Determination of the kinetics and influence of the mercury ion on papain catalytic activity

Abstract

The effect of the mercury ion on papain activity of the substrate casein was investigated. Mercury ions (Hg²⁺) at low concentrations induced an increase of papain activity, but decreased it at high concentrations, confirming a typical hormesis phenomenon. Papain activity increased to a maximum of 111.03% at a concentration of 10⁻⁶ mol L⁻¹ Hg²⁺, but was almost completely deactivated at concentrations above 10⁻⁴ mol L⁻¹ Hg²⁺. The conformational changes in papain structure because of the interaction of Hg²⁺ and papain were studied using synchrotron radiation circular dichroism, attenuated total reflectance Fourier-transform infrared and intrinsic fluorescence spectroscopies, and the catalytic behavior of the enzyme was studied using kinetic analysis. At up to 10⁻⁴ mol L⁻¹ concentrations, Hg²⁺ significantly decreased the α-helix content of papain and increased the random coil content so that the papain with a lower affinity to substrate was nearly completely inactivated. However, papain activity increased with an increase of the α-helix content and decrease of random coils when the Hg²⁺ concentration at 10⁻⁶ mol L⁻¹. There were different modification mechanisms of papain activity with different concentrations of Hg²⁺. At a concentration of 10⁻⁶ mol L⁻¹ Hg²⁺, Hg²⁺ was acting as an efficacious activator, and the impact could be classified as a non-competitive type. At a concentration of 10⁻⁴ mol L⁻¹ Hg²⁺, the inhibition of Hg²⁺ on papain was found to be a competitive and uncompetitive mixed type, and Hg²⁺ at this concentration bound to the enzyme molecule leading to the loss of enzyme activity. As a result, the detection limit of papain was 10⁻⁴ mol L⁻¹ and has a potential application for determining low doses of Hg²⁺.