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

RINALDI, DÉBORA; FERREIRA-GOMES, MARIELA S.; SAFFIOTI NICOLÁS A.; MANGIALAVORI IRENE C.; ONTIVEROS, MALLKU; ROSSI, JUAN PABLO

La Plata

Congreso; XLVII reunión anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

The photoaffinity labeling is used to covalently bind the ligands to the proteins to determine their spatial arrangement and structure. These ligands can be natural analogs and allow us to study their binding site. This approach uses a photoactivatable probe, which implies the incorporation of two important functionalities; a unit of specificity, responsible for the reversible binding to the target protein, and a photoreactive group, which allows photoinductible permanent binding to the target protein. In this work, we describe a laboratory exercise to easily demonstrate the permanent photo-inducible binding of a photoactivatable probe, Azido-ruthenium (AzRu), which is used to identify and characterize the Ca2+ binding sites of proteins. AzRu is a photoactivable probe similar to Ca2+ formed by Ru2+, specific unit, and by Azide, the photoreactive group. Therefore, AzRu binds covalently and specifically to the Ca2+ binding proteins after exposure to ultraviolet radiation at 290 nm.The AzRu properties were studied for the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and the plasma membrane Ca2+-ATPase (PMCA). PMCA and SERCA use the hydrolysis of ATP as an energy source to transport Ca2+ from the cytoplasm to the extracellular medium or the reticulum lumen, respectively. The effect of AzRu on Ca2+-ATPase activity was evaluated, using a low-cost colorimetric assay. The Ca2+-ATPases were incubated for 15 minutes with different concentrations of AzRu with or without UV irradiation. Then, both the non-irradiated and the UV-irradiated samples were diluted with the appropriate reaction mixture for the assayed activity. Both PMCA and SERCA were only inhibited by AzRu when the samples were irradiated with UV, suggesting that photoactivation leads to an irreversible binding of the probe to the enzyme.In this way, students demonstrate that AzRu can be linked in a reversible or irreversible (covalently) way to the Ca2+ binding site in a protein, using an easy, low-cost method.Acknowledgments. 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).