Fluorometric determination of bacterial protease activity using fluorescein isothiocyanate-labeled proteins as substrates.
Intact fluorescein isothiocyanate-labeled proteins have relatively low background fluorescence at excitation and emission wavelengths of 495 and 525 nm, respectively. Degradation of these substrates leads to exposure of covalently linked fluorescein isothiocyanate molecules and to a concomitant increase in relative fluorescence at these wavelengths. The increase in relative fluorescence is proportional to the degree of protein degradation. This phenomenon provides the basis for a sensitive assay for bacterial protease activity. There is no requirement for the removal of undegraded substrate from the assay mixture prior to the measurement of fluorescence. Assays can be performed in 96-well microtiter trays, enabling a large number of samples and their respective controls to be processed simultaneously and repeated determinations of fluorescence values may be made on the same assay.
Homer
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Hunterian Dental Research Unit, London Hospital Medical College, United Kingdom.
Beighton
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D001439:Bacteroides / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; C039217:fluorescein isothiocyanate bovine serum albumin; D012710:Serum Albumin, Bovine; D010450:Endopeptidases; D016650:Fluorescein-5-isothiocyanate