INTERACTION OF RUTHENIUM RED WITH CA-2+-BINDING PROTEINS

The interaction of ruthenium red, [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]Cl6·4H2O, with various Ca2+-binding proteins was studied. Ruthenium red inhibited Ca2+ binding to the sarcoplasmic reticulum protein, calsequestrin, immobilized on Sepharose 4B. Furthermore, ruthenium red bound to calsequestrin with high affinity (Kd = 0.7 μm; Bmax = 218 nmol/mg protein). The dye stained calsequestrin in sodium dodecyl sulfatepolyacrylamide gels or on nitrocellulose paper and was displaced by Ca2+ (Ki = 1.4 mm). The specificity of ruthenium red staining of several Ca2+-binding proteins was investigated by comparison with two other detection methods, 45Ca2+ autoradiography and the Stainsall reaction. Ruthenlum red bound to the same proteins detected by the 45Ca2+ overlay technique. Ruthenium red stained both the erythrocyte Band 3 anion transporter and the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum. Ruthenium red also stained the EF hand conformation Ca2+-binding proteins, calmodulin, troponin C, and S-100. This inorganic dye provides a simple, rapid method for detecting various types of Ca2+-binding proteins following electrophoresis. © 1990.