Microplate assay for determination of cathepsin D activity based on quantification of a specific and stable peptide released from hemoglobin: VV-hemorphin-7 Original Research Article

Cathepsins are key enzymes in mediating turnover of cytosolic proteins. In the context of cancer progression, those most actively studied include cathepsins D and B which have been implicated in processes such as growth and metastasis of many types of cancer. For more than 10 years, their roles as tumor marker and prognostic indicators have been studied, especially in breast cancer. Most of the studies relating the role of cathepsin D in cancer used immunological detection methods to determine the level of enzyme but do not reflect enzyme activity. Moreover, one of the problems in understanding cathepsin D clinical studies is that immunoassays may employ antibodies against the different form of the antigen. As an alternative, this work describes an indirect method to assess the active form of cathepsin D based on ELISA quantification of a specific and stable product of hemoglobin hydrolysis: VV-hemorphin-7. The procedure described here allows a low detection limit (ca. 5×10−9 M) and thus can represent an original approach to evaluate cathepsin D activity in biological samples.