AFFINITY AND STOICHIOMETRY OF CALCIUM-BINDING BY ARSENAZO-III

Arsenazo III forms a 1:1 complex with Ca. The affinity constant of arsenazo III for Ca (pKCa) was determined by titrating purified arsenazo III with standard Ca solutions. The method of evaluation used allows correct pKCa values even in the presence of .mu.mol amounts of contaminating Ca. The pKCa is influenced by the following factors: in the neutral pH range the apparent pKCa increases strongly with pH; alkali ions bind weakly to arsenazo III and .mu.mol concentrations cause a decrease in the apparent pKCa; the Mg affinity of arsenazo III, although much lower than the Ca affinity, increases strongly with pH in the neutral range (at pH 7.0 the Ca affinity of arsenazo III is not appreciably altered by up to 2 mM Mg); St and Ba form weaker complexes with arsenazo III than Ca, but much stronger complexes than Mg; the apparent pKCa decreases with increasing buffer concentration in the mmol range. The pKCa of arsenazo III is so high that, unless the arsenazo III concentration greatly exceeds the Ca concentration, a considerable fraction of the total arsenazo III is in the Ca complexed form. Because of this, arsenazo III responds nonlinearly to all but the lowest Ca concentrations; quantitation of the Ca concentration can readily be done from the mass action law provided that the pKCa is determined under the actual experimental conditions. Arsenazo III is a reliable Ca indicator if the experimental conditions, particularly pH, are well controlled.