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

Abstract

Papain is a protease that consists of ?-helical and ?-sheet domains which unfold almost independently. Both, papain considerable thermal stability and sodium dodecyl sulphate (SDS) resistance 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 thermal inactivation resistance when it is compared to stem papain with rather high activation energy (Ea) of 223 ? 16 kJmol-1 and Tm50 value of 79 ? 2 ?C. SDS resistance of fruit papain was estimated by SDS-PAGE analysis and activity staining. It has been noted that, in the presence of SDS, unless heat energy was applied in order to unfold papain, the protein remained active. Furthermore, it has been proven via Fourier transform infrared spectroscopy (FT-IR) that ?-helical domain of fruit papain is more prone to unfolding at elevated temperatures and in the presence of SDS then ?-sheet rich domain. Thermal denaturation of papain without detergent present led to accelerated formation of aggregation specific intermolecular ?-sheets as compared to native protein. Presented results are both, of fundamental and application importance.