Increase in Fluorescence upon the Hydrolysis of Tyrosine Peptides: Application to Proteinase Assays

✍ Scribed by A.G. Peranteau; P. Kuzmic; Y. Angell; C. Garciaecheverria; D.H. Rich

• Book ID: 102561862
• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 304 KB
• Volume: 227
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.1276

✦ Synopsis

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 gag (^{129-135}), Ser-Gln-Asn-Tyr-ProIle-Val, was followed continuously at excitation and emission wavelengths 275 and (305 \mathrm{~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 (\left(p-\mathrm{NO}_{2}\right))-GluAla-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. 1995 Academic Press, Inc.