Back to Search
Start Over
Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes.
- Source :
-
The Analyst [Analyst] 2015 Jan 07; Vol. 140 (1), pp. 340-5. - Publication Year :
- 2015
-
Abstract
- A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.
Details
- Language :
- English
- ISSN :
- 1364-5528
- Volume :
- 140
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- The Analyst
- Publication Type :
- Academic Journal
- Accession number :
- 25386732
- Full Text :
- https://doi.org/10.1039/c4an01731e