Glutathione Mixed Disulfides Research Articles

Hemoglobin S-thiolation during peroxide-induced oxidative stress in chicken blood

Protein S-thiolation or protein–glutathione mixed disulfide (PSSG) occurs when cells are exposed to oxidative stress, and has been implicated in several cellular functions. The S-thiolation of hemoglobin as well as other abundant proteins is proposed to participate as a redox buffer, being part of the antioxidant protection system of the cell during the oxidative challenge. We studied the oxidative stress caused by peroxides (H2O2, cumene and tert-butyl hydroperoxide) on chicken blood by measuring the thiol/disulfide status. Chicken blood under peroxide treatment showed a time- and concentration-dependent increase in glutathione disulfide (GSSG) and PSSG. GSSG peaked immediately after treatment (1 min), while PSSG increased progressively over time, showing a maximum after about 30 min. The system recovered after 140 min of incubation, with GSSG and PSSG then barely reaching control values. The S-thiolation of hemoglobin was monitored under nondenaturing PAGE, and the fraction of S-thiolated hemoglobin, or Hb A1, rose in a dose-dependent fashion and was proportional to total S-thiolation, measured as PSSG. This significant correlation indicates that hemoglobin is the major S-thiolated protein in chicken erythrocytes treated with peroxides. The present work shows the behavior of chicken blood under peroxide treatment; it anticipated that chicken hemoglobin thiol groups can actively participate in the redox processes of erythrocytes exposed to oxidative stress, and that hemoglobin is the major S-thiolated protein. This further corroborates the hypothesis that abundant proteins, such as hemoglobin, may take part in the cellular antioxidant defense system.

NMR Reveals a Novel Glutaredoxin–Glutaredoxin Interaction Interface

Glutaredoxins (Grx) represent a large family of glutathione (GSH)-dependent oxidoreductases that catalyse the reduction of disulfides or glutathione mixed disulfide. Grx domains from pathogenic bacteria and plant Grxs have been recently reported to target specific peroxiredoxins (Prxs). The specificity that triggers the interaction between Grx and Prx is poorly understood and is only based on the structure of Haemophilus influenzae Prx-Grx hybrid (hyPrx5). We report here an NMR study of the Populus tremula Grx C4 that targets a P.tremula D-type II Prx. We show that Grx C4 specifically self-associates in a monomer-dimer equilibrium with an apparent K(d) of ca 2.6 mM. Grx C4 homodimer was docked under experimental restraints. The results reveal a novel Grx-Grx interface that is unrelated to the hyPrx5 Grx-Grx dimer interface. Chemical-shift perturbations and 15N spin-relaxation measurements show that the auto-association surface comprises both the active site and the GSH binding site. Reduced GSH is demonstrated to bind reduced Grx with a K(d) of ca 8.6 mM. The potential biological significance of the new Grx-Grx interaction interface is discussed.

Catalysis of Thiol/Disulfide Exchange: GLUTAREDOXIN 1 AND PROTEIN-DISULFIDE ISOMERASE USE DIFFERENT MECHANISMS TO ENHANCE OXIDASE AND REDUCTASE ACTIVITIES

Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative disulfides in scrambled ribonuclease and protein-glutathione mixed disulfides 30-180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.

S-glutathionylation in human platelets by a thiol–disulfide exchange-independent mechanism

Protein–glutathione mixed disulfide formation was investigated in vitro by exposure of human platelets to the thiol-specific oxidant azodicarboxylic acid-bis-dimethylamide (diamide). We found that diamide causes a decrease in the reduced form of glutathione (GSH), paralleled by an increase in protein–GSH mixed disulfides (S-glutathionylated proteins), which was not accompanied by any significant increase in the basal level of glutathione disulfide (GSSG). The increase in the appearance of S-glutathionylated proteins was inversely correlated with ADP-induced platelet aggregation. Platelet cytoskeleton was analyzed by SDS–PAGE followed by Western immunoblotting with anti-GSH antibody. The main S-glutathionylated cytoskeletal protein proved to be actin, which accounts for 35% of the platelet total protein content. Our results suggest that neither GSSG formation nor a consequent thiol–disulfide exchange mechanism is involved in actin S-glutathionylation of human platelets exposed to diamide. Instead, a mechanism involving the initial oxidative activation of actin thiol groups, which then react with GSH to the protein–GSH mixed disulfides, makes it likely that platelet actin is S-glutathionylated without any significant increase in the GSSG content.

Inhibition of basal and interleukin-1-induced VCAM-1 expression by phospholipid hydroperoxide glutathione peroxidase and 15-lipoxygenase in rabbit aortic smooth muscle cells

Cytokines or hydroperoxides upregulate cell adhesion molecules (CAM) in early stages of atherosclerosis. VCAM-1 expression was therefore investigated in rabbit aortic smooth muscle cells (SMC) stably transfected either with phospholipid hydroperoxide glutathione peroxidase (PHGPx; SMC PHGPx) as a hydroperoxide-reducing enzyme or with 15-lipoxygenase (15-LOX; SMC LOX) as a hydroperoxide-producing enzyme. Transfected cells showed up to 3-fold enhanced PHGPx and a marked LOX activity, respectively, that was absent in controls. Intracellular hydroperoxides were 6-fold higher in SMC LOX than in SMC or SMC PHGPx. Intracellular protein thiols were decreased by 50 and 90% in SMC PHGPx and SMC LOX, respectively. Glutathione mixed disulfides were tentatively increased from SMC via SMC PHGPx to SMC LOX, accordingly. Thiol reduction with tris(2-carboxyethyl)phosphine completely restored protein thiols in SMC PHGPx, whereas in SMC LOX only 60% of control values were recovered. Basal VCAM-1 mRNA levels were decreased by 50% in SMC PHGPx and 75% in SMC LOX. VCAM-1-inducibility was abrogated in SMC LOX but not in SMC PHGPx. Accordingly, NFκB-driven reporter gene activation by IL-1 was unaffected in SMC PHGPx but abolished in SMC LOX. The data confirm that PHGPx overexpression dampens CAM expression either by lowering stimulatory hydroperoxides or by using hydroperoxides for protein modification. But hydroperoxides, when constitutively overproduced as in SMC LOX, inhibit CAM expression and render cells refractory to IL-1 stimulation likely due to oxidation of protein thiols of the signaling system.

Functional glutaredoxin (thioltransferase) activity in rat brain and liver mitochondria

Glutaredoxin (Grx) is a specific and efficient catalyst of glutathione-dependent deglutathionylation of protein-SS–glutathione mixed disulfides. Grx has been identified in brain cytosol, but the presence of activity in subcellular organelles has not been reported. Increases in protein glutathionylation are likely to occur in mitochondria during oxidative stress and it is, therefore, important to know if this organelle contains the enzyme activity needed to reverse such protein thiolation. Grx-like activity in the P1 supernatant from rat brain and liver was doubled in the presence of Triton-X 100 suggesting a releasable pool of Grx. Brain and liver homogenates were subfractionated into cytosolic, mitochondrial and microsomal fraction, their purity determined by biochemical assay and EM and assayed for Grx-like activity. The data presented demonstrate that mitochondria contain functional Grx-like activity.

Hydrogen peroxide removal and glutathione mixed disulfide formation during metabolic inhibition in mesencephalic cultures.

Compromised mitochondrial energy metabolism and oxidative stress have been associated with the pathophysiology of Parkinson's disease. Our previous experiments exemplified the importance of GSH in the protection of neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. This study further defines the role of oxidative stress during energy inhibition and begins to unravel the mechanisms by which GSH and other antioxidants may contribute to cell survival. Treatment of mesencephalic cultures with 10 microM buthionine sulfoximine for 24 h depleted total GSH by 60%, whereas 3 h exposure to 5 mM 3-amino-1,2,4-triazole irreversibly inactivated catalase activity by 90%. Treatment of GSH-depleted cells with malonate (40 mM) for 6, 12 or 24 h both potentiated and accelerated the time course of malonate toxicity, however, inhibition of catalase had no effect. In contrast, concomitant treatment with buthionine sulfoximine plus 3-amino-1,2,4-triazole in the presence of malonate significantly potentiated toxicity over that observed with malonate plus either inhibitor alone. Consistent with these findings, GSH depletion enhanced malonate-induced reactive oxygen species generation prior to the onset of toxicity. These findings demonstrate that early generation of reactive oxygen species during mitochondrial inhibition contributes to cell damage and that GSH serves as a first line of defense in its removal. Pre-treatment of cultures with 400 microM ascorbate protected completely against malonate toxicity (50 mM, 12 h), whereas treatment with 1 mM Trolox provided partial protection. Protein-GSH mixed disulfide formation during oxidative stress has been suggested to either protect vulnerable protein thiols or conversely to contribute to toxicity. Malonate exposure (50 mM) for 12 h resulted in a modest increase in mixed disulfide formation. However, exposure to the protective combination of ascorbate plus malonate increased membrane bound protein-GSH mixed disulfides three-fold. Mixed disulfide levels returned to baseline by 72 h of recovery indicating the reversible nature of this formation. These results demonstrate an early role for oxidative events during mitochondrial impairment and stress the importance of the glutathione system for removal of reactive oxygen species. Catalase may serve as a secondary defense as the glutathione system becomes limiting. These findings also suggest that protein-GSH mixed disulfide formation under these circumstances may play a protective role.

NMR structure of oxidized glutaredoxin 3 from Escherichia coli

A high precision NMR structure of oxidized glutaredoxin 3 [C65Y] from Escherichia coli has been determined. The conformation of the active site including the disulphide bridge is highly similar to those in glutaredoxins from pig liver and T4 phage. A comparison with the previously determined structure of glutaredoxin 3 [C14S, C65Y] in a complex with glutathione reveals conformational changes between the free and substrate-bound form which includes the sidechain of the conserved, active site tyrosine residue. In the oxidized form this tyrosine is solvent exposed, while it adopts a less exposed conformation, stabilized by hydrogen bonds, in the mixed disulfide with glutathione. The structures further suggest that the formation of a covalent linkage between glutathione and glutaredoxin 3 is necessary in order to induce these structural changes upon binding of the glutathione peptide. This could explain the observed low affinity of glutaredoxins for S-blocked glutathione analogues, in spite of the fact that glutaredoxins are highly specific reductants of glutathione mixed disulfides.

NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: implications for the enzymatic mechanism

Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of protein disulfide groups and glutathione-containing mixed disulfide groups via an active site Grx-glutathione mixed disulfide (Grx-SG) intermediate. The NMR solution structure of the Escherichia coli Grx3 mixed disulfide with glutathione (Grx3-SG) was determined using a C14S mutant which traps this intermediate in the redox reaction. The structure contains a thioredoxin fold, with a well-defined binding site for glutathione which involves two intermolecular backbone-backbone hydrogen bonds forming an antiparallel intermolecular β-bridge between the protein and glutathione. The solution structure of E. coli Grx3-SG also suggests a binding site for a second glutathione in the reduction of the Grx3-SG intermediate, which is consistent with the specificity of reduction observed in Grxs. Molecular details of the structure in relation to the stability of the intermediate and the activity of Grx3 as a reductant of glutathione mixed disulfide groups are discussed. A comparison of glutathione binding in Grx3-SG and ligand binding in other members of the thioredoxin superfamily is presented, which illustrates the highly conserved intermolecular interactions in this protein family.

24] Nonenzymatic colorimetric assay of glutathione in the presence of other mercaptans

Publisher Summary This chapter discusses the nonenzymatic colorimetric assay of glutathione in the presence of other mercaptans. Direct assays of total mercaptans are based on chromogenic reactions of sulfhydryl groups with electrophilic reagents. Such assays have obvious limitations in terms of specificity. Other methods are time-consuming and are based on glutathione reductase-coupled assays, with interferences of mixed disulfides of glutathione and enzyme inhibitors or on chromatographic techniques. Reliable chromatographic methods are relatively complex and require expensive materials. Such observations underline the need for faster and easier methodology, especially when large series of biological samples must be processed on a daily basis. The chapter describes a colorimetric procedure that takes advantage of a glutathione (GSH)-specific reaction in the absence of a coupling enzyme. The chapter discusses the advantages and limitations of this methodology. The assay—which is based on the colorimetric measurement of 7-trifluoromethyl-4-thioquinolone at the 400-nm wavelength—has the unique advantage to be glutathione specific in the absence of enzyme. Another advantage is that a colorimetric estimation of total sulfhydryl groups can be obtained at the 356-nm wavelength, before the addition of sodium hydroxide to the mixture of thioethers produced at pH 7.8. The sensitivities of the two assays are very similar and given the stability of chromophoric products, large series of samples can be processed within 20–30 min for subsequent absorbance measurement.

Loss of GSH, Oxidative Stress, and Decrease of Intracellular pH as Sequential Steps in Viral Infection

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products. The reduced tripeptide was quantitatively recovered in the culture medium of the cells. Since the GSH loss in infected cells was not blocked by methionine, a known inhibitor of hepatocyte GSH transport, a nonspecific leakage through the plasma membrane is proposed. UV-irradiated Sendai virus gave the same results, confirming that the major loss of GSH was due to membrane perturbation upon virus fusion. Consequent to the loss of the tripeptide, an intracellular pH decrease occurred, which was due to a reversible impairment of the Na+/H+ antiporter, the main system responsible for maintaining unaltered pHi in those cells. At the end of the infection period, a rise in both pHi value and GSH content was observed, with a complete recovery in the activity of the antiporter. However, a secondary set up of oxidative stress was observed after 24 h from infection, which is the time necessary for virus budding from cells. In this case, the GSH decrease was partly due to preferential incorporation of the cysteine residue in the viral proteins and partly engaged in mixed disulfides with intracellular proteins. In conclusion, under our conditions of viral infection, oxidative stress is imposed by GSH depletion, occurring in two steps and following direct virus challenge of the cell membrane without the intervention of reactive oxygen species. These results provide a rationale for the reported, and often contradictory, mutual effects of GSH and viral infection.

1H NMR Studies of the Conformational Properties of Somatostatin, Reduced Somatostatin and Somatostatin-Glutathione Mixed Disulfides

The conformational properties of somatostatin, a tetradecapeptide hormone with a disulfide bond joining cysteine residues at positions 3 and 14, and of the reduced dithiol form of somatostatin, the two somatostatin–glutathione single mixed disulfides and somatostatin–glutathione double mixed disulfide were investigated in aqueous solution by one- and two-dimensional1H NMR. Resonances were assigned using connectivities derived from 2D TOCSY and ROESY spectra.1H–1H ROEs together with the temperature coefficients for the chemical shifts of amide NH resonances for the native disulfide form of somatostatin are consistent with previous conclusions that somatostatin exists as an equilibrium of several rapidly interconverting conformations; however, the ROE results provide more information about the conformations than was obtained from previous studies. In particular, CαHi–NHi+2 ROEs together with NHi–NHi+1 ROEs identify specific β-turns in the family of low-energy conformations. The NMR results indicate that reduced somatostatin and the somatostatin–glutathione mixed disulfides have some of the same β-turn conformational features as cyclic somatostatin, but that the populations of the conformations having the β-turns are less for the acyclic peptides.

An HPLC radiotracer method for assessing the ability of L-cysteine prodrugs to maintain glutathione levels in the cultured rat lens.

To apply a high performance liquid chromatographic radiotracer method to test a variety of L-cysteine prodrugs and one dipeptide prodrug for their ability to synthesize glutathione in cultured rat lenses. Rat lenses were incubated for 48 h in a medium containing [14C(U)]-glycine and prodrugs. Following homogenization and derivatization, lens extracts were analyzed to determine the extent of biosynthetic incorporation of this labeled amino acid into [14C]-glutathione using high performance liquid chromatography with radioisotope and ultraviolet absorption detection. All of the thiazolidine prodrugs contained masked sulfhydryl groups to stabilize them against air oxidation. L-buthionine-(S,R)-sulfoximine-an inhibitor of the first step in glutathione biosynthesis-was present in media containing the dipeptide prodrug. In all cases, a large [14C]-labeled peak eluted just prior to [14C]-glutathione. This peak had some characteristics of the mixed disulfide of glutathione and L-cysteine, viz., L-cysteine/glutathione disulfide, but requires further investigation in order to be positively identified. Of the eleven L-cysteine prodrugs investigated, the most effective was 2(R,S)-methylthiazolidine-4(R)-carboxylic acid, which increased the rate of [14C]-glutathione biosynthesis 35% over that of the controls. A number of other L-cysteine prodrugs were somewhat effective, increasing glutathione synthesis 5-30% over the controls, while several L-cysteine prodrugs were totally ineffective. The only dipeptide prodrug investigated, viz., gamma-L-glutamyl-L-cysteine ethyl ester, increased the biosynthesis of [14C]-glutathione 18% over control. Biosynthetic rates based on ultraviolet absorption of the derivatized glutathione demonstrated a similar pattern, the compounds most effective in synthesizing [14C]-glutathione generally yielding the highest ultraviolet glutathione concentrations and the ineffective compounds showing the lowest concentrations. 2(R,S)-methylthiazolidine-4(R)-carboxylic acid, 2(R,S)-n-propylthiazolidine-4(R)-carboxylic acid and N-acetyl-L-cysteine were the only compounds that were statistically significant in yielding higher levels of both ultraviolet and radioactive glutathione as compared to their respective controls. Thus, these prodrugs have very promising anti-cataract potential.

No-induced oxidative stress and glutathione metabolism in rodent and human cells

Nitric oxide (NO.), a radical species produced by many types of cells, is known to play a critical role in both regulatory processes and cell defense, yet it may also participate in collateral reactions, leading to DNA damage and cell death in both NO-generating and neighboring cells. Glutathione has been shown to protect cells from the toxic effects of free radicals and reactive oxygen species. The goal of this study was to investigate whether differences in glutathione metabolism could account for the resistance or sensitivity to cell killing by NO. The cytotoxic effect of NO- was examined in CHO-AA8 (Chinese Hamster Ovary) cells and TK6 (human lymphoblastoid) cells pretreated with L-buthionine SR-sulfoximine (BSO), a potent inhibitor of γ-glutamylcysteine synthetase, and with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), an irreversible inhibitor of glutathione reductase. The consequences resulting from the depletion of glutathione levels and from the arrest of oxidoreduction allowed us to show the involvement of glutathione in protecting cells from NO. and to investigate the importance of changes in glutathione metabolism on NO-induced toxicity. In CHO-AA8 cells, we found that treatment with NO. resulted in the oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG) and to mixed glutathione disulfides (GSSR). The resulting depletion of GSH stimulated its de novo synthesis, enabling the cells to resist killing by NO-. A slight difference in GSH metabolism was observed in TK6 cells. NO- led to an increase in GSSG levels similar to that observed in CHO-AA8 cells, however, a decrease in GSH levels, no change in GSSR levels, and higher levels of toxicity were also found, suggesting that NO-treated TK6 cells are not as competent in GSH homeostasis as CHO cells. We conclude that GSH is involved in protecting cells from killing by NO- and that both de novo synthesis of GSH and GSSG reduction are important in maintaining an adequate level of protection for the cells.

Determination of thiols and disulfides using high-performance liquid chromatography with electrochemical detection

Low-molecular-mass thiols, such as glutathione (GSH), and their associated disulfides are ubiquitous in nature, and based upon the many known functions of these compounds, their identification and accurate measurement is essential. Our objectives were to develop a simple method for the simultaneous measurement of thiols and disulfides in biological samples using HPLC with dual electrochemical detection (HPLC-DED). Particular emphasis was placed on the applicability to a wide variety of important GSH-related thiols and disulfides, including γ-Glu-Cys, Cys-Gly, their disulfides, and the mixed disulfide of glutathione and cysteine (CSSG), validation on different types of biological samples, maintenance of chromatographic resolution and reproducibility with routine and extended use, and enhancement of assay sensitivity. To this end, optimal HPLC conditions including mobile phase, column, and electrode polishing procedures were established and the method was applied to, and validated on a variety of biological samples. This improved methodology should prove to be a useful tool in studies on the metabolism of GSH and other thiols and disulfides and their role in cellular homeostasis and disease processes.

Dissecting the mechanism of protein disulfide isomerase: catalysis of disulfide bond formation in a model peptide.

As a model for understanding how protein disulfide isomerase (PDI) catalyzes disulfide bond formation in proteins, its action on a 28-residue disordered peptide containing only two cysteine residues has been examined. Disulfide formation in the peptide using the chemical reaction with small molecule thiol/disulfide reagents, such as oxidized and reduced glutathione or cystamine and cysteamine, occurs in two steps, via two alternative intermediate mixed disulfides between the reagent and either peptide cysteine residue. All thiol/disulfide forms of the peptide could be trapped and quantified, so the rates of their interconversion could be measured. Catalytic amounts of PDI increased the rates of these reactions. All rate enhancements were independent of the concentration of the peptide, indicating that it bound to PDI with an apparent Km of less than 3 microM. In the presence of glutathione, PDI accelerated the formation of both single mixed disulfide species, plus their subsequent rearrangement to form the peptide disulfide bond, but not interchange of the mixed disulfide glutathione between the two cysteine residues. In contrast, PDI did not catalyze the reaction of the reagent cystamine with the reduced peptide to form the mixed disulfide, nor the interchange of this mixed disulfide between cysteine residues, but did catalyze the subsequent intramolecular step of peptide disulfide bond formation to a similar extent as with the glutathione mixed disulfide. These effects on the two steps involving the mixed disulfides with glutathione or cystamine accounted for much of the overall catalytic effect of PDI on disulfide bond formation in the peptide, indicating that direct transfer of disulfide bonds from PDI to the peptide occurred less frequently. These findings demonstrate the utility of using such peptides as PDI substrates and have implications for the mechanism of action of PDI.

Oxidative stress induced by administration of the neuroleptic drug haloperidol is attenuated by higher doses of haloperidol

The effect of haloperidol administration on lipid peroxidation and glutathione/protein thiol homeostasis in the brain was examined 4 h following subcutaneous administration of a single dose of haloperidol; 1.0, 1.5, 2.0 or 2.5 mg/kg b.wt. Glutathione (GSH) levels decreased significantly in cortex, striatum and midbrain after haloperidol administration. Maximal decreases of GSH was observed in the striatum. The depleted GSH was recoverable as protein glutathione mixed disulfide (Pr-SSG) with concomitant loss of protein thiols (Pr-SH) in all the regions of the brain examined. Administration of 1.5 mg/kg b.wt of haloperidol resulted in significant depletion of GSH in striatum and midbrain as compared to that after administration of the lower dose of 1.0 mg/kg b.wt. of haloperidol. However, administration of higher doses of haloperidol (2.0 and 2.5 mg/kg b.wt.) did not result in greater depletion of GSH; the GSH levels were not significantly different from that observed following the administration of 1.5 mg/kg b.wt. of haloperidol. However, Pr-SSG levels increased dose-dependently following haloperidol administration. The total GSH recovered as sum of GSH and Pr-SSG was significantly higher than controls in striatum and midbrain following administration of higher doses of haloperidol, namely, 2.0 and 2.5 mg/kg b.wt. The depleted GSH was not recoverable as glutathione disulfide (GSSG), GSSG levels were not significantly different from controls 4 h after administration of 1.5 mg/kg b.wt. of haloperidol. The levels of malondialdehyde (indicative of lipid peroxidation) increased significantly as compared to control levels (280-220%) following administration of 1.0 and 1.5 mg/kg b.wt. of haloperidol. Thereafter, the malondialdehyde levels in brain regions decreased and were only (186-150%) of control levels after administration of 2.0 and 2.5 mg/kg b.wt. of haloperidol, respectively. The present study demonstrates that administration of low doses of haloperidol results in depletion of GSH and increased levels of malondialdehyde. However, administration of higher doses of haloperidol results in attenuation of peroxidative damage with concomitant increase in the total GSH recovered as sum of free GSH and GSH bound to protein thiols (Pr-SSG).

Glutathione mixed disulfides and heterogeneity of chicken hemoglobins

1. 1. Adult chicken hemoglobins Hb A and Hb D interact with glutathione disulfide, GSSG. The major hemoglobin, Hb A, forms at least two new components, termed GHb AI and GHb AII, and Hb D forms at least one, GHb DI. 2. 2. At pH 8.0 and 5°C, glutathione disulfide (GSSG) in a molar excess of 50 x took 6 days to complete the reaction, although at pH 8.6 and 41°C only 1 hr was needed, where the hemoglobins Hb A and Hb D were converted to their most mobile forms GHb AII and GHb DI. 3. 3. Slight molar excess (2.7 GSSG/Hb, pH 7.4, 41°C), reacting for 1 hr, showed extensive formation of GHb AI and GHb AII. 4. 4. Electrophoretic patterns, from the reaction products of 54 GSSG/Hb excess at different times, showed a marked pH dependence. 5. 5. Titration with pCMB ( p-chloromercuribezoic acid) of DTE (dithioerythrytol)-reduced samples showed 8.0 ± 0.4 ( N = 5) -SH (sulfhydryl) per tetramer. In hemolysates not reacted with DTE, 6.0 ± 0.4 ( N = 3) -SH were detected. 6. 6. DTE-reduced and GSSG-reacted hemoglobins showed 4.6 ± 0.5 ( N = 7) -SH and 1.5 ± 0.4 ( N = 6) -SH, respectively, as titrated by DTNB, pH 8.0. DTE-reduced hemoglobins showed four fast-reacting -SH groups, no longer present in GSSG-reacted hemoglobins. 7. 7. Our data indicate that chicken GHb AI and GHb DI probably have two glutathionyl residues per tetramer whereas GHb AII has four. 8. 8. The exceptionally high glutathione content in chicken erythrocytes (Smith J. E. 1974 J. Lab. clin. Med. 83, 444–450.), combined with the easy formation of glutathionyl hemoglobin, certainly seem to be the most common cause of the so-called “additional heterogeneity” of chicken hemoglobins (Lee K. S. Chuang P. C. and Cohen B. H. 1975. Biochim. biophys. Acta 427, 178–196).

Further evidence for independent folding of domains in serine proteases.

The folding pathway of pancreatic serine proteases was clarified from kinetic studies on the refolding of the glutathione-mixed disulfide derivative of bovine neochymotrypsinogen. Neochymotrypsinogen is prepared from a limited proteolysis of native chymotrypsinogen A by cleavage at Tyr146-Thr147 (Duda and Light (1982) J. Biol. Chem. 257, 9866-9871). The mixed disulfide methodology (Odorzynski and Light (1979) J. Biol. Chem. 254, 4291-4295) was necessary to successfully refold chymotrypsinogen and neochymotrypsinogen. Mixtures of the chromatographically purified amino- and carboxyl-terminal polypeptides of neochymotrypsinogen, as the mixed disulfide derivatives, were refolded at varying molar ratios of the polypeptides. The regeneration of native structure was followed as a function of time from activity measurements and from the regain of the molecular weight of the zymogen. The rate data fit first-order kinetics. The kinetic analysis is compatible with a folding mechanism that supports (a) independent folding of the amino- and carboxyl-terminal domains; (b) identical rates of folding of each domain; and (c) the rate-limiting step is the formation of the interdomain disulfide. The formation of a stable complex of the folded domains was favored by complementary hydrophobic and hydrogen bonding interactions and the formation of the last disulfide bond. The geometric arrangement of the active site residues was regained and the zymogen could be converted to the active enzyme, namely, alpha-chymotrypsin.

Metabolism of acetaminophen by cultured rat hepatocytes: Depletion of protein thiol groups without any loss of viability

Over the course of 4 hr, the metabolism of acetaminophen (APAP) by cultured rat hepatocytes resulted in a depletion of protein thiols and an accumulation of oxidized glutathione (GSSG) in the medium. With 20 mM APAP, arylation and the formation of glutathione mixed disulfides accounted for a loss of 22% of the total protein thiols in the absence of any loss of viability. With 20 mM APAP and an inhibition of glutathione reductase by 1,3-(2-chloroethyl)-1-nitrosourea (BCNU), protein thiols were depleted by 40% by arylation and the formation of glutathione mixed disulfides, again without a loss of viability. With 20 mM APAP and BCNU in the presence of 20 mM deferoxamine, there was still little or no cell killing after 8 hr despite a loss now of almost 60% of the total protein thiols. These data do not support the hypothesis that a depletion of protein thiols is related to the toxicity of APAP. One millimolar APAP and BCNU killed 60% of the hepatocytes within 4 hr. In this circumstance, the loss of protein thiols was not attributable to either arylation by APAP metabolites or the formation of glutathione mixed disulfides. The antioxidant N, N'-diphenyl-phenylenediamine prevented the cell killing and the loss of protein thiols, a result implicating a role for lipid peroxidation in the depletion of protein-bound thiols. However, protein thiol depletion under these circumstances is not necessarily related to the lethal cell injury and most likely represents an epiphenomenon of the peroxidation of cellular lipids.