Caspase 3/7 are cysteine/aspartic acid-specific protease and play key effector roles in apoptosis in mammalian cells1–4.
The split luciferin approach enables the generation of D-luciferin through the reaction of D-Cysteine and 6-amino-2-cyanobenzothiazole (NH2-CBT). The D-Cysteine amino acid has been caged with a caspase 3/7 specific peptidic DEVD sequence. The modification of the amino group on D-Cysteine prohibits reaction with the counterpart NH2-CBT since the 1,2-aminothiol is required for the reaction with the cyano group. After cleavage of the peptide by Caspase 3/7 free D-Cysteine is liberated and can further react with NH2-CBT to form Luciferin and then bioluminescence through oxydation by Luciferase (Figure 1). Luminescence is thus proportional to the amount of active caspase 3/7.5
One of the advantages of the split luciferin approach is improved cell penetration property of CBT derivatives in comparison to full luciferin scaffold. The Caspase 3/7 quantification kit is also more sensitive than fluorescence-based caspase assays. The assay uses a substrate containing the DEVD sequence that is selective for caspase 3 and 7.6
This technology has been developed to measure caspase 3/7 acitivity in live cells but also in living animals. The caspase 3/7 quantification kit provide a very high signal to background ratio allowing sensitive detection of caspase 3/7 activity.
The assay provides a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence linked to D-Cysteine Z-Asp-Glu-Val-Asp-D-Cys (Z-DEVD-(D-Cys)), and 6-amino-2-cyanobenzothiazole (NH2-CBT).