A new assay method for DNase by fluorescence polarization and fluorescence intensity using DNA-ethidium bromide complex as a sensitive substrate.
The fluorescence polarization method was applied for the determination of DNase-activity using ethidium bromide-labeled DNA as a substrate. This assay was based on a decrease in fluorescence intensity or fluorescence polarization value, which reflected degraded molecular size resulting from the specific cleavage of DNA by the enzyme. DNase activity was quantitatively determined above 0.1 ng/ml of the enzyme. A decrease in fluorescence intensity or polarization value brought about by DNase was inhibited by the specific inhibitors of DNase such as EDTA, pyrophosphate, and thymidine-3',5'-diphosphate. In addition, specificity of the change of polarization value was established using other enzymes. Since this method was simple, rapid and sensitive, it is applicable for both quantitative and qualitative assays as well as for the kinetic analysis of the enzyme.
D004247:DNA; D003851:Deoxyribonucleases / Q000032:analysis* / Q000037:antagonists & inhibitors; D004996:Ethidium; D005454:Fluorescence Polarization; D008832:Microchemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D004247:DNA; D003851:Deoxyribonucleases; D004996:Ethidium