Equation 7 indicates that the optimal total ligand concentration [L tot ] opt is determined by the two binary binding affinities instead of cooperativity (Figure ). Substituting Eq. 8 in Eq. 4 provides a scheme to optimize the optimal fraction f opt 3 and D depict the 2D projection of f opt 3 with a fixed cooperativity α and a fixed K A-L , respectively. Generally, f opt 3 'benefits' the least by enhancing binding affinity between ligand and the stronger binding protein. When binding affinities between ligand and the two proteins are highly skewed (i.e.,