Spectrophotometric determination of reaction stoichiometry and equilibrium constants of metallochromic indicators. III. Antipyrylazo III complexing with Ca2+ and acetylcholine receptor protein.
Stoichiometries, equilibrium constants and optical extinction coefficients of calcium-antipyrylazo III (An) complexing are determined with the analytical method described in article I of this series. Spectrophotometric Ca titrations of An at the wavelengths 595 and 710 nm indicate overall dissociation equilibrium constants for the complexes CaAn, CaAn2 and Ca2An to be 4.5 x 10(-4) M, 1.1 x 10(-8) M2 and 1.5 x 10(-6) M2, respectively, extrapolated to zero ionic strength. Ca titrations of solutions containing An plus acetylcholine receptor protein give clear evidence that An binds to the protein to a large extent in the presence of Ca2+; furthermore, addition of acetylcholine results in release of protein-bound Ca and An. This is the first reported indication that antipyrylazo III binds to biological material and questions the usefulness of this dye as a Ca indicator in biological systems.
D000818:Animals; D001391:Azo Compounds / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D004557:Electric Organ / Q000378:metabolism; D007700:Kinetics; D009282:Naphthalenesulfonates / Q000378:metabolism*; D011485:Protein Binding; D011950:Receptors, Cholinergic / Q000378:metabolism*; D013053:Spectrophotometry; D014101:Torpedo
D001391:Azo Compounds; D009282:Naphthalenesulfonates; D011950:Receptors, Cholinergic; C016238:antipyrylazo III; D002118:Calcium