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  • As shown in B the recombinant

    2024-06-07

    As shown in B, the recombinant human 15-LOX-1 (30nM) showed a time-dependent increase in fluorescent signal, and signal development was almost completed within 20min in the presence of 50μM arachidonic apigenin and 5μM DHR. For both purified enzyme and cell lysates, the enzyme activities disappeared without arachidonic acid or after the addition of the 15-LOX-1 enzyme inhibitor NDGA. HEK293T cell lysates that did not express 15-LOX-1 (mock) also had no enzyme activity, like the 15-LOX-1-expressing lysates in the absence of arachidonic acid (). The experiment indicated that the observed fluorescent signal was 15-LOX-1 and arachidonic acid dependent. In addition, the blank well showed a very slow increase in fluorescent signal owing to DHR autoxidation in the air. The fluorimetric assay was sensitive and had a dynamic range from 3 to 120nM for human 15-LOX-1, and the concentrations of arachidonic acid and DHR were optimized to 50 and 5μM, respectively ( Fig. S3). Enzyme assays for determination were carried out for purified 15-LOX-1 under similar conditions, except that the concentration of arachidonic acid varied from 1 to 100μM. Using the fluorescence-based method, a of 2.85±0.21μM was calculated through Lineweaver–Burk plots, and it is in fair agreement with previously published values obtained using spectrophotometric and chemiluminescence methods () , . To validate our assay system, 12-HpETE and 15-HpETE, the products of human 15-LOX-1, were tested for their oxidation capacity on DHR. As presented in , 12-HpETE and 15-HpETE could cause a linear fluorescence increase by directly oxidizing nonfluorescent DHR to the highly fluorescent dye rhodamine 123. The apigenin experiment demonstrated the principle of the fluorimetric assay for 15-LOX-1: the increase in fluorescence was based on the production of 12-HpETE and 15-HpETE (). Although continuous fluorescence assays are beneficial to enzyme kinetics study, they are time consuming and unsuitable for the high-throughput screening of a chemical library. So, we evaluated the feasibility of an endpoint method. To validate this new assay further, three well-known nonselective inhibitors of human 15-LOX-1, NDGA, quercetin, and fisetin, were evaluated against 15-LOX-1 in both 96- and 384-well microplate formats (). Recombinant human 15-LOX-1 or cell lysates were incubated with various concentrations of compounds in assay buffer (50mM Tris, pH 7.4) with 5μM DHR for 3min at room temperature. The reaction was started by quickly adding arachidonic acid solution (final concentration 50μM). After incubation for 15min at room temperature, the plate was read using an excitation wavelength of 500nm and an emission wavelength of 536nm through a Synergy Multi-Mode microplate reader (BioTek). The inclusion of 1.5% dimethyl sulfoxide in the assay as a result of inhibitor and fluorogenic probe DHR addition inhibited the reaction less than 5% (data not shown). As presented in , in our fluorimetric assay, NDGA, quercetin, and fisetin behaved as potent inhibitors of human 15-LOX-1, with IC values of 0.088, 0.26, and 0.23μM, respectively, which were similar to previously reported data from an Fe/xylenol orange-based screening method . The IC values calculated from the 384-well microplate were comparable to those from the 96-well microplate. All our results indicated that the newly developed fluorimetric assay was adaptable for high-throughput compound screening.
    Introduction Lipoxygenases (LOs) are enzymes catalyzing the positional as well as stereo-specific introduction of molecular oxygen into 1,4-pentadiene structures found in unsaturated fatty acids such as arachidonic acid or linoleic acid [1], [2], [3]. The positional specificity of the introduction of molecular oxygen in arachidonic acid is commonly used to classify lipoxygenases. Thus, 5-LO introduces oxygen at carbon 5 of arachidonic acid and 12-LO at carbon 12. Lipoxygenases are distributed throughout the animal, plant and fungi kingdom as well as in several prokaryotes [4], [5], [6]. The mammalian 15-LO (EC 1.13.11.33) was first purified and characterized 1975 [7]. Later on it was demonstrated that the enzyme showed a fairly restricted expression pattern with the airway epithelium [8], eosinophils [9], reticulocytes [7], macrophages [10], dendritic cells [11], human mast cells [12] and Hodgkin lymphoma [13] as being the most prominent sites of expression. It has also been shown that the expression can be regulated by inflammatory cytokines such as interleukin-4 (IL-4) and IL-13 [8], [10], [11], [13]. The regulation of 15-LO-1 by pro-inflammatory cytokines is in line with the role of 15-LO-1 in the formation of pro-inflammatory mediators such as the eoxins [14]. These potent mediators have been described to be formed in cells involved in airway inflammation such as eosinophils and mast cells [14]. Several studies have demonstrated increased expression and activity of 15-LO-1 in the bronchial mucosa of patients with asthma compared with control subjects [15], [16]. Furthermore, mice deficient of 12/15-LO had an attenuated allergic airway inflammation compared to wild-type controls [17], [18]. On the other hand, the enzyme can also under certain conditions be involved in the formation of lipoxins which possess anti-inflammatory actions [19]. A second 15-LO was discovered in 1997 and it is hence named 15-LO-2 whereas the first reported 15-lipoxygenase is referred to as 15-LO-1.