Glutathione reoxidation

Fig. 7. Oxidative refolding of reduced RNase Tl. Reoxidation conditions were 0.1 M Tris-HCl, pH 7.8, 0.2 Af guanidinium chloride, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, 0.2 mM EDTA, and 2.5 nM RNase Tl at 25°C. The kinetics of oxidative refolding were followed by the increase in tryptophan fluorescence intensity at 320 nm ( ), by an unfolding assay (Kiefhaber el ai, 1990b) that measures the formation of native protein molecules (A), and by the increase in the intensity of the band for native RNase Tl in native polyacrylamide gel electrophoresis ( ). Fluorescence emission in the presence of 10 mM reduced dithioerythritol to block disulfide bond formation (O). The small decrease in signal after several hours is caused by slight aggregation of the reduced and unfolded protein. (From Schonbrunner and Schmid (1992).

Fig. 8. Acceleration of the oxidative refolding of RNase T1 by PPI and PDI. The increase in fluorescence at 320 nm is shown as a function of the time of reoxidation. The final conditions were 2.5 fiM RNase T1 in 0.1 Af Tris-HCl, 0.2 M GdmCl, 2 mM EDTA, 3 mAf glycine, 0.4 mAf oxidized glutathione, and 4 mAf reduced glutathione at pH 7.8 and 25°C. Reoxidation ( ) in the absence of PPI and PDI, (O) in the presence of 1.4 tiM PPI, (A) in the presence of 1.6 fiM PDI, and (A) in the presence of both 1.6 fiM PDI and 1.4 /uAf PPI. In all experiments more than 90% of the observed kinetics were well approximated by single first-order processes, as indicated by the continuous lines. The respective time constants (t) are ( ) t = 4300 sec, (O) r = 2270 sec, (A) t = 1500 sec, (A) T = 650 sec. In all cases the initial fluorescence signal was about 10% of the final emission of the native protein. From Schonbrunner and Schmid (1992).

Reoxidation of pro-a chains of types I and II procollagen into triple helical procollagen was studied using various concentrations of reduced (GSH) and oxidized (GSSG) glutathione as an oxidizing system in the reaction mixture. Pure protein disulfide-isomerase (PDI 5ng) was used in the reaction. 

Electron transfer between pyridine nucleotides and disulfide compounds is catalyzed by several fiavoproteins and three of these are well characterized. Lipoamide dehydrogenase functions in the oxidative decarboxylation of a-keto acids catalyzing the reoxidation of reduced lipoate by NAD+ (18, 19). Glutathione reductase catalyzes electron transfer between NADPH and glutathione ZO-22). Thioredoxin reductase catalyzes the reduction of thioredoxin by NADPH (5) thioredoxin is a protein of 12,000 molecular weight containing a single cystine residue which is the electron acceptor S3). 

The importance of EH2 in catalysis by lipoamide dehydrogenase and glutathione reductase has been demonstrated by rapid reaction spectrophotometry. It is produced upon reduction with NADH or NADPH, respectively, in the dead time of the instrument (ca. 3 msec) and is rapidly reoxidized by lipoamide or glutathione at rates commensurate with catalysis (24, 50, 54) ... 

The stability of EH2 is very species dependent. All of the above results refer to the pig heart enzyme and, where tested, to other mammalian species. It was initially reported that no long wavelength absorption was observed upon reduction of E. coli enzyme with NADH 109), but reduction by 1 equivalent of NADH or dihydrolipoamide leads to the formation of 25% of the maximal 2-electron-reduced species 108) and similar results are obtained with the Azotobacter enzyme 114)- That this species is the catalytically important one in the E. coli enzyme as well as in the mammalian enzyme has also been demonstrated 50). Reduction with dihydrolipoamide in the rapid reaction spectrophotometer at 2° results in the full formation of EH2 followed by the slow k = 13 min, 1 mAf dihydrolipoamide) four-electron reduction. The spectrum of EHa generated in this way is shown in Fig. 7 and is identical with that of the pig heart enzyme. The 2-electron-reduced form, EHj of lipoamide dehydrogenase of spinach 99) may be somewhat unstable however, spectrally it is difficult to distinguish between instability and formation of the EHa-NADH complex (see above) on the basis of available spectral data. Either phenomenon could lead to inhibition by excess NADH. In glutathione reductase it is possible that the complex can be rapidly reoxidized by glutathione 53). 

Fig. 5.19. Time dependence of the reoxidation of ribonuclease in the presence of oxidized and reduced glutathione (from Hantgan et a/., 1974). Key (O), fraction of activity ( ), absorbance (A), fluorescence ( ), number of SH groups per protein molecule. SH groups disappear very rapidly prior to the variations of the other signals. The slower rate corresponds to the return of enzymatic activity.

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