Equilibrium binding properties of the metallochromic indicator Arsenazo III (AIII) were characterized by Ca2+ and acid/base titration. Free calcium was measured directly with Ca2+ ion-sensitive electrodes. Absorbance changes were measured by both a conventional scanning spectrophotometer and a dual wavelength spectrophotometer. Acid/base titration of AIII in conjunction with Ca2+ ion-sensitive electrode measurement and absorbance changes indicate that pH can change AIII absorbance through a change of the K(D) for Ca-AIII formation, and, in addition, that there is a pH-specific component that is not dependent on Ca-AIII formation. THe dissociation constant (K(D)) of AIII varied not only with pH, but with ionic strength and AIII concentration. Studies conducted to examine AIII-Ca stoichiometry resulted in different initial conclusions, depending on the method of analysis. Log delta A-log AIII relations were in accord with previously published results, which indicate that more than one AII binds to one Ca2+ ion. But Job plots. Scatchard analysis, and Hill plots all indicated 1:1 binding. THe method of absorbance measurement, i.e., scanning or dual wavelength did not influence the results. these findings were reconciled on the basis of changes in K(D) with AIII concentration and ionic strength. In 200 mM KCl, K(D) of AIII-Ca varies by a factor of 7 between 10(-4.3) and 10(-3) M AIII. Thus, a disproportionately large amount of Ca-AIII is formed as AIII concentration is increased, which results in slopes greater than unity for log delta A-log AIII relations
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