Nashed et
al., 1990), whereas the former antibodies (X24 and J539) bind
their homologous polysaccharide anywhere along the antigenic chain
(Glaudemans, 1987; Glaudemans et al., 1993). It can be seen in Fig. 5and Fig. 6that the standard entropy change
associated with the binding of each of these two classes of antibodies
shows a distinct correlation with the chain length of their ligands;
the anti-dextran antibodies show a nearly constant negative standard
entropy change of association (a factor opposing the binding of ligand
to the antibody) after the ligand has reached the size of the
disaccharide (i.e. methyl
-isomaltoside). For the
anti-galactan antibodies on the other hand, the negative standard
entropy change of association continues to increase up to the
pentasaccharide level. For the former case it was previously shown that
the upstream (Pavliak et al., 1993) terminal glucopyranosyl
residue of the homologous
(1
6)-gluco-oligosaccharides fits
into a tight cavity-like pocket of the antibody with the penultimate
glucosyl residue protruding from that pocket into the solution
(Glaudemans et al., 1989, 1994a, 1994b; Nashed et
al., 1990). On the other hand, for the anti-galactans the
oligosaccharide hapten was shown to bind on the surface of the antibody
combining area, with larger haptens making increasing linear contact
(Glaudemans, 1987, 1991; Glaudemans et al., 1984) with the
antibody surface. Thus, in the former case, the loss of degrees of
freedom may hinder most in haptens having up to two glucosyl residues,
whereas in the latter case the entropic opposition to binding continues
to increase up to the pentasaccharide level.
Figure 5: Relationship between the standard entropy change of binding of both IgA W3129 and IgA X24 and the chain length of their respective homologous haptens.
Figure 6: Relationship between the standard entropy change of binding of both IgA 16.4.12E and IgA J539 and the chain length of their respective homologous haptens.
Hydrophobicity has been
defined as the tendency of nonpolar substances to aggregate in aqueous
solution so as to minimize their interaction with water molecules
(Ben-Naim, 1980). It is to be expected that the binding of any ligand
to its antibody, even those ligands that we consider as hydrophilic,
for instance multiple hydroxyl-bearing haptens such as
(oligo)saccharides, could have some hydrophobic component. Indeed, in a
case where both hydrogen bonding and significant hydrophobic
interactions had been found by x-ray diffraction analysis of the
antibody having the immunodeterminant bound to its binding site (Cygler et al., 1991), it has been shown that antigen-antibody
interactions can also have favorable entropy contributions (Sigurskjold
and Bundle, 1992) to the binding. In another case, thermodynamic
van't Hoff analysis of the interaction between di- and
trinitrophenyl ligands with monoclonal antibody MOPC 315 revealed the
interaction to have positive H° and
S° values, and the interaction was thus deduced to be
hydrophobically driven (Tanaka et al., 1986). Furthermore,
study of the binding of D-glucose to yeast hexokinase showed
the enthalpy change (
H°) to be nearly zero over the
temperature range studied, so that the change in heat capacity was also
found to be essentially zero (Takahashi et al., 1981).
We
have found that the interaction of meGal
in binding to
anti-galactan IgA X24 is moderately influenced by high salt
concentration of the medium. Thus it appears unlikely that large
hydrophobic contributions to the interaction play a role (Breslow,
1991; Tropea et al., 1992). Because the anti-galactan
immunoglobulins of this family all show the same binding
characteristics (Glaudemans, 1987), this would also hold for the
interaction of IgA J539 with its homologous oligosaccharide
determinant. This agrees in turn with our observation that this binding
shows enthalpy and entropy changes that are comparable with those of
IgA X24.
In the case of the dextran-anti-dextran system, due to the paucity of antibodies, we could not acquire independent information on the nature of the binding, such as the influence of high electrolyte concentration on the affinity. However, our finding that a high negative enthalpy change accompanies the association strongly indicates that hydrophobic effects are unlikely to dominate the interaction between antibody and bound ligand in this case as well.
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