Chemistry Dept., College of Science, University of Babylon, Iraq Hilla City, Babylon Governorate, p.o. 51002, Iraq.
Anal Biochem. 2023 Jul 15;673:115192. doi: 10.1016/j.ab.2023.115192. Epub 2023 May 22.
This paper reports a sensitive method for assaying xanthine oxidase (XO) enzyme activity. XO produces hydrogen peroxide (HO) and superoxide anion radicals (O), promoting the development of oxidative stress-related diseases, and is inhibited by various plant extracts. XO activity is quantified by incubating enzyme samples with an appropriate xanthine concentration as the substrate. The proposed method requires XO activity to be quantified based on HO generation using a 3,3',5,5'-tetramethylbenzidine (TMB)-HO system catalyzed by cupric ions. After a 30-min incubation at 37 °C, sufficient cupric ion and TMB amounts are added. The assay produces optical signals that can be visually recognized or detected with a UV-visible spectrometer. A direct correlation was found between XO activity and the absorbance at 450 nm of the resulting di-imine (dication) yellow product. The proposed method uses sodium azide to prevent catalase enzyme interference. The new assay's function was confirmed using the TMB-XO assay and a Bland-Altman plot. The resulting correlation coefficient was 0.9976. The innovative assay was relatively precise and comparable to the comparison protocols. In conclusion, the presented method is very efficient at measuring XO activity.
本文报道了一种灵敏的黄嘌呤氧化酶（XO）酶活性测定方法。XO 产生过氧化氢（HO）和超氧阴离子自由基（O），促进氧化应激相关疾病的发展，并被各种植物提取物抑制。通过将酶样品与适当浓度的黄嘌呤作为底物孵育，定量 XO 活性。该方法要求基于 HO 的产生来定量 XO 活性，HO 的产生是通过铜离子催化的 3,3',5,5'-四甲基联苯胺（TMB）-HO 系统实现的。在 37°C 孵育 30 分钟后，加入足够量的铜离子和 TMB。该测定产生光学信号，可以通过肉眼观察或使用紫外-可见分光光度计检测。在 450nm 处，产生的二亚胺（二价阳离子）黄色产物的吸光度与 XO 活性之间存在直接相关性。该方法使用叠氮化钠来防止过氧化氢酶的干扰。新测定法的功能通过 TMB-XO 测定和 Bland-Altman 图得到了验证。得到的相关系数为 0.9976。该创新测定法具有相对较高的精密度，与比较方案相当。总之，所提出的方法在测量 XO 活性方面非常有效。
