Measuring the Ca2+-Binding Kinetics of Proteins

Abstract

Ca2+-binding proteins (CBPs) are instrumental in the control of Ca2+ signaling. For the transduction of a change in intracellular Ca2+ concentration into a cellular biochemical or biophysical action, it is necessary for Ca2+ to bind specific Ca2+-binding proteins (CBPs) that relay the Ca2+ signal. The competition for Ca2+ between the various CBPs plays an essential and direct role in this transduction and in the resulting biochemical message. Therefore, a thorough understanding of Ca2+ signaling necessitates appreciating the kinetic properties of all the relevant CBPs. Unfortunately, most conventional techniques used to measure Ca2+-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 Ca2+-binding properties of CBPs following flash photolysis of caged Ca2+. We introduce here the protocols that are necessary for the data collection associated with this technique.