Categoría 3: Corresponsabilidad de la sociedad civil

To a sample of deoxygenated metal-free (apo-) MT 1a, solid 113CdCl2 was added directly to the solution at a ratio that would lead to complete metalation for either of the

isolated domains (3 or 4 Cd2+ per β or α-rhMT, respectively), or the full MT protein (7 Cd2+ per βα-rhMT 1a). The solution was neutralized by the stepwise addition of deoxygenated concentrated ammonium hydroxide. MT spontaneously remetalates when neutralized. The solution is then rigorously evacuated to remove any oxygen that may have entered the solution. A fine G-25 Sephadex size-exclusion column was buffer equilibrated with 5 mM ammonium formate that had been pH adjusted to 7.4 with deoxygenated concentrated ammonium hydroxide. The isotopically enriched MT sample was then desalted into the 5 mM ammonium formate solution, pH 7.4 in order to remove any excess salt of 113Cd2+ ions that may be present. The solution was then concentrated to 8 mL using an Amicon stirred ultrafiltration cell model 8200. The MT solution was further concentrated to 1-2 mL using an Amicon stirred ultrafiltration cell model 8010. The final concentration was determined by UV absorption spectroscopy through a thousand fold dilution of an aliquot of the isotopically enriched MT sample.

2.3 Instrumental Techniques

Metallothionein binds a wide array of metals, ranging from physiologically crucial metals, such as Zn2+ and Cu+, to the toxic metals, such as Cd2+ and Hg2+. We outline several techniques, which have been critical to the understanding of metal binding. Critical to this thesis include UV absorption, circular dichroism and nuclear magnetic resonance spectroscopy, as well as electrospray ionization mass spectrometry. A brief overview of these techniques and the information that can be extracted from them is discussed. A section describing the computational modeling that was used to calculate the qualitative models of supermetalated MT is also presented.

2.3.1 UV absorption spectroscopy

Metallothionein exhibits a number of characteristic spectroscopic signatures that depend on the type of metal bound. Because of the lack of either aromatic amino acids or a defined secondary structure, such a β sheet or an α helix, in the apo-form (1-2) it is far easier to measure spectroscopic properties that are entirely dependent on both the type and number of metals bound, than is the case with many metalloproteins (3-5). UV- visible absorption and circular dichroism spectroscopies have been the key techniques in

the determination of the relative affinities of metallothioneins for metal ions, the mode of metal coordination and possible stoichiometries of these metals. In particular, the lack of aromatic amino acid residues allows UV absorption spectroscopy to monitor the ligand- to-metal charge transfer bands above 220 nm without interference. These techniques are widely used to monitor metalation reactions of d10 metals, such as Zn2+, Cd2+, Hg2+, Cu+ and Ag+ (6-11). Typically, a titration with a metal ion leads to a change in the UV absorption spectrum of the solution demonstrating either binding of the metal to the metal free protein, or, alternatively displacement of an already present metal. The former is used to determine binding stoichiometries for high affinity metals, while the latter can be used to determine binding affinity and possible mixed metal species. If, however, the incoming metal ion has a low binding affinity, then caution should be taken in directly correlating a spectroscopically-determined maximum with a particular metal binding stoichiometry.

Figure 2-3. UV absorption spectroscopy of rabbit liver metallothionein 2 (βα-

rlMT-2) as a function of increasing additions of aliquots of Cd2+. The concentration of protein was 10 μM, while spectra were recorded at pH 7. There are 11 lines: line 1, apo-βα-rlMT-2 at pH 2; line 2, apo-βα-rlMT-2 at pH 7; lines 3-11 for apo-βα-rlMT-2 with different concentrations of Cd2+ (1.06, 2.14, 3.5, 5.0, 5.95, 6.05, 6.92, 7.61, and 8.82 molar equivalent). It should be noted that as the amount of Cd2+ added to the solution increases, the absorbance at 250 nm increases as well. The inset represents changes in the intensity as monitored by the ligand-to-metal charge transfer band of cadmium thiolate at 250 nm, as a function of molar equivalents of Cd2+ added. Adapted from (12) with permission from ASBMB Copyright 1987.

Figure 2-3 shows a classical titration of rabbit liver apoMT-2 with Cd2+ monitored by UV-absorption spectroscopy between 210 and 290 nm (12). Initially, the absorbance is associated with the protein backbone, the metal-free apo-MT. However, upon addition of Cd2+ a shoulder forms at 250 nm, which corresponds to the ligand-to-metal charge transfer of cadmium-thiolate bonds. The UV absorbance at 250 nm is at a maximum at 7 Cd2+ atoms, corresponding to fully metalated Cd7-βα-MT-2. Complete binding of the Cd2+ ions is the result of the high affinity of cysteinyl sulfurs for Cd2+, 6.0x1021 – 1025.6 M-1 (13-14). While the stoichiometry of metal binding may be extracted from UV absorption spectra, it is the CD spectra that clearly show changes in the metal-binding sites indicative of a transition from individual CdS4 sites to the formation of the polynuclear clusters (15). The maximum of seven Cd2+ per βα-MT has been confirmed by ESI-MS measurements for MTs from many different mammalian sources, for example, human MT (16-17). In the case shown in Figure 2-3, we see a slight reduction in absorbance at 250 nm, which has been interpreted as due to non-specific Cd2+ associating with the protein. That these ions do not change the CD band envelope morphology is strong evidence that the Cd7S20 domain structure is not directly affected.

While metallothionein is key in the cellular homeostasis of both Zn2+ and Cu+, as d10 metals, their spectroscopic signatures are essentially limited to the ligand-to-metal charge transfer bands (thiolate to Zn2+ and thiolate to Cu+). On the other hand, metalation of metallothionein using metal ions with an incomplete d shell allows dd transitions to be monitored. These transitions are sensitive to the coordination geometry of the metal atom, and coupled with information provided by the ligand-to-metal charge transfer bands, can allow assignment of both the coordinating ligands and the metal binding geometry. In the following three examples of Co2+, Ni2+ and Fe2+, a maximum metal binding stoichiometry of 7 metal atoms has been determined spectroscopically. The spectral data for the binding of Co2+ to metallothionein demonstrates that the protein enforces a distorted tetrahedral arrangement in which the α-domain has more tetrahedral character than the β-domain (18-21). The spectroscopic data for Ni2+ binding to metallothionein does not have distinct dd transitions, however, the spectrum was similar to Ni2+ bound to azurin, and this suggests a tetrahedral coordination. Fe2+ has also been shown to bind in a tetrahedral manner, with a dd transition in the near infrared

region (22). While these metals may not be naturally associated with the protein, their spectroscopic characteristics are useful in determining the flexibility of the protein in accommodating different metals.