Nawar Yaseen Mohsin, Halit Demir, Mahmoud Hussein Hadwan, Asad M Hadwan, Rawaa M Mohammed
{"title":"利用单溴比曼测量过氧化氢酶活性的新型荧光方法","authors":"Nawar Yaseen Mohsin, Halit Demir, Mahmoud Hussein Hadwan, Asad M Hadwan, Rawaa M Mohammed","doi":"10.1007/s10895-024-03991-4","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":15800,"journal":{"name":"Journal of Fluorescence","volume":null,"pages":null},"PeriodicalIF":2.6000,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"A New Fluorescent Method for Measuring Peroxiredoxin Enzyme Activity Using Monobromobimane.\",\"authors\":\"Nawar Yaseen Mohsin, Halit Demir, Mahmoud Hussein Hadwan, Asad M Hadwan, Rawaa M Mohammed\",\"doi\":\"10.1007/s10895-024-03991-4\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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. 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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.
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.