Simultaneous measurement of two cations with the fluorescent dye indo-1

The fluorescent chelator Indo-1 can simultaneously provide information about two ions, such as Ca2+ and Ba2+, if spectra after binding can be distinguished from each other and from the ion-free dye. In the case of extracellular Ca2+ and Ba2+, the three spectra were found to be unique and independent, in the sense that no one of them could be approximated by any combination of the other two. Upon binding Ba2+, Indo-1 experienced a peak shift from 470 to 455 nm, whereas the peak for Ca(2+)-bound Indo-1 occurred at 405 nm. The computer could distinguish a contribution from Ba2-bound Indo-1 just as well when it was in a mixture containing contributions from free dye and Ca(2+)-bound Indo-1. The program used all the spectral data in a rapid noniterative calculation. It found the unique combination of Ca(2+)-bound, Ba(2+)-bound, and metal-free Indo-1 spectra which best matched the data. The spectral weighting factors which formed the best combination spectrum then corresponded to the relative concentrations of the dye in the three states. From these and the dissociation constants for Ca2+ and Ba2+, it was possible to calculate both [Ca2+] and [Ba2+] from a single data spectrum. The method was also applied to Mn2+, a metal which does not generate a unique spectrum, but which quenches Indo-1.(ABSTRACT TRUNCATED AT 250 WORDS)