Walmsley T A, Abernethy M H, Fitzgerald H P
Department of Clinical Biochemistry, Christchurch Hospital, New Zealand.
Clin Chem. 1987 Oct;33(10):1928-31.
The reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) with thiols is sensitive to daylight, in particular to ultraviolet radiation at wavelengths around 325 nm. Exposure to light at the absorbance maximum of the yellow product (the thionitrobenzoate ion) at 410 nm had no effect on the reaction. The light-sensitive species is apparently the DTNB, because a spectral-irradiation experiment showed that the wavelength of light that produced the maximum rate of absorbance change coincided with the peak absorbance of DTNB, and it was well separated from the thionitrobenzoate absorbance peak. Ascorbate is ineffective as a stabilizer and can produce an apparent increase in the rate of DTNB destruction. In a practical example we found the light interference to be severe when hydrolysis of propionylthiocholine by plasma cholinesterase (EC 3.1.1.8) was measured after a 20-min incubation. The apparent cholinesterase activity in clear glass or plastic tubes exposed to diffuse daylight could be decreased to 25% of the value obtained for samples in light-excluded tubes. We recommend the reaction be carried out in artificial room light, with total elimination of daylight, because window glass does not sufficiently attenuate 325-nm wavelength irradiation.
5,5'-二硫代双(2-硝基苯甲酸)(DTNB，埃尔曼试剂)与硫醇的反应对日光敏感，尤其对波长约为325 nm的紫外线辐射敏感。在黄色产物(硫代硝基苯甲酸根离子)在410 nm处的最大吸光度下曝光对反应没有影响。光敏物质显然是DTNB，因为光谱辐照实验表明，产生最大吸光度变化速率的光波长与DTNB的峰值吸光度一致，并且与硫代硝基苯甲酸根的吸光度峰分得很开。抗坏血酸作为稳定剂无效，并且会使DTNB的破坏速率明显增加。在一个实际例子中，我们发现，在20分钟孵育后测量血浆胆碱酯酶(EC 3.1.1.8)对丙酰硫代胆碱的水解时，光干扰很严重。暴露在漫射日光下的透明玻璃管或塑料管中的表观胆碱酯酶活性可能会降至避光管中样品所得值的25%。我们建议反应在人造室内光线下进行，完全消除日光，因为窗户玻璃不能充分衰减325 nm波长的辐射。
