Caspase 9 Substrate 1, chromogenic

Product Name: Caspase 9 Substrate 1, chromogenic

Sequence One Letter Code: Ac-LEHD-pNA

Sequence Three Letter Code: Ac-LEHD-pNA

Chemical Formula:C29H38N8O11

Molecular Weight: 674.7

Purity: 95%

Form: Lyophilized

Storage Conditions: - 20 °C Protected from light

Research Area: peptide substrate

Conjugation: Conjugated

Conjugation Type: Conjugates

Code Nacres: NA.26

Application: Caspase-9 Substrate 1, chromogenic, is a pNA-based peptide substrate developed for colorimetric detection of caspase-9 activity in intrinsic apoptosis research. Caspase-9 functions as an initiator caspase within the apoptosome complex, activating downstream executioner caspases. Proteolytic cleavage releases 4-nitroaniline, which generates an absorbance signal detectable at approximately 405–408 nm. This substrate is well suited for enzymatic assays, kinetic characterization, and inhibitor screening focused on upstream mitochondrial apoptotic signaling mechanisms.

Current Research: Caspase-9 Substrate 1, chromogenic is a peptide-based pNA (4-nitroaniline)–conjugated substrate designed for colorimetric detection of caspase-9 activity, a key initiator protease in the intrinsic (mitochondrial) apoptosis pathway. The peptide incorporates a caspase-9 recognition motif linked to pNA, enabling direct spectrophotometric monitoring of enzymatic cleavage. Upon proteolysis, 4-nitroaniline (pNA) is released, producing a measurable absorbance increase at approximately 405–408 nm, allowing quantitative assessment using standard microplate readers. Biological Context Caspase-9 plays a central role in intrinsic apoptosis. In response to mitochondrial outer membrane permeabilization (MOMP), cytochrome c is released into the cytosol, where it associates with Apaf-1 and procaspase-9 to form the apoptosome complex. This assembly facilitates autocatalytic activation of caspase-9, which then cleaves and activates downstream executioner caspases such as caspase-3 and caspase-7. Because caspase-9 acts upstream in the apoptotic cascade, its activity serves as a key indicator of mitochondrial pathway engagement. Mechanism of Detection In the intact substrate, the peptide–pNA conjugate exhibits minimal absorbance at 405 nm. Upon cleavage at the caspase-9 recognition sequence: The peptide bond adjacent to pNA is hydrolyzed. Free pNA is liberated. Accumulation of pNA produces a time-dependent increase in absorbance. The rate of absorbance increase correlates directly with caspase-9 enzymatic activity, enabling both endpoint and kinetic measurements. Research Applications 1. Intrinsic Apoptosis Monitoring The substrate is widely used to measure caspase-9 activation in cell lysates following mitochondrial stress, DNA damage, oxidative injury, or chemotherapeutic treatment. 2. Kinetic Characterization Purified recombinant caspase-9 can be analyzed to determine kinetic parameters such as Km and Vmax, supporting mechanistic studies of apoptosome-mediated activation. 3. Inhibitor Screening The chromogenic format is well suited for evaluating selective caspase-9 inhibitors. Reduced pNA release allows calculation of IC₅₀ values and assessment of inhibitory potency. 4. Pathway Differentiation Studies By pairing caspase-9 substrate assays with executioner caspase substrates (e.g., caspase-3), researchers can distinguish upstream initiation events from downstream effector activation. Advantages Simple colorimetric readout No requirement for fluorescence instrumentation Continuous kinetic monitoring capability Compatible with purified enzyme and cell lysate assays Suitable for medium-throughput screening applications Experimental Considerations Optimal assay conditions typically include reducing agents (e.g., DTT) to maintain caspase activity and appropriate buffer composition near physiological pH. Because caspase-9 activation often depends on apoptosome assembly, assay configuration should reflect whether recombinant enzyme or cell lysate systems are used. Inclusion of selective inhibitors can confirm assay specificity.