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Azido ruthenium, a Ca2+-likephotoactivatable reagent used in biophysical chemistry teachingOntiveros, M*; Saffioti, N*; Rinaldi, D; Delfino, JM; Rossi, JP;Mangialavori, IC; Ferreira-Gomes, M. Departamentode Química Biológica and Instituto de Química y Fisicoquímica Biológicas ?Prof.Alejandro Paladini?, (IQUIFIB, UBA-CONICET), Facultad de Farmacia y Bioquímica,Universidad de Buenos Aires. irenem@qb.ffyb.uba.ar; msferreiragomes@qb.ffyb.uba.ar. *Both authors contributed equally to this work. Photoaffinity labelingenables to covalently bind ligands to proteins to determine their relativespatial arrangement. Analogues of natural ligands illuminate structuralfeatures of binding sites. A photoactivatable probe implies the incorporationof two important functionalities: (i) a unit that imparts specificity,responsible for the reversible binding to the target protein, and (ii) a photoreactive functional group, allowing photo inducible permanent binding. Wedescribe here a laboratory exercise to straightforwardly demonstrate the lightdependent binding of the photo probe azido ruthenium (AzRu) for identifying theCa2+ binding sites in a protein. Ru2+ mimics Ca2+,the specific unit, and azide is the photoreactive moiety. Hence, AzRu bindsspecifically and covalently to Ca2+ binding proteins after exposureto ultraviolet radiation at 290 nm. AzRu was assayed withthe sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and the plasmamembrane Ca2+-ATPase (PMCA). Both pumps use the hydrolysis of ATP asthe energy source to drive Ca2+ from the cytoplasm to theextracellular medium or the SR/ER lumen, respectively. The effect of AzRu on Ca2+-ATPaseactivity was evaluated, using a low-cost colorimetric assay. Each enzyme wasincubated for 15 min with AzRu at different concentrations with or without UVirradiation. Afterwards, both non-irradiated and UV-irradiated samples wereappropriately diluted for the enzyme assay. Both pumps show inhibition by AzRuonly when UV-irradiation takes place. In this way, using an easy, low-costmethod students are able to carry out the key experiment demonstrating the causativelink between photoactivation and irreversible binding of the probe to theenzyme. Acknowledgements. This work was supportedby grants, facilities and materials provided by the Department of BiologicalChemistry, School of Pharmacy and Biochemistry, University of Buenos Aires(FFyB-UBA), and by the National Research Council (CONICET).

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