The synthesis, purification, and evaluation of a chromophoric substrate for pepsin and other aspartyl proteases: Design of a substrate based on subsite preferences

摘要: Abstract A convenient chromophoric assay for porcine pepsin has been developed using a new synthetic substrate. The sequence of this substrate was chosen based on the known subsite preferences enzyme. peptide contains phenylalanyl-p-nitrophenylalanine at reactive site. Cleavage bond yields change in absorbance 310 nm between 1700 and 2000 per mole. This allows kinetic data to be obtained readily accurately. products cleavage have identified by isolation fragment high-performance liquid chromatography. Values kcat, Km, k cat K m 94 ± 6 s−1, 0.13 .04 , 815 210 s−1/m −1 were pH 3.0 37°C. is soluble over range from 2 7, thus facilitating determination dependence parameters. also valuable studying inhibition pepsin.