Fluorogenic peptide substrates for assay of aspartyl proteinases.
Via a combination of chemical and enzymatic synthesis, new hexapeptide substrates convenient for use in activity assessment of several aspartyl proteinases--porcine pepsin, human pepsin, gastricsin, and cathepsin D--were prepared. These peptide derivatives, o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala-p-nitroanilide and N-(o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala)-N'-2,4-dinitrophenyl ethylenediamine, contain a fluorescent o-aminobenzoyl moiety as well as p-nitroaniline or N-2,4-dinitrophenyl ethylenediamine--the groups that cause fluorescence quenching. Aspartyl proteinases hydrolyze the Phe-Phe peptide bond in the substrates, which diminishes quenching due to separation of the fluorescent and quenching moieties and leads to an increase in the fluorescence intensity of o-aminobenzoyl residue. Abz-Ala-Ala-Phe-Phe-Ala-Ala-Ded, being fairly well hydrolyzed by HIV proteinase, might be used for assay of this enzyme.
Filippova
I Y
IY
Department of Chemistry, Moscow State University, Russia.
Lysogorskaya
E N
EN
Anisimova
V V
VV
Suvorov
L I
LI
Oksenoit
E S
ES
Stepanov
V M
VM
D000595:Amino Acid Sequence; D062365:Aminobenzoates / Q000737:chemistry*; D000818:Animals; D002402:Cathepsin D / Q000032:analysis*; D005470:Fluorometry; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009842:Oligopeptides / Q000737:chemistry*; D010434:Pepsin A / Q000032:analysis*; D013552:Swine
C101257:2-aminobenzoyl-alanyl-alanyl-phenylalanyl-phenylalanyl-alanyl-alanyl-4-nitroanilide; D062365:Aminobenzoates; C101258:N-(2-aminobenzoyl-alanyl-alanyl-phenylalanyl-phenylalanyl-alanyl-alanyl)-N'-2,4-dinitrophenylethylenediamine; D009842:Oligopeptides; D010434:Pepsin A; C020557:gastricsin; D002402:Cathepsin D