Increase in fluorescence upon the hydrolysis of tyrosine peptides: application to proteinase assays.
The intrinsic fluorescence of tyrosine increases by a factor of approximately two when the carboxy group is liberated from a peptide bond by hydrolysis. The increase in fluorescence provides a novel way to monitor the hydrolysis of native tyrosine peptides that contain only proteinogenic amino acids. Thus, for example, the hydrolysis by HIV-1 proteinase of a heptapeptide viral protein fragment gag129-135, Ser-Gln-Asn-Tyr-Pro-Ile-Val, was followed continuously at excitation and emission wavelengths 275 and 305 nm. The fluorescence increase is magnified by at least a factor of a thousand when a resonance energy quencher, such as paranitrophenylalanine, is in the vicinity. For example, the peptide Lys-Ala-Arg-Val-Tyr-Phe(p-NO2)-Glu-Ala-Nle-NH2 [Richards et al. (1990) J. Biol. Chem. 265, 7733], widely used for spectrophotometric assays of the HIV-1 proteinase, yields a substrate:product fluorescence ratio greater than 1:1000. Tyrosine-containing substrates of pepsin and trypsin showed similar behavior. The detection limit of the present method is at least one order of magnitude lower than absorbance assays of p-nitrophenylalanine peptides.
Peranteau
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AG
School of Pharmacy, University of Wisconsin, Madison 53706, USA.
Kuzmic
P
P
Angell
Y
Y
García-Echeverría
C
C
Rich
D H
DH
D015683:Gene Products, gag / Q000378:metabolism; D016333:HIV Protease / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry*; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D009842:Oligopeptides; D010446:Peptide Fragments; D054301:gag Gene Products, Human Immunodeficiency Virus; C095540:gag protein (129-135); C040239:4-nitrophenylalanine; D014443:Tyrosine; D010649:Phenylalanine; D010447:Peptide Hydrolases; D016333:HIV Protease