Abstract
Cathepsin D is an aspartyl protease of lysosomal origin and functions in a variety of roles including protein turnover, catabolism of peptide hormones, antigen processing and presentation, and neoplastic disease. In breast cancer, the level of cathepsin D has been linked to metastasis and prognosis for survivability. Many of these studies concerning the role of cathepsin D in cancer have used immunological detection methods to determine the level of enzyme. These indirect methods to assess the cathepsin D level may not reflect enzyme activity accurately. The significance of cathepsin D to physiological and pathophysiological processes suggests that rapid and sensitive methods for determining cathepsin D activity would contribute to a more complete assessment of this enzyme in its various roles. This work describes a procedure to determine cathepsin D activity based on hydrolysis of fluorescently labeled hemoglobin and employs capillary electrophoresis to separate and measure the products of reaction. A single major cleavage product, representing the first 32 residues of the hemoglobin α-chain, appeared after a very short incubation time (less than 10 min) and was used to determine activity. The procedure described here requires very small sample volumes, has a low detection limit (approximately 10-9 M) and thus represents an additional approach to determine cathepsin D activity in biological samples.
Original language | English (US) |
---|---|
Pages (from-to) | 2945-2951 |
Number of pages | 7 |
Journal | ELECTROPHORESIS |
Volume | 20 |
Issue number | 14 |
DOIs | |
State | Published - 1999 |
Keywords
- Capillary electrophoresis
- Cathepsin D
- Protease activity
ASJC Scopus subject areas
- Analytical Chemistry
- Biochemistry
- Clinical Biochemistry