Determination of CRIP2-Cu+ Dissociation Constant

The dissociation constant (KD) of hsCRIP2 for Cu⁺ was determined through competition assays with the chromogenic ligand bathocuproine disulfonate BCS ([Cu⁺(BCS)₂]³⁻ β₂’ formation constant 10^20.8 M⁻², ε_{483 nm} 13000 M⁻¹ cm⁻¹) (95 (link)) as previously shown (96 (link)). Immediately before the assay, CRIP2 was fully reduced by incubation with 5 mM TCEP on ice for 3 h, and the reducing agent was removed using a 3 kDa Centricon (Millipore Sigma). Cu⁺ solutions were generated in situ from CuSO₄ in the presence of ascorbate. Briefly, 10 μM Cu⁺, 25 μM BCS in buffer 25 mM HEPES pH 8, 150 mM NaCl, 10 mM ascorbic acid were titrated with 1 - 30 μM purified CRIP2, incubated 5 min at room temperature, and the 300-800 nm absorption spectra were recorded. CRIP2-Cu⁺ ${\mathrm{K}}_{\mathrm{D}}$ was calculated by curve-fitting the experimental data to the equilibrium equations [1] and [2] (97 (link)), where M is the metal, L is the competing ligand and P are the Cu-sites in the protein. Reported errors are asymptotic standard errors provided by the fitting software (KaleidaGraph; Synergy).