H-Gly-Phe-AMC is a fluorogenic dipeptide substrate with an AMC reporter for monitoring protease activity. The Gly-Phe motif presents a hydrophobic cleavage site recognized by chymotrypsin-like enzymes. Researchers quantify fluorescence release to assess catalytic rates. Applications include enzyme-assay design, inhibitor screening, and substrate optimization.
CAT No: R2403
CAS No:201852-70-2
Synonyms/Alias:H-Gly-Phe-AMC;201852-70-2;Gly-Phe-AMC;L-Phenylalaninamide, glycyl-N-(4-methyl-2-oxo-2H-1-benzopyran-7-yl)-;(2S)-2-[(2-aminoacetyl)amino]-N-(4-methyl-2-oxochromen-7-yl)-3-phenylpropanamide;MFCD00152091;DTXSID60426782;HY-P4425;DA-74146;FG110504;CS-0654071;(2S)-2-(2-AMINOACETAMIDO)-N-(4-METHYL-2-OXOCHROMEN-7-YL)-3-PHENYLPROPANAMIDE;(S)-2-(2-Aminoacetamido)-N-(4-methyl-2-oxo-2H-chromen-7-yl)-3-phenylpropanamide;
H-Gly-Phe-AMC is a synthetic peptide substrate featuring a glycine-phenylalanine dipeptide linked to 7-amino-4-methylcoumarin (AMC), a widely used fluorogenic leaving group. As a member of the peptide-AMC conjugate family, it serves as a highly sensitive tool for monitoring protease activity, particularly in enzymology and biochemical assays. The compound's design allows for the release of a fluorescent signal upon enzymatic cleavage, enabling real-time detection and quantification of proteolytic activity. Its specificity and robust signal output make it a valuable asset in both fundamental research and applied bioscience settings, where precise measurement of enzyme kinetics and substrate specificity is essential.
Protease Activity Assays: H-Gly-Phe-AMC is extensively utilized as a fluorogenic substrate in the quantitative analysis of protease enzymes, including those with chymotrypsin-like or other aromatic residue specificity. Upon enzymatic hydrolysis, AMC is liberated, resulting in a measurable increase in fluorescence. This feature allows researchers to monitor enzyme kinetics in real time, facilitating the characterization of protease activity, inhibitor screening, and the study of enzyme regulation in diverse biological systems. The sensitivity of the fluorescence readout enables detection of even low levels of enzymatic activity, making it ideal for high-throughput screening and detailed mechanistic studies.
Enzyme Kinetics Studies: In biochemical research, the compound is a preferred substrate for determining kinetic parameters such as Km and Vmax of proteolytic enzymes. The rapid and quantifiable release of AMC upon peptide bond cleavage allows for precise measurement of reaction rates under varying substrate and enzyme concentrations. This capability supports the elucidation of catalytic mechanisms and aids in the comparison of enzyme variants or mutant forms, thus providing critical insights into structure-function relationships within protease families.
Inhibitor Screening: The use of H-Gly-Phe-AMC as a reporter substrate is fundamental in the screening and characterization of protease inhibitors. By measuring changes in fluorescence in the presence of potential inhibitory compounds, researchers can rapidly assess inhibitor potency, selectivity, and mode of action. This approach is widely adopted in pharmaceutical research and lead optimization, where efficient identification of candidate molecules that modulate protease activity is a primary objective.
Substrate Specificity Profiling: The dipeptidyl structure of Gly-Phe-AMC enables its application in profiling the substrate preferences of various proteases. By comparing the hydrolysis efficiency of this substrate with related peptide-AMC conjugates, investigators can delineate the substrate recognition motifs of target enzymes. Such studies are instrumental in mapping protease specificity, guiding the design of custom substrates or inhibitors, and advancing understanding of proteolytic processes in physiological and pathological contexts.
Cell-Free Biochemical Assays: In addition to its use in purified enzyme systems, the substrate is compatible with cell-free extracts or semi-purified samples, supporting the assessment of protease activity in more complex biological matrices. Its high sensitivity and straightforward fluorescence-based detection facilitate the analysis of proteolytic events in tissue lysates, subcellular fractions, or recombinant expression systems. This versatility enhances its value in broad-spectrum protease research, enabling applications ranging from basic enzymology to drug discovery and functional genomics.
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