Ac-IEAR-pNA is a chromogenic peptide substrate with p-nitroanilide coupled to a cleavage-sensitive IEAR motif. The acetyl cap modulates solubility and ensures consistent assay performance. Researchers monitor p-nitroaniline release to determine protease kinetics. Applications include enzyme-substrate profiling, inhibitor screening, and mechanistic enzymology.
CAT No: R2780
Ac-IEAR-pNA is a synthetic peptide substrate widely utilized in biochemical research for the quantitative assessment of protease activity, particularly caspases and related cysteine proteases. Structurally, it features an acetylated N-terminus and a para-nitroanilide (pNA) moiety at the C-terminus, which serves as a chromogenic reporter. This specific peptide sequence, IEAR, is designed to mimic natural cleavage sites recognized by certain proteases, making the compound a valuable tool for elucidating proteolytic mechanisms and kinetic parameters. Its chromogenic properties enable sensitive and real-time detection of enzyme activity, supporting a broad range of enzymology, apoptosis, and cell signaling studies.
Enzyme activity assays: Ac-IEAR-pNA is primarily employed as a substrate in spectrophotometric assays to monitor the catalytic activity of caspase enzymes and related proteases. Upon cleavage by the target enzyme, the pNA group is released, producing a measurable yellow color that can be quantified at 405 nm. This direct readout allows researchers to determine enzyme kinetics, substrate specificity, and inhibitor potency under controlled in vitro conditions, facilitating detailed mechanistic studies of protease function.
Apoptosis research: The peptide substrate is frequently used in apoptosis studies to quantify caspase activation in cell lysates or purified enzyme systems. Because the IEAR sequence corresponds to preferred cleavage motifs for certain executioner caspases, its hydrolysis serves as a reliable indicator of apoptotic protease activation. By enabling precise measurement of caspase activity, the substrate supports investigations into cell death pathways, regulatory mechanisms, and the effects of potential modulators on programmed cell death.
Inhibitor screening: Ac-IEAR-pNA is instrumental in high-throughput screening protocols aimed at identifying and characterizing small molecule or peptide-based inhibitors of caspase and related protease families. The chromogenic assay format allows for rapid and sensitive detection of changes in enzymatic activity in response to test compounds, supporting lead identification and structure-activity relationship studies in drug discovery and chemical biology research.
Protease specificity profiling: Researchers utilize this substrate to profile the substrate selectivity and cleavage preferences of various cysteine proteases beyond caspases. By comparing the hydrolysis rates of Ac-IEAR-pNA with those of other peptide substrates, scientists can delineate enzyme specificity, map active site requirements, and refine substrate design for biochemical assays or inhibitor development.
Cell-free assay development: The compound is also valuable in the design and optimization of cell-free assay systems for protease research. Its defined peptide sequence and chromogenic functionality enable robust, reproducible, and scalable assay formats suitable for basic research, automated workflows, and the validation of new experimental platforms. This versatility makes it a preferred choice for laboratories seeking reliable tools for quantitative protease analysis across diverse research applications.
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