Protein and acidosis alter calcium-binding and fluorescence spectra of the calcium indicator indo-1

The fluorescent indicator indo-1 is widely used to monitor intracellular calcium concentration. However, quantitation is limited by uncertain effects of the intracellular environment on indicator properties. The goal of this study was to determine the effects of protein and acidosis on the fluorescence spectra and calcium dissociation constant (Kd) of indo-1. With 350 nm excitation light, the ratio of indo-1 fluorescence in the absence versus the presence of saturating Ca2+ at wavelength lambda (S lambda) and Kd increased with [protein]. At pH 7.3, Kd, S400, and S470, which were 210 nM, 0.033, and 1.433 in the absence of protein, increased to 808 nM, 0.161, and 2.641, respectively, by adding proteins from frog muscle and to 638 nM, 0.304, and 3.039, respectively, by adding proteins from rat heart. Effects of protein on indo-1 fluorescence were reduced at higher [indo-1]. Acidosis (pH 6.3) had separate effects, which were additive to those of protein: in the absence of protein, acidosis increased Kd to 640 nM; frog muscle proteins further increased Kd to 1700 nM. Acidosis also changed S lambda slightly. In summary, interaction with protein or protons alters indo-1 calcium-binding and fluorescence. These findings are consistent with several previous studies and suggest that indo-1 calibration constants need to be derived in the presence of appropriate types of protein, ratio of [indo-1]/[protein], and pH.