A rapid inhibitive assay for the determination of heavy metals using α-chymotrypsin; a serine protease
Abstract
Agricultural areas in Malaysia depend upon unpolluted source of water and hence need continuous monitoring. An enzyme-based assay for the detection of xenobiotics is a promising low cost and rapid method for monitoring. An enzymatic-heavy metal assay has been developed using α-chymotrypsin; a serine protease. This enzyme is assayed by using casein as a substrate with Coomassie dye to track the complete hydrolysis of casein. In the absence of inhibitors, casein is hydrolysed to completion and the solution is brown. Eight heavy metals were tested on the α- chymotrypsin bioassays and in the presence of Hg2+, Cr2+ and Zn2+ at the final concentration of 5mg/L, hydrolysis of casein is inhibited and the solution remains blue. The assay was sensitive to
Hg2+, Cr2+ and Zn2+ with an IC50 (the concentration of toxicant giving 50% inhibition) of 1.34 mg/L, 1.974 mg/L and 2.49 mg/L, respectively. The calculated limits of quantitation (LOQ) value for Hg2+, Cr2+ and Zn2+ were 0.012 mg/L, 0.324 mg/L and 0.098 mg/L, respectively. The calculated LOD values for Hg2+, Cr2+ and Zn2+ were 0.003 mg/L, 0.320 mg/L and 0.074 mg/L, respectively. The results of this study indicate that this assay has the potential to be used as a tool in biosensor development for the determination of heavy metals in various samples.
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