Calcium sequestration by isolated sarcoplasmic reticulum: real-time monitoring using ratiometric dual-emission spectrofluorometry and the fluorescent calcium-binding dye indo-1.

This study demonstrates a simple, rapid, and reproducible microassay for real-time monitoring of Ca2(+)-sequestration by isolated sarcoplasmic reticulum (SR) using ratiometric dual-emission spectrofluorometry and the fluorescent calcium-binding dye indo-1. The SR membranes were isolated by differential centrifugation and suspended in a medium including Ca2+, indo-1, ATP and oxalate. As Ca2+ was sequestered by SR, Ca2(+)-bound indo-1 fluorescence decreased equivalently but reciprocally to the increase in Ca2(+)-free indo-1 fluorescence. The kinetic and thermodynamic properties of Ca2(+)-transport measured fluorometrically were similar to those measured radiometrically by 45Ca2+, with the exception that the former monitors changes in free Ca2+ whereas the latter monitors total Ca2+. An estimate of the maximal rate of change in total Ca2+ could be made by multiplying the maximal rate of change in free Ca2+ by the ratio of initial total Ca2+ to free Ca2+ concentration.