HyPer7: High-level bacterial expression and kinetics showing significant oxidation by peroxynitrite and hypochlorous acid

IF 8.2 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Edlaine Linares , Fernando R. Coelho , Natalia E. Lemos , Thiago G.P. Alegria , Luis ES. Netto , Ohara Augusto
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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.

Abstract Image

HyPer7:高水平细菌表达和动力学显示过氧亚硝酸盐和次氯酸的显著氧化。
基因编码的HyPer家族探针被认为对H2O2具有特异性。然而,由于在优化条件下研究它们与其他生物相关氧化剂的反应性的研究有限,这种观点可能为时过早。过氧亚硝酸盐是特别感兴趣的,因为它对胱氨酸残基的反应性,包括参与细胞信号传导的酶的催化胱氨酸。在这里,我们报道了一种新的质粒pET-15b-HyPer7的构建,使HyPer7的高水平细菌表达成为可能。纯化后的HyPer7对H2O2 (2-20 μM)的光谱特性和敏感响应(F500/F400)与先前报道的数据一致。相比之下,HyPer7 (1 μM)对过氧亚硝酸盐和次氯酸(10-50 μM)也有响应,但对H2O2的响应效率较低。在所有情况下,主要氧化产物与HyPer7二硫化物形成一致,如紫外-可见和荧光光谱所证明的,以及还原SDS-PAGE;过氧亚硝酸盐和次氯酸也能产生二次产物。在pH为7.4,25οC的磷酸盐缓冲液中,不同浓度的H2O2 (10-200 μM)或过氧亚硝酸盐(10-90 μM)对Hyper7 (1 μM)氧化的停流动力学允许测定不同数量级的二阶速率常数,kH2O2=(3.60±0.50)x105 M-1 s-1和kONOOH=(2.15±0.03)x 104 M-1 s-1。次氯酸动力学更为复杂,需要进一步研究;初步估计表明速率常数约为106 M-1 s-1。总的来说,我们的结果表明HyPer7对H2O2的特异性比之前假设的要低。由于类似的氧化还原敏感结构域,这一结论可能适用于HyPer家族的其他成员。
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来源期刊
Free Radical Biology and Medicine
Free Radical Biology and Medicine 医学-内分泌学与代谢
CiteScore
14.00
自引率
4.10%
发文量
850
审稿时长
22 days
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.
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