Almashhedy Lamia A, Hadwan Mahmoud Hussein, Abbas Khudhair Dunia, Kadhum Mohammed A, Hadwan Asad M, Hadwan Muntadhar M
Chemistry Dept., College of Science, University of Babylon, Hilla City, Babylon Governorate, 51002, Iraq.
Department of Medical Laboratory Techniques, Al-Mustaqbal University College, 51001, Hillah, Babylon, Iraq.
Talanta. 2023 Feb 1;253:123899. doi: 10.1016/j.talanta.2022.123899. Epub 2022 Sep 2.
Spectrophotometric methodologies have been used to assess glutaminase activity, for which coloured complexes have been developed that measure spectrophotometry across the visible spectrum using different reagents. The present paper describes a precise, simple and reliable procedure for quantifying glutaminase activity, which is a key enzyme in glutamine hydrolysis and also involved in glutamine metabolism regulation. The procedure presented here measures glutaminase activity by incubating glutaminase enzyme at 37 °C for 20 min with a glutamine substrate dissolved in a buffer (pH 8.6). The enzymatic reaction contains suitable activity of glutamate oxidase, which acts to convert glutamate to hydrogen peroxide and 2-oxoglutarate. To terminate the enzymatic activity, a working solution containing pyridine-2,6-dicarboxylic (PDA) acid and ammonium vanadate (AV) was added following incubation. Oxo-peroxo-pyridine-2,6-dicarboxylato-vanadate (OPDV), a stable orange-coloured chelate complex measuring 435 nm spectrophotometrically, was produced by the interaction between the generated hydrogen peroxide and the supplied reagent. Using the response surface methodology (RSM) as an indicator of the assay's accuracy, we employed the Box-Behnken design (BBD) to improve the method's design (the OPDV-Glutaminase assay). Improvement factors were the volume of working reagent solution (PDA/AV), volume of glutamate oxidase solution (GO), and incubation time. In matched samples, this novel method was verified against a Bland-Altman plot assessment of glutaminase activity using the indophenol methodology. A correlation value of 0.99 between the two methods' comparisons showed that the novel protocol was equally applicable to the reference method.
分光光度法已被用于评估谷氨酰胺酶活性，为此开发了有色络合物，使用不同试剂在可见光谱范围内进行分光光度测量。本文描述了一种精确、简单且可靠的谷氨酰胺酶活性定量方法，谷氨酰胺酶是谷氨酰胺水解中的关键酶，也参与谷氨酰胺代谢调节。这里介绍的方法是将谷氨酰胺酶与溶解在缓冲液（pH 8.6）中的谷氨酰胺底物在37℃孵育20分钟来测量谷氨酰胺酶活性。酶促反应中含有适量的谷氨酸氧化酶活性，其作用是将谷氨酸转化为过氧化氢和2-氧代戊二酸。为终止酶活性，孵育后加入含有吡啶-2,6-二羧酸（PDA）和偏钒酸铵（AV）的工作溶液。所产生的过氧化氢与所提供的试剂相互作用产生了氧代过氧吡啶-2,6-二羧基钒酸酯（OPDV），这是一种稳定的橙色螯合物，通过分光光度法在435nm处进行测量。使用响应面方法（RSM）作为测定准确性的指标，我们采用Box-Behnken设计（BBD）来改进该方法的设计（OPDV-谷氨酰胺酶测定法）。改进因素包括工作试剂溶液（PDA/AV）的体积、谷氨酸氧化酶溶液（GO）的体积和孵育时间。在匹配的样本中，使用吲哚酚法通过Bland-Altman图评估对该新方法进行了谷氨酰胺酶活性验证。两种方法比较的相关值为0.99，表明该新方案与参考方法同样适用。
