Azido ruthenium, a Ca2+-like photoactivatable reagent used in biophysical chemistry teaching.

ONTIVEROS, M; SAFFIOTI, N; RINALDI, D.E; DELFINO, JM; ROSSI, J.P.; MANGIALAVORI, I.; FERREIRA GOMES, M

La PLata

Congreso; XLVII Reunion Anual de la Sociedad Argentina de Biofisica; 2018

Photoaffinity labeling enables to covalently bind ligands to proteins to determine their relative spatial arrangement. Analogues of natural ligands illuminate structural features of binding sites. A photoactivatable probe implies the incorporation of two important functionalities: (i) a unit that imparts specificity, responsible for the reversible binding to the target protein, and (ii) a photo reactive functional group, allowing photo inducible permanent binding. We describe here a laboratory exercise to straightforwardly demonstrate the light dependent binding of the photo probe azido ruthenium (AzRu) for identifying the Ca2+ binding sites in a protein. Ru2+ mimics Ca2+, the specific unit, and azide is the photoreactive moiety. Hence, AzRu binds specifically and covalently to Ca2+ binding proteins after exposure to ultraviolet radiation at 290 nm. AzRu was assayed with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and the plasma membrane Ca2+-ATPase (PMCA). Both pumps use the hydrolysis of ATP as the energy source to drive Ca2+ from the cytoplasm to the extracellular medium or the SR/ER lumen, respectively. The effect of AzRu on Ca2+-ATPase activity was evaluated, using a low-cost colorimetric assay. Each enzyme was incubated for 15 min with AzRu at different concentrations with or without UV irradiation. Afterwards, both non-irradiated and UV-irradiated samples were appropriately diluted for the enzyme assay. Both pumps show inhibition by AzRu only when UV-irradiation takes place. In this way, using an easy, low-cost method students are able to carry out the key experiment demonstrating the causative link between photoactivation and irreversible binding of the probe to the enzyme. Acknowledgements. This work was supported by grants, facilities and materials provided by the Department of Biological Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires (FFyB-UBA), and by the National Research Council (CONICET).