Abstract

Calsequestrin, a high-capacity, intermediate-affinity, calcium-binding protein present in the lumen of sarcoplasmic reticulum, undergoes extensive calcium-induced conformational changes at neutral pH that cause distinct intrinsic fluorescence changes. The results reported in this work indicate that pH has a marked effect on these calcium-induced intrinsic fluorescence changes, as well as on calorimetric changes produced by the addition of Ca 2+ to calsequestrin. The addition of Ca 2+ at neutral pH produced a marked and cooperative increase in calsequestrin intrinsic fluorescence. In contrast, at pH 6.0 calsequestrin's intrinsic fluorescence was not affected by the addition of Ca 2+, and the same intrinsic fluorescence as that measured in millimolar calcium at neutral pH was obtained. The magnitude and the cooperativity of the calcium-induced intrinsic fluorescence changes decreased as either [H +] or [K +] increased. The evolution of heat production, determined by microcalorimetry, observed upon increasing the molar ratio of Ca 2+ to calsequestrin in 0.15 M KCl, decreased markedly as the pH decreased from pH 8.0 to pH 6.0, indicating that pH modifies the total heat content changes produced by Ca 2+. We propose that protons bind to calsequestrin and induce protein conformational changes that are responsible for the observed proton-induced intrinsic fluorescence and calorimetric changes.