Journal of Environmental Biology

The development of an inhibitive determination method for zinc using a serine protease

 

M.Y. Shukor*¹, N.A. Baharom¹, N.A. Masdor², M.P.A. Abdullah¹, N.A. Shamaan¹, J.A. Jamal³ and M.A. Syed¹

¹Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia

²Biodiagnostic and Biosafety Programme, Biotechnology Research Centre, Malaysian Agriculture and Research Development Institute, PO.Box 1230, 50774 Kuala Lumpur, Malaysia

³Department of Pharmacy, Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia

(Received: December 18, 2007; Revised received: June 10, 2008 ; Accepted: June 20, 2008)

Abstract: A new inhibitive heavy metals determination method using trypsin has been developed. The enzyme was assayed using the casein-Coomassie-dye-binding method. In the absence of inhibitors, casein was hydrolysed to completion and the Coomassie-dye was unable to stain the protein and the solution became brown. In the presence of metals, the hydrolysis of casein was inhibited and the solution remained blue. The bioassay was able to detect zinc and mercury with IC₅₀ (concentration causing 50% inhibition) values of 5.78 and 16.38 mg l^-1 respectively. The limits of detection (LOD), for zinc and mercury were 0.06 mg l^-1 (0.05-0.07, 95% confidence interval) and 1.06 mg l^-1 (1.017-1.102, 95% confidence interval), respectively. The limits of quantitation (LOQ) for zinc and mercury were 0.61 mgl^-1 (0.51-0.74 at a 95% confidence interval) and 1.35 mg l^-1 (1.29-1.40 at a 95% confidence interval), respectively. The IC₅₀ value for zinc was much higher than the IC₅₀ values for papain and Rainbow trout, but was within the range of Daphnia magna and Microtox^TM. The IC₅₀ value for zinc was only lower than those for immobilized urease. Other toxic heavy metals, such as lead, silver, arsenic, copper and cadmium, did not inhibit the enzyme at 20 mg l^-1. Using this assay, we managed to detect elevated zinc concentrations in several environmental samples. Pesticides, such as carbaryl, flucythrinate, metolachlor, glyphosate, diuron, diazinon, endosulfan sulphate, atrazine, coumaphos, imidacloprid, dicamba and paraquat, showed no effect on the activity of trypsin relative to control (One-way ANOVA, F_{12, 26} = 0.3527, p> 0.05). Of the 17 xenobiotics tested, only (sodium dodecyl sulphate) SDS gave positive interference with 150 % activity higher than that of the control at 0.25% (v/v).

Key words: Tryspsin, Serine protease, Inhibitive determination method

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