Interaction of mercury and copper on papain and their combined inhibitive determination

• Hg2+ and Cu2+ exhibited typical hormetic effect on papain catalytic activity.
• Cu2+ exhibited more sensitive to papain than Hg2+ in the combined buffers.
• Inhibition of 10−4 mol/L Cu2+ + 10−4 mol/LHg2+ on papain is a noncompetitive type.
• Papain has a potential application for low doses of Hg2+ and Cu2+ determination.
• Detection limit of Hg2+ inhibitive determination by papain biosensor is 10−4 mol/L.

Influence and interaction of mercury ion (Hg2+) and copper ion (Cu2+) on papain activity in casein hydrolysis were investigated. Single Hg2+ or Cu2+ at low concentrations induced an increase in papain activity, but decreased it at high concentrations, confirming a typical hormesis phenomenon. The interaction of Hg2+ and Cu2+ at various concentration combinations showed that the binary interaction of 10−8 mol/L Cu2+ and 10−6 mol/L Hg2+ (Binary union S) buffer was of synergistic nature, while 10−4 mol/L Cu2+ and 10−4 mol/L Hg2+ (Binary union I) buffer was of competitive inhibition. The conformational changes in papain structure due to the interaction of binary metal ions were studied by ATR-FTIR, UV–vis and intrinsic fluorescence spectroscopies, also the changes of papain catalytic behavior were studied through kinetic analysis. Decreasing of α-helix content with increasing in intermolecular β-sheet aggregates content in Binary union I buffer resulted in an inactivation of papain activity by 57.2% and lower affinity for casein. On the contrary, papain activity increased with α-helix content increasing and intermolecular β-sheet aggregates content decreasing in Binary union S buffer. The competitive interaction between Cu2+ and Hg2+ on papain activity was found at higher concentrations (≥10−4 mol/L), and the inhibition of the binary metal ions on papain was of a noncompetitive type.