title: A fluorimetric method for the determination of pepsin activity.
abstract: An intramolecularly quenched fluorogenic peptide containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (Eddnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Lys-Pro-Ile-Glu-Phe-Phe-Arg-Leu-Eddnp, was hydrolyzed by purified human pepsin, gastricsin, and gastric juice uniquely at the Phe-Phe bond. Kinetic parameters determined for purified pepsin were K(m)=0.68+/-0.11 microM; k(cat)=6.3+/-0.16s(-1); k(cat)/K(m)=9.26s(-1) microM(-1); Gastricsin showed K(m)=2.69+/-0.18 microM; k(cat)=0.03+/-0.005s(-1); k(cat)/K(m)=0.011s(-1) microM(-1). Gastric juice (21 samples) from subjects without gastric disorders at endoscopy examination showed activities varying from 0.0008 to 9.72 micromolml(-1)min(-1). Pepstatin A inhibition of gastric juice enzymatic activity was complete at 3.4x10(-5)M (final concentration) inhibitor. In the proposed method the presence of a unique scissile bond in the synthetic substrate provides a direct ratio between enzymatic activity and amount of substrate hydrolyzed, and a unique step reaction facilitates the use of this assay for the determination of the activity of aspartic proteinases in biological fluids and during enzyme purification procedures.
authors.affiliation: Departamento de Ciências Biológicas, Faculdade de Medicina do Triângulo Mineiro, 38015-050, Uberaba, Brazil. bioq@dcb.fmtm.br
authors.forename: Roseli Aparecida
authors.initials: RA
authors.lastname: da Silva Gomes
authors.forename: Rodrigo Pedutti
authors.initials: RP
authors.lastname: Batista
authors.forename: Andréia
authors.initials: A
authors.lastname: Costa de Almeida
authors.forename: Daurin Narciso
authors.initials: DN
authors.lastname: da Fonseca
authors.forename: Luis
authors.initials: L
authors.lastname: Juliano
authors.forename: Valdemar
authors.initials: V
authors.lastname: Hial
mesh_terms: D005470:Fluorometry / Q000379:methods*; D005750:Gastric Juice / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D010434:Pepsin A / Q000032:analysis*; D010436:Pepstatins / Q000494:pharmacology; D013997:Time Factors
chemical_list: D010436:Pepstatins; D010434:Pepsin A; C020557:gastricsin