The pH dependence and the binding equilibria of the calcium indicator--arsenazo III.

The underlying principles of binding equilibria of arsenazo III with Ca2+ and Mg2+ are presented. Ca2+ and Mg2+ can bind arsenazo III in several different protonated forms depending on pH. The binding affinities of these different protonated forms of arsenazo III with Ca2+ increase in the order of H4A4- less than H3A5- less than H2A6- and with Mg2+, H4A4- congruent to H3A5- less than H2A6-. Arsenazo III is not membrane bound. The sensitivity ratio of arsenazo III with Ca2+ to arsenazo III with Mg2+ is close to two orders of magnitude. Arsenazo III and its complexes are extremely sensitive to pH changes. With 5 microM arsenazo III, the minimum detectable amount of Ca2+ can be as low as 0.08 microM. Contrary to current belief, we found that Mg2+ can bind to arsenazo III in a slightly acidic medium. Potential applications of arsenazo III to the study of membrane Ca2+ transport are also discussed.