TPEN and BAPTA spectrophotometric analysis. BAPTA absorbance shift after Zn²⁺ or Ca²⁺ binding in the presence or absence of TPEN was determined by spectrophotometric analysis (250–350-nm scan). (A) The spectrum of 0.2 mM TPEN in the measurement solution shown as the blue solid line. The spectrum of 0.15 mM BAPTA in the measurement solution shown as the black solid line. The combination of 0.15 mM BAPTA and 0.2 mM TPEN spectrum shown as the black dash/dot line. (B) The addition of 0.15 mM ZnCl₂ into 0.15 mM BAPTA in the measurement solution caused a reduction in BAPTA absorbance (blue solid line) compared with the unbound BAPTA spectrum around 292 nm (black solid line). The addition of 0.2 mM TPEN into the Zn²⁺-bound BAPTA yielded a spectrum shown as the black dash/dot line. (C) Determination of the extinction coefficient (Δε) for Ca²⁺ or Zn²⁺ binding to BAPTA: Ca²⁺ standard or ZnCl₂ was added step-wise to the 0.15-mM BAPTA solution to obtain 0.01-, 0.025-, 0.05-, 0.075-, 0.1-, 0.15-, and 0.2-mM final concentrations. The Ca²⁺ and Zn²⁺ additions to BAPTA were repeated twice. Determination of Δε for Ca²⁺ or Zn²⁺ binding to BAPTA in the presence of TPEN: TPEN stock solution was added into 0.15 mM BAPTA in the measurement solution to reach a final concentration of 0.2 mM. Ca²⁺ standard or ZnCl₂ was added step-wise to the 0.15-mM BAPTA/0.2-mM TPEN solution to obtain 0.01-, 0.025-, 0.05-, 0.075-, 0.1-, 0.15-, and 0.2-mM final concentrations. The Ca²⁺ and Zn²⁺ additions to 0.15 mM BAPTA/0.2 mM TPEN were repeated twice. Values of BAPTA absorbance at 292 nm were plotted using GraphPad Prism software as a function of increasing Ca²⁺ or Zn²⁺ concentration. The linear portion of the additions (0–0.10 mM) was used to estimate the extinction coefficients by performing linear regression analysis.