Abstract
We have developed two microtiter plate assays to quantify the deoxyribonuclease activity in biological fluids. Both assays are based on hydrolysis of biotinylated and fluorescein-labeled DNA substrates, with subsequent immunochemical detection of non-digested DNA. The assay based on hydrolysis of 974 bp PCR product labeled with biotinylated forward and fluorescein-labeled reverse primers is more sensitive (0.05 U/ml) and convenient for quantifying the DNase activity in biological fluids than the assay based on hydrolysis of double-labeled 20 bp oligonucleotide. The DNase activity in urine and blood plasma of healthy donors was measured using the PCR product-based assay. Urine samples revealed greater activity, 1.49+/-1.41 U/ml; blood plasma DNase I-like activity was 0.36+/-0.20 U/ml. DNase II-like activity was not detected in the plasma samples. The data obtained confirm that DNase I-like enzymes are responsible for the majority of deoxyribonuclease activity in blood plasma.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / pharmacology
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Avidin / chemistry
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Biotinylation
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Body Fluids / enzymology*
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Calibration
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Catalysis / drug effects
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Cell Line, Tumor
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DNA / chemistry
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DNA / metabolism
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Deoxyribonuclease I / blood
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Deoxyribonuclease I / metabolism
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Deoxyribonuclease I / urine
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Deoxyribonucleases / blood*
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Deoxyribonucleases / metabolism
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Deoxyribonucleases / urine*
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Edetic Acid / pharmacology
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Endodeoxyribonucleases / blood
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Endodeoxyribonucleases / metabolism
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Endodeoxyribonucleases / urine
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Enzyme-Linked Immunosorbent Assay
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Female
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Fluorescein / chemistry
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Horseradish Peroxidase / chemistry
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Humans
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Immunoassay / methods
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Immunochemistry
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Male
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Oligodeoxyribonucleotides / chemistry
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Oligodeoxyribonucleotides / genetics
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Oligodeoxyribonucleotides / metabolism
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RNA, Ribosomal, 18S / genetics
Substances
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Actins
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Oligodeoxyribonucleotides
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RNA, Ribosomal, 18S
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Avidin
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DNA
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Edetic Acid
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Horseradish Peroxidase
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Deoxyribonucleases
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Endodeoxyribonucleases
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Deoxyribonuclease I
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deoxyribonuclease II
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Fluorescein