The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA

The apparent calcium association constants (K′_Ca) of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and 1,2-bis[2-bis(o-amino-5-bromophenoxy)ethane-N,N,N′,N′-tetraacetic acid (dibromo-BAPTA) were measured using the method described by Bers (Am. J. Physiol. 242 (1982) C404-408). The purity of the three ligands determined from the x-intercept of Scatchard plots were 96.3%, 79.0% and 97.3% for EGTA, BAPTA and dibromo-BAPTA, respectively. The impurity of BAPTA was found to be water by drying several samples to constant weight. Increasing temperature from 1 to 36°C led to an increase in K′_Ca which was of similar magnitude for the three ligands. Increasing ionic strength from 0.104 to 0.304 M led to a reduction of K′_Ca in all cases, though EGTA was affected much less than BAPTA or dibromo-BAPTA. Experimental results were compared with values of K′_Ca calculated from the individual association constants of the ligands for calcium and protons which were modified for the experimental conditions using the Debye-Hückel limiting law and the Van't Hoff Isochore to correct for ionic strength and temperature, respectively. The experimental values of K′_Ca of EGTA agree well with those in the literature and with the calculated values. Good agreement was also found between the experimental and calculated values of K′_Ca for the temperature and ionic strength dependence of BAPTA and dibromo-BAPTA.