Re-Examination of the Apparent Binding Constant of Ethylene Glycol Bis(β-Aminoethyl Ether)-N,N,N',N'-Tetraacetic Acid with Calcium around Neutral pH12

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Abstract
The apparent binding constant (Kapp(Ca-G)) for GEDTA (ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, EGTA) to calcium was determined under conditions of biological significance in the presence of various kinds of pH-buffering agents, using murexide or tetramethylmurexide as a Ca indicator. The value of Kapp(Ca-G) at pH 6.80 was 1.0 × 106M−1 at an ionic strength of 0.114 at 20°C, irrespective of the type of pH-buffering ions. This value is similar to that of Allen, Blinks and Prendergast (1977) (Science196, 996–998), but still half that calculated from the results of Schwarzenbach, Senn and Anderegg (1957) (Helv. Chim. Acta40, 1886–1900). The value of Kapp(Ca-G) varied according to the following equation as the ionic strength (I) was varied from 0.039 to 0.264: log Kapp(Ca-G)=6.460−[2□I/(1+□I)−0.4×1] (pH 6.80, 20°C) The discrepancy between the present results and previous ones (Ogawa, Y. (1968) J. Biochem. 64, 255–257) may have been due to inadequate regulation of the temperature of the reaction medium in the previous determinations, during which an increase in the temperature of the solution may have occurred. An increase of temperature causes a decrease in the pH of the solution in the presence of histidine, imidazole or Tris-maleate, but causes very little change of pH in the presence of phosphate or maleate. The association rate constant for GEDTA with calcium was determined by the stopped-flow method in solutions containing 100 mM KCl and 20 mM pH-buffering ions at 20°C: the values obtained were 1.4 × 106M−1s−1 in the presence of MOPS-KOH at pH 6.80; 3.0 × 106 M−1 s−1 with imidazole at pH 6.80; 1.0 × 106 sc−1s−1 with Tris-maleate at pH 6.80.