Arsenazo I and tetramethylmurexide as optical calcium indicators

Abstract

Calcium-binding stoichiometry, dissociation equilibrium constants at zero ionic strength ( K 0), and molar extinction difference coefficients (Δϵ λ) at the wavelength λ of the metallochromic indicators arsenazo I (ArsI) and tetramethylmurexide (TMX) were reevaluated with a computerized method based on mass conservation and thermodynamic consistency checks. This new method is shown to provide a more critical assessment of the assumed calcium-dye complexing model than is afforded by the commonly used reciprocal-plot method. The analyses of spectrophotometric Ca titrations confirm that both dyes form only 1:1 complexes in aqueous solution. For TMX, K 0 = 1.3 × 10 −3 m and Δϵ 480 = 1.5 × 10 4 m −1 cm −1; for ArsI, K 0 = 5.8 × 10 −3 m and Δϵ 562 = 1.8 × 10 4 m −1 cm −1 at pH 7.0 and T = 293°K. The discriminatory power of the analytical method is demonstrated by comparison of these results with those found for a different dye, arsenazo III, which complexes Ca in 1:1, 1:2, and 2:1 forms.