Fluorometric polyethyleneglycol-peptide hybrid substrates for quantitative assay of protein disulfide isomerase
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Fluorometric polyethyleneglycol-peptide hybrid substrates for quantitative assay of protein disulfide isomerase. / Christiansen, Camilla; Hilaire, Phaedria M. St.; Winther, Jakob R.
I: Analytical Biochemistry, Bind 333, Nr. 1, 2004, s. 148-155.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - Fluorometric polyethyleneglycol-peptide hybrid substrates for quantitative assay of protein disulfide isomerase
AU - Christiansen, Camilla
AU - Hilaire, Phaedria M. St.
AU - Winther, Jakob R.
PY - 2004
Y1 - 2004
N2 - 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.
AB - 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.
KW - Disulfides
KW - Glutathione
KW - Humans
KW - Kinetics
KW - Oligopeptides
KW - Oxidation-Reduction
KW - Polyethylene Glycols
KW - Protein Disulfide-Isomerases
KW - Substrate Specificity
U2 - 10.1016/j.ab.2004.06.027
DO - 10.1016/j.ab.2004.06.027
M3 - Journal article
C2 - 15351291
VL - 333
SP - 148
EP - 155
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -
ID: 43973530