Comparison of the Characteristics of Four Metallochromic Dyes as Potential Calcium Indicators for Biological Experiments1

Abstract

The characteristics of four calcium indicators, murexide(MX), tetramethylmurexide(TMX), arsenazo III (Az) and antipyrylazo III (Ap), were examined to assess their applicability for biological experiments. Az has the following serious disadvantages, although it has a high sensitivity to Ca: (i) nonlinearity of absorbance change against the concentration of Ca due to a change in the structural composition of its complex (2 : 1 complex to 1 : 1 complex) in the range below 10 μM Ca²⁺, and due to the high apparent binding constant to Ca at 20 μM or higher Ca²⁺; (ii) severe interference by Mg²⁺; (iii) rather low association and dissociation rate constants with Ca which are not sufficient under conditions where Ca²⁺ to be determined in the system cannot be disturbed by Az; (iv) inhibitory effect on Ca uptake by bullfrog fragmented sarcoplasmic reticulum. Ap has similar disadvantages, although Ap reacts with Ca faster than Az. (i) Ap suffers from interference by Mg²⁺ even though the effect of Mg²⁺ can be minimized at the expense of reduced sensitivity. (ii) Plots of absorbance change versus the concentration of Ca deviate from linearity around 20 μM Ca²⁺ or higher. (iii) Ap is not very sensitive at concentrations which would not disturb Ca²⁺ in the system to be determined. TMX is twice as sensitive as MX. However, TMX may be more permeable through membranes than MX in the presence of ATP. MX has no disadvantages except for its low sensitivity to Ca²⁺.