The interaction of Ca2+-binding proteins with the carbocyanine dye stains-all

The interaction of the carbocyanine dye Stains-all with the Ca²⁺-binding proteins calmodulin, troponin C, and parvalbumin has been monitored by means of absorption spectra and CD. In the absence of Ca²⁺, complexes with Stains-all of all three proteins exhibit at high dye: protein mole ratios an intense J absorption band at 600–650 nm, which is associated with a characteristic CD spectrum. In the cases of calmodulin and troponin C, the J-band is progressively lost as the dye: protein ratio decreases and is replaced by bands of the γ and β types at 450–550 nm, which likewise give rise to characteristic CD spectra. For parvalbumin, only the J-band is observed; its intensity is undiminished at the lowest dye: protein ratios examined. In the presence of excess Ca²⁺ the J-band is lost for all three proteins. For calmodulin and troponin C it is replaced by σ- and β-bands; in the case of parvalbumin the bound dye is released. A tentative model has been proposed to account for these observations.