Kinetic Properties of DM-Nitrophen Binding to Calcium and Magnesium

Caged-Ca2+ compounds such as nitrophenyl-EGTA (NP-EGTA) and DM-nitrophen (DMn) are extremely useful in biological research, but their use in live cells is hampered by cytoplasmic [Mg2+]. We determined the properties of Ca2+ release from NP-EGTA and DMn by using Oregon green BAPTA-5N to measure changes in [Ca2+] after ultraviolet flash photolysis in vitro, with or without Mg2+ present. A large fraction (65%) of NP-EGTA, which has a negligible Mg2+ affinity, uncages with a time constant of 10.3 ms, resulting in relatively slow increases in [Ca2+]. Uncaging of DMn is considerably faster, but DMn has a significant affinity for Mg2+ to complicate the uncaging process. With experimentally determined values for the Ca2+ and Mg2+ binding/unbinding rates of DMn and NP-EGTA, we built a mathematical model to assess the utility of NP-EGTA and DMn in rapid Ca2+-uncaging experiments in the presence of Mg2+. We discuss the advantages and disadvantages of using each compound under different conditions. To determine the kinetics of Ca2+ binding to biologically relevant Ca2+ buffers, such as Ca2+-binding proteins, the use of DMn is preferable even in the presence of Mg2+.