Covalent Association of Protein Disulfide Isomerase with Recombinant Human Interleukin 2in Vitro

Abstract

Protein disulfide isomerase has broad specificity in the catalysis of the formation and rearrangement of native disulfide bonds in proteins. This enzyme has two independent thioredoxin-like active sites (-CGHC-) and a peptide binding site. However, the mechanisms involving the catalytic processes are not clearly understood. It was reported that the enzyme associates with scrambled pancreatic ribonuclease Ain vitro,and with misfolded human lysozymein vivo.In the present study, recombinant human interleukin 2 has been chosen to probe the reaction intermediate in the reaction with the enzyme. We have identified and characterized a covalent associate formedin vitroby SDS-PAGE and Western blot analysis. This associate has a molecular weight of 71–72 kDa, the approximate sum of the molecular weights of the enzyme and the substrate. Western blot analysis confirmed that it formed via an intermolecular disulfide bond. Upon treatment with 2-mercaptoethanol, this bond was cleaved.