Mohsin Nawar Yaseen, Demir Halit, Hadwan Mahmoud Hussein, Hadwan Asad M, Mohammed Rawaa M
Faculty of Science, Department of Chemistry, Van Yüzüncü Yıl Üniversitesi, Van, Turkey.
Chemistry Department, College of Science, University of Babylon, Babylon Governorate, Hilla City, PO, 51002, Iraq.
J Fluoresc. 2024 Oct 23. doi: 10.1007/s10895-024-03991-4.
A novel fluorometric method is presented for accurately quantifying peroxiredoxin (Prx) enzyme activity in vitro. The rate-limiting step in the Prx-catalyzed reaction is the dissociation of peroxide. To avoid interference from catalase, we developed an assay using tert-butyl hydroperoxide (t-BOOH) as a substrate for specific Prx activity measurement. The assay involves incubating the enzyme substrates 1,4-dithio-DL-threitol (DTT) and t-BOOH in a suitable buffer at 37 °C for 10 min in a known volume of Prx enzyme. Following incubation, the reagent monobromobimane (mBB) is added to terminate the enzymatic reaction and produce a fluorescent product. Prx activity is subsequently determined by measuring thiol fluorescence, with reaction conditions optimized using a Bland-Altman plot. The efficacy of this novel protocol was rigorously validated by comparing Prx activity measurements from paired samples with those generated by a reference assay. A correlation coefficient of 0.995 was observed between the two methods, demonstrating superior precision and reliability compared to existing methods. The mBB-Prx protocol offers a significant safety advantage by using t-BOOH as a substrate for Prx activity measurement. As catalase does not catalyze t-BOOH dissociation, including sodium azide is unnecessary. Moreover, the method obviates the need for concentrated acids to terminate the Prx enzymatic reaction, as the mBB reagent efficiently inhibits Prx activity. This streamlined approach simplifies the assay and significantly improves its safety and usability, providing users with a reliable and convenient tool. The convenience of this method allows users to focus on their research without worrying about safety or complex procedures.
本文介绍了一种用于体外准确量化过氧化物还原酶（Prx）酶活性的新型荧光测定方法。Prx催化反应中的限速步骤是过氧化物的解离。为避免过氧化氢酶的干扰，我们开发了一种使用叔丁基过氧化氢（t-BOOH）作为底物来测量特定Prx活性的测定方法。该测定方法包括在已知体积的Prx酶中，于37°C下在合适的缓冲液中将酶底物1,4-二硫代-DL-苏糖醇（DTT）和t-BOOH孵育10分钟。孵育后，加入试剂单溴代双马来酰亚胺（mBB）以终止酶促反应并产生荧光产物。随后通过测量硫醇荧光来确定Prx活性，并使用布兰德-奥特曼图优化反应条件。通过将配对样品的Prx活性测量值与参考测定法产生的值进行比较，严格验证了这种新方案的有效性。两种方法之间观察到的相关系数为0.995，表明与现有方法相比具有更高的精度和可靠性。mBB-Prx方案通过使用t-BOOH作为Prx活性测量的底物，具有显著的安全优势。由于过氧化氢酶不催化t-BOOH解离，因此无需添加叠氮化钠。此外，该方法无需使用浓酸来终止Prx酶促反应，因为mBB试剂可有效抑制Prx活性。这种简化的方法简化了测定过程，显著提高了其安全性和可用性，为用户提供了一种可靠且便捷的工具。该方法的便利性使用户能够专注于研究，而无需担心安全问题或复杂的操作步骤。
