©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reaction of Cd-Metallothionein with EDTA
A REAPPRAISAL (*)

(Received for publication, September 7, 1994; and in revised form, December 16, 1994)

Tong Gan Amalia Munoz C. Frank Shaw III David H. Petering (§)

From the Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The ligand substitution reaction of EDTA with Cd(7)-metallothionein (Cd(7)-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 beta-domain cluster. NMR and ultrafiltration kinetic analysis of this reaction using 5.6 mM Cd as Cd(7)-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(7)-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 proteinbulletEDTA adducts, followed by their breakdown into products. Kinetic measurements were also made for the reactions of the isolated Cd(4)-alpha- and Cd(3)-beta-domains with EDTA. The Cd(4) 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(3)-beta reacted with EDTA like the faster rate process associated with the Cd(7)-protein. The observed rate constants for the reaction of Cd(7)-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.


INTRODUCTION

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)(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(7)-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 beta-domain, Cd(3)S(9) cluster was substantially less than that for cadmium ions in the alpha-domain, Cd(4)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(7)-MT could be converted to Cd(6)-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.


EXPERIMENTAL PROCEDURES

Materials

EDTA, Trizma (Tris base), beta-mercaptoethanol, and D(2)O were purchased from Sigma and were of the highest quality available. Tetrakis(acetonitrile)copper(II) hexofluorophosphate was purchased from Aldrich. CdO was obtained from Oak Ridge National Laboratories and converted into CdCl(2) with concentrated, reagent grade HCl. Subtilisin (peptidase type VIII), Trizma base, and various Sephadex products were obtained from Sigma.

Isolation of Zinc Metallothionein and Preparation of Cadmium Metallothionein

Zinc metallothionein was isolated from rabbit livers after injection with 2 ml of 0.15 M ZnSO(4) each day for 7 days. Sephadex G-75 and high pressure liquid DEAE 5PW chromatography was then employed to separate Zn-MT and its two isoforms as described previously(9) . CdO was dissolved in 2 N HCl, and its pH was adjusted to 3 with Trizma base. Using either CdCl(2) or CdCl(2), Cd(7)-MT 2 was prepared by adding the cadmium chloride solution dropwise into buffered Zn-MT 2 with stirring. Chelex-100 was added to remove zinc and excess cadmium. More than 95% of the zinc was displaced upon forming the Cd-MT product according to atomic absorption spectroscopy. Finally, Cd(7)-MT 2 was concentrated using a stirred cell ultrafiltration system fit with a YM-2 membrane and stored at -4 °C until further use. Metallothionein concentration was determined by the absorbance of apoMT at 220 nm ( = 47,300 M cm) in 0.05 N HCl(10) . Cadmium was measured by atomic absorption spectroscopy, and the sulfhydryl content in the protein was determined by the 5,5`-dithio-bis(2-nitrobenzoate) reaction ( = 13, 600 M cm)(11) . Only MT with ratios of metal to protein and thiol to metal greater than or equal to 6.7 and 2.8, respectively, were used in experiments.

Preparation of Cd(4)-alpha- and Cd(3)-beta-Domains

Purified Zn(7)-MT 2 was used to prepare the (Cd or Zn)(4)-alpha-domain(12) . After acidification to pH 3.0, the protein was chromatographed over a Sephadex G-25 column using 0.05 N HCl as eluant buffer in order to separate the apoMT from other products of the reaction. The absorbance at 220 nm for each of the eluting fractions was analyzed, and the fractions corresponding to the apoMT 2 were pooled. The sulfhydryl concentration of the apoMT was analyzed with the DTNB reaction in order to determine the necessary amounts of either cadmium or zinc to add. The following metal ion reconstitution procedure for the protein was carried out in an anaerobic chamber. 2 mM beta-mercaptoethanol was added to avoid the oxidation of the thiol groups. Then, the pH of solution was increased back to 7.4 using solid Trizma base. Since Cd preferentially binds to the alpha-domain of the MT, 4 equivalents of Cd were slowly added. This solution was incubated for 1.5 h. Subsequently, subtilisin (enzyme:protein, 1:20), were added to the solution to degrade the peptide segment corresponding to the beta-domain. Twenty hours was allotted for this reaction.

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 alpha-domain fractions pooled, and the material concentrated again for use.

The Zn(3)-beta-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 beta-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(3))(4). After 1.5 h, subtilisin was added to the solution, and the mixture was incubated for 17-20 h. Cu(6)-beta-domain was isolated after chromatography over Sephadex G-50. To release copper bound to the beta-domain fragment, the pH of this solution was decreased below 0.5. After separation of the apobeta-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 apobeta-domain, in the presence of dithiothreitol. Finally, the Zn(3)-beta-domain was separated from other products of the reaction using Sephadex G-50 column chromatography. It was converted to Cd(3)-beta by reaction with a stoichiometric concentration of Cd followed by incubation with Chelex 100 resin to remove displaced Zn.

Both Cd(4)-alpha and Cd(3)-beta were characterized by atomic adsorption spectrophotometry and sulfhydryl analysis using DTNB. Routinely alpha- and beta-domain complexes gave SH and cadmium ratios of 2.67 ± 0.24 and 3.06 ± 0.2, respectively.

NMR Methods

Cd NMR spectra were recorded at 106 MHz on a GE GN-500 spectrometer with continuous broadband proton decoupling. Cd(7)-MT 2 was placed in a 10-mm tube with 15% D(2)O to provide the field frequency lock. The pulse sequence consisted of a 15-µs pulse, a 168-ms acquisition time, and a 400-ms delay time. Chemical shifts were reported in parts per million downfield from the Cd resonance of 0.1 M Cd(ClO(4))(2) in D(2)O.

NMR Titration Experiment

A 12,288-scan Cd NMR spectrum of 1.2 ml of 0.8 mMCd(7)-MT 2 was taken at 35 °C in the absence of EDTA. Then spectra were obtained under similar conditions in the presence of ratios of Cd to EDTA of 7:1 to 7:7. Each spectrum was obtained after the mixture was incubated overnight to let the reaction go to completion. The Cd peaks were manually integrated. For these experiments the buffer was 20 mM Tris, pH 7.5. EDTA was independently set at pH 7.5, and the pH of the final solution was measured as 7.5.

Kinetic Experiments

NMR Spectroscopy

The Cd NMR spectrum of 1.2 ml of 1 mMCd-MT 2 was recorded for 10,240 scans as a reference. Then EDTA was added to make the cadmium:EDTA ratio 1:1 or 1:10. The NMR spectra were collected every 10,240 scans for a total of about 10.5 h at 25 °C.

UV Spectrophotometry

Reactions were carried out using 20 µM cadmium in Cd-MT 2 with 1-25 mM EDTA at 25 °C, pH 7.5. Reactions were monitored at 265 nm because EDTA absorbs at 254 nm, the wavelength of maximum absorbance for the cadmium-thiolate clusters. All samples were run against blanks containing the same concentration of all reactants, excluding protein. They were allowed to proceed to completion, and end point absorbances (A) were measured after 1-2 days. Data plots of ln(A(t)- A) versus time were created by a Quattro Pro program. Reactions were biphasic and were analyzed by a standard algorithm as described previously(13) . Analogous kinetic experiments were done using Cd(4)-alpha- and Cd(3)-beta-domains derived from MT 2 in place of Cd(7)-MT 2. Spectra were recorded on a Beckman DU 70 spectrophotometer.

Ultrafiltration

Reaction mixtures consisting of 5.6 mM cadmium in Cd(7)-MT 2 and 56 mM EDTA were allowed to react for various times at 25 °C. After each period of incubation, Amicon Centricon-10 microconcentrator tubes were used to rapidly separate the EDTA-bound cadmium from residual Cd-MT. During centrifugation, these tubes, with a nominal 10-kDa molecular weight retention limit, completely retained native Cd(7)-MT. Cadmium concentrations were determined by atomic absorption spectrophotometry. Centricon kinetic data were analyzed using the same algorithms applied to the UV spectrophotometric data, except that µg of cadmium/ml in the filtrate was substituted for 265-nm absorbance as the time-dependent variable.

Sephadex Gel Filtration

To confirm that ligand exchange had occurred in reactions of cadmium-protein with EDTA, 1.0-ml aliquots were removed from the reaction mixture and placed onto a Sephadex G-50 column (1 times 25 cm) equilibrated with 20 mM Tris-HCl, 0.1 M KCl at pH 7.5 and room temperature. The reaction was sampled over a 245-min interval. The kinetics of the reactions were then determined by plotting observed changes in the Cd concentration over time for the chromatographic fractions corresponding to MT and EDTA.


RESULTS

EDTA Titration of Cd(7)-MT

Cd NMR spectroscopy was used to monitor the titration of Cd(7)-MT 2 with EDTA. A NMR spectrum was recorded without EDTA as a control. Then 1-7 equivalents of EDTA were added per 7 equivalents of cadmium in Cd(7)-MT 2 to carry out a titration. Fig. 1displays the NMR spectra for the EDTA titration of Cd(7)-MT 2. After the first equivalent of EDTA was added per MT molecule or per 7 equivalents of cadmium, peak areas from both alpha-cluster (I, V, VI, VII) and beta-cluster resonances (II, III, IV) diminished. The intensities of beta-cluster peaks decreased by 40%, whereas those of the alpha-cluster peaks declined only by 12%. The decreases in intensity for particular peaks within the alpha-cluster or the beta-cluster were indistinguishable. After the addition of 3 mol of EDTA/7 mol of cadmium, only 38% of the alpha-cluster peak intensity had been lost, but all three Cd peaks from the beta-cluster had disappeared. With the titration of the seventh equivalent of EDTA, all Cd peaks from both the alpha- and the beta-cluster had disappeared. Fig. 2summarizes the titration results, showing the sum of the integrated peak intensities in the 3- and 4-Cd clusters as a function of EDTA concentration. Although the titration of the Cd(3)S(9) cluster signals was completed first, it had only a modest preference for reaction with EDTA in comparison with the Cd(4)S cluster.


Figure 1: NMR titration of Cd(7)-MT with EDTA. The ratio of EDTA to MT is indicated nextto each spectrum. Conditions included 5.6 mMCd as Cd(7)-MT and 20 mM Tris, pH 7.5, at 35 °C.




Figure 2: Summary of the NMR titration of Cd(7)-MT with EDTA. The sum of integrated peak intensities of Cd(4)S () and Cd(3)S(9) clusters () is shown.



NMR Kinetic Analysis of the Reaction of Cd(7)-MT 2 with EDTA

Kinetic studies were done that utilized NMR to follow the reaction of Cd(7)-MT with EDTA. The molar ratio of EDTA to cadmium was 1:1 or 10:1. One reference NMR spectrum of the protein was measured in the absence of EDTA. After the reaction was initiated, one spectrum was recorded every 1.5 h for a total of 10.5 h. For reactions using 1:1 molar ratios of cadmium and EDTA, the Cd NMR signals lost some of their intensity rapidly in the first 1.5 h. Then, the reaction became very slow for the remaining 9 h of observation. When the samples were checked by NMR 1 week later, Cd NMR signals still could be observed, but the intensities were further diminished. In the reaction involving the larger molar ratio of EDTA to cadmium, the Cd NMR signals lost their intensities rapidly and almost completely according to the spectrum accumulated during the first 1.5-h interval (Fig. 3). Cd resonances II, III, and IV of the beta-cluster disappeared, whereas peaks I, V, VI, and VII of the alpha-cluster also lost most of their NMR intensity. The residual intensities were only 15% of those in the reference spectrum. After the second 1.5-h period, all the NMR signals disappeared. Twelve h after the reaction of EDTA and Cd(7)-MT was initiated, the mixture was loaded on a G-50 column to examine the distribution of cadmium between protein and EDTA. Surprisingly, there were two cadmium peaks in the column effluent (Fig. 4), one representing cadmium in Cd-MT 2 and accounting for 26% of the total cadmium. The other corresponded to the migration of cadmium-EDTA with 74% of the total cadmium.


Figure 3: Time-dependent NMR spectra of the reaction of Cd(7)-MT with EDTA. 5.6 mMCd as Cd(7)-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(7)-MT with EDTA in Fig. 3(t = 3 h). box, 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 times 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 times 10 s and 5.5 times 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(7)-MT at NMR concentrations of reactants determined by ultrafiltration. 5.6 mM Cd as Cd(7)-MT 2 was reacted with 56 mM EDTA in 20 mM Tris at 25 °C and pH 7.4.



UV Spectrophotometric Analysis of the Reaction of Cd(7)-MT 2 with EDTA

Reactions of Cd(7)-MT 2 with EDTA were carried out under pseudo-first-order conditions using 20 µM Cd as Cd(7)-MT and 1-25 µM EDTA. Reactions were monitored for absorbance changes at 265 nm in the region of the cadmium-thiolate absorption band. Fig. 6shows a typical kinetic profile obtained by plotting ln(A(t) - A) versus time. The kinetics could be described by two pseudo-first-order rate constants, k and k, which depend on EDTA concentration (Fig. 7, a and b). Each shows a hyperbolic dependence of k on EDTA concentration indicative of a mechanism that includes a binding step as part of the pathway. In addition, there is a component of the rate constant that is independent of EDTA concentration as shown by the non-zero intercept of both graphs. Empirically, the EDTA concentration dependence of each kinetic phase can be described by the following equation.


Figure 6: Semilog plot of the rate of reaction of Cd(7)-MT with EDTA at spectrophotometric concentrations of reactants measured by the change in absorbance at 254 nm. 20 µM cadmium as Cd(7)-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(7)-MT on EDTA concentration. a, k(1) observed; b, k(2) 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(D) for the faster and slower steps of 3.8 times 10 s, 3.5 times 10 s, 9.7 times 10M and 3.6 times 10 s, 1.1 times 10 s, and 2.3 times 10M, 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(7)-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) times 10 s and (7.5 ± 0.2) times 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) times 10 s and (7.2 ± 0.4) times 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(7)-MT with EDTA monitored by gel filtration chromatography. 20 µM cadmium as Cd(7)-MT was reacted with 10 mM EDTA under the conditions listed in Fig. 6. , cadmium remaining in MT; box, 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 times 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 times 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(7)-MT concentration as did the fraction of reaction in step 1. As protein concentration increased, k(1) first increased and then became constant. In contrast, k(2) was largest at the smallest concentration of Cd(7)-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.



Reaction of Isolated Cadmium Domains with EDTA

The reactions of Cd(4)-alpha and Cd(3)-beta-domains at various concentrations of EDTA were followed over time spectrophotometrically and by gel filtration of samples of the reaction mixtures. The reactions of the alpha-cluster were biphasic. Fig. 9, a and b, summarizes the rate constants that were derived from analysis of the kinetic results for the reaction. The faster rate process, constituting about 30% reaction at all concentrations of EDTA and independent of EDTA concentration, was characterized by a first-order rate constant of 1.4 times 10 s. The slower process displayed a hyperbolic rate dependence on EDTA, consistent with , in which k = 5.5 times 10 s and K = 1.0 times 10M. That the spectrophotometric results represented conversion of reactants to products was verified using gel filtration chromatography as above. The reaction of the Cd(3)-beta-domain with EDTA was described by a single-rate constant that displayed a hyperbolic dependence on EDTA concentration as in . For this rate process k = 4.6 times 10 s and K = 7.5 times 10M.


Figure 9: Dependence of the rate constants for reaction of Cd(4)-alpha and Cd(3)-beta with EDTA on EDTA concentration. Observed rate constants determined by UV spectrophotometry are shown. a, Cd(4)-alpha, faster () (k(1)) and slower (box) (k(2)) components; b, Cd(3)-beta. 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.




DISCUSSION

The reaction of Cd(7)-MT with EDTA has been reexamined by several techniques. Following the approach of other workers to monitor the Cd NMR spectrum of Cd(7)-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 beta-domain decreased in tandem as did resonances in the alpha-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 beta-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 beta-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 beta-cluster is thermodynamically less stable than the alpha-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(7)-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(7)-MT with EDTA, the reaction of each domain with EDTA was examined. Two rate processes were observed when Cd(4)-alpha 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(7)-MT with EDTA. The reaction of Cd(4)-alpha 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(3)S(9) cluster analogous to the Cd(3)S(9) cluster present in the intact beta-domain. This species reacts relatively rapidly with EDTA to form an EDTAbulletCd(3)-alpha 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(3)-beta 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(3)-alpha.

The much smaller k` describing the dissociation into products of EDTAbulletCd(3)-alpha in comparison with EDTAbulletCd(3)-beta might be caused by the presence of the two extra sulfhydryl groups in the Cd(3)-alpha-domain. Their potential to bind Cd may stabilize EDTAbulletCd(3)-alpha against dissociation into products.

The faster and slower rate processes seen in the reaction of Cd(7)-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(4)-alpha and to the dissociation of the Cd(3)-beta-EDTA adduct (k`) that is resolved in the hyperbolic term for Cd(3)-beta. 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(4)-alpha. 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(7)-MT is inhibited in the holoprotein.



In particular, it is hypothesized that the beta-domain is unreactive in the dimer; as a result MT reacts primarily via the Cd(4)-alpha cluster, albeit with a smaller observed rate constant (Table 1). Given the apparent preference of Cd to bind in the alpha-domain exemplified in the method to prepare the Cd(4)-alpha domain, it is hypothesized that Cd migrates rapidly from the Cd(3)S(9) cluster into apo-alpha-domains as they form during the reaction of the Cd(4)-alpha with EDTA () (12) .

If this occurs, the whole reaction could be rate-limited by ligand substitution of the thiolate groups of Cd(3)-alpha 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 beta-domain and one of the four Cd ions in the Cd(4)-alpha cluster. Yet, at the smallest concentration of EDTA 80-100% of the total reaction proceeded as if the alpha-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 alpha-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 EDTAbulletprotein 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(5),Zn-MT, which shows that MT molecules in each unit cell are present as head-to-tail, alpha- to beta-linked dimers(15) . More directly, information on the gel filtration behavior of MT indicates that dimerization occurs with an association constant of 3.0 times 10^4M(16) . Using this constant, one calculates that the equilibrium of protein species shifts from 81% monomer at 5 µM Cd(7)-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.


FOOTNOTES

*
The authors acknowledge the support of NIH Grant ES-04026. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: MT, metallothionein; alpha and beta, alpha-domain and beta-domain, respectively, of metallothionein; DEAE, diethylaminoethyl; DTNB, 5,5`-dithio-bis(2-nitrobenzoic acid); Cd(7)-MT and Zn(7)-MT, metallothionein in which all seven binding sites are occupied by cadmium or zinc, respectively; Cd, Zn-MT, metallothionein whose seven binding sites are occupied by a mixture of cadmium and zinc; MT 1 and MT 2, the two major isoprotein forms of rabbit metallothionein.


ACKNOWLEDGEMENTS

We acknowledge the skilled guidance of Dr. Eugene DeRose in the early NMR experiments and the exploratory experiments of Daniel Lemkuil.


REFERENCES

  1. Winge, D. R., Premakumar, R., and Rajagopalan, K. V. (1978) Arch. Biochem. Biophys. 188, 466-475 [Medline] [Order article via Infotrieve]
  2. Oh, S. H., Deagen, J. T., Whanger, P. D., and Weswig, P. H. (1978) Am. J. Physiol. 243, E282-E285
  3. Otvos, J. D., Petering, D. H., and Shaw, C. F., III (1989) Comm. Inorg. Chem. 9, 1-35
  4. Gale, G. R., Smith, A. B., Atkins, L. M., and Jones, M. M. (1985) Res. Commun. Chem. Pathol. Pharmacol. 49, 6334-6338
  5. Li. T.-Y., Kraker, A. J., Shaw, C. F., III, and Petering, D. H. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6334-6338 [Abstract]
  6. Dalgarno, D. C., and Armitage, I. M. (1986) in Advances in Inorganic Biochemistry, 6 (Eichhorn, G. L., and Marzilli, L. G., eds) pp. 113-138, Elsevier, New York
  7. Nicholson, J. K., Sadler, P. J., and Va s á k, M. (1987) in Metallothionein II (K ä gi, J. H. R., and Kojima, Y., eds) pp. 191-201, Birkh ä user Verlag, Basel, Switzerland
  8. Vasquez, F., and Vasák, M. (1988) Biochem. J. 253, 611-614 [Medline] [Order article via Infotrieve]
  9. Minkel, D. T., Poulsen, K., Wielgus, S., Shaw, C. F., III, and Petering, D. H. (1980) Biochem. J. 191, 475-485 [Medline] [Order article via Infotrieve]
  10. Buhler, R. H. O., and K ä gi, J. H. R. (1979) in Metallothionein (K ä gi, J. H. R., and Nordberg, M., eds) pp. 211-220, Birkh ä user Verlag, Basel, Switzerland
  11. Ellman, G. (1959) Arch. Biochem. Biophys. 82, 70-77 [Medline] [Order article via Infotrieve]
  12. Winge, D. R. (1991) Methods Enzymol. 205, 438-447 [Medline] [Order article via Infotrieve]
  13. Pattanaik, A., Bachowski, G., Laib, J., Lemkuil, D., Shaw, C. F., III, Petering, D. H., Hitchcock, A., and Saryan, L. (1992) J. Biol. Chem. 267, 16121-16128 [Abstract/Free Full Text]
  14. Carr, J. D., Libby, R. A., and Margerum, D. W. (1967) Inorg. Chem. 6, 1083-1088
  15. Robbins, A. H., McRee, D. E., Williamson, M., Collett, S. A., Xuong, N. H., Furey, W. F., Wang, B. C., and Stout, C. D (1991) J. Mol. Biol. 221, 1269-1293 [CrossRef][Medline] [Order article via Infotrieve]
  16. Otvos, J. D., Liu, X., Li, H., Shen, G., and Basti, M. (1993) in Metallothionein III (Suzuki, K. T., Imura, N., and Kimura, M., eds) pp. 57-74, Birkh ä user Verlag, Basel, Switzerland
  17. Rivera, H., Bruck, M. A., Aposhian, H. V., and Fernando, Q. (1991) Chem. Res. Toxicol. 4, 572-580 [Medline] [Order article via Infotrieve]

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