Structural Changes of Almost Completely Inactivated Papain with Higher Concentration of Parachloromercuribenzoic Acid

Abstract

A hydrolysis of casein by papain was carried out to explore interaction between parachloromercuribenzoic acid (PCMB) and papain through Attenuated Total Reflection Flourier transformed Infrared Spectroscopy (ATR-FTIR), Trp intrinsic fluorescence spectroscopy and molecular docking analysis. Papain activity was almost completely inactivated when PCMB concentration reached to 10–4 mol/L or higher. The content of α-helix was decreased from 36.79% to 19.79% even up to 17%. On the other hand, the content of β-sheet was decreased from 13.41% to 11.57% by a little degree of 1.84%. However, the contents of β-turn, random coil and intermolecular β-sheet aggregates were elevated by different degree of 2.67%, 8.87% and 7.3%, respectively at the papain exposure of 10–4 mol/L PCMB solution. PCMB could bind to papain mainly through hydrophobic force and hydrogen bonding. The hydrogen bond between PCMB and Gly66; while, the hydrophobic bonds between PCMB and Trp26 in the ligand-binding pocket of papain might be the key functional groups for the inhibition of papain activity. The L-domain of papain was destructed, leading to the inactivation and fluorescence quenching of papain. The inhibition mechanism of papain activity by 10–4mol/L PCMB was involved the formation of hydrophobic force and hydrogen bond between PCMB and papain. This attempt successfully brought forth the application of computational approaches to dissect the inhibition mechanism and directed design in enzyme engineering area.