Mzi

] Control HOOjiM NOC-15

] Control HOOjiM NOC-15

Asc alone Asc/Cu5MT Asc/Cu5MT/SOD

Incubation Conditions

Fig. 2. Nitric oxide-dependent reconstitution of apo-ZnSOD by Cu-MT. The intramolecular transfer of copper from c115-MT to apo-ZnSOD in the presence and absence of 100 fiM NOC-15 was performed as outlined in protocol 2. (A) Resultant SOD activity measured on the basis of the inhibition of superoxide-driven nitroblue tetrazolium reduction. Insets: Corresponding SOD activity gels developed after native polyacrylamide gel electrophoresis was applied to the samples. (B) Similar incubation with Cu5-MT and apo-ZnSOD (both 1 fiM) with and without 100 fiM NOC-15 but conducted in the presence of 40 11M ascorbate (Asc) as a reporter molecule of copper-dependent redox cycling. Redox-active copper catalyzes the one-electron oxidation of ascorbate, which was measured by electron paramagnetic resonance (EPR) of ascorbate radical. Data represent the magnitude of the EPR ascorbate radical signal recorded over 8 min. Inset: Magnitude of the ascorbate radical signals obtained with cu5-MT alone in the absence (a) and presence (b) of the NO donor; the magnitudes were essentially indistinguishable from background signal obtained in the absence of any MT. Note that the transfer of copper from MT to apo-ZnSOD was not accompanied by any enhanced redox activity of copper.

Asc alone Asc/Cu5MT Asc/Cu5MT/SOD

Incubation Conditions

Fig. 2. Nitric oxide-dependent reconstitution of apo-ZnSOD by Cu-MT. The intramolecular transfer of copper from c115-MT to apo-ZnSOD in the presence and absence of 100 fiM NOC-15 was performed as outlined in protocol 2. (A) Resultant SOD activity measured on the basis of the inhibition of superoxide-driven nitroblue tetrazolium reduction. Insets: Corresponding SOD activity gels developed after native polyacrylamide gel electrophoresis was applied to the samples. (B) Similar incubation with Cu5-MT and apo-ZnSOD (both 1 fiM) with and without 100 fiM NOC-15 but conducted in the presence of 40 11M ascorbate (Asc) as a reporter molecule of copper-dependent redox cycling. Redox-active copper catalyzes the one-electron oxidation of ascorbate, which was measured by electron paramagnetic resonance (EPR) of ascorbate radical. Data represent the magnitude of the EPR ascorbate radical signal recorded over 8 min. Inset: Magnitude of the ascorbate radical signals obtained with cu5-MT alone in the absence (a) and presence (b) of the NO donor; the magnitudes were essentially indistinguishable from background signal obtained in the absence of any MT. Note that the transfer of copper from MT to apo-ZnSOD was not accompanied by any enhanced redox activity of copper.

2. Cu-MT containing various molar ratios of copper to MT ranging from Cup MT to Cuio-MT can be prepared by incubating Zn-MT (freshly reduced as described in protocol 1) in the presence of varying concentrations of CUSO4 in 50 mM phosphate buffer. For example, to prepare Q15-MT 40 \lM Zn-MT is incubated with 200 ¡xM CuS04 in 50 mM phosphate buffer containing 800 ¡xM sodium ascorbate for 20 min at room temperature. Ascorbate is included to maintain copper ions in the cuprous (Cu+) state required for binding to MT. The much higher affinity of MT for copper relative to zinc assures the rapid equilibration and stoichiometric addition of copper to MT at subsaturating copper concentrations.

3. Coincubations of Cu-MT and apo/ZnSOD are performed by combining Cu-MT with apo/ZnSOD (both at 10 ¡xM) in 0.1 ml of 50 mM phosphate buffer and incubating for 2 hr at room temperature. Oxidants, NO generators, or other thiol-active agents can be added to parallel tubes to assess the requirement of SH modification for copper transfer. EDTA (0.1 mM, final concentration) is added at the end of incubation to prevent further reconstitution. After incubation the activity of SOD can be directly measured by the inhibition of superoxide-driven nitroblue tetrazolium reduction20 or by SOD activity gels developed after native polyacrylamide gel electrophoresis.21

Experiments with Intact Cells. The modification of MT SH groups has been implicated as a mechanism to account for the reported antioxidant activity of MT, as well as modulation of metal binding and release. However, describing the quantitative relationships between MT oxidation/nitrosation and MT function within cells is often compromised by lack of sensitivity and the problems inherent in complex mixtures of intracellular protein that contain relatively small amounts of MT. Therefore, chromatographic separation of MTs in conjunction with sensitive biochemical analysis of cysteine redox status is required to achieve fully quantitative results under these conditions. Protocol 3, given below, describes our approach to assess the redox status of MT in intact cells.

Protocol 3: NO-Dependent Modification of Metallothionein Thiols in Intact HL-60 Cells

1. MT can be induced in actively growing HL-60 cells by treatment with 150 ¡xM ZnCl2 in RPMI 1640 containing 12% (v/v) fetal bovine serum (FBS) at 37° under a 5% C02 atmosphere for 48 hr. ZnCl2 is removed by centrifugation and the recovered cells can then be returned to culture for exposure to SH-modifying conditions, such as the NO donor NOC-15 (1 mM) for 1 hr.

2. Cells (lxlO8 per treatment) are then recovered by centrifugation (400g, 10 min, 4°), washed twice with cold phosphate-buffered saline (PBS), and

20 J. F. Ewing and D. R. Janero, Anal. Biochem. 232, 243 (1995).

21 C. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276 (1971).

subjected to sonication for 1 min in 1 ml of ^-saturated 50 mM phosphate buffer, pH 7.8, containing 2 mM EDTA, using a 4710 series ultrasonic homogenizer (Cole-Palmer Instrument, Chicago, IL).

3. The resulting homogenate is cleared by centrifugation at 40,000g for 20 min at 4°. The supernatant is then applied to a Sephadex G-75 column (1 x 40 cm) and eluted with N2-saturated 10 mM Tris-HCl, 2 mM EDTA, pH 7.8. The column is run at 4° at a flow rate of 0.5 ml/min and fractions (0.5 ml) are collected.

4. The SH group content of each fraction can be determined by adding a 2O-/1I aliquot of each fraction to 5 /xl of 10 mM dithiodipyridine in 0.5 M sodium acetate, pH 4.0. After incubation at room temperature for 30 min the absorbance of each sample is read at 343 nm, using a SpectroMate UV-Vis fiberoptic micro-spectrophotometer (World Precision Instruments) equipped with a 15-/U.1 capillary microcuvette with internal reflecting surface and a resulting optical band path of 10 cm.

5. In addition, the presence of MT protein in the fractions can be assessed by an immuno-dot-blot procedure.22 Briefly, a 20-/U.1 aliquot of each fraction is mixed with 20 /A of 3% (v/v) glutaraldehyde and applied to a nitrocellulose membrane, using a Bio-Dot microfiltration apparatus (Bio-Rad, Hercules, CA). The membrane is blocked with 5% (w/v) fat-free milk in TBST [50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 0.05% (v/v) Tween 20] for 1 hr and then incubated with E9 mouse monoclonal anti-MT antibody (Dako, Carpintería, CA)(1:500 in TBST). After washing (six times with TBST), horseradish peroxidase-conjugated polyclonal goat anti-mouse IgG (PharMingen, San Diego, CA) is added for 1 hr (1:5000 in TBST). The signal is developed by enhanced chemiluminescence, using a SuperSignal West Pico chemiluminescence kit (Pierce Chemical, Rockford, IL) and exposure to X-ray film.

The first hurdle to overcome is the relative lack of sensitive biochemical methods to measure small changes in the relatively low amounts of MT available in cells. Various fluorigenic thiol-specific probes such as ThioGlo (Covalent Associates, Woburn, MA) are available but we have found their specific application to MT to be problematic. Such probes appear to grossly underestimate the amount of available thiols, presumably because of difficulty in removing bound metal ions that may competitively inhibit ThioGlo interaction with SH groups. In addition, nearly all the MT cysteines within the two thiolate clusters represent contiguous (Cys-Cys) or vicinal (Cys-X-Cys) pairs. Therefore, reaction between ThioGlo and one cysteine may limit the accessibility of SH-active reagent to the neighboring cysteine. Alternatively, a significant self-quenching of fluorescence may also limit

22 C. A. Mizzen, N. J. Cartel, W. H. Yu, P. E. Fraser, and D. R. McLaughlin, J. Biochem. Biophys.

fluorescence yield when multiple reporter molecules exist in close proximity to each other within the thiolate clusters of MT.

We, therefore, have chosen to apply traditional spectrophotometric titration with the thiol reagent 2,2'-dithiodipyridine (DTDP) combined with contemporary fiberoptic-based sensitive spectrophotometry. We utilize a SpectroMate UV-Vis fiberoptic microspectrophotometer (World Precision Instruments) supplied with a 15-/xl capillary microcuvette with internal reflecting surface and a resulting optical band path of 10 cm. This provides for highly sensitive measurement of weakly absorbing samples in microvolumes.

The second difficulty is to specifically measure MT SH groups in the presence of much higher amounts of glutathione (GSH) and high molecular weight (HMW) protein thiols. For this, we have taken advantage of the small size of MT and applied gel-filtration chromatography to resolve an MT-enriched fraction separate from other proteins as well as low molecular weight thiols. Figure 3 shows typical elution profiles of DTDP-reactive thiols contained in cell lysates derived from intact control HL-60 cells (solid stars) and cells treated with the MT inducer zinc for 24 hr (solid circles). Note the prominent appearance of an intermediate peak of SH groups between HMW proteins and glutathione observed after zinc pre-treatment, which most likely represents MT cysteines. It is important, however, to firmly establish the presence of MT protein by an independent measure. To this end immunological identification of MT protein, which is likely to be independent of its redox status, may be performed. Both monoclonal and polyclonal MT-specific antibodies have been used.23'24 As shown in Fig. 3B, treatment of HL-60 cells with zinc causes a manyfold increase in the amount of immunoreactive MT (detected with E9 monoclonal antibody; Dako) within fractions corresponding to the intermediate peak of thiols. No immunoreactivity is detected in untreated control cells. In addition, MT immunoreactivity is never observed in the HMW or GSH fractions. This confirms that MT is confined to these intermediate fractions. This alone, however, does not exclude the presence of other SH-containing proteins similar in molecular mass to MT within this fraction. This issue can be resolved, at least in part, by quantifying the total protein content in the fraction and comparing its molar content of SH groups with the predicted 20:1 ratio in intact MTs.

After characterizing this peak as representing MT thiols, assessment of redox status after oxidative/nitrosative challenge can be performed. Figure 3A demonstrates that the amount of cysteines within the MT peak is decreased by about 70% after exposure of live zinc-pretreated cells to a donor of nitric oxide, NOC-15. Essentially no change is observed in other protein thiols, and the decrement in GSH is less than that observed in MT, suggesting that MT cysteines

23 B. Jansani and M. E. Elmes, Methods Enzymol. 205, 95 (1991).

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