Azido ruthenium: A new photoreactive probe for calcium-binding proteins

Ca²⁺, involved in almost all processes of cell life, mediates its activity through reversible interaction with specific binding sites in proteins. Although several Ca²⁺-dependent activities are known, many of the proteins responsible remain unidentified. Here we describe the synthesis, purification, characterization and potential uses of a new Ca²⁺-like reagent, azido ruthenium (AzRu), which can be photoactivated. AzRu strongly inhibits Ca²⁺ -dependent activities. AzRu can be used to probe proteins in solution or embedded in membranes. AzRu has no effect on Ca²⁺-independent or Mg²⁺-dependent activity. After exposure to ultraviolet irradiation, AzRu binds covalently and specifically to Ca²⁺ -binding proteins, thus providing a new approach for identifying and purifying Ca²⁺-binding proteins, for characterizing their Ca²⁺-binding sites and for exploring previously unknown Ca²⁺-dependent processes. In this protocol we also include a description of the preparation of [¹⁰³Ru]AzRu, which can be used for labeling Ca²⁺-binding sites in proteins and identifying previously unknown Ca²⁺-binding proteins. The preparation of AzRu takes approximately 2-3 days.