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 (K(d)) of a fluorescent dye indicator to Ca²⁺. To elucidate the sensitivity of K(d) of an NMR-sensitive Ca²⁺ indicator, 5-fluoro-1,2-bis(2amino-phenoxy)ethane N,N,N',N'-tetraacetic acid (5F-BAPTA) to proteins, and compare with that of a dye indicator, Fura-2, we measured K(d) 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 K(d) 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, K(d) of 5F-BAPTA was not markedly changed by ALD (298.4 ± 3.4 nM without ALD (n = 8), 385.1 ± 2.7 nM (n = 4) with 50 mg/ml ALD). BCP (50 mg/ml) also significantly increased K(d) of Fura-2 (928.5 ± 3.3 nM, n = 4, P < 0.05), but did not change K(d) of 5F-BAPTA (316.0 ± 2.9 nM, n = 4). These results indicate that K(d) 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.