The synthesis, purification, and evaluation of a chromophoric substrate for pepsin and other aspartyl proteases: design of a substrate based on subsite preferences.
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 for this enzyme. The peptide contains a phenylalanyl-p-nitrophenylalanine sequence at the reactive site. Cleavage of this bond yields a change in absorbance at 310 nm of between 1700 and 2000 per mole. This allows kinetic data to be obtained readily and accurately. The products of cleavage have been identified by isolation of a peptide fragment by high-performance liquid chromatography. Values of kcat, Km, and kcat/Km of 94 +/- 6 s-1, 0.13 +/- .04 mM, and 815 +/- 210 s-1/mM-1 were obtained at pH 3.0 and 37 degrees C. The peptide is soluble over the pH range from 2 to 7, thus facilitating determination of the pH dependence of the kinetic parameters. The substrate is also valuable in studying the inhibition of pepsin.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002863:Chromogenic Compounds / Q000138:chemical synthesis* / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis*; D010434:Pepsin A / Q000378:metabolism*; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013552:Swine
D002863:Chromogenic Compounds; D009842:Oligopeptides; C041723:prolyl-threonyl-glutamyl-phenylalanyl-4-nitrophenylalanyl-arginyl-leucine; D010450:Endopeptidases; D010434:Pepsin A