New fluorescent reagents specific for Ca²⁺-binding proteins

Abstract

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.

Introduction

Ca²⁺ carries information pivotal to cell life and death via its interactions with specific Ca²⁺-binding sites in proteins. As a ligand, Ca²⁺ has special flexibility and is able to bind to sites with irregular geometry, which makes its binding to proteins particularly easy [1], [2].

Several reagents which interact specifically with Ca²⁺-binding proteins and inhibit their activity have previously been reported. Ruthenium red, [(NH₃)₅Ru–O–Ru(NH₃)₄–O–Ru(NH₃)₅]Cl₆·4H₂O (RuR), a water-soluble hexavalent cation [3], [4], [5], competes for the Ca²⁺-binding site [5]. Ruthenium amine binuclear complex (Ru360), a derivative of RuR and inhibitor of the mitochondrial Ca²⁺ uniporter [6] and some other Ca²⁺-binding proteins [4]. However, these compounds are not very specific and cannot be used for localizing Ca²⁺-binding sites.

Recently, we developed a novel photoreactive reagent, Azido Ruthenium (AzRu), which specifically and irreversibly binds to calcium-binding proteins upon UV irradiation, and strongly inhibits their Ca²⁺-dependent activities [4], [7]. AzRu was used as a photoreactive reagent to characterize the Ca²⁺-binding sites of the mitochondrial voltage-dependent anion channel (VDAC) [8].

Several fluorescent and bioluminescent Ca²⁺ indicators have been developed that enable the monitoring of changes in Ca²⁺ concentration in defined cell compartments (e.g. organelles and cytosolic sub-regions) [9], [10]. In this study, we report on the development of fluorescent indicators which are specific for Ca²⁺-binding proteins, rather than the cation, Ca²⁺. These compounds constitute a new class of fluorescent reagents which can be used to specifically inhibit the activity of Ca²⁺-binding proteins, and to monitor their binding to Ca²⁺-binding proteins thus serving as a novel tool in the study of Ca²⁺-dependent processes.