title: 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.
abstract: 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)
authors.affiliation: Central Research Division, Pfizer Inc., Groton, Connecticut 06340.
authors.forename: K F
authors.initials: KF
authors.lastname: Geoghegan
authors.forename: M J
authors.initials: MJ
authors.lastname: Emery
authors.forename: W H
authors.initials: WH
authors.lastname: Martin
authors.forename: A S
authors.initials: AS
authors.lastname: McColl
authors.forename: G O
authors.initials: GO
authors.lastname: Daumy
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017364:Collagenases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D010450:Endopeptidases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007546:Isoquinolines; D020781:Matrix Metalloproteinase 1; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000378:metabolism*; D010504:Periodic Acid / Q000378:metabolism / Q000494:pharmacology*; D012083:Renin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D007546:Isoquinolines; D010455:Peptides; D010504:Periodic Acid; D012694:Serine; C017475:lucifer yellow; C009288:metaperiodate; D010450:Endopeptidases; D012083:Renin; D017364:Collagenases; D020781:Matrix Metalloproteinase 1