Edlaine Linares , Fernando R. Coelho , Natalia E. Lemos , Thiago G.P. Alegria , Luis ES. Netto , Ohara Augusto
{"title":"HyPer7: High-level bacterial expression and kinetics showing significant oxidation by peroxynitrite and hypochlorous acid","authors":"Edlaine Linares , Fernando R. Coelho , Natalia E. Lemos , Thiago G.P. Alegria , Luis ES. Netto , Ohara Augusto","doi":"10.1016/j.freeradbiomed.2025.09.042","DOIUrl":null,"url":null,"abstract":"<div><div>Genetically encoded probes of the HyPer family are regarded as specific for H<sub>2</sub>O<sub>2</sub>. However, this view may be premature due to the limited studies examining their reactivity with other biologically relevant oxidants under optimized conditions. Peroxynitrite is of particular interest due to its reactivity towards Cys residues, including the catalytic Cys of enzymes involved in cellular signaling. Here, we report the construction of a new plasmid, pET-15b-HyPer7, enabling high-level bacterial expression of HyPer7. The purified HyPer7 presented spectroscopic properties and sensitive response (F<sub>500</sub>/F<sub>400</sub>) to H<sub>2</sub>O<sub>2</sub> (2–20 μM) consistent with previously reported data. In contrast, HyPer7 (1 μM) also responded to peroxynitrite and hypochlorous acid (10–50 μM), though less efficiently than to H<sub>2</sub>O<sub>2</sub>. In all cases, the predominant oxidation product was consistent with HyPer7 disulfide formation, as evidenced by UV–Vis and fluorescence spectra, along with reducing SDS–PAGE; peroxynitrite and hypochlorous acid also yielded secondary products. Stopped-flow kinetics of Hyper7 (1 μM) oxidation by various H<sub>2</sub>O<sub>2</sub> (10–200 μM) or peroxynitrite concentrations (10–90 μM) allowed determination of second-order rate constants that differed by one order of magnitude, k<sub>H2O2</sub>= (3.60 ± 0.50) x10<sup>5</sup> M<sup>−1</sup> s<sup>−1</sup> and k<sub>ONOOH</sub>= (2.15 ± 0.03) x 10<sup>4</sup> M<sup>−1</sup> s<sup>−1</sup>, in phosphate buffer, pH 7.4 at 25<sup>ο</sup>C. Hypochlorous acid kinetics proved more complex and require further studies; initial assessments suggest a rate constant in the order of 10<sup>6</sup> M<sup>−1</sup> s<sup>−1</sup>. Overall, our results indicate that HyPer7 is less specific to H<sub>2</sub>O<sub>2</sub> than previously assumed. This conclusion likely extends to other members of the HyPer family due to the similar redox-sensitive domain.</div></div>","PeriodicalId":12407,"journal":{"name":"Free Radical Biology and Medicine","volume":"241 ","pages":"Pages 459-468"},"PeriodicalIF":8.2000,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Free Radical Biology and Medicine","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0891584925009992","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Genetically encoded probes of the HyPer family are regarded as specific for H2O2. However, this view may be premature due to the limited studies examining their reactivity with other biologically relevant oxidants under optimized conditions. Peroxynitrite is of particular interest due to its reactivity towards Cys residues, including the catalytic Cys of enzymes involved in cellular signaling. Here, we report the construction of a new plasmid, pET-15b-HyPer7, enabling high-level bacterial expression of HyPer7. The purified HyPer7 presented spectroscopic properties and sensitive response (F500/F400) to H2O2 (2–20 μM) consistent with previously reported data. In contrast, HyPer7 (1 μM) also responded to peroxynitrite and hypochlorous acid (10–50 μM), though less efficiently than to H2O2. In all cases, the predominant oxidation product was consistent with HyPer7 disulfide formation, as evidenced by UV–Vis and fluorescence spectra, along with reducing SDS–PAGE; peroxynitrite and hypochlorous acid also yielded secondary products. Stopped-flow kinetics of Hyper7 (1 μM) oxidation by various H2O2 (10–200 μM) or peroxynitrite concentrations (10–90 μM) allowed determination of second-order rate constants that differed by one order of magnitude, kH2O2= (3.60 ± 0.50) x105 M−1 s−1 and kONOOH= (2.15 ± 0.03) x 104 M−1 s−1, in phosphate buffer, pH 7.4 at 25οC. Hypochlorous acid kinetics proved more complex and require further studies; initial assessments suggest a rate constant in the order of 106 M−1 s−1. Overall, our results indicate that HyPer7 is less specific to H2O2 than previously assumed. This conclusion likely extends to other members of the HyPer family due to the similar redox-sensitive domain.
期刊介绍:
Free Radical Biology and Medicine is a leading journal in the field of redox biology, which is the study of the role of reactive oxygen species (ROS) and other oxidizing agents in biological systems. The journal serves as a premier forum for publishing innovative and groundbreaking research that explores the redox biology of health and disease, covering a wide range of topics and disciplines. Free Radical Biology and Medicine also commissions Special Issues that highlight recent advances in both basic and clinical research, with a particular emphasis on the mechanisms underlying altered metabolism and redox signaling. These Special Issues aim to provide a focused platform for the latest research in the field, fostering collaboration and knowledge exchange among researchers and clinicians.