Fluorometric polyethyleneglycol-peptide hybrid substrates for quantitative assay of protein disulfide isomerase.
In eukaryotic cells the enzyme protein disulfide isomerase (PDI) is responsible for the formation and reshuffling of disulfide bonds in secretory proteins. The reaction carried out by PDI involves interaction with a highly complex mixture of polypeptide molecules that are in the process of folding. This means that PDI activity is typically measured in the context of a globular protein folding pathway. The absence of small, well-defined substrates for the quantitation of both oxidation and reduction reactions constitutes an inherent problem in the analysis of PDI activity. We describe a new type of substrate for PDI where two cysteine-containing oligopeptides are connected by an onameric ethylene glycol linker. We term such hybrid compounds PEGtides. The oligopeptides are each marked with a fluorescent aminobenzoic acid and a quenching nitrotyrosine group, respectively. The reversible formation of an intramolecular disulfide bond between fluorophore-containing and quencher-containing peptide segments results in a redox-dependent fluorescence signal. We find a model compound of this type to be a highly sensitive substrate for PDI both in oxidation and in reduction assays under steady state conditions. These aspects should make substrates of this type generally applicable for assaying PDI and other thiol-disulfide exchange enzymes.
Christiansen
Camilla
C
Carlsberg Laboratory, Department of Physiology, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Denmark.
St  Hilaire
Phaedria M
PM
Winther
Jakob R
JR
D004220:Disulfides / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry*; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols / Q000737:chemistry*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry*; D013379:Substrate Specificity
D004220:Disulfides; D009842:Oligopeptides; D011092:Polyethylene Glycols; D019704:Protein Disulfide-Isomerases; D005978:Glutathione