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.