DETERMINATION OF FURA-2 DISSOCIATION-CONSTANTS FOLLOWING ADJUSTMENT OF THE APPARENT CA-EGTA ASSOCIATION CONSTANT FOR TEMPERATURE AND IONIC-STRENGTH

The accurate calibration of Fura-2 fluorescence in living cells is dependent upon the apparent dissociation constant (Kd) of Fura-2 for Ca2+. If Ca-EGTA calibration buffers are used to construct an in vitro calibration curve, then the calculated value of the apparent Ca-EGTA association constant (K'CaEGTA) will have an important influence on the Kd of Fura-2 and thus the calculated free [Ca2+] in cells. In order to simulate experimental conditions, the individual proton and Ca2+ association constants for EGTA in these experiments were adjusted for both ionic strength and temperature using a semi-empirical form of the Debye-Huckel limiting law and the Van't Hoff isochore, respectively, as described by Harrison and Bers [7]. The modified individual binding constants were then employed in the calculation of K'CaEGTA using the SPECS computer program of Fabatio. At pH = 7.05, ionic strength = 0.15 M, temp = 20-degrees-C, K'CaEGTA = 3.232 x 10(6) M-1; at pH = 6.84, temp = 36-degrees-C, K'CaEGTA = 1.652 x 10(6) M-1. These values differed substantially from those obtained with unadjusted individual association constants. Calibration buffers of varying [Ca2+] were prepared using the corrected values of K'CaEGTA, and Fura-2 fluorescence ratios were measured during superfusion of these buffers in the experimental chamber at both 20-degrees-C and 37-degrees-C. The Kd of Fura-2 for Ca2+ was determined to be 236 nM at 20-degrees-C and 285 nM at 37-degrees-C, utilizing the value of K'CaEGTA adjusted by the method of Harrison and Bers. These new Kd constants can be applied in conjunction with in vivo measurements of R'max and R'min to achieve accurate quantitation of cytoplasmic free Ca2+ in a variety of cell types under physiological conditions.