The oxidation of rabbit liver metallothionein-II by 5,5′-dithiobis(2-nitrobenzoic acid) and glutathione disulfide

The Oxidation of Rabbit Liver Metallothionein-II by 5,5’-Dithiobis(2-Nitrobenzoic Acid) and Glutathione Disulfide M. Meral Savas, C. Frank Shaw III, and David H. Petering Department of Chemistry, The University of Wisconsin-Milwaukee: Milwaukee, Wisconsin

ABSTRACT Because metallothionein (MT) may undergo thiol-disulfide or other redox reactions under certain cellular conditions, the partially and completely oxidized products of the reactions of Cd,MT-II with the electrophile 5,5’-dithiobis(2-nitrobenzoic acid), ESSE, and oxidized glutathione, GSSG, were characterized. Reaction with the stoichiometric quantity of ESSE (1 ESSE per MT thiolatel generates monomeric and polymeric MTs with three types of disulfide bonds: intra- and intermolecular CyS-SCy linkages and a small number (2-3/MT) of mixed disulfides, CyS-SE, involving thionitrobenzoate (ES-). Reaction with substoichiometric quantities of ESSE (0.02 or 0.1 per MT thiolate) causes the formation of intra- and intermolecular CyS-SCy disulfides, but no mixed disulfides. In the latter reactions, two equivalents of ES- are released per mole of ESSE, but the release is described by a single first-order rate constant (k = 3.0 f 0.5 set-‘1. Substantial amounts of cadmium remained bound to the MT monomers and polymers after reaction with the substoichiometric quantities. Despite the Cd bound to the MT after reaction with 0.1 ESSE per MT thiolate, no ‘iiCd NMR signals were detected, indicating rapid equilibration of the remaining metal ions among the disrupted binding sites. Large excesses of the endogenous aliphatic disulfide, GSSG, displace Zn+2 from Zn,-MT slowly. The reaction is complete after 24 hours with 5000 PM GSSG, but only 25% complete after 72 hours with 250 PM GSSG. Approximately one Cd+2 is displaced rapidly from CdTMT by 5000 PM GSSG and half as much by 250 PM GSSG, but no further reaction occurs. It is unlikely that GSSG oxidation of MTs would be physiologically significant.

ABBREVIATIONS MT, metallothionein; ESSE, 5,5’-dithiobis(2nitrobenzoic GSSG, oxidized glutathione; GSH, reduced glutathione;

acid); ES-, 5-thio-2nitrobenzoate; DTDP, 2,2’-dipyridyl disulfide; DTT,

Address reprint requests and correspondence to: Professor C. Frank Shaw III, Department Chemistry, The University of Wisconsin-Milwaukee, Milwaukee, WI 53201-0413. Journal of Inorganic Biochemistry, 52,235249 (1993) 0 1993 Elsevier Science Publishing Co., Inc., 655 Avenue

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of the Americas, NY, NY 10010 0162-0134/93/$6.00

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A4. A4. Savas et al.

dithiothreitol; t-BuOOH, t-butyl hydroperoxide; MOPS, 4-morpholinopropanesulfonic atomic absorption spectroscopy; HMW, high molecular weight; LMW, low molecular

acid; AAS, weight.

INTRODUCTION Metallothionein (MT) is a low-molecular-weight, cysteine-rich metal-binding protein [l-3]. The cysteine thiolates of MT are known to be the reactive sites for metal ions, alkylating agents, and radical species in cells. MT has been suggested to play important roles in essential metal metabolism [4], heavy metal detoxification [5], and radical scavenging [6] in cells and living organisms. Most of the research on MT has concentrated on its structure [l-3, 7-121. Mammalian MT binds 7 Zn” or Cd” ions, . each tetrahedrally coordinated to 4 cysteine thiolate ligands. It has been shown that MT folds into two domains [7-91, each consisting of about 30 amino acids. The a-domain at the COOH terminus contains 4 bivalent metal ions and 11 cysteines and the P-domain at the NH, terminus contains 3 bivalent metal ions and 9 cysteines. Relatively less research has been done on the chemical reactivity of MT and the relationship of its reactivity to is functions and structure. Kinetic studies of reactions of MT with chromophoric electrophiles and chelating agents have contributed a great deal to our knowledge and understanding of its reactivity [4, 13-171. Among these species ESSE (5,5-dithiobis(2-nitrobenzoic acid)) has been studied extensively [13, 14, 171. ESSE slowly reacts with the metal-coordinated thiolates of MT forming disulfides and releasing ES (5-thio-2nitrobenzoate) with an absorption maximum at 412 nm that is used to monitor the reaction [13, 14, 171. In effect the reaction is a redox process in which ESSE is reduced and the protein sulfhydryls are oxidized [l&22]. It is known that ES is one of the products of this reaction, but the fate of MT and the metals bound to it are not known. The protein sulfhydryls might form either mixed disulfides to ES, MT(S-SE), or cysteine disulfides (CyS-SCy) which, in turn, may be intra- or intermolecular. In general, the reaction of a low molecular weight thiol with a disulfide reagent usually involves two steps: RSH + ES-SE + RS-SE + ES- + H+,

(la)

RS-SE+RSH-tRS-SR+ES+H+.

(lb)

The reaction of a protein thiol with a disulfide often stops at mixed disulfide (Eq. (la)) because the formation of an intermolecular disulfide is thermodynamically unfavorable due to stereochemical barriers that prevent specific alignment of protein thiols for dimerization. The second step (Eq. (lb)) is favored in the presence of excess, unhindered RSH or when another thiol is nearby. In the case of MT, the existence of 20 closely spaced cysteines could favor the formation of inter- or intramolecular disulfides (RS-SR). The stoichiometry of the reaction will differ in the limiting cases corresponding to one-step or two-step reactions: Cd&MT

+ 20 ES-SE ---) MT(S-S&I

+ 7 Cd”* + 20 ES-,

Cd&MT

+ 10 ES-SE -+ MT@ - S)io + 7Cd-2

+ 20 ES-.

(2) (3)

OXIDATION

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237

Therefore, to characterize the nature of the oxidized protein and the stoichiometry of the reaction between MT and ESSE, we studied this reaction by “‘Cd NMR spectroscopy, size-exclusion chromatography, and UV-VIS spectroscopy. The corresponding reactions of MT with the endogenous disulfide, oxidized glutathione (GSSG) were also examined to determine whether they might have any physiological significance.

EXPERIMENTAL Materials 5,5’-Dithiobis(2-nitrobenzoic acid) (ESSE), t-butyl hydroperoxide (t-BuOOH), and Sephadex G-50 were purchased from Sigma, 2,2’-dipyridyl disulphide (DTDP) from Lancaster Synthesis, dithiothreitol (DTT) from P-L Biochemicals, KH,PO, from Fisher, and GSSG, NH,HCO,, and MOPS buffer from Aldrich Chemical Co. The “‘Cd0 was purchased from Oak Ridge National Laboratories. Zn,MTII was isolated from rabbit liver as described elsewhere [23]. ” ’ Cd NMR Experiments “‘Cd0 was dissolved in excess of 2 M HCl and evaporated is a water bath while adding water frequently to raise the pH to about 3. Zn,MT-II was reconstituted with “’ Cd by adding ” 'CdCl, dropwise while stirring. The displaced Zn+* ions and excess Cd+* ions were removed with Chelex-100 (the pH of the Chelex was preadjusted to 7.4). A sample was analyzed chromatographically and spectroscopically to ascertain that the product was monomeric ” ’ Cd,MT-II and that all the Cd present was bound to MT. ” ‘Cd NMR spectra were recorded at 106 MHz on a General Electric GN-500 spectrometer. Phosphate buffer, pH 7.4, was selected for these experiments because ESSE is less soluble in other common buffers such as Tris-HCl, MOPS, or NH,CO,. 10% D,O was added to the samples for field-frequency lock. Chromatographic

Separations, Metal and Disulfide Analyses

Reactions were carried out under anaerobic conditions at 25” C in phosphate buffer. Protein concentrations were of the same magnitude as for the NMR experiments. The reactions were monitored spectroscopically at 412 nm. The reaction mixtures were then introduced into a G-50 Sephadex column (1.5 X 52 cm) eluted with 10 mM MOPS, pH 7.4. Fractions corresponding to various polymerized MTs, native MT, Cd+*, and the Cd+2 complexes of thionitrobenzoate were pooled and designated I, II, III, and IV, respectively. The pooled fractions were analyzed for metals by atomic absorption spectroscopy (AAS) and for protein content using a UV-VIS spectrophotometer (Perkin-Elmer h-6). The fractions were also tested for their SH content using DTDP [241. The NMR samples, after measuring their spectra, were also applied to a G-50 Sephadex column (1.5 x 52 cm) to separate the products for further analysis as described above. These experiments were repeated three times. Each reaction mixture was kept under an Ar atmosphere until it was applied to the chromatography column for separation. The presence of mixed disulfides (CySSE) was determined by the absorbance at ca. 325 nm due to the thionitrobenzoate group linked to the protein and,

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more quantitatively, using D’TT to release the intensely colored chromophore, ES, detected at 412 nm. Extinction Coefficient of Oxidized apo-MT To determine whether oxidation would alter the extinction coefficient of the apo-protein, 2.9 PM MT (57 PM SH) was acidified to pH 3 and treated with Chelex-100 to remove the free Zn ions. The apo-MT solution was transferred to a cuvette and UV spectra were obtained after adding 1, 2, 5, and 10 equivalents of t-BuOOH to the sample: 10 t-BuOOH + MT(SH)zo -+ oxidized MT + 10 t-BuOH + 10 H,O. The absorbance at 220 nm was unchanged during the oxidation, indicating that for fully oxidized MT, as for apo-MT, the extinction coefficient is 47,300 Mm’ cm-‘. Kinetic Experiments The solutions of 14.3 PM Cd,MT-II (100 PM Cd+*; 286 PM SH) and 5.7 PM ESSE (1:0.02 Thiolate:ESSE) were mixed in the UV cell at time zero and immediately placed in a Perkin-Elmer A-6 UV-VIS spectrophotometer. The ES-) was meaabsorbance change at 412 nm (habs of 5-thio-2-nitrobenzoate, sured over time against an equivalent amount of ESSE as reference. Temperature was maintained at 25.0 kO.l”C by a circulating thermostated water bath. The reactions were carried out in 5 mM Tris/HCl + 100 mM KC1 buffer at pH 7.4 to be consistent with our original published data [13, 141. The data were analyzed as first-order reactions by plotting AX-A, versus time on a semilogarithmic scale. Glutathione

Disulfide Reactions

The reactions of Zn,MT-II and Cd,MT-II with another disulfide reagent, GSSG, were also carried out at 25°C and under an Ar atmosphere. Typically, 14 /.LM protein and 250 and 5000 PM GSSG were employed. After 1.5, 24, and 48 hr (also 72 hr for 250 PM GSSG), the reaction mixtures were applied to a G-50 Sephadex column (0.5 x 25 cm), equilibrated with 10 mM NH,HCO, buffer at pH 7.2. The fractions were analyzed for metals by AAS and characterized by UV-VIS spectroscopy. Control experiments involving Cd,-MT in the absence of any GSSG were also fractionated to insure that the metal losses observed were a consequence of the GSSG and not due to degraded protein. RESULTS AND DISCUSSION ESSE Reactions “‘Cd7MT-11 was prepared from Zn,MT-II as described in the experimental section. The “‘Cd NMR spectrum of a typical Cd,MT-II sample (1.14 mM protein, 22.86 mM SH) is shown in Figure l(a). The seven resonances correspond to seven discrete metal binding sites in the protein [81. The ” rCd NMR spectrum of the reaction mixture of ESSE (10 mM) and “‘Cd,MT-1IcO.57 mM protein) (1:l protein thiolate:ESSE ratio), shown in Figure l(b), was measured

OXIDATION

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239

P-0 .

(a)

660

660

640

620

600

PPM

FIGURE 1. “‘Cd NMR spectra of (a) Cd,MT-II, 1.14 mM protein (22.9 mM SH); (b) Cd,MT-II, 0.57 mM protein + 10 mM ESSE; (c) Cd,MT-II, 1.14 mM protein + 2 mM ESSE. The ESSE reagent was added as a solution in 100 mM K,HPO, at pH 7.4.

beginning 30 min after mixing. All seven peaks have disappeared completely indicating that protein had undergone significant chemical changes accompanied by a loss of the original metal binding sites. Reactions of ESSE with MT are kinetically biphasic [13] and the fast step is associated with the a-domain [14]. For that reason, it is of interest to study the reaction with a surfeit of ESSE, since the reaction should be localized in the more reactive domain. Thus, the NMR experiment was repeated with the concentration of ESSE lowered to 2 mM while the protein concentration was 1.14 mM (22.9 mM SH), so that protein thiolate to ESSE ratio was 1:0.1(2 ESSE per MT). The “‘Cd NMR spectrum of this mixture, measured beginning 15 min after mixing (Fig. l(c)), also did not have any detectable “lCd resonances corresponding to the intact clusters. No additional Cd+’ peak(s) were detected in the range -80 to 600 ppm. This absence suggests that two or more labile Cd+2 species were undergoing rapid exchange and that a mechanism exists for altering both clusters after the initial attack of the ESSE at the kinetically more labile a-cluster. A question of particular interest is whether the complete loss of the NMR signals at the 1:l and 1:O.l thiolate:ESSE ratios was caused by complete removal of the bound Cdf2 or by disruption of the signals due to labilization of the Cd+’ caused by perturbation of the metal-thiolate cluster structures. To characterize the reaction products, oxidized protein samples generated using l:l, l:O.l, and 1:0.02 MT thiolate:ESSE ratios, were analyzed by gel-filtration chromatography.

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The chromatograms (Fig. 2) were obtained by analyzing for Cd by AAS, for CyS-SE mixed disulfides (325 nm absorbance), and for protein concentration (220 nm absorbance) by UV-VIS spectroscopy. The extinction coefficient at 220 nm for fully oxidized MT prepared by treating apo-MT at pH 3 with t-BuOOH was found to be 47,300 M- ’ cm- ‘. This is experimentally equal to the value for apo-protein and indicates that the total MT content (oxidized and reduced) in a sample can be determined using A,,, and the value ~~~~~ = 47,300 M-’ cm-‘. The results are summarized in Table 1. A chromatogram for MT oxidized with a 1:l thiolate:ESSE ratio is shown in Figure 2(a). In two of three independent trials, no Cd was found in the high molecular weight (HMW) fractions (Peak I) or the monomeric MT fractions (Peak II, arrow). In the third trial, a small amount of Cd (- 1 Cd/protein) was detected in the HMW peak and none in the monomeric MT fractions. Nearly all of the Cd+* (93 + 7% for the three trials) appeared in the two low molecular weight (LMW) peaks (III and IV). An absorbance at 325 nm indicates the presence of a thionitrobenzoate disulfide (CyS-SE) in the fraction. Species containing mixed CyS-SE disulfide bonds were detected in peaks I and II (Fig. 2(a)). The presence of the broad HMW protein peak (I in Fig. 2(a)), which tails off, is indicative of the formation of polymerized MTs. Since no polymeric MT was present in the sample before the reaction, the crosslinking is a consequence of an ESSE-induced redox process. The UV-VIS spectrum of the HMW fractions (peak I) is shown in Figure 3(a). If the resulting disulfide bonds were exclusively between the cysteines of the proteins (intra- or intermolecular), only a weak absorbance (250 nm, E = 322 M-’ cm-‘) due to CyS-SCy linkages would be observed. If all the ESSE reacted to form mixed disulfides (CyS-SE), a very strong absorbance slightly shifted from the maximum of ESSE (324 nm, E = 21,000 M-’ cm-‘) is expected. Although the weak absorbance around 328 nm demonstrates that some mixed disulfides are present, conversion of the absorbance to concentration using e324 indicates that very few of the expected disulfides are of the MT(S-SE) type. Treatment of the protein with excess DTI to release any ES bound as disulfide provides a more sensitive measurement of the CJyS-SE concentration. A small increase, was observed due to the release of ES- and yielded a value of 1.7 rt 0.6 AA412, CyS-SE linkage per protein molecule. The second peak underwent similar spectral changes, from which an ES-/protein ratio of 3.4 + 2.5 was calculated. The HMW and the monomeric MT fractions were reacted with excess ESSE to find the extent of the oxidation. No reaction with ESSE was observed in either case indicating that all of the thiolates were in the form of either CyS-SE or . CyS-SCy disulfide bonds. The UV spectrum of the pooled peak IV fractions from the chromatogram (Fig. 2(a)) showed a band at 409 nm due to the ES present. The band maximum is slightly shifted from 412 nm, the characteristic maximum for ES-. In a separate experiment, it was observed that adding Cd+* to ES- causes a blue shift of its 412 nm electronic absorption band. From the molar absorptivity of ES- at 412 nm (E = 13,600 M-’ cm-‘) and the AAS measurement of the Cd+* concentration, the ES/Cd ratio is estimated as 2.7 + 0.2. This indicates the formation of a Cd-thiolate complex, Cd,(ES),-“. Examples of model complexes with various stoichiometries, including Cd,(ES),0-2 for which the ES/Cd ratio is 2.5, have been reviewed [25, 261. Although these complexes are larger

OXIDATION

6_

8

OF RABBIT LIVER METALLOTHIONEIN-II

(a)

241

IV

7-

26 T) c 5: 48 3 32-

II

I

l-

III

,JA+ ,

01 0

20

p-k___

40

60

100

60

120

1 .O

6 (W

I

s-

)

i i II

4L? 9 33

Ill

2l0t 0

20

40

60

60

100

1

16 14-

w

I

12-

E

10-

8

6-

'

6-

Ill

4-

ElutionVolume,ml

FIGURE 2. Gel-filtration chromatograms of reaction mixtures of Cd,MT-II (0.57 mM protein) in 100 mM phosphate buffer, pH 7.4; with thiolate:ESSE ratios of (a) 1:l; (b) 1:O.l; (c) 1:0.02. G-SO Sephadex column (1.5 X 52 cm), eluted with 10 mM MOPS, pH 7.4. Arrow designates the position of the 10,000 MW band as calibrated with cytochrome-C. Cd (-1 and A,,, (-O-+X

242

M. M. Savas et al.

TABLE 1. Chromatographic Analysis of Reaction Products of Cd,MT-II oxidized by ESSE (G-50 Sephadex Column (1.5 x 50 cm), 10 mM MOPS buffer, pH 7.4)”

Peak

1:l

I II III IV

0 0 16 + 2 77 + 9

I II

1.1 + 0.6 3.4 f 2.5

Metal Thiolate:ESSE 1:O.l Cd% 51+4 21+2 13+ 1 15f3

Ratios 1:0.02 49 + 6 31+ 10 21 k4

ES/Protein

I II

I II

-

0 0

Cd/Protein 3.7 + 1.6 1.5 + 1.0

8.7h 6.7b

SH/Protein 9.0 + 3.7 5.7 f 4.0

18.3 + 1.3 13.8b

a All values are mean + ESD of three trials, except as indicated. b Only one trial is included.

than aquated Cd+’ which elutes as peak III, both the aquated and complexed cadmium are smaller than the low molecular weight cut-off of Sephadex G-SO. The fact that they do not co-elute as expected for the molecular sieving mechanism represents the operation of other, secondary chromatographic mechanisms such as electrostatic and hydrophobic interactions. These weaker fractionation mechanisms, which can alter the ideal behavior, are often overlooked. To separate and analyze the reaction products when ESSE was the limiting reagent, three MT samples oxidized with 1:O.l thiolate:ESSE ratio were also applied to a G-50 Sephadex column. As seen from a typical chromatogram (Fig. 2(b)), there were three overlapping HMW protein peaks (collectively designated I> which had 51 + 4% of the total Cd and a monomeric MT peak (II) which contains 21 + 2% of the total Cd. The LMW peaks (III and IV) were present, as observed in the first reaction, and contained 13 rt 1 and 15 & 3% of the total Cd, respectively. The UV-VIS spectra of both peaks I and II had shoulders at 250 nm (Fig. 3(b)). To determine if this shoulder is due to Cd-S bonding (A,,, = 245 nm [271) or CyS-SCy (Amax= 250 nm [28]) bonding, the solutions were treated with concentrated HCl. The 250 nm shoulders diminished significantly for each peak, which is strong evidence that the 250 nm shoulders were largely due to S-Cd charge transfer transition and that Cd was bound to the protein through metal thiolate linkages. The concentration of the bound Cd was found from the difference molar absorptivity at 250 nm (E = 14,500 M-’ cm-- ’ Cd-‘) [27]. The concentration of the protein was calculated from the molar absorptivity of the acidified protein backbone at 220 nm (E = 47,300 M-’ cm-’ [29]. For peaks I and II, the Cd/protein ratios were found to be 3.7 _C1.6 and 1.5 f 1.0, respec-

OXIDATION

OF RABBIT LIVER METALLOTHIONEIN-II

243

0.16 (a)

326 nm

0.02-

1

-0.021 150

200

250

200

250

, 300

350

400

450

5

400

450

E50

1.6 (b)

0.40.2-

-0.2 1 150

300 35b Wavelength, nm

FIGURE 3. UV-VIS spectrum of the protein fractions (peak I and peak II in each case had similar spectral features). (a) Peak I from Figure 2(a); (b) peak I from Figure 2(b); spectra of peak I and II from Figure 2(c) were similar. tively. The concentrations of Cd determined using AAS agree within the experimental error with the values determined from difference molar absorptivity showing that all the Cd in the peaks were bound to the protein. Thiol analysis using DTDP indicated there were 9.0 + 3.7 and 5.7 f 4.0 SH per protein molecule in peaks I and II, respectively. Peak III did not have any spectral features in the UV-VIS range and is attributed to free Cd+‘. The UV-VIS spectrum ‘of the last peak (IV) had the characteristic strong band at 407 nm, from which the ES-/Cd ratio was found to be 5.5 &-2.8. Since Cd-thiolates with coordination numbers greater than 4 have not been reported [25, 261, this result indicates the presence of a complex Cd,(SE),-” species and excess ES-.

244

M. M. Savas et al.

In experiments where the concentration of ESSE was further reduced so that the thiolate:ESSE ratio was 1:0.02, the reaction mixture had a chromatographic profile (Fig. 2(c)) which was similar to that of Figure 2(b). The first two peaks (I and II) contained 79.0 + 8.5% of the total Cd and the last peak (III) contained 21.4 + 4.0. The fractions were also tested for SH, protein, and ES contents as described before. The SH/protein ratio was calculated as 18.3 _t 1.3 for peak I indicating that only about 2 of the 20 cysteines were involved in disulfide bonding. The absence of the 324 nm band (due to CyS-SE bond) in the UV-VIS spectra of peak I and II indicates that the disulfide bonds were of CyS-SCy type. Figure 3(b) shows a representative spectrum for peaks I and II. The LMW peak (III) had a band at 407 nm due to the ES formed, from which the ES/Cd ratio was calculated to be 5.7. Because the previous kinetic studies of ESSE-MT reactions were carried out with large, pseudo-first-order excesses of ESSE [13, 14, 171, it was of interest to monitor the reaction when protein is in excess and ESSE is limiting (Fig. 4). The reaction was completed in less than 20 min and analysis as a first-order reaction revealed a single exponential step with a rate constant of 3.0 + 0.5 X lo-’ s-‘: Rate = k I [MT]. This corresponds to the fast, first-order rate constant of the complex, four-term rate law observed with excess ESSE, Rate = k,,[MT] + k,,[MTI[ESSEl

+ k,,[MTl + k,,[MTl[ESSEl,

k,, = 1.26 x 1O-3 s-l, k,, = 1.75 M-’ SK’, k,, = 4.20 x 1O-4 s-l, k,, = 0.12 M-’ s-l [9]. The present result is consistent with the more complex rate law since at limiting ESSE concentrations, the second-order terms become negligible and the faster first-order term should dominate as observed. The absorbance change due to ES- released should discriminate between the two possible stoichiometries given in Eqs. (2) and (3), since one ES- is produced per ESSE in Eq. (2) and two are produced in Eq. (3). The absorbance change (AA 412= 0.2 for 1:0.02 thiolate:ESSE ratio when 14.3 PM Cd,MT-II (100 PM Cd+‘) and 5.7 PM ESSE react) corresponds to the release of two ES per ESSE, consistent with Eq. (3) as the correct stoichiometry. The fact that two ES are released at the same rate indicates that the second step, the formation of a CyS-SCy disulfide bond according to Eq. (lb), occurs more rapidly than the initial, rate-limiting electrophilic attack according to Eq. (la). While this work was in progress, Huang and Cismowski [30] reported that when ESSE is limiting, monophasic reactions are observed for native and modified MTs. The rates correspond to the faster of the two reaction rates observed by Li et al. when ESSE is present in excess [131. Although Huang and Cismowski’s experimental result agrees with our present finding, they attributed the single reaction step observed with limiting ESSE to the P-cluster 1301.Our previous examination of the reaction of isolated@-cluster shows that it reacts at the fast rate, suggesting that it is more labile toward ESSE [14]. While the p-cluster is clearly more reactive than the cy-cluster with respect to scrambling of the Cd+* ions in certain NMR experiments, the mechanisms for that process are not expected to resemble those for attack of ESSE and other reagents on

OXIDATION

OF RABBIT LIVER METALLOTHIONEIN-II

0.22--

245

-----p__ (a)

0.08-l 0

5

1

10

1'5

25

35

30

4

--------Pep___ r

0.1 I

lE-050

5

10

15

20 25 Time, mln

30

35

40

FIGURE 4. Kinetic analysis of a reaction mixture of 14.3 PM Cd,MT-II and ESSE with 1:0.02 thiolate:ESSE ratio. (a) Absorbance change at 412 nm vs time; (b) first-order semi-log plot of A, - A, vs time. 5 mM tris/HCI + 100 mM KC1 buffer, pH 7.4, at 25°C.

the sulfhydryl ligands or MT-bound metal ions. Indeed, Otvos and Li [31] have observed that NTA reacts biphasically with Zn,MT and, based on the reaction of (Zn,),(Ag&MT, they assigned the faster step to the a-cluster. The relative lability of each cluster toward various reagents is likely to be controlled by specific steric and electronic factors that will vary from reagent to reagent. In some cases the kinetically more reactive cluster may actually be more stable thermodynamically toward a given ligand or electrophile. GSSG Reactions Oxidized and reduced glutathione (GSSG, GSH) are found in most cells 132,331. Glutathione is present primarily in the reduced form, consistent with its principle function as a cellular reducing agent, but the equilibrium concentration of GSSG might drive metal release from MT by a process analogous to Eq. (2) or

246

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TABLE 2. Chromatographic Analysis of Metals Released by GSSG Oxidation of Zn,MT-II and Cd,MT-II (G-50 Sephadex Column (0.5 X 25 cm), 10 mM NH,HCO, buffer, pH 7.4)” % Metals MT-bound Metal Cd

GSSG ( PM) 250

Zn

250

Cd

5000

Zn

5000

Time (hr) 1.5 24 48 72 1.5 24 48 72 1.5 24 48 1.5 24 48

a Peaks 1 and II were divided arbitrarily at an elution polymerized MTs in peak I; Cd+* in Peak III.

peak I 48.8 f 1.8 60.2 f 1.6 67.7 + 0.4

55.0 * 7.9 51.9 + 0.5

volume

Displaced

peak II

peak III

98.1 f 0.3 45.9 f 3.1 31.7 & 3.2 31.7 rt 6.0 100 91.3 $3.9 89.8 + 0.5 76.3 f 0.8 89.5 It 3.2 30.5 + 5.9 36.3 + 7.8 81.8 i 4.9 1.5 + 1.0 0

1.9+0.4 5.2 + 1.4 3.9 * 0.1 4.2 & 2.1 0 7.9 & 2.7 10.2 * 0.4 21.5 & 1.2 8.0 + 5.7 14.2 + 1.7 9.3 + 3.5 17.0 f 4.9 95.8 + 4.2 98.5 + 1.5

of 4.0 mL. Native MT elutes in peak II;

(3). Therefore, the reaction between MT and GSSG was also investigated. GSSG is an aliphatic disulfide, whereas ESSE has aromatic rings with electronwithdrawing nitro and carboxylate substituents. Two different concentrations of GSSG (250 PM, 5000 PM) were reacted with Zn,Mt-II or Cd,MT-II (14.3 PM protein, 286 PM SH) for 1.5, 24, and 48 hr (also 72 hr for 250 PM GSSG) at 25°C. The reactions were carried out under an Ar atmosphere to minimize spurious oxidation. For each combination of time, [GSSG], and substrate, three independent reaction mixtures were analyzed by gel-filtration chromatography, Table 2. The results of these studies indicate that 250 PM glutathione does not effectively oxidize Zn,MT-II and does not release the metals even after 48 hr. Even with a large excess of GSSG, 5000 PM, the reaction required 24 hr to effect complete Zn removal (Fig. S(a)). The reaction of GSSG with Cd,MT-II, on the other hand, does not result in significant release of metals even at 5000 PM GSSG and 72 hr (Fig. 5(b)). A small and constant fraction of Cd’* (I 10%) was removed after 1.5 hr, but no further change occurred over the next 72 hr. The greater ease with which Zn is released may be thermodynamic rather than kinetic since Zn has a smaller binding constant with MT, compared to that of Cd, yet electrophiles such as gold thiomalate or ESSE have similar rate constants for reaction with Zn,MT and Cd, MT. The Cd,MT-II polymerizes slowly in the presence of 5000 PM GSSG, as indicated by the evolution of a new high MW shoulder on the monomeric MT band in the gel-filtration chromatogram (Fig. 5(b)). A 250 nm shoulder, much broader than the S-Cd charge transfer band, appears in the UV spectrum and does not disappear upon acidification. This behavior indicates that disulfide bond formation is the mechanism of MT polymerization in this case.

OXIDATION

OF RABBIT LIVER METALLOTHIONEIN-II

247

0.6

0.1 0

0

1

2

3

4

5

6

7

8

9

10

Elution Volume, ml

FIGURE 5. Gel chromatograms of reaction mixtures of (a) Zn,MT-II; (b) Cd,MT-II (14.3 PM protein), and 5000 PM GSSG. G-50 Sephadex column (0.5 x 25 cm), eluted with 10 mM NH,HCO,,

pH 7.4. 1.5 hr (--H-I, 24 hr (-O-),48

hr (-A-_) and 72 hr (-*I.

CONCLUSIONS The difference in reactivity of the two disulfide reagents, ESSE and GSSG, towards MT is interesting. GSSG is much less reactive even when it is present in excess over the protein thiolate concentration. The aromatic rings and electron withdrawing substituents of ESSE make it more electrophilic and, hence, more reactive to the nucleophilic attack by the MT thiolates. The role of steric effects is less obvious since GSSG has bulkier substituents, but they are more flexible than the aromatic groups of ESSE. From the small extent and slow rate of metal release from MT found here, it seems unlikely that oxidation by GSSG is a significant factor in the release of MT-bound metal ions in cellular homeostasis.

248

M. M. Savas et al,

The results of the ESSE chromatography experiments indicate that when the protein thiolate concentration significantly exceeds that of ESSE (1:O.l or 1:0.02), several polymerized MT species form. These aggregates contain only CyS-SCy type disulfide bonding and no mixed disulfide (CyS-SE) bonding, indicated by a lack of any spectral features beyond 300 nm. Cd was found to be still bound to the remaining monomeric MT and the polymerized MT species. The extent of Cd loss and thiolate oxidation exceeded that expected from the stoichiometry of ESSE used. This suggests that partial disruption of the cluster structures by ESSE leads to further oxidation and metal loss during the chromatographic separations. The effects observed here are greater than that observed by Minkel et al. [23] upon prolonged exposure of MT to aerobic conditions. After reaction with 0.1 ESSE per MT thiolate, the "'Cd NMR signals of the clusters were lost completely, despite the presence of MT-bound Cd ions. This finding demonstrates that the absence of “‘Cd NMR signals cannot be interpreted as the complete loss of MT-bound cadmium unless strong corroborating evidence is presented. When the protein thiolate to ESSE ratio was l:l, Cd was removed completely, or nearly so, from the protein, since 87-100% was found in the LMW fractions. The formation of mixed disulfides (CyS-SE) was demonstrated by the observation of a UV band at 328 nm in the spectrum of the oxidized protein. The formation of mixed disulfides only at higher ESSE concentrations may be attributed to the relative rate of ESSE attack at successive thiolates compared to the rate of formation of CyS-SCy disulfide bonds according to Eq. (la> and (lb). Higher ESSE concentrations will favor mixed disulfide bonds over the protein disulfide bonds, consistent with the experimental observations. It has been suggested that MT may function as a reversibly oxidized cellular target for O,- and OH mediated damage [34-361. The results of the present study using ESSE as the oxidant demonstrate facile formation of both intraand intermolecular CyS-SCy linkages, consistent with the proposed biological function. This work was suppotied by the National Institutes of Health (Grant No. ES-04026). We thank Dr. Pu Chen and Mr. Jun Xiao for their help in obtaining the “‘Cd NMR spectra.

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