New fluorescent substrates for metalloendopeptidases with internal quenching of fluorescence

p-Nitroanilides of antranyloyltripeptides of the general structure Abz-Ala-Ala-P'1-pNA (P'1 = Phe, Leu, Ile, Val) containing intramolecularly quenched fluorescent groups (Abz is a fluorogenic group and pNA is a quencher of fluorescence) were prepared by combination of chemical and enzymatic methods. Thermolysin and metalloproteinases from Legionella pneumophila and Thermoactinomyces species were shown to hydrolyse Ala-P'1 bond of the peptides with simultaneous 4-7 fold increase in fluorescence. Kinetic parameters for enzymatic hydrolysis of the substrates were determined. Metalloendopeptidases can be assayed in the presence of serine proteinases (of the subtilisin type) using Abz-Ala-Ala-Ile-pNA or Abz-Ala-Ala-Val-pNA.