PROTEOLYSIS AS A MEASURE OF THE FREE-ENERGY DIFFERENCE BETWEEN CYTOCHROME-C AND ITS DERIVATIVES

Limited cleavage of oxidized and reduced horse heart cytochrome c (Cyt c) and the azide complex of Cyt c by proteinase K at room temperature yields a single cut within the central loop (36-60 in the sequence). Using an assay that allows spectroscopic evaluation of the fraction of intact protein as a function of time, together with a simple kinetic model for proteolysis, fluctuation opening of the loop can be related to the free energy of the corresponding protein. This allows us to est imate quantitatively the free energy difference between the oxidized f orm of Cyt c and other states using proteolysis as a probe. The result s we obtain indicate that oxidized Cyt c is 2.0 kcal mol(-1) less stab le than the reduced form, and 0.07 kcal mol(-1) is more stable than th e Cyt c: azide complex at 25 degrees C. These values agree in magnitud e with results from hydrogen exchange and unfolding studies, suggestin g that the stability of a protein can be directly related to its struc tural dynamics.