Capillary electrophoretic determination of cathepsin D activity using Oregon Green-labeled hemoglobin.
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 alpha-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.
Chu
Q
Q
Doheny Eye Institute, Los Angeles, CA, USA.
Jones
S
S
Zeece
M
M
D002402:Cathepsin D / Q000032:analysis*; D002460:Cell Line; D019075:Electrophoresis, Capillary; D006454:Hemoglobins; D006801:Humans; D018482:Muscle, Skeletal / Q000378:metabolism
D006454:Hemoglobins; D002402:Cathepsin D