New fluorescent reagents specific for Ca²⁺-binding proteins

Ca²⁺ carries information pivotal to cell life and death via its interactions with specific binding sites in a protein. We previously developed a novel photoreactive reagent, azido ruthenium (AzRu), which strongly inhibits Ca²⁺-dependent activities. Here, we synthesized new fluorescent ruthenium-based reagents containing FITC or EITC, FITC-Ru and EITC-Ru. These reagents were purified, characterized and found to specifically interact with and markedly inhibit Ca²⁺-dependent activities but not the activity of Ca²⁺-independent reactions. In contrast to many reagents that serve as probes for Ca²⁺, FITC-Ru and EITC-Ru are the first fluorescent divalent cation analogs to be synthesized and characterized that specifically bind to Ca²⁺-binding proteins and inhibit their activity. Such reagents will assist in characterizing Ca²⁺-binding proteins, thereby facilitating better understanding of the function of Ca²⁺ as a key bio-regulator.