Influence and binary interaction of mercury and copper ions on papain activity in the hydrolysis of casein were investigated,respectively.Single mercury or copper ion(Hg2+,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 each concentration combination was carried out,showing that the binary interaction between Cu and Hg in 10-8 mol/L Cu2++10-6 mol/L Hg2+(Binary union S)buffer was of synergistic nature,but competitive inhibition in 10-4 mol/L Cu2++Hg2+(Binary union Ⅰ)buffer.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,and the changes in enzyme catalytic behavior were also studied by kinetic analysis.At the combination of 10-4 mol/L Cu2++Hg2+,Binary union I significantly decreased the α-helix content of papain and increased β-sheet aggregated and random coil contents so that the activity of papain with lower affinity for substrate was inactivated by 57.2%.On the contrary,the papain activity increased with increase of the β-helix content and decrease of β-sheet aggregated and random coil contents when papain exposed in the Binary union S buffer.The Cu2+exhibited more sensitive to papain than Hg2+in the combined buffers.The competitive interaction between Cu and Hg on papain activity was found at higher concentration(i.e.,Hg2+or Cu2+≥ 10-4 mol/L),and the inhibition of the binary metal ions on the enzyme was a noncompetitive type.This study corresponded to evaluating the interaction effects of heavy metal ions on enzyme activity confirmed the possibility of the determination of mercury in the presence of other heavy metal ions for environmental analysis.