Site-directed double fluorescent tagging of human renin and collagenase (MMP-1) substrate peptides using the periodate oxidation of N-terminal serine. An apparently general strategy for provision of energy-transfer substrates for proteases.

Periodate in neutral aqueous solution rapidly converts N-terminal Ser or Thr to an alpha-N-glyoxylyl moiety that can serve as the locus for incorporation of a modifying group [Geoghegan, K. F., and Stroh, J. G. (1992) Bioconjugate Chem. 3, 138-146. Gaertner, H. F. et al. (1992) Bioconjugate Chem. 3, 262-268]. The usefulness of this procedure has been further illuminated in a route to "energy-transfer" substrates for endoproteases. Each such substrate is an oligopeptide cleavable by a proteinase, but modified (usually at its termini) with two chromophores that form an energy donor-acceptor pair. Production of these substrates is an exercise in double site-directed peptide modification. The new route is composed of three steps, beginning from an unprotected peptide in which a sequence recognized by the pertinent enzyme is placed between N-terminal Ser and C-terminal Lys. Lys may not occur elsewhere in the peptide. Periodate oxidation converts the N-terminal Ser to an alpha-N-glyoxylyl group, which is then allowed to form a hydrazone with the carbohydrazide derivative Lucifer Yellow CH, a hydrophilic fluor with a large Stokes shift (excitation maximum, 425 nm; emission maximum, 525 nm). Finally, the modified peptide is allowed to react with 5-carboxytetramethylrhodamine succinimidyl ester. This reaction selectively modifies the epsilon-amino group of C-terminal Lys, the only amino group remaining in the peptide. 5-Carboxytetramethylrhodamine strongly (> 90%) quenches Lucifer Yellow fluorescence by resonance energy transfer in the intact substrate, but enzyme-catalyzed cleavage eliminates the quenching. The resulting increase in fluorescence may be used to follow the hydrolytic reaction.(ABSTRACT TRUNCATED AT 250 WORDS)