B. Allosteric reactions (Hill Plot) 1. A protein has a binding affinity for its ligand of Ka = 2 x 105 M-1 at pH 5.0 and 25°C. (i) At what concentration of the ligand is half of the protein bound? (iii) At what ligand concentration would 80% of the protein be bound? what fraction of the protein is bound at a ligand concentration of 1.25 μM? when the pH is raised to 6.5 the Kd increased to 20μM. Is the binding tighter or weaker at this pH compared to 5.0? Explain your answer. (iv) (v What amino acid residuels) could be responsible for the observed change in the binding affinity with change in pH?