A new substrate for porcine pepsin possessing cryptic fluorescence properties.
A fluorescent substrate for porcine pepsin, 50-dimethylaminonaphthalene-1-sulfonyl (Dns)-Ala-Ala-Phe-Phe-3-[4-(N-CH3)-pyridyl]propyl-1-oxy ester has been synthesized. It is stable, soluble from pH 1 to 7, and is readily hydrolyzed by pepsin with values of 288 (+/- 40) s-1 for kcat, 0.039 mM (+/- 0.005) for Km, and 7510 s-1 mM-1 (+/- 500) for kcat/Km in sodium formate, pH 3.1. Kinetic studies were carried out by following the increased fluorescence (300-nm excitation, 525-nm emission) as hydrolysis occurred. The products of hydrolysis were identified and established that the peptide bond between the phenylalanine residues is cleaved by pepsin. The inhibition of pepsin catalysis by pepsinogen (1-12) activation peptide was studied in order to compare the inhibition of the reaction of pepsin with Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ with that obtained by the standard milk-clotting assay. The inhibition results were comparable. Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ should be a valuable tool for studies of pepsin because of its solubility over an extended pH range, its excellent turnover rate, and the ease with which the hydrolysis can be followed.
D000818:Animals; D003619:Dansyl Compounds / Q000138:chemical synthesis*; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006868:Hydrolysis; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis*; D010434:Pepsin A / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D009842:Oligopeptides; C037953:dansyl-alanyl-alanyl-phenylalanyl-phenylalanine-3-(4-(N-methyl)pyridyl)-1-propionate; D010434:Pepsin A