Measuring the Ca²⁺-binding kinetics of proteins

Ca(2+)-binding proteins (CBPs) are instrumental in the control of Ca(2+) signaling. For the transduction of a change in intracellular Ca(2+) concentration into a cellular biochemical or biophysical action, it is necessary for Ca(2+) to bind specific Ca(2+)-binding proteins (CBPs) that relay the Ca(2+) signal. The competition for Ca(2+) between the various CBPs plays an essential and direct role in this transduction and in the resulting biochemical message. Therefore, a thorough understanding of Ca(2+) signaling necessitates appreciating the kinetic properties of all the relevant CBPs. Unfortunately, most conventional techniques used to measure Ca(2+)-binding kinetics are too slow to determine accurately the fast binding kinetics of most CBPs. To address this problem, we have developed an ultrafast in vitro technique for measuring the Ca(2+)-binding properties of CBPs following flash photolysis of caged Ca(2+). We introduce here the protocols that are necessary for the data collection associated with this technique.