A fluorescence-based assay for ribonuclease A activity.
A sensitive assay for ribonuclease A activity based on the relief of fluorescence quenching within a defined oligomeric substrate (5' fluorescein-AAAArUAAAA-3'-rhodamine) is described. The substrate can be produced using an automated nucleic acid synthesizer and commercially available reagents. Together with a nonfluorescent cosubstrate (5'-dimethoxytrityl-AAAArUAAAA), the compound can be used to determine kinetic constants for the first step (transphosphorylation) of the ribonuclease-catalyzed reaction. These measurements should be useful for structure-based analyses of ribonuclease activity since a crystal structure has been determined for a closely analogous enzyme-inhibitor complex.
James
D A
DA
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, M5S 3H6, Canada.
Woolley
G A
GA
D005452:Fluoresceins; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D015345:Oligonucleotide Probes / Q000378:metabolism; D009841:Oligonucleotides; D012235:Rhodamines; D012259:Ribonuclease, Pancreatic / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity
C115609:5'-fluorescein-AAAArUAAAA-3'-rhodamine; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D012235:Rhodamines; D012259:Ribonuclease, Pancreatic