Evidence of β-sheet structure induced kinetic stability of papain upon thermal and sodium dodecyl sulfate denaturation

Papain is a protease that consists of alpha-helical and beta-sheet domains that unfold almost independently. Both, considerable thermal stability and sodium dodecyl sulfate (SDS) resistance of papain have been shown. However, the ability of each domain to unfold upon thermal and SDS denaturation has never been studied. This work shows that fruit papain has slightly higher resistance to thermal inactivation when compared to that of stem papain with a rather high activation energy (E-a) of 223 +/- 16 kJ mol(-1) and a T(m)50 value of 79 +/- 2 degrees C. The SDS resistance of fruit papain was estimated by SDS-PAGE analysis and activity staining. It was noted that, in the presence of SDS the protein remained active, unless heat energy was applied in order to unfold papain. Furthermore, it was proven via Fourier transform infrared spectroscopy (FT-IR) that an alpha-helical domain of fruit papain is more prone to unfolding at elevated temperatures and in the presence of SDS then a beta-sheet rich domain. Thermal denaturation of papain without detergent present led to accelerated formation of aggregation specific intermolecular beta-sheets as compared to native protein. The presented results are of both fundamental and practical importance.