Figure 5. Divalent metal cation binding by Zn(II.

)Har. Fluorescence spectroscopic titration of Zn(II)Har with ferrous ions and manganese ions in HEPES (5 mM, pH 7.0) containing 250 mM NaCl in the presence of TCEP (2 mM). Change in fluorescence emission intensity of Zn(II)Har (8 µM) at 305 nm upon addition of 0 – 3.3 molar equivalent Fe²⁺ (A) and 0 – 8 molar equivalent Mn²⁺ (B), with each set of presented data being averaged from three individual titrations. Apparent dissociation constants (K_d) were estimated by fitting the titration data using the biochemical analysis program Dynafit [31]. λ_ex = 275 nm.