Assay of disulfide oxidase and isomerase based on the model of hirudin folding.
Oxidative folding of fully reduced hirudin (R-Hir, six cysteines) undergoes two distinct stages. A first stage of nonspecific disulfide formation promoted by oxidase converts R-Hir to form 3-disulfide scrambled hirudins (X-Hir) as obligatory intermediates. A second stage of disulfide shuffling catalyzed by isomerase converts X-Hir to the native hirudin (N-Hir). The model of hirudin folding is utilized here to develop an assay system for measuring the activity of disulfide oxidase and isomerase, using high-performance liquid chromatography (HPLC) quantification of R-Hir, X-Hir, and N-Hir. The oxidase assay measures the ability of an oxidase to promote R-HirX-Hir conversion. The molar specific activity is expressed as mol ofR-Hir decrease per mol of oxidase per min. The isomerase assay measures the ability of an isomerase to catalyze X-HirN-Hir transformation. The molar specific activity is expressed as mol ofN-Hir increase per mol of isomerase per min. Alternatively, the recovery of N-Hir in the isomerase assay can be determined by its alpha-thrombin inhibitory activity. Using both HPLC and activity-based assay, we have measured the relative oxidase and isomerase activity of reduced and oxidized glutathione, Cys, Cys-Cys, and reduced and oxidized protein disulfide isomerase (PDI). The molar specific activity of reduced PDI was shown to be 0.1+/-0.01 U, which is consistent with documented data obtained by the scrambled RNase-A-based assay. These proposed assay methods provide alternatives to the limited option of methodologies currently available for measuring oxidase and isomerase activities. A major merit of the proposed assay system is the potential to accommodate the analysis of biological samples.
Lu
Bao-Yuan
BY
Research Center for Protein Chemistry, Institute of Molecular Medicine, The University of Texas, Houston, TX 77030, USA.
Chang
Jui-Yoa
JY
D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006629:Hirudins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors
D004220:Disulfides; D005978:Glutathione; D006629:Hirudins; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors