An Inhibitive Determination Method for Heavy Metals Using Bromelain, A Cysteine Protease

Abstract

A heavy-metal assay has been developed using bromelain, a protease. The enzyme is assayed using casein as a substrate with Coomassie dye to track completion of hydrolysis of casein. In the absence of inhibitors, casein is hydrolysed to completion, and the solution is brown. In the presence of metal ions such as Hg²⁺ and Cu²⁺, the hydrolysis of casein is inhibited, and the solution remains blue. Exclusion of sulfhydryl protective agent and ethylenediaminetetraacetic in the original assay improved sensitivity to heavy metals several fold. The assay is sensitive to Hg²⁺ and Cu²⁺, exhibiting a dose–response curve with an IC₅₀ of 0.15 mg l⁻¹ for Hg²⁺ and a one-phase binding curve with an IC₅₀ of 0.23 mg l⁻¹ for Cu²⁺. The IC₅₀ value for Hg²⁺ is found to be lower to several other assays such as immobilized urease and papain assay, whilst the IC₅₀ value for Cu²⁺ is lower than immobilized urease, 15-min Microtox™, and rainbow trout.