Rate of release of Ca2+ following laser photolysis of the DM-nitrophen-Ca2+ complex

The determination of the rate of release of Ca2+ by pulsed photolysis of the photolabile chelator DM-nitrophen is important for its use in time-resolved physiological studies: the rate of substrate or effector release should be faster than the processes they initiate. Flash photolysis of DM-nitrophen using a 50-ns pulse from a frequency-doubled ruby laser (with emission at 347 nm having energy of ca. 10-20 mJ) yields short-lived photochromic or aci-nitro intermediates. At pH 6.9, double-exponential decay of a photochromic intermediate was observed for DM-nitrophen itself and its Ca2+ complex (tau 1/2 values of 24 and 570 microseconds, and 32 and 220 microseconds respectively), while only monoexponential decay of the DM-nitrophen-Mg2+ complex was detected (tau 1/2 = 31 microseconds). Only the photochemistry of DM-nitrophen-Ca2+ was found to be pH sensitive (monoexponential decay, tau 1/2 approximately 115 microseconds at pH 7.9 and 8.9). Use of the Ca(2+)-sensitive metallochromic dye antipyrylazo III in conjunction with pulsed photolysis of DM-nitrophen-Ca2+ enabled an upper limit of the half-time of release of Ca2+ to be established of ca. 180 microseconds (the rate of association of Ca2+ with the dye was probably rate determining). The rate of Ca2+ photorelease may, however, be faster than this. Thus, the DM-nitrophen-Ca2+ complex releases Ca2+ on photolysis sufficiently rapidly for the study of many Ca(2+)-dependent physiological processes with improved kinetic resolution over conventional mixing methods.