Shukor M Y, Baharom N A, Masdor N A, Abdullah M P A, Shamaan N A, Jamal J A, Syed M A
Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
J Environ Biol. 2009 Jan;30(1):17-22.
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 mg l(-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. 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).
一种使用胰蛋白酶的新型重金属抑制测定方法已被开发出来。该酶采用酪蛋白 - 考马斯染料结合法进行测定。在没有抑制剂的情况下，酪蛋白被完全水解，考马斯染料无法对蛋白质染色，溶液变为棕色。在有金属存在的情况下，酪蛋白的水解受到抑制，溶液仍保持蓝色。该生物测定法能够检测锌和汞，其IC50（引起50%抑制的浓度）值分别为5.78和16.38 mg l(-1)。锌和汞的检测限（LOD）分别为0.06 mg l(-1)（0.05 - 0.07，95%置信区间）和1.06 mg l(-1)（1.017 - 1.102，95%置信区间）。锌和汞的定量限（LOQ）分别为0.61 mg l(-1)（95%置信区间下为0.51 - 0.74）和1.35 mg l(-1)（95%置信区间下为1.29 - 1.40）。锌的IC50值远高于木瓜蛋白酶和虹鳟鱼的IC50值，但在大型溞和Microtox的范围内。锌的IC50值仅低于固定化脲酶的IC50值。其他有毒重金属，如铅、银、砷、铜和镉，在20 mg l(-1)时不会抑制该酶。使用这种测定方法，我们成功检测到了几个环境样品中升高的锌浓度。农药，如西维因、氟氰菊酯、异丙甲草胺、草甘膦、敌草隆、二嗪农、硫酸硫丹、莠去津、蝇毒磷、吡虫啉、麦草畏和百草枯，相对于对照对胰蛋白酶的活性没有影响（单因素方差分析，F(12,26)= 0.3527，p> 0.05）。在所测试的17种外源化合物中，只有（十二烷基硫酸钠）SDS产生了正干扰，在0.25%（v/v）时活性比对照高150%。
