The effects of proteins on [Ca2+] measurement: different effects on fluorescent and NMR methods

Previous reports showed that the presence of proteins shifts the apparent dissociation constant (Kd) of a fluorescent dye indicator to Ca2+. To elucidate the sensitivity of Kd of an NMR-sensitive Ca2+ indicator, 5-fluoro-1,2-bis(2-amino-phenoxy)ethane N,N,N'N'-tetraacetic acid (5F-BAPTA) to proteins, and compare with that of a dye indicator, Fura-2, we measured Kd of Fura-2 or 5F-BAPTA using Ca-EGTA buffer with or without proteins. Aldolase (ALD) or bovine cardiac protein (BCP) extracted from bovine hearts was used at concentrations of 10, 25, or 50 mg/ml. ALD significantly increased the apparent Kd of Fura-2 to Ca2+ from 164.1 +/- 5.6 nM (mean +/- SE, N = 8) to 757.2 +/- 2.1 nM (n = 4, P < 0.05) at the concentration of 50 mg/ml. In contrast, Kd of 5F-BAPTA was not markedly changed by ALD (298.4 +/- 3. nM without ALD (n = 8), 385.1 +/- 2.7 nM (n = 4) with 50 mg/ml ALD). BCP (50 mg/ml) also significantly increased Kd of Fura-2 (928.5 +/- 3.3 nM, n = 4, P < 0.05), but did not change Kd of 5F-BAPTA (316.0 +/- 2.9 nM, n = 4). These results indicate that Kd of 5F-BAPTA is much less sensitive to the presence of proteins than Fura-2, and that 19F-NMR coupled with 5F-BAPTA is a more robust method to measure intracellular Ca2+ concentration than a fluorescent method with Fura-2.