angiotensinogen and 4-nitrophenylalanine

Fluorogenic substrates for endopeptidases are often prepared by attaching fluorophores and quenchers to opposite ends of the peptide substrates. Here, we describe a new approach by incorporating tryptophan and p-nitrophenylalanine into peptides to give fluorogenic substrates composed entirely of alpha-amino acids. Two advantages are apparent: (1) They can be prepared on a peptide synthesizer; no synthetic expertise is required. (2) Both ends of these peptides are free; other residues can be attached to increase their solubilities or to label them with affinity ligands. We tested the applicability of this new approach by developing a continuous assay for renin, a protease with unusually stringent substrate specificity. The fact that new fluorogenic peptides exhibited similar kinetic properties as those of known renin substrates suggests that this new method should be generally applicable to other proteases.

Topics: Amino Acid Sequence; Angiotensinogen; Animals; Endopeptidases; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Oligopeptides; Phenylalanine; Renin; Spectrometry, Fluorescence; Substrate Specificity; Swine; Tryptophan; Tyrosine