KINETIC-ANALYSIS OF THE MECHANISM AND SPECIFICITY OF PROTEIN-DISULFIDE ISOMERASE USING FLUORESCENCE-QUENCHED PEPTIDES

Protein-disulfide isomerase (PDI) is an abundant folding catalyst in t he endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond is omerizations, We have synthesized a library of disulfide-linked fluore scence-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sens itive peptide substrates of PDI, Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino ac id sequencing of the bead-linked peptides revealed substantial similar ities. Several of the peptides were synthesized in solution, and a qua ntitative characterization of pre-steady state kinetics was carried ou t. Interestingly, a greater than 10-fold difference in affinity toward PDI was seen for various substrates of identical length. As opposed t o conventional PDI assays involving larger polypeptides, the starting material for this assay is homogenous. It is furthermore simple and hi ghly sensitive (requires less than 0.5 mu g of PDI/assay) and thus ope ns the possibility for quantitative determination of PDI activity and specificity.