A fluorimetric method for the determination of pepsin activity.
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.
da Silva Gomes
Roseli Aparecida
RA
Departamento de Ciências Biológicas, Faculdade de Medicina do Triângulo Mineiro, 38015-050, Uberaba, Brazil. bioq@dcb.fmtm.br
Batista
Rodrigo Pedutti
RP
Costa de Almeida
Andréia
A
da Fonseca
Daurin Narciso
DN
Juliano
Luis
L
Hial
Valdemar
V
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
D010436:Pepstatins; D010434:Pepsin A; C020557:gastricsin