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

Abstract

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.