Abstract
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.
References
May 1, 1979·Analytical Biochemistry·A YaronE Katchalski-Katzir
Nov 1, 1976·Proceedings of the National Academy of Sciences of the United States of America·P G Richman, J S Fruton
Apr 15, 1977·The Journal of Organic Chemistry· Wang S-SJ Meienhofer
Sep 1, 1975·Proceedings of the National Academy of Sciences of the United States of America·G P Sachdev, J S Fruton
Mar 14, 1972·Biochemistry·G P SachdevJ S Fruton
Jul 18, 1972·Biochemistry·M W Hunkapiller, J H Richards
Apr 1, 1974·Proceedings of the National Academy of Sciences of the United States of America·P S Sampath-Kumar, J S Fruton
Apr 1, 1973·Analytical Biochemistry·A M Felix, M H Jimenez
Feb 1, 1968·Proceedings of the National Academy of Sciences of the United States of America·R E Humphreys, J S Fruton
May 1, 1968·Biochemistry·K Inouye, J S Fruton
Jun 1, 1968·Biochemistry·T R Hollands, J S Fruton
Nov 1, 1969·Biochemistry·G P Sachdev, J S Fruton
Nov 10, 1970·Biochemistry·G P Sachdev, J S Fruton
Mar 1, 1972·Analytical Biochemistry·J Garden, A M Tometsko
Jul 1, 1966·Biochemistry·K InouyeJ S Fruton
Jun 1, 1967·Biochemistry·K Inouye, J S Fruton
Feb 3, 1970·Biochemistry·W M Vaughan, G Weber
Feb 1, 1983·The Biochemical Journal·B M DunnC Pham
Apr 11, 1980·Biochimica Et Biophysica Acta·P MartinB R Dumas
Sep 15, 1980·Molecular and Cellular Biochemistry·J S Fruton
Dec 8, 1981·Biochemistry·B M DunnA Hsu
Apr 1, 1982·Archives of Biochemistry and Biophysics·B M Dunn
Nov 1, 1963·The Biochemical Journal·W R GRAY, B S HARTLEY
Mar 20, 1938·The Journal of General Physiology·R M Herriott
Sep 20, 1938·The Journal of General Physiology·M L Anson
Citations
Dec 30, 1996·Journal of Biochemical and Biophysical Methods·A A Gershkovich, V V Kholodovych
Oct 23, 1984·Biochemistry·B M Dunn, A L Fink
Nov 1, 1993·Bioconjugate Chemistry·K F GeogheganG O Daumy
Feb 1, 1988·Journal of Neuroscience Research·A V Azaryan, A A Galoyan
Feb 23, 1990·Science·E D MatayoshiJ Erickson