کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10536304 | 962067 | 2005 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Assay of disulfide oxidase and isomerase based on the model of hirudin folding
دانلود مقاله + سفارش ترجمه
دانلود مقاله ISI انگلیسی
رایگان برای ایرانیان
کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله

چکیده انگلیسی
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-Hir  X-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-Hir  N-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 α-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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 339, Issue 1, 1 April 2005, Pages 94-103
Journal: Analytical Biochemistry - Volume 339, Issue 1, 1 April 2005, Pages 94-103
نویسندگان
Bao-Yuan Lu, Jui-Yoa Chang,