A fluorimetric method for the determination of pepsin activity.

Autor: da Silva Gomes RA; Departamento de Ciências Biológicas, Faculdade de Medicina do Triângulo Mineiro, 38015-050, Uberaba, Brazil. bioq@dcb.fmtm.br, Batista RP, Costa de Almeida A, da Fonseca DN, Juliano L, Hial V
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2003 May 01; Vol. 316 (1), pp. 11-4.
DOI: 10.1016/s0003-2697(03)00025-3
Abstrakt: 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.
Databáze: MEDLINE
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