Cytochrome C Functionalized with CPP

Initially, equine cytochrome C (Sigma-Aldrich, Saint Louis, MO, USA) was solubilised in phosphate-buffered saline (pH 8.0) at a concentration of 0.83 mM. Subsequently, a 5-fold excess of the linker was dissolved in dimethyl sulfoxide (DMSO) and added to the cytochrome C solution at room temperature for a duration of 30 min. Without further purification, a 10-fold excess of purified cell-penetrating peptide (CPP) was dissolved in phosphate-buffered saline and added to the cytochrome C solution. The resulting mixture was incubated for 1 h at room temperature. Excess linkers and peptides were removed from the reaction mixture using a spin column with a molecular weight cut-off of 5000. The conjugation of CPP to cytochrome C was confirmed by liquid chromatography-mass spectrometry (LCMS).