A fluorescent oligopeptide energy transfer assay with broad applications for neutral proteases.
A fluorescent peptide substrate to explore the protease specificity for the amino acid regions C- and N-terminal to the cleavage site has been designed. Intramolecular quenching of indole fluorescence by an N-terminal dansyl group separated by six amino acid residues forms the basis of this assay. For a particular enzyme, specificity can be designed into the peptide sequence by means of the number of residues that separate the two chromophores. In the present instance, the heptapeptide Dns-Gly-Lys-Tyr-Ala-Pro-Trp-Val is used to assay angiotensin converting enzyme (ACE), Astacus protease, carboxypeptidase A, alpha-chymotrypsin, and trypsin, all of which cleave the peptide in accord with their known specificity: Trypsin and Astacus protease hydrolyze only the Lys-Tyr and Tyr-Ala bonds, respectively. alpha-Chymotrypsin primarily cleaves the Tyr-Ala bond while ACE makes three successive dipeptidyl cleavages from the C-terminus. Carboxypeptidase rapidly hydrolyzes first the Trp-Val and then the Pro-Trp bond. For all of the enzymes, catalytic activity (kcat/Km) is in the range from 10(5) to 10(6) M-1 s-1. Hydrolysis causes a fluorescence increase in the 310 to 410 nm region of 8.6- to 13.6-fold depending on the enzyme that is assayed. Assays can be designed based on the increase in tryptophan fluorescence or by individual product analyses using thin-layer or high-performance liquid chromatography. The specificity and sensitivity of such internally quenched fluorescent oligopeptides would seem to be ideal for the assay of specific endoproteases.
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Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts.
Auld
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D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000032:analysis; D043422:Carboxypeptidases A; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D002918:Chymotrypsin / Q000032:analysis; D004735:Energy Transfer; D005453:Fluorescence; D006801:Humans; D006868:Hydrolysis; D008722:Methods; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000032:analysis*; D007703:Peptidyl-Dipeptidase A / Q000032:analysis; D014357:Trypsin / Q000032:analysis
D009842:Oligopeptides; C062673:dansyl-glycyl-lysyl-tyrosyl-alanyl-prolyl-tryptophyl-valine; D002268:Carboxypeptidases; D010447:Peptide Hydrolases; D007703:Peptidyl-Dipeptidase A; D043422:Carboxypeptidases A; D002918:Chymotrypsin; D014357:Trypsin