Trajkovska Snezana, Tosheska Katerina, Aaron Jean Jacques, Spirovski Filip, Zdravkovski Zoran
Department of Medical and Experimental Biochemistry, Medical Faculty, Ss. Ciril and Metodius University, 50 Divizija 6, 1000 Skopje, R. Macedonia.
Luminescence. 2005 May-Jun;20(3):192-6. doi: 10.1002/bio.820.
Firefly luciferase (EC 1.13.12.5) (FL) is the key enzyme in the firefly bioluminescence method (FB), which is widely used to determine the viability of living cells. The FB method can also be applied to monitoring the influence of different pollutants, such as pesticides. Firefly luciferase is a hydrophobic enzyme and its activity depends on the type of solvent, pH and substances present in the reaction mixture. The influence of three aromatic pesticides, including fenoxaprop-p-ethyl (I), diclofop-methyl (II) and metsulfuron methyl (III), on the enzyme activity was indirectly evaluated through the measurement of emitted light in the bioluminescence reaction, expressed in relative luminescence units (RLU). The reaction mixture used in the bioluminescence measurements consisted of: Tris buffer (pH 7.75), adenosine triphosphate (ATP) and ATP monitoring reagent, where FL is present. Ethanol-water solutions of each pesticide were then added at concentrations of 2.4 x 10(-4)-2.4 x 10(-8) mol/L. The FL activity inhibition factors (FL In%) were determined. The FL activity was maximally inhibited in the presence of all pesticides under study at a concentration of 2.4 x 10(-4) mol/L and was lowered by about 15-26% for pesticide I at concentrations of 2.4 x 10(-5)-2.4 x 10(-8) mol/L, whereas pesticides II and III, applied in the same concentration range, showed smaller FL inhibition values (5.3-20%). The pesticide degradation products (obtained after a 1 month period), measured in the same experimental conditions, in most cases exhibited a much less inhibitory effect on the enzyme activity than the corresponding initial pesticide.
萤火虫荧光素酶（EC 1.13.12.5）（FL）是萤火虫生物发光法（FB）中的关键酶，该方法广泛用于测定活细胞的活力。FB方法也可用于监测不同污染物（如农药）的影响。萤火虫荧光素酶是一种疏水性酶，其活性取决于溶剂类型、pH值和反应混合物中存在的物质。通过测量生物发光反应中发射的光（以相对发光单位（RLU）表示），间接评估了三种芳香族农药，包括精恶唑禾草灵（I）、禾草灵（II）和甲磺隆（III）对酶活性的影响。生物发光测量中使用的反应混合物包括：Tris缓冲液（pH 7.75）、三磷酸腺苷（ATP）和ATP监测试剂，其中存在FL。然后以2.4×10⁻⁴ - 2.4×10⁻⁸ mol/L的浓度添加每种农药的乙醇 - 水溶液。测定了FL活性抑制因子（FL In%）。在所有研究的农药存在下，浓度为2.4×10⁻⁴ mol/L时，FL活性受到最大抑制；对于农药I，在浓度为2.4×10⁻⁵ - 2.4×10⁻⁸ mol/L时，FL活性降低了约15 - 26%，而在相同浓度范围内应用的农药II和III，对FL的抑制值较小（5.3 - 20%）。在相同实验条件下测量的农药降解产物（1个月后获得），在大多数情况下，对酶活性的抑制作用比相应的初始农药小得多。
