Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives

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Abstract
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 estimate quantitatively the free energy difference between the oxidized form of Cyt c and other states using proteolysis as a probe. The results we obtain indicate that oxidized Cyt c is 2. 0 kcal mol−1 less stable than the reduced form, and 0. 07 kcal mol−1 is more stable than the Cyt c: azide complex at 25°C. These values agree in magnitude with results from hydrogen exchange and unfolding studies, suggesting that the stability of a protein can be directly related to its structural dynamics.