title: Determination of DNase activity by degradation of ethidium bromide-DNA complexes using a fluorescence plate reader.
abstract: The long known toxicity of free chromatin mediated by histones regained attention after discovery of neutrophil extracellular traps (NETs). Free histones from necrotic cells or NETs can damage prokaryotic and eukaryotic cells and are responsible for the aggravation of a growing list of diseases. DNases degrade the toxic chromatin polymer to nucleosomes and efficiently reduce local high histone concentrations. Therefore, DNase activity as a biomarker is of growing interest in basic and clinical research. Here a detailed one-step protocol is presented that allows rapid and sensitive detection of DNases down to 400 fg/μl per reaction based on the detection of fluorescent ethidium bromide/DNA complexes in a 96-well plate reader. The flexible protocol uses an internal standard for background correction and allows convenient and reliable data analysis using common laboratory equipment and chemicals without elaborate preparations. The DNase activity of a sample is clearly defined by substrate amount, incubation time, and (if appropriate) a DNase standard for absolute quantification in Kunitz units per milligram sample protein. Quantitative kinetic determination is possible within less than 1h down to 5 pg DNases/μl per reaction.
authors.affiliation: Comprehensive Heart Failure Center (CHCF), Universitätsklinikum Würzburg, 97078 Würzburg, Germany; Medizinische Klinik und Poliklinik I, Universitätsklinikum Würzburg, 97078 Würzburg, Germany. Electronic address: vogel_b@ukw.de.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Vogel
authors.affiliation: Universitätsklinik und Poliklinik für Innere Medizin III, Universitätsklinikum Halle (Saale), 06120 Halle (Saale), Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Frantz
mesh_terms: D002138:Calibration; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D004996:Ethidium / Q000737:chemistry*; D007700:Kinetics; D013050:Spectrometry, Fluorescence
chemical_list: D004247:DNA; D003851:Deoxyribonucleases; D004996:Ethidium
keywords: DNase activity; DNase1; Ethidium bromide fluorescence; Free chromatin