The thermal stability of pepsin in a strong acid medium as a function of pH has been investigated using differential scanning calorimetry (DSC), UV absorbance, polyacrylamide gel electrophoresis (PAGE) and MALDI-TOF MS methods. The "two independent two-state transitions model" with view of physiological function of pepsin was used for analyzing thermal denaturation curves. The transition temperature (T-m) values ranging from 305.15 to 319.15K for the first transition and from 322.15 to 349.15K for the second transition in the examined pH range implicating the higher stability at pH 4 are in good agreement with MALDI-TOF MS results. The corresponding maximum, Delta G degrees(25), was stability obtained at pH 4 with values of 65.3 kJ mol(-1). (C) 2013 Elsevier B.V. All rights reserved.