𝔖 Bobbio Scriptorium
✦   LIBER   ✦

A continuous fluorescent assay for measuring protease activity using natural protein substrate

✍ Scribed by Wendy H. Farmer; Zhengyu Yuan

Publisher
Elsevier Science
Year
1991
Tongue
English
Weight
564 KB
Volume
197
Category
Article
ISSN
0003-2697

No coin nor oath required. For personal study only.

✦ Synopsis

A continuous caseinolytic activity assay has been developed and characterized with trypsin, a serine protease, and transin, a metalloproteinase. Beta-casein labeled with both N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and fluorescein isothiocyanate (FITC) is used as the substrate in this assay. The effect of proteolysis of the substrate is a reduction of the intermolecular energy transfer from DACM to FITC. The caseinolytic activity is then monitored by the fluorescence increase. The activities of both proteases obey Michaelis-Menten kinetics with Km = 1.6 +/- 0.2 microM for trypsin and Km = 13.2 +/- 1.9 microM for transin. Protease concentrations as low as 10 ng/mL can be utilized. The pH dependence of the caseinolytic activity has been determined for both enzymes.

πŸ“œ SIMILAR VOLUMES

BODIPY–α-Casein, a pH-Independent Protei
BODIPY–α-Casein, a pH-Independent Protein Substrate for Protease Assays Using Fluorescence Polarization
✍ Sylvia Z. Schade; Michael E. Jolley; Brian J. Sarauer; Lloyd G. Simonson πŸ“‚ Article πŸ“… 1996 πŸ› Elsevier Science 🌐 English βš– 123 KB

the molecular mass of the labeled molecule, the greater BODIPY-a-casein is a new fluorescent protein subthe motion (rotation) will be and the lower the emitted strate designed for fluorescence polarization studies fluorescence polarization value. When a relatively large to measure proteolytic activi