An assay for proteinases and their inhibitors based on DNA/ethidium bromide fluorescence.
Proteinases and their inhibitors have become the subject of intense research interest recently, since they control a multitude of very important biological processes, from the development of lambda phage to hypertension in humans. We have developed a simple and sensitive assay for detecting the activity of proteinases and of their proteinase inhibitors. The assay is based on ethidium bromide fluorescence, according to the following principles: (i) Ethidium bromide increases its fluorescence by 25-fold when it intercalates between base pairs of double-stranded DNA. (ii) Histones prevent this large increase in fluorescence by binding with high affinity to DNA thus blocking ethidium bromide intercalation. (iii) A proteinase that digests histones will make more DNA available for ethidium bromide intercalation, thereby producing an increase of fluorescence. Proteinase activity can easily be determined, in the presence of a DNA/histone complex, from the rate of ethidium fluorescence increase. In contrast, activity of a proteinase inhibitor is quantitated by the inhibition of fluorescence gain in the presence of a known amount of proteinase. This assay is rapid, simple, inexpensive, and, at the same time, accurate and sensitive enough to allow quantitation of nanogram amounts of various broad-specificity proteinases and their inhibitors. We show some possible applications of the assay (i) in testing column fractions during protein purifications, (ii) quantitation of alpha 1-antitrypsin in human serum, and (iii) detection of proteinase activity in cell extracts.
Severini
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Department of Biochemistry, University of Alberta, Edmonton, Canada.
Morgan
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AR
D004247:DNA / Q000737:chemistry*; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry*; D005453:Fluorescence; D005470:Fluorometry; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D011480:Protease Inhibitors / Q000032:analysis*; D013379:Substrate Specificity
D006657:Histones; D011480:Protease Inhibitors; D004247:DNA; D010450:Endopeptidases; D004996:Ethidium