(Received for publication, September 7, 1994; and in revised form, December 16, 1994)
From the
The ligand substitution reaction of EDTA with
Cd-metallothionein (Cd
-MT) has been
reinvestigated. NMR titration of the
Cd-protein with EDTA
showed that the ligand interacts preferentially and cooperatively with
Cd
ions in the
-domain cluster. NMR and
ultrafiltration kinetic analysis of this reaction using 5.6 mM Cd
as
Cd
-MT and 56
mM EDTA indicated that cadmium-EDTA formed less rapidly than
Cd peak intensity declined. Spectrophotometric and gel
filtration studies of the reaction with 20 µM Cd
as Cd
-MT with various
concentrations of EDTA revealed biphasic kinetics with much larger rate
constants than observed in the NMR experiments. The fraction of total
ligand substitution occurring in each kinetic step varied with EDTA
concentration. The EDTA concentration dependence of both kinetic steps
was consistent with the initial formation of protein
EDTA adducts,
followed by their breakdown into products. Kinetic measurements were
also made for the reactions of the isolated Cd
-
- and
Cd
-
-domains with EDTA. The Cd
domain
reacted with EDTA with biphasic kinetics, in which one Cd
was removed rapidly with first-order kinetics, which were
zero-order in EDTA. The other three reacted with kinetics like those
for the slower step of the holoprotein. Cd
-
reacted
with EDTA like the faster rate process associated with the
Cd
-protein. The observed rate constants for the reaction of
Cd
-metallothionein with EDTA and the fraction of reaction
in the faster rate process were sensitive to protein concentration.
These results are consistent with the hypothesis that the monomer-dimer
equilibrium of the protein controls its kinetic reactivity with EDTA.
Metallothionein, a 61-amino acid protein with a high density of
cysteinyl sulfhydryl groups, is the principal steady state binding site
for Cd entering liver, kidney, and other tissues.
After injection of a bolus of Cd
to rodents, the
metal ion distributes within tissues such as liver and induces the de novo synthesis of apoMT(
)(1) . As the MT
concentration increases, it competes successfully with other
cadmium-binding sites in the cells to form Cd-MT and later Cd, Zn-MT.
Thereafter, a cycle of biodegradation and new protein synthesis
maintains cadmium in the cell in a steady state bound largely to MT (2) . Although it is assumed that MT is the preferential
binding site in cells for Cd
because of its large
thermodynamic affinity for the metal ion, the chemistry underlying the
reactions between Cd-MT and other metal-binding ligands has received
only modest attention(3) . Similarly, in the effort to design
chelating agents that detoxify cells of Cd
, little
effort has been devoted to determining the properties of their
reactions with cadmium-metallothionein(4, 17) .
One
approach to understanding the metal exchange properties of
metallothionein has been to study the reaction of Cd-MT
with competing ligands such as EDTA, which have multiple amino and
carboxylate groups as found in proteins and peptides. An early study
indicated that most of this reaction occurs with slow
kinetics(5) . A subsequent
Cd NMR study using
Cd
-MT 1 showed that
Cd
signals were lost exclusively from the 3-metal cluster when
substoichiometric amounts of EDTA were added to the protein, leading to
the interpretation that the thermodynamic affinity of cadmium ions in
the
-domain, Cd
S
cluster was substantially
less than that for cadmium ions in the
-domain,
Cd
S
cluster(6) . However, in another
study using excess EDTA the removal of only about 1 cadmium ion was
observed by product analysis over a 3-h period(7) . Yet,
another NMR investigation suggested that in the presence of EDTA,
Cd
-MT could be converted to
Cd
-MT(8) .
The lack of a unified understanding of this reaction has prompted its reexamination. This has led to the development of a revised model for differential metal-cluster reactivity.
The
solution was taken from the anaerobic chamber and concentrated using a
stirred cell ultrafiltration unit (membrane YM-1). The concentrated
solution was loaded onto a Sephadex G-50 column, and 5 mM Tris-HCl at pH 7.4 was used as eluant buffer. The Cd content of each fraction was analyzed by atomic absorption
spectrophotometry, the
-domain fractions pooled, and the material
concentrated again for use.
The Zn-
-domain of MT 2
was prepared by similar procedures according to the method described by
Winge with minor modifications(12) . ApoMT 2 was obtained from
Zn-MT 2 by incubation at pH 3, followed by Sephadex G-50 chromatography
in 0.05 N HCl to isolate the protein. In order to avoid
oxidation of apoMT or the Cu(I)-containing product below, 5 mM
-mercaptoethanol was added to the apoprotein and anaerobic
conditions were used. Upon neutralization of the protein solution with
solid Tris, it was reacted with 6 equivalents of Cu(I)
(NCCH
)
. After 1.5 h,
subtilisin was added to the solution, and the mixture was incubated for
17-20 h. Cu
-
-domain was isolated after
chromatography over Sephadex G-50. To release copper bound to the
-domain fragment, the pH of this solution was decreased below 0.5.
After separation of the apo
-domain from copper during Sephadex
G-25 chromatography at pH 0.5 and its adjustment to pH 7.4 with solid
Tris, 3 equivalents of zinc were added to the apo
-domain, in the
presence of dithiothreitol. Finally, the Zn
-
-domain
was separated from other products of the reaction using Sephadex G-50
column chromatography. It was converted to Cd
-
by
reaction with a stoichiometric concentration of Cd
followed by incubation with Chelex 100 resin to remove displaced
Zn
.
Both Cd-
and
Cd
-
were characterized by atomic adsorption
spectrophotometry and sulfhydryl analysis using DTNB. Routinely
-
and
-domain complexes gave SH and cadmium ratios of 2.67 ±
0.24 and 3.06 ± 0.2, respectively.
Figure 1:
NMR titration of Cd
-MT with EDTA. The ratio of EDTA to MT is
indicated nextto each spectrum. Conditions included
5.6 mM
Cd
as
Cd
-MT and 20 mM Tris, pH 7.5, at 35
°C.
Figure 2:
Summary of the NMR titration of Cd
-MT with EDTA. The sum of integrated peak
intensities of
Cd
S
(
) and
Cd
S
clusters (
) is
shown.
Figure 3:
Time-dependent NMR spectra of the reaction
of Cd
-MT with EDTA. 5.6 mM
Cd as Cd
-MT was reacted with 5.6 mM EDTA for 3 h in the NMR spectrometer to yield average spectra for
the period 0-1.5 h and 1.5-3.0 h designated as spectra t = 1.5 and t = 3 h. Conditions were as
in Fig. 1.
Figure 4:
Sephadex G-75 separation of the components
of the mixture stemming from the reaction of Cd
-MT with EDTA in Fig. 3(t = 3 h).
, separation made at t = 12
h, 254-nm absorbance;
, cadmium
concentration.
To examine
this reaction further, 0.8 mM protein, the same concentration
as that used in the NMR study, was mixed with a ratio of EDTA to
cadmium of 10:1, and the rate of cadmium-EDTA formation was observed by
Centricon filtration. The result is shown on a semilog plot of cadmium
remaining in MT versus time in Fig. 5. In the first 0.5
h of the reaction, cadmium bound to EDTA accounted for 23% of the total
cadmium. Then during the next 11.5 h, product formation became slow,
with 57% of the reaction completed in 12 h. The second process was
characterized by a first-order rate constant of 1.5
10
s
and accounted for 84% of the
cadmium-EDTA found. This reaction was also followed
spectrophotometrically and was characterized by two rate constants, 1.9
10
s
and 5.5
10
s
. Evidently, the loss of NMR
intensity during the reaction, as described in Fig. 3, proceeded
more rapidly than cadmium-EDTA was formed.
Figure 5:
Semilog plot of the rate of Cd loss from Cd
-MT at NMR concentrations of reactants
determined by ultrafiltration. 5.6 mM Cd
as
Cd
-MT 2 was reacted with 56 mM EDTA in 20 mM Tris at 25 °C and pH 7.4.
Figure 6:
Semilog plot of the rate of reaction of
Cd-MT with EDTA at spectrophotometric concentrations of
reactants measured by the change in absorbance at 254 nm. 20 µM cadmium as Cd
-MT 2 was reacted with 5 mM EDTA
in 5 mM Tris, 0.1 M KCl at pH 7.4 and 25
°C.
Figure 7:
Dependence of spectrophotometric rate
constants and fraction of absorbance change in faster reaction of
Cd-MT on EDTA concentration. a, k
observed; b, k
observed; c,
fraction of reaction occurring in faster rate process. The reaction
conditions are as in Fig. 6. The solid lines in a and b are the best fit
simulations.
Fitting Fig. 7, a and b, to this
equation one obtains values for k, k`, and K for the faster and slower steps of 3.8
10
s
, 3.5
10
s
, 9.7
10
M and 3.6
10
s
, 1.1
10
s
, and 2.3
10
M, respectively.
Fig. 7c shows the fraction of reaction that occurred in the faster step as a function of EDTA concentration. Interestingly, this graph also describes a hyperbola with a maximal extent of reaction of 0.6.
To
determine whether these rate processes observed spectrophotometrically
represented product formation, the rate profile for the reaction of 20
µM cadmium as Cd-MT with 10 mM EDTA
was determined by both spectrophotometric and gel filtration
procedures. As seen in Fig. 8, when this reaction is monitored
for loss of cadmium from MT and formation of cadmium-EDTA, a biphasic
conversion of reactants to products was observed, which was described
by two pseudo-first-order rate constants, (2.6 ± 0.1)
10
s
and (7.5 ± 0.2)
10
s
with 59% of the
overall reaction occurring in the faster step. For comparison, under
the same conditions the spectrophotometric analysis of the reaction
yielded the rate constants (2.2 ± 0.1)
10
s
and (7.2 ± 0.4)
10
s
with 49% of the reaction
allocated to the faster rate process. Thus, in contrast to the NMR
experiments, under these conditions changes in absorbance at 254 nm
represented conversion of reactant to product.
Figure 8:
Kinetics of reaction of Cd-MT
with EDTA monitored by gel filtration chromatography. 20 µM cadmium as Cd
-MT was reacted with 10 mM EDTA
under the conditions listed in Fig. 6.
, cadmium remaining
in MT;
, cadmium present as
cadmium-EDTA.
The
spectrophotometric and gel filtration results were apparently
inconsistent with those in Fig. 5, for the rate constant and
fraction of reaction in the slower step at 25 mM EDTA in Fig. 7was 9 10
s
and 34%, respectively. In contrast, at an even larger
concentration of EDTA, 56 mM, the rate constant for the slow
step, which constituted 89% of the overall reaction, was 1.5
10
s
(Fig. 5). Therefore,
it was hypothesized that the rate constant and the associated fraction
of reaction varied with protein concentration because that was the one
obvious difference in the two experiments. The kinetics of reaction
were monitored spectrophotometrically as a function of MT
concentration. The hypothesis was correct as seen in Table 1, for
the rate constants of both reactions changed as a function of
Cd
-MT concentration as did the fraction of reaction in step
1. As protein concentration increased, k
first
increased and then became constant. In contrast, k
was largest at the smallest concentration of Cd
-MT
and then fell off as protein concentration was increased. Finally, the
fraction of reaction of the faster step declined when the protein
concentration rose.
Figure 9:
Dependence of the rate constants for
reaction of Cd-
and Cd
-
with EDTA on
EDTA concentration. Observed rate constants determined by UV
spectrophotometry are shown. a, Cd
-
, faster
(
) (k
) and slower (
) (k
) components; b, Cd
-
.
20 µM cadmium was reacted with EDTA under conditions
described in Fig. 6. The solid lines in a and b are the best fit simulation.
The reaction of Cd-MT with EDTA has been
reexamined by several techniques. Following the approach of other
workers to monitor the
Cd NMR spectrum of
Cd
-MT, the titration of this protein with
EDTA was carried out as illustrated in Fig. 1(6, 7) . Spectra showed that
Cd resonances associated with metal ions in the
-domain decreased in tandem as did resonances in the
-domain.
That is, each cluster appeared to react cooperatively with EDTA in an
all or nothing fashion. After the addition of 7 mol of EDTA/7 g atoms
of cadmium, the Cd-MT spectrum was completely abolished. Notably, even
after the first addition of EDTA (1 EDTA/7 cadmium), the intensity of
resonances in both clusters was reduced, with the signals from the
-domain more affected. To some extent these results resemble a
previously published titration of
Cd-MT with EDTA, which
was also observed by NMR spectroscopy(6) . In that experiment,
effects were observed only on
-domain resonances, perhaps because
the titration was carried out at pH 9.0. According to the present
experiment done at pH 7.5, both clusters interact with EDTA even at
substoichiometric concentrations of the competing ligand, tempering the
conclusion that the
-cluster is thermodynamically less stable than
the
-cluster(6) . The differential rates of loss of NMR
intensity and decrease in MT-bound cadmium described in Fig. 3Fig. 4Fig. 5agree with the finding of
Nicholson et al., who observed that only one cadmium ion was
removed from Cd
-MT over a 3-h period when 0.5-2.0
mM protein was reacted with a 12-30 mM excess
of EDTA relative to protein(7) . Thus, in short term
experiments changes in
Cd peak heights do not directly
represent cadmium extraction. Instead, it is hypothesized that
localized ligand substitution between carboxylate or amino groups of
EDTA and sulfhydryl ligands of the protein leads to exchange broadening
of the
Cd resonances in the EDTA cluster adducts. This
interaction is then followed by complete ligand substitution and
formation of the cadmium-EDTA product.
Support for this mechanism comes from kinetic studies of the reaction using UV spectrophotometry and gel filtration chromatography (Fig. 6Fig. 7Fig. 8). The reaction rate was biphasic when monitored by these methods. The rate law for each step () is comprised of an EDTA-independent component, which apparently involves a dissociation or rearrangement process in the protein and another process in which 1 equivalent of EDTA/mol of MT binds to the protein before ligand substitution is complete.
To
simplify the composite biphasic reaction of Cd-MT with
EDTA, the reaction of each domain with EDTA was examined. Two rate
processes were observed when Cd
-
reacted with EDTA;
one was independent of EDTA concentration and comprised a fixed
percentage of the total reaction approximately equal to 1
Cd
. The other followed a hyperbolic rate law. A
mechanism consistent with this rate law is illustrated in and and in Fig. 10.
Figure 10:
Model for the reaction of
Cd-MT with EDTA. The reaction of Cd
-
with
EDTA is shown.
It is hypothesized that the Cd ion with the asterisk (Fig. 10) is removed in the dissociation step (k), leaving a Cd
S
cluster analogous
to the Cd
S
cluster present in the intact
-domain. This species reacts relatively rapidly with EDTA to form
an EDTA
Cd
-
adduct, characterized by K
. Formation of products follows in a
first-order process (k`), which might represent the
rate-limiting displacement of the third thiolate ligand of one of the
cadmium ions leading to rapid chelation of all of the cadmium ions in
the cluster. This mechanism is analogous to that describing the ligand
substitution reaction of EDTA with metal ions bound to
polyamines(14) .
Cd-
reacts according to a
single hyperbolic rate law, consistent with an initial equilibrium
binding of EDTA to the cluster, followed by its breakdown as described
in and Fig. 10for Cd
-
.
The much smaller k` describing the dissociation into
products of EDTACd
-
in comparison with
EDTA
Cd
-
might be caused by the presence of the
two extra sulfhydryl groups in the Cd
-
-domain. Their
potential to bind Cd
may stabilize
EDTA
Cd
-
against dissociation into products.
The faster and slower rate processes seen in the reaction of
Cd-MT with EDTA can be assigned to reactions of each
cluster by comparison with the reactions of the independent domains
with EDTA (Table 2). The faster process has rate constants (k, k`) similar to the EDTA-independent term for
Cd
-
and to the dissociation of the
Cd
-
-EDTA adduct (k`) that is resolved in the
hyperbolic term for Cd
-
. The slower rate process finds
no counterpart for its EDTA-independent term in the kinetics of the
isolated domains. However, its k` value is similar to that for
Cd
-
. The isolated domain forms much stronger adducts
with EDTA than do the domains in the intact protein. Apparently, EDTA
binding to either cluster in Cd
-MT is inhibited in the
holoprotein.
In particular, it is hypothesized that the -domain
is unreactive in the dimer; as a result MT reacts primarily via the
Cd
-
cluster, albeit with a smaller observed rate
constant (Table 1). Given the apparent preference of
Cd
to bind in the
-domain exemplified in the
method to prepare the Cd
-
domain, it is hypothesized
that Cd
migrates rapidly from the
Cd
S
cluster into apo-
-domains as they form
during the reaction of the Cd
-
with EDTA () (12) .
If this occurs, the whole reaction could be rate-limited by
ligand substitution of the thiolate groups of Cd-
by
EDTA (). This would lead to the large fraction of reaction
occurring with the smaller rate constant that was observed at higher
concentrations of protein. When MT is present in smaller
concentrations, the monomer protein increases in concentration, and
EDTA can successfully bind to both domains in a concentration-dependent
fashion, directly removing Cd
from both clusters.
In fact, under the conditions of Fig. 7, a maximum of of the
Cd reacted with the faster rate constant as the EDTA
concentration was increased beyond 10 mM (Fig. 7c). According to the results above, these
include the three Cd
ions of the
-domain and one
of the four Cd
ions in the Cd
-
cluster. Yet, at the smallest concentration of EDTA 80-100% of
the total reaction proceeded as if the
-domain were the sole site
of reaction. Furthermore, the distribution of reaction between fast and
slow steps also shifted, favoring the slower rate process
characteristic of the
-domain cluster, as the concentration of
protein in the reactions increased (Table 1). These results are
consistent with the hypothesis that protein dimer formation and
EDTA
protein adduct formation compete with one another to
determine the characteristics of this ligand substitution reaction.
Support for the possibility that MT dimerizes in solution comes from
the x-ray structure of Cd,Zn-MT, which shows that MT
molecules in each unit cell are present as head-to-tail,
- to
-linked dimers(15) . More directly, information on the gel
filtration behavior of MT indicates that dimerization occurs with an
association constant of 3.0
10
M
(16) . Using this constant,
one calculates that the equilibrium of protein species shifts from 81%
monomer at 5 µM Cd
-MT to 17% at 500 µM protein (Table 1). It is over this same range of
concentrations that the two observed rate constants and the fractional
contribution of their respective rate processes to the overall reaction
undergo substantial change.