The former utilizes a FRET (fluorescent resonance energy transfer)-based peptide substrate and the latter, a fluorophore labeled substrate. Both substrates emit at the green emission wavelength of 520 nm (excitation at 490 nm); consequently, background fluorescence is lower. As well, both substrates provide minimal (if any) cross-reaction with other cathepsins.
The FRET peptide in the SensoLyte® 520 kit employs the QXL™ 520/HiLyte Fluor™ 488 pair for optimal quenching of the intact substrate. Hydrolysis of the FRET substrate by active Cathepsin L results in increase of HiLyte Fluor™ 488 fluorescence. Additionally, the fluorescent signal from HiLyte Fluor™ 488 is stable at low pH, the pH at which Cathepsin L activity is optimal. This homogeneous assay can be used for the detection of Cathepsin L activity in biological samples or in purified enzyme preparations. The SensoLyte® Rh110 Cathepsin L Activity Assay Kit provides a convenient assay for screening Cathepsin L inhibitors or assay of enzyme activity using Rh110 (rhodamine 110). Upon cleavage by Cathepsin L, this substrate generates Rh110 emitting bright green fluorescence.
Cathepsin L, a lysosomal endopeptidase, is a member of the papain-like family of cysteine proteinases (1). It is involved in the promotion of tumor cell invasion (2), antigen processing and turnover of intracellular and secreted proteins (3). It also plays a major role in growth regulation, bone resorption and metastasis (4, 5).
1. Barrett, A.J. et al. Meth Enzymol 80, 535 (1981).
2. Gottesman, M.M. et al. Biochem 20, 1659 (1981).
3. Ishidoh, K. et al. Biochem Biophys Res Commun 217, 624 (1995).
4. Kane, S.E. et al. Semin Cancer Biol 1, 127 (1990).
5. Kirschke, H. et al. Eur J Cancer 36, 787 (2000).
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