New fluorescent reagents specific for Ca(2+)-binding proteins

Ca(2+) 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 Ca(2+)-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 Ca(2+)-dependent activities but not the activity of Ca(2+)-independent reactions. In contrast to many reagents that serve as probes for Ca(2+), FITC-Ru and EITC-Ru are the first fluorescent divalent cation analogs to be synthesized and characterized that specifically bind to Ca(2+)-binding proteins and inhibit their activity. Such reagents will assist in characterizing Ca(2+)-binding proteins, thereby facilitating better understanding of the function of Ca(2+) as a key bio-regulator.