ACID-BASE AND CALCIUM-BINDING PROPERTIES OF THE FLUORESCENT CALCIUM INDICATOR INDO-1

Indo-1, a tetracarboxylate fluorescent probe, is commonly used for monitoring intracellular calcium levels. Indo-1 is calibrated at pH > 7, whereas intracellular pH is, in many cases, below pH 7. Therefore, the behaviour of the probe was studied at these pH values. We present the acid-base and calcium-binding properties of the molecule, as revealed by absorption and fluorescence spectroscopy, within the pH range 3 - 8. Two protonated forms of Indo-1 are described. The corresponding acidity constants are determined (i.e. pKa = 6.2, pKa = 4.35). The existence of a protonated form in the physiological domain could seriously limit quantitative applications of the 'ratio' method, which is generally used for intracellular measurements. An improved physicochemical model is proposed, and the three-dimensional structures of the single protonated and calcium-bound forms of Indo-1 are given. © 1990.