Caspase 3 (Apopain) Substrate 1m, fluorogenic

Product Name: Caspase 3 (Apopain) Substrate 1m, fluorogenic

Sequence One Letter Code: Ac-DEVD-AMC

Sequence Three Letter Code: Ac-Asp-Glu-Val-Asp-AMC

Cas No: 169332-61-0

Chemical Formula:C30H37N5O13

Molecular Weight: 675.7

Purity: 95%

Form: Lyophilized

Storage Conditions: - 20 °C Protected from light

Research Area: peptide substrate

SMILES: CC1=CC(=O)OC2=C1C=CC(=C2)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)C

IUPAC: (4S)-4-[[(2S)-2-acetamido-3-carboxypropanoyl]amino]-5-[[(2S)-1-[[(2S)-3-carboxy-1-[(4-methyl-2-oxochromen-7-yl)amino]-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-oxopentanoic acid

INCHIKEY: ALZSTTDFHZHSCA-RNVDEAKXSA-N

INCHI:

InChI=1S/C30H37N5O13/c1-13(2)26(35-27(44)18(7-8-22(37)38)33-29(46)19(11-23(39)40)31-15(4)36)30(47)34-20(12-24(41)42)28(45)32-16-5-6-17-14(3)9-25(43)48-21(17)10-16/h5-6,9-10,13,18-20,26H,7-8,11-12H2,1-4H3,(H,31,36)(H,32,45)(H,33,46)(H,34,47)(H,35,44)(H,37,38)(H,39,40)(H,41,42)/t18-,19-,20-,26-/m0/s1

Source / Species:

Conjugation: Conjugated

Conjugation Type: Fluorescent dyes

Code Nacres: NA.26

Application: Caspase-3 (Apopain) Substrate 1m is a fluorogenic peptide substrate optimized for sensitive detection of executioner caspase activity in apoptosis research. It is efficiently cleaved by caspase-3 (Km ≈ 9.7 µM) and caspase-7 (Km ≈ 11 µM), enabling reliable profiling of apoptotic signaling in biochemical and cell-based assays. Upon proteolytic cleavage, the AMC (7-amino-4-methylcoumarin) fluorophore is released, generating a strong fluorescent signal measurable by fluorimetry (Ex 340–350 nm, Em 440–460 nm). This substrate supports quantitative assessment of caspase activation, kinetic analysis, and inhibitor screening in studies of programmed cell death.

Current Research: Caspase-3 (Apopain) Substrate 1m is a fluorogenic peptide substrate specifically optimized for sensitive detection of executioner caspase activity, particularly caspase-3 and caspase-7, in apoptosis research. The peptide sequence is designed to be efficiently recognized and cleaved by these proteases, with reported kinetic parameters of approximately Km ≈ 9.7 µM for caspase-3 and Km ≈ 11 µM for caspase-7, supporting reliable enzymatic profiling in both purified systems and complex biological samples. Upon proteolytic cleavage at the caspase recognition site, the 7-amino-4-methylcoumarin (AMC) fluorophore is released from the peptide backbone. Free AMC produces a strong fluorescent signal measurable by fluorimetry, typically at excitation 340–350 nm and emission 440–460 nm, enabling quantitative and kinetic monitoring of caspase activation. Biological Context Caspase-3 and caspase-7 are terminal executioner caspases activated downstream of intrinsic and extrinsic apoptotic pathways. Once activated, they cleave a wide range of structural and regulatory proteins, driving hallmark features of apoptosis such as: DNA fragmentation Cytoskeletal breakdown Membrane blebbing Formation of apoptotic bodies Accurate measurement of caspase-3/7 activity is therefore a central endpoint in programmed cell death studies. Mechanism of Detection Caspase-3 Substrate 1m incorporates a canonical caspase-3/7 recognition motif linked to AMC. In its intact form, fluorescence is quenched due to conjugation within the peptide structure. Enzymatic cleavage liberates AMC, resulting in: Increased fluorescence intensity Time-dependent signal accumulation Direct correlation between fluorescence output and protease activity This format enables continuous kinetic monitoring without additional detection reagents. Research Applications 1. Apoptosis Quantification in Cell Lysates The substrate is widely used to measure caspase activation following treatment with chemotherapeutics, oxidative stress, cytokines, or death receptor agonists. Increased fluorescence indicates activation of the executioner caspase cascade. 2. Real-Time Kinetic Enzyme Assays In purified enzyme systems, Substrate 1m supports determination of kinetic parameters (Km, Vmax) and catalytic efficiency, enabling mechanistic studies of caspase activity. 3. Inhibitor Screening and Drug Discovery The fluorogenic readout is compatible with high-throughput screening platforms for identifying caspase inhibitors or modulators of apoptotic signaling. Competitive or irreversible inhibitors can be evaluated based on reduction in AMC release. 4. Comparative Apoptotic Pathway Analysis By combining caspase-3 Substrate 1m with initiator caspase substrates (e.g., caspase-8 or caspase-9), researchers can dissect pathway hierarchy and confirm executioner caspase engagement. Advantages High sensitivity and low background signal Continuous kinetic monitoring capability Compatibility with microplate fluorimeters Suitable for biochemical and cell-based assays Quantitative and reproducible measurement of caspase-3/7 activity Experimental Considerations Assay performance depends on appropriate buffer composition (typically containing reducing agents such as DTT) and optimal substrate concentration relative to Km. Fluorescence measurements should be performed under consistent temperature and pH conditions to ensure reproducibility. Inclusion of selective caspase inhibitors can confirm assay specificity.