Noves aportacions a l'estudi de la capacitat coordinant de les metal·lotioneïnes: el catió Ag(I) com a substitut de Cu(I) en les Cu(I)-MT i l'espectrometria de masses (ESI-MS) com a tècnica essencial i complementària a l'espectroscòpia òptica

Les metalotioneines (MT) son unes proteines de baix pes molecular i elevat contingut en residus de cisteina que es caracteritzen per la seva extremada habilitat para enllacar tant cations de metalls essencials, com Zn(II) i Cu(I), com de metalls toxics (Cd(II), Hg(II)). En una primera part daquest treball sanalitza la utilitzacio del catio Ag(I) com a sonda del catio Cu(I) en lestudi de les Cu-MT, i en consequencia es compara detalladament el comportament dambdos metalls enfront la isoforma 1 de MT de mamifer. Aquesta proteina, aixi com els seus fragments constitutius a i b, sha obtingut per enginyeria genetica amb la finalitat dassegurar al maxim el seu grau de puresa. Lestudi de la capacitat coordinant de MT 1, aMT 1 i bMT 1 enfront Ag(I) sha dut a terme mitjancant tecniques despectroscopia electronica (CD i UV-Vis) i de masses (ESI-MS). Aquesta darrera tecnica ha requerit la incorporacio dun metode de separacio previ a la determinacio de la massa, per tal deliminar la presencia de tampo a la mostra. Lelectroforesi capil·lar (CZE) acoblada a lESI-MS ha demostrat ser una bona opcio. Amb la finalitat de validar lacoblament CZE-ESI-MS, en primer lloc sha aplicat lesmentat acoblament a lestudi del desplacament del Zn(II) en Zn7-MT 1 per Cd(II) a pH 7, proces ja conegut en les MT de mamifer que procedeix de manera sequencial i isomorfa. Aquest estudi demostra que la tecnica CZE permet una bona separacio del tampo de la solucio que conte la proteina, a la vegada que ofereix una deteccio precisa de lestequiometria de les especies que coexisteixen en solucio. En base a aquests resultats, sha seguit en paral·lel -mitjancant CD, UV-Vis i CZE-ESI-MS- la valoracio de Zn7-MT 1, Zn4-aMT 1 i Zn3-bMT 1 amb AgClO4 a pH 7.5 i 2.5. Els resultats mostren que al llarg de les valoracions es forma un elevat nombre despecies, tant heterometal·liques AgxZny-MT com homometal·liques Agx-MT, i que en la majoria dels punts de valoracio coexisteixen diverses especies. Tambe sha observat que el zinc exerceix un efecte plantilla en les 3 proteines estudiades, ja que el grau destructuracio despecies Ag-MT de la mateixa estequiometria depen de manera important de que lespecie inicial sigui Zn-MT o apo-MT. La comparacio Ag-Cu permet concloure que els unics casos en que lio Ag(I) es podria utilitzar com a substitut de Cu(I) sense causar alteracions estructurals significatives es dona en les especies heterometal·liques M4Zn5-MT i M7Zn3-MT (M= Ag, Cu). En la segona part del treball sha estudiat una nova MT de Drosophila melanogaster, denominada MTO, amb lobjectiu de contribuir a un criteri funcional de classificacio de les MT en Zn- o Cu-tioneina. La proteina MTO, que disposa de 12 Cys en la seva cadena aminoacidica, es biosintetitza com a Zn4-MTO en medis rics en Zn, mentre que en medis enriquits en Cu sobte lespecie Cu9-MTO. El conjunt de resultats, tant in vivo com in vitro, permet classificar la MTO com una Cu-tioneina, es a dir, amb preferencia coordinativa respecte aquest metall en medis fisiologics. Tambe, per primera vegada shan aportat evidencies estructurals sobre la participacio dels ions clorur en la coordinacio dions metal·lics en MT, concretament en les especies M4-MTO (M = Zn, Cd). Metallothioneins (MT) are ubiquitous low molecular weight cysteine-rich metalloproteins, which characterize by their extremely high binding abilities towards essential metal cations, such as Zn(II) and Cu(I), as well as toxic cations like Cd(II) and Hg(II). In the first stage of this work the use of Ag(I) as a probe for Cu(I) in Cu-MT studies is analysed, and thus, the behaviour of both metal ions when binding to the mammalian MT 1 isoform has been compared. This protein, as well as their constitutive a and b fragments, was obtained by genetic engineering in order to assure the highest purity degree. The binding abilities of Ag(I) to MT 1, aMT 1 and bMT 1 have been studied by using optical spectroscopy (CD, UV-vis) and mass spectrometry (ESI-MS) techniques. The latter required the use of a separation method previous to the mass determination in order to remove the buffer present in the sample. With the aim of validating the CZE-ESI-MS coupling, we first studied the Zn(II)/Cd(II) replacement in Zn7-MT 1 at pH 7, a well known system in mammalian MT that proceeds by means of a sequential and isomorphous Zn/Cd replacement. This study proves that the previous coupling allow a good separation of the protein from the buffer, while achieving a precise detection of the stoichiometries of the coexisting species in solution. Based on these results, the titrations of Zn7-MT 1, Zn4-aMT 1 and Zn3-bMT 1 with AgClO4 at pH 7.5 and 2.5 were monitored in parallel by CD, UV-vis and CZE-ESI-MS techniques. The results obtained throughout the titration show formation of a high number of heterometallic AgxZny-MT as well as homometallic Agx-MT species. Also, at each titration point, there is coexistence of several species. It has also been shown the Zn template effect in the 3 proteins studied as revealed by the different folding found for the Ag-MT species of the same stoichiometry formed either from Zn-MT or from apo-MT. Comparison of the behaviour of Ag(I) and Cu(I) toward mammalian MT 1 allows us to conclude that the only cases in which the Ag(I) ion can be used as a probe for Cu(I) without causing significant structural alterations are in the heterometallic M4Zn5-MT and M7Zn3-MT (M= Ag, Cu) species. In the second stage of this work, a new Drosophila melanogaster MT, named MTO, has been studied with the aim of contributing to a new functional criterion of classifying MT as Zn- or Cu-thioneins. The MTO protein, which includes 12 Cys residues in the aminoacidic chain, was biosynthesised as Zn4-MTO in a Zn enriched media, while in a Cu supplemented media the Cu9-MTO species was obtained. The overall results, both in vivo and in vitro, allow us to propose classification of MTO as a Cu-thionein, in agreement with the coordination preference shown by the protein toward this metal ion. Furthermore, unprecedented structural evidences showing the participation of chloride ions as ligands of the metal ions in MT is reported. This was found in the case of M4-MTO (M = Zn, Cd) species.