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. 2009 May 12;284(28):19090–19100. doi: 10.1074/jbc.M109.007021

FIGURE 5.

FIGURE 5.

Determination of apparent Ca2+ affinities of cTn and thin filament HCM mutants by fluorescence. Steady state fluorescence measurements excited the IAANS probe at 330 nm, and emission was detected at 450 nm. A, cTn complexes containing HCM HcTnC mutants that were doubly labeled at cysteines 35 and 84. B, TF-containing HCM HcTnC mutants that were singly labeled at cysteine 35. The WT, A8V, E134D, and D145E had the cysteine 84 mutated allowing labeling at a single site. Fluorescence spectral changes were recorded during the titration of microliter amounts of CaCl2 to a 2-ml experimental volume. The data were fitted to a version of the Hill equation that accounted for the spectral changes occurring at low Ca2+ concentration. The Ca2+ affinities are reported in Table 2 as pCa50 values ±S.E. Each point represents an average of four to five experiments and is expressed as mean ± S.E.

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