NITROPHENYL-EGTA, A PHOTOLABILE CHELATOR THAT SELECTIVELY BINDS CA2+ WITH HIGH-AFFINITY AND RELEASES IT RAPIDLY UPON PHOTOLYSIS

The synthesis and properties of a caged calcium are described. The compound is an ortho-nitrophenyl derivative of EGTA. It is synthesized in 10 steps and with 24% overall yield. The photosensitive chelator, nitrophenyl-EGTA, has a K(d) value for Ca2+ of 80 nM and for Mg2+ of 9 mM. Upon exposure to UV radiation (almost-equal-to 350 nm), the chelator is cleaved, yielding iminodiacetic acid photoproducts with low Ca affinity (K(d) = 1 mM). The quantum yield of photolysis of nitrophenyl-EGTA in the presence of Ca2+ is 0.23 and in the absence of Ca2+ is 0.20. In experiments with chemically skinned skeletal muscle fibers, a fully relaxed fiber equilibrated with nitrophenyl-EGTA-Ca2+ Complex, in the presence of 1 mM free Mg2+, maximally contracted after a single flash from a frequency-doubled ruby laser (347 nm). Half-maximal tension was achieved in 18 ms at 15-degrees-C. Nitrophenyl-EGTA provides a tool for the investigation of the mechanism of Ca2+-dependent physiological processes, since under conditions of normal intracellular Ca2+ and Mg2+ concentrations, only Ca2+ is bound by the photolabile chelator and on illumination released rapidly and in high photochemical yield.