SPR binding data for UN2A with S100A1 at pH 6.0. (A) Binding affinity of S100A1 increases at pH 6.0. SPR experiments were repeated at pH 6.0 at pCa 3.0. The signal for 100 µM S100A1 increases from ∼800 RU at pH 7.4 to ∼2,400 RU at pH 6.0. The calculated binding affinity (K_D) increased from 21.6 μM at pH 7.4 to 5.0 μM at pH 6.0. (B) Ca²⁺ dependence of S100A1 binding occurs at lower pH similar to pH 7.4. SPR-binding experiments were repeated at a range of Ca²⁺ concentrations, from pCa 10 to pCa 3 at pH 6.0. Similar to at pH 7.4, negligible binding was observed at pCa 10 and 7 with increased binding at pCa 5, and the strongest binding observed at pCa 3. RU, response units.