Figure 5.

Competitive binding of metals and MYR to BSA. (A) Effect of increasing amounts of MYR on the zinc-binding capacity of BSA. ITC curves for titrations of 333 μM Zn²⁺ into 25 μM BSA in the presence and absence of varying amounts (0–5 molar equiv) of MYR in 50 mM Tris/50 mM NaCl (pH 7.2). The fits (Figure S8) allowed estimates of the ratio of site A availability, as shown in (B). A clear downward trend was observed. A 4:1 MYR:Zn molar ratio suppressed occupation of site A almost completely. A second binding site was also affected by fatty acid binding (see D). (C) ITC curves for titrations of 500 μM MYR into 12.5 μM BSA or Zn₁BSA. These titrations were carried out in H₂O because of the insufficient solubility of MYR in Tris buffer. The fits (Figure S9) correspond to a model with one set of binding sites to estimate the stoichiometry for the highest-affinity sites. This equaled 5.0 ± 0.3, and the average log K was 6.3 ± 0.4 in the absence and 5.9 ± 0.4 in the presence of Zn²⁺. More complex fits were possible, but the data were insufficient to justify them. (D) ¹¹¹Cd NMR spectra of Cd₂BSA recorded in the absence and presence of 5 molar equiv of MYR. Peaks A and B were both suppressed by MYR.