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Detection of Protease Activity Using a Fluorescence-Enhancement Globular Substrate
- Source :
- BioTechniques, Vol 20, Iss 2, Pp 286-291 (1996)
- Publication Year :
- 1996
- Publisher :
- Future Science Ltd, 1996.
-
Abstract
- Bovine serum albumin (BSA) highly derivatized with fluorescein isothiocyanate (FITC, isomer I) served as a fluorescent enhancement substrate to measure protease activity. In the native globular BSA structure, the fluorescence of the lysine-conjugated fluorescein moieties was quenched 98%. Proteolytic digestion of highly derivatized BSA with Pronase® resulted in fluorescence enhancement of 4300%. Both α-chymotrypsin and proteinase K yielded lower but similar fluorescence enhancement values of 2880% and 2800%, respectively. Digestion of the fluorescein-BSA substrate with trypsin, which required basic amino acids for activity, showed fluorescence enhancement of 1480% reflecting the fluorescein-lysine thiocarbamyl linkage. When derivatized substrate was pretreated with a thiol-reducing agent prior to incubation with proteases, a relatively small increase in fluorescence was noted relative to the untreated substrate except in the case of Pronase. The minimum sensitivity of proteolytic activity, based on a comparison of untreated and reduced FITC25BSA was 32 × 10−6 units for 1 ng proteinase K, 1 × 10−3 units for 1 ng α-chymotrypsin and 10 × 10−3 units for Pronase and trypsin (1 ng each). The fluorescence enhancement assay was suited for sensitive intensity measurements or as an endpoint assay to detect protease activity.
- Subjects :
- Biology (General)
QH301-705.5
Subjects
Details
- Language :
- English
- ISSN :
- 19409818 and 07366205
- Volume :
- 20
- Issue :
- 2
- Database :
- Directory of Open Access Journals
- Journal :
- BioTechniques
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.f2c8230554e944b095fd3582f93c77e4
- Document Type :
- article
- Full Text :
- https://doi.org/10.2144/96202rr06