This chapter describes the method used successfully to study the Ca2+-binding properties of various high-affinity Ca2+-binding proteins. This method employs equilibrium dialysis in combination with metal chelators to regulate free metal concentration. There are only two parameters measured in this experiment: the free metal concentration and the amount of metal bound at that concentration. For the system discussed in the chapter, the free metal concentration is not measured, but is set using a calculated mixture of Ca2+ and ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA) at a given pH, ionic strength, and temperature. Metal chelators are used to regulate the free metal ion concentration. The second parameter measured is the amount of bound Ca2+ and this is determined by the distribution of 45Ca between the inside and outside of the dialysis bags. Data are presented in the chapter using the equilibrium dialysis-Ca2+ chelator technique. These data are compared to the data obtained directly using a Ca2+ electrode, because data obtained by this alternate technique are independent of any assumptions about EGTA-binding constants. Because both the techniques give exactly the same results, two conclusions can be drawn: (1) the EGTA constants used in the calculations are correct and (2) EGTA itself does not affect Ca2+ binding to calmodulin.
ASJC Scopus subject areas
- Molecular Biology