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 intracellul ar calcium concentration. However, quantitation is limited by uncertai n effects of the intracellular environment on indicator properties. Th e goal of this study was to determine the effects of protein and acido sis on the fluorescence spectra and calcium dissociation constant (K-d ) of indo-1. With 350 nm excitation light, the ratio of indo-1 fluores cence in the absence versus the presence of saturating Ca2+ at wavelen gth lambda (S-lambda) and K-d increased with [protein]. At pH 7.3, K-d , S-400, and S-470, 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 a dding proteins from frog muscle and to 638 nM, 0.304, and 3.039, respe ctively, 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 abs ence of protein, acidosis increased K-d to 640 nM; frog muscle protein s further increased K-d to 1700 nM. Acidosis also changed S-lambda sli ghtly. In summary, interaction with protein or protons alters indo-1 c alcium-binding and fluorescence. These findings are consistent with se veral previous studies and suggest that indo-1 calibration constants n eed to be derived in the presence of appropriate types of protein, rat io of [indo-1]/[protein], and pH.