Synthesis and characterisation of Thio-H, a new excitation and emission ratioable fluorescent Ca2+/Mg2+ indicator with high brightness

A new fluorescent ratiometric indicator for Ca2+ and Mg2+, tricaesium salt of {2-[bis(carboxymethyl)amino]-5(5-phenylthiophen-2-yl) phenoxy} acetic acid, Thio-H, has been synthesised and evaluated for its cation binding properties via fluorimetric titrations. Thio-H has the interesting property of being both excitation and emission ratioable, expanding the range of possible applications. The in vitro dissociation constant, K-d, measured at 20degreesC in 100 mM KCl, pH 7.20 for the Ca2+-Thio-H complex is 45 +/- 13 muM; for Mg2+-Thio-H a K-d value of 5.6 +/- 0.6 mM is found. The free form of Thio-H has a high fluorescence quantum yield (0.74), which decreases to 0.50 and 0.65 upon binding to Ca2+ and Mg2+, respectively. Measurements under physiological conditions show that increasing the temperature from 20 to 37degreesC decreases the affinity of Thio-H for Ca2+. Changing the pH from 7.05 to 7.40 does not affect the K-d value of the Mg2+ complex but it increases somewhat the affinity of the probe for Ca2+. In the presence of 1 mM Mg2+, the Ca2+ affinity of Thio-H decreases (K-d = 126 +/- 46 muM at 20degreesC, pH 7.05). Time-resolved fluorescence measurements confirm that the inflection point in the fluorimetric titration curve can be correctly assigned to K-d. Since the dissociation constant for the Mg2+-indicator complex falls within the intracellular [Mg2+] domain, while the K-d value for Ca2+ is well above the basal Ca2+ levels, Thio-H has potential applications as a Mg2+ indicator. The low affinity for Ca2+ can be exploited for detecting intracellular Ca2+ levels in the micromolar range, on condition that [Mg2+](i) remains practically constant during the measurements. Thio-H provides an excellent addition to the commercial ratiometric low-affinity Ca2+ indicators Mag-fura-2, Mag-fura-5, Mag-indo-1, Fura-FF, and BTC.