Field trials on heavy metals using alpha-chymotryopsin enzyme assay

Abstract

An inhibitive enzyme assay using α-chymotrypsin was developed. This serine protease was assayed using Bradford-protease casein assay system. This assay is sensitive to several metals such as mercury Hg²⁺, Zn²⁺, and Cr⁶⁺. The IC₅₀ (concentration of toxicant giving 50% inhibition) of Hg²⁺, Zn²⁺, and Cr⁶⁺ is 1.34 mg/l, 2.49 mg/l and 2.19 mg/l respectively. The principle of the protein assay using casein as a substrate relies upon the inability of the Bradford dye to stain polypeptide with less than a molecular weight of 2 kDa. In the presence of heavy metals that can inhibit this enzyme, casein is not being degraded and it is stained by the Bradford dye reagent giving a blue colour. On the other hand, in the absence of inhibitors, casein is hydrolyzed to polypeptides with molecular weight of 2 kDa and below which can’t be stained by Bradford dye-binding reagent. Therefore, solution remains brown in colour. The synergistic effect of combined heavy metals was studied and the results obtained shown that there were elevation of percentage of inhibition several folds. The combination of Hg²⁺ (0.3 mg/l) with Zn²⁺ (0.8 mg/l), Hg²⁺ (0.3 mg/l) with Cr⁶⁺ (1.8 mg/l), Zn²⁺ (0.8 mg/l) with Cr⁶⁺ (1.8 mg/l) increased 15.6, 73.0, 78.8 % respectively. Biomonitoring of heavy metals using an inhibitive α-chymotripsin assay was carried out. There sites for biomonitoring were Prai Industrial Areas, Bukit Tengah Industrial Area, Juru Industrial Area, Melaka River, Kuyoh River and Endau Rompin National Park. Many of the samples gave positive inhibitory effect on this enzyme. Those samples were analysed by inductively coupled plasma-optical emission spectrometry (ICP-OES) for confirmation of the presence of heavy metals that inhibited this enzyme.