[Kamiya Biomedical] Ac-VEID-AFC (Fluorogenic caspase-1, 3, 6 substrate)

[Kamiya Biomedical] Ac-VEID-AFC (Fluorogenic caspase-1, 3, 6 substrate)

 

 

 

Cat. No.: AC-007 (10 mg)

 

Chemical Name: Ac-Val-Glu-Ile-Asp-AFC

Molecular Weight: 727.69

 

Description:

Peptide substrate labeled at the carboxy end with AFC (7-amino-4-trifluoromethyl coumarin). Designed to measure Caspase-6 activity in vitro.

Introduction: Caspase-6 (also known as Mch2) is a member of the caspase family of cysteine proteases involved in apoptosis. It is a member of the Group III caspases (6, 8, and 9) which prefer the (L/V)EXD sequence as a substrate. Caspase-6 prefers a hydrophobic amino acid at P4, along with caspases-1 and -4, as opposed to the preference for Asp seen with caspases-2, -3, and -7. This is at odds with the gene sequence alignment that predicts Caspase-6 is more closely related to caspases-3 and -7 than to caspase-1. The preference by Caspase-6 for ßbranched amino acids in P4 fits well with the one known natural substrate, lamin A, and distinguishes it from caspases-1 and -4. Reconstitution experiments indicate that Caspase-6 activates caspases-3 and -7 and is therefore part of the proteolytic cascade that initiates apoptosis.

Principal: A synthetic peptide substrate, Ac-Val-Glu-IleAsp, has been labeled with AFC (7-amino-4trifluoromethyl coumarin) at the carboxy end. AFC is a fluorescent molecule whose release from the substrate can be used to measure Caspase-6 activity. Caspase-6 activity in the sample is proportional to the amount of free AFC produced.

When AFC is attached to the peptide substrate, it produces a blue fluorescence upon exposure to UV light (400 nm). Caspase-6 enzymatically cleaves the AFC-substrate and releases free AFC, which produces a yellow- green fluorescence at 505 nm when exposed to UV light.

AFC has two advantages over other fluorogenic labels. The wide Stokes’ shift between bound and free AFC enables the substrate to be both chromogenic (yellow-green color visible to the naked eye) and fluorogenic (emission at 505 nm). The wide Stoke’s shift also makes the assay more sensitive.

Specificity: Highly specific substrate for Caspase-6. May be weak substrate for Caspases-1 and -3.

Applications: For in vitro assays of Caspase-6 activity. Can be used with purified or partially purified enzymes or possibly with crude cell lysates (if the Caspase-6 Inhibitor is included to determine background protease activity).

Protocol: Fluorometer calibration: The fluorometer is calibrated using known concentrations of free AFC (Excitation = 400 nm, Emission = 505 nm) to generate a standard curve of fluorescence versus µmoles AFC.

Samples: Can be either purified or partially purified enzyme preparations. Application to crude cell lysates has not been confirmed. If crude cell lysates are to be assayed, the nonspecific protease background must be determined using our Caspase-6 Inhibitor (Cat. No. AB-007).