利用单溴比曼测量过氧化氢酶活性的新型荧光方法

IF 2.6 4区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Nawar Yaseen Mohsin, Halit Demir, Mahmoud Hussein Hadwan, Asad M Hadwan, Rawaa M Mohammed
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引用次数: 0

摘要

本文介绍了一种新的荧光测定法,用于准确量化体外过氧化物歧化酶(Prx)的酶活性。Prx 催化反应的限速步骤是过氧化物的解离。为了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)作为底物的检测方法,用于特异性地测量 Prx 活性。该检测方法是将酶底物 1,4-二硫代-DL-苏糖醇(DTT)和叔丁基过氧化氢(t-BOOH)放入适当的缓冲液中,与已知体积的 Prx 酶在 37 ℃ 下孵育 10 分钟。孵育后,加入试剂单溴比曼(mbb)以终止酶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,并利用布兰-阿尔特曼曲线图对反应条件进行优化。通过比较成对样本的 Prx 活性测量值和参考测定法产生的测量值,严格验证了这种新方案的有效性。两种方法之间的相关系数为 0.995,显示出与现有方法相比更高的精确度和可靠性。mBB-Prx 方案使用 t-BOOH 作为 Prx 活性测定的底物,具有显著的安全性优势。由于过氧化氢酶不会催化 t-BOOH 的解离,因此无需加入叠氮化钠。此外,由于 mBB 试剂能有效抑制 Prx 活性,因此该方法无需使用浓酸来终止 Prx 酶反应。这种精简的方法简化了检测过程,大大提高了安全性和可用性,为用户提供了可靠而方便的工具。这种方法的便利性使用户可以专注于他们的研究,而不必担心安全性或复杂的程序。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A New Fluorescent Method for Measuring Peroxiredoxin Enzyme Activity Using Monobromobimane.

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.

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来源期刊
Journal of Fluorescence
Journal of Fluorescence 化学-分析化学
CiteScore
4.60
自引率
7.40%
发文量
203
审稿时长
5.4 months
期刊介绍: Journal of Fluorescence is an international forum for the publication of peer-reviewed original articles that advance the practice of this established spectroscopic technique. Topics covered include advances in theory/and or data analysis, studies of the photophysics of aromatic molecules, solvent, and environmental effects, development of stationary or time-resolved measurements, advances in fluorescence microscopy, imaging, photobleaching/recovery measurements, and/or phosphorescence for studies of cell biology, chemical biology and the advanced uses of fluorescence in flow cytometry/analysis, immunology, high throughput screening/drug discovery, DNA sequencing/arrays, genomics and proteomics. Typical applications might include studies of macromolecular dynamics and conformation, intracellular chemistry, and gene expression. The journal also publishes papers that describe the synthesis and characterization of new fluorophores, particularly those displaying unique sensitivities and/or optical properties. In addition to original articles, the Journal also publishes reviews, rapid communications, short communications, letters to the editor, topical news articles, and technical and design notes.
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