Fluorogenic Peptide Substrates for Assay of Aspartyl Proteinases

Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography

“…The crucial role played by the aspartic acid residues in the catalytic mechanism is evident from the pH value of 4.0-4.5 for the 50% activity observed here, which approximately corresponds to the pK' of aspartic acid. The values derived for the KM and kcat are within the range of values for other aspartic proteases using the same substrate, however they are higher than those observed for human cathepsin D (19 …”

“…Due to the broad pH range tested, three buffer systems were employed; 0.2 M Glycine-HC1 (pH 2.0), 0.2 M citrate-phosphate (pH 2.5-7.0) and 0.1 M Tris-HC1 (pH 7.5-8.0). Kinetic parameters were measured by the hydrolysis of a synthetic fluorogenic peptide containing at the extremities the chromophore o-aminobenzoyl (Abz) and its quenching partner ethylenediaminidinitrophenol (Eddnp) which are separated by 8 amino acid residues including two consecutive phenyalanine residues (19). Cleavage of the peptide between these hydrophobic residues results in separation of the two peptide fragments, and a consequent dequenching of the Abz which leads to an increase in the fluorescence signal.…”

A Heme-binding Aspartic Proteinase from the Eggs of the Hard TickBoophilus microplus

“…THAP was also assayed against several different synthetic fluorogenic and chromogenic substrates. Substrates for the main classes of acidic proteinases (cathepsin L, cathepsin B, cathepsin G, and cathepsin D) were assayed (27,28). Only Abz-AIAFFSRQ-EDDnp, a substrate based on the sequence susceptible to enzymes such as pepsin and related aspartic proteinases (28), was hydrolyzed by THAP (Table II).…”

“…Substrates for the main classes of acidic proteinases (cathepsin L, cathepsin B, cathepsin G, and cathepsin D) were assayed (27,28). Only Abz-AIAFFSRQ-EDDnp, a substrate based on the sequence susceptible to enzymes such as pepsin and related aspartic proteinases (28), was hydrolyzed by THAP (Table II). As expected for a cathepsin D-like enzyme, analysis of the peptide after hydrolysis revealed that cleavage occurred between the two Phe residues (data not shown).…”

Capillary electrophoretic determination of cathepsin D activity using Oregon Green-labeled hemoglobin

“…Solomayer et al [19] have suggested that both total amount and activity of cathepsin D may be of importance in the metastasis process. Most recently, fluorogenic peptide substrates were developed for assessing the activity of cathepsin D and other important aspartyl proteinases such as HIV protease [29,30]. These substrates are based on fluorophore-labeled peptides (7±12 mers) in which the fluorescence is substantially quenched until the internal PhePhe bond is cleaved by the enzyme.…”