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Timestamp: 2019-04-25 08:50:25+00:00

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It has been speculated, as well, that MT can play an active role in maintaining metal ion homeostasis through delivery or sequestration of important endogenous metal ions. For example, Maret and Vallee11 and Jacob and co-workers12 have suggested that MT can deliver zinc to a variety of enzymes that require this metal for biological activity. Similarly, we have shown that MT can deliver copper ion to zinc-replete apo-superoxide dismutase (apo-SOD) in a nitric oxide-dependent fashion.13 Alternatively, apometallothionein can serve to remove zinc from specific sites on proteins, such as caspase and glyceraldehyde-3-phosphate dehydrogenase and, thus, activate their function by removal of the zinc-dependent inhibition.14 All these studies have invoked a critical role for redox regulation in determining metal binding and delivery by MT. Remarkably, MTs are not prone to the traditional posttranslational modifications that typically modulate protein function. No evidence of MT phosphorylation, palmitoylation, farnesylation, or controlled proteolysis has been presented. This leaves the thiolate clusters and their redox conversions as a likely mode for regulation of MT function. Thus, knowledge of redox conversion of MT cysteines is central to understanding the regulatory roles and pathways of this important metal-binding protein. The purpose of this chapter is to describe contemporary approaches to assess the redox status of MT cysteines and their relationship to MT metal sequestration and release.
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availability of large amounts of pure protein permits the application of conventional analytical techniques to study metal binding and cysteine redox status in cell-free model systems. In fact, such preparations of purified MT are available commercially. We have found, however, that the redox state of many of these available MTs needs to be carefully characterized before use. It is commonly observed that the number of reduced cysteines is significantly lower than that theoretically predicted by the amino acid sequence (20 mol of SH groups per mole of MT). Therefore, an obligatory treatment of MTs with a reducing agent such as dithiothreitol (DTT) is required in order to reconstitute the full complement of cysteines. It is necessary to be cautious and verify that the number of cysteines after this treatment is indeed close to the theoretical maximum for the amount of protein present. Moreover, systematic monitoring of MT SH groups needs to be conducted during its storage. We have, indeed, observed that some MTs stored for prolonged periods of time fail to reconstitute the full complement of cysteines after DTT treatment, suggesting their oxidation beyond simple disulfides. A detailed protocol for the preparation of reduced MT and characterization of thiol group status is given below.
1. Five milligrams of rabbit Zn-MTII (Sigma, St. Louis, MO) is dissolved in 4.165 ml of 10 mM Tris-HCl, pH 8, to yield an approximately 200 /iM MT stock solution and frozen in 200-/ul aliquots at —80°. Tris buffer and all other solutions are prepared with Chelex-treated water to remove any adventitious metals. Solutions are also purged for 5 min with N2 to remove dissolved oxygen. These precautions minimize the spontaneous oxidation of MT during storage.
2. To prepare freshly reduced MT, 10 /xl of 100 mM DTT prepared in water is added to 200 /¿I of MT stock and incubated at room temperature for 30 min.
3. The resultant mixture is split into two equal portions and applied to the tops of Microcon YM-3 centrifuge filters (3000 MW cutoff; Millipore, Bedford, MA) and centrifuged at 1300g for 30 min at 4°. The filtrate is discarded and another 100 fj.1 of fresh 10 mM Tris, pH 8.0, is added to the top of the filter. This is repeated for a total of three centrifugations and the final retentates are combined, transferred to a clean microcentrifuge tube, and brought to 200 /¿I with 10 mM Tris buffer.
4. The exact protein concentration of MT should be determined by preparing a 100-fold dilution of MT in 0.1 M HC1 and measuring the absorbance at 220 nm (£220 = 48,200 M"1 cm-1). Recovery of MT is, on the basis of original weight, approximately 30-50%.

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