Methods for determining cardiac sarcoplasmic reticulum Ca2+ pump kinetics from fura 2 measurements

We explored the use of four methods for analyzing real and simulated fura 2 measurements of Ca2+ uptake by membrane vesicles derived from the sarcoplasmic reticulum (SR) of cardiac muscle. Uptake velocity was calculated 1) directly from the raw data, 2) after segmenting the raw data and averaging the data points in each segment, 3) after smoothing of the raw data by moving-window averaging, and 4) by Savitsky-Golay convolution. Methods 2, 3, and 4 could be used to determine maximum uptake velocity, the Hill coefficient, and the Ca2+ concentration at half-maximal pump velocity from Ca2+ concentration vs. time and velocity curves that were too noisy to analyze directly. Data analysis using these methods should have general applicability to biological experiments, especially those in which large numbers of measurements are made. The fluorometric method we describe for measuring Ca2+ uptake by cardiac SR vesicles opens up the possibility of studying SR function from very small starting tissue samples.