Formation Of Protein Carbonyl Groups Research Articles

The Effect of Hemp Cake (Cannabis sativa L.) on the Characteristics of Meatballs Stored in Refrigerated Conditions.

Hemp cake, a by-product of cold pressing oil from hemp seeds, is a nutritious ingredient that could be used for the production of new or reformulated meat products. The aim of this study was to determine the influence of inclusion of 0.9%, 2.6%, 4.2%, and 7.4% (w/w) hemp cake (Cannabis sativa L.) on the physicochemical and textural properties, oxidation, and sensory acceptance of cooked and vacuum-packed meatballs during refrigerated storage. The addition of 7.4% hemp cake enhanced the amount of dry matter and reduced the content of water. Lightness (L*) and redness (a*) values reduced significantly with higher levels of hemp supplementation. Regardless of the amount of hemp additive, pH, color parameters did not differ significantly during the 12 days of storage. Hemp cake significantly decreased protein and lipid oxidation: the inhibitory effect of adding 7.4% hemp cake on protein carbonyl group formation and TBARS values reached 11.16% and 36.5%, respectively, after 10 days of storage. Sensory analysis revealed that meatballs prepared with 0.9% and 2.6% hemp cake gained higher overall scores. The results indicate that hemp cake, a material considered mainly as waste, may be destined for food purposes and be an alternative ingredient for the production of sustainable meat products.

KARNOZİNİN SAĞLIK ÜZERİNE ETKİLERİ

Rus bilim insanları tarafından 1900 yılında keşfedilen karnozin (β-alanin-L-histidin) sadece hayvan dokularında bulunmaktadır. Karnozin birçok biyokimyasal değişikliklerin eşlik ettiği patolojileri (protein oksidasyonu, glikasyon, ileri glikozilasyon ürünlerinin (AGE’lerin) oluşumu ve çapraz bağlanma vb.) engelleme potansiyeline sahiptir. AGE'lerin üretimi ve protein karbonil gruplarının oluşumu yaşlanmada, diyabette, diyabetin sekonder komplikasyonlarında ve nörodejeneratif durumlarda önemli rol oynamaktadır. Karnozinin antiglikasyon özelliği, zararlı karbonillere karşı reaktivitesi, çinko ve bakır şelasyon aktivitesi, düşük toksisitesi ve yaşa bağlı protein karbonil stresine karşı tepkisi hastalıklardan koruyucu ve hastalıkları engelleyici özellik göstermesine neden olmaktadır. Tüm bu muhtemel etkilerinden dolayı insanlarda kronik hastalıkların önlenmesi ve tedavisinde karnozinin rolünü tespit etmek için daha fazla çalışmaya ihtiyaç bulunmaktadır. Bu derleme karnozinin besinlerle veya suplemantasyon olarak alınması durumunda gerçekleşen bazı biyolojik rolleri hakkında bilgi vermek amacıyla planlanmış ve yürütülmüştür.

Phenelzine reduces the oxidative damage induced by peroxynitrite in plasma lipids and proteins

Peroxynitrite is a reactive nitrogen species produced in the intravascular compartment from superoxide anion and nitric oxide. Peroxynitrite destroys blood plasma proteins and membranes of red blood cells and of platelets. This explains why excessive production of peroxynitrite contributes to diseases and to ageing. Therapeutics that antagonize peroxynitrite may delay ageing and the progression of disease. We developed an in vitro assay that allows the investigation of the oxidative damage caused by peroxynitrite in the intravascular compartment. This assay correlates the damage with the rate of formation of protein carbonyl groups, 3-nitrotyrosine (3-NT) and thiobarbituric acid reactive substances. Using this assay, we evaluated the ability of phenelzine, a scavenger of reactive aldehydes, to antagonize the effects of peroxynitrite. Herein, we showed that phenelzine significantly decreased the lipid peroxidative damage caused by peroxynitirite in blood plasma and platelets. Moreover, it inhibited carbonyl group and 3-NT formation in blood plasma and platelet proteins.

Canavanine Alters ROS/RNS Level and Leads to Post-translational Modification of Proteins in Roots of Tomato Seedlings

Canavanine (CAN), a structural analog of arginine (Arg), is used as a selective inhibitor of inducible NOS in mammals. CAN is incorporated into proteins’ structure in the place of Arg, leading to the formation of aberrant compounds. This non-protein amino acid is found in legumes, e.g., Canavalia ensiformis (L.) DC. or Sutherlandia frutescens (L.) R.Br. and acts as a strong toxin against herbivores or plants. Tomato (Solanum lycopersicum L.) seedlings were treated for 24–72 h with CAN (10 or 50 μM) inhibiting root growth by 50 or 100%, without lethal effect. We determined ROS level/production in root extracts, fluorescence of DAF-FM and APF derivatives corresponding to RNS level in roots of tomato seedlings and linked CAN-induced restriction of root growth to the post-translational modifications (PTMs) of proteins: carbonylation and nitration. Both PTMs are stable markers of nitro-oxidative stress, regarded as the plant’s secondary response to phytotoxins. CAN enhanced H2O2 content and superoxide radicals generation in extracts of tomato roots and stimulated formation of protein carbonyl groups. An elevated level of carbonylated proteins was characteristic for the plants after 72 h of the culture, mainly for the roots exposed to 10 μM CAN. The proteolytic activity was stimulated by tested non-protein amino acid. CAN treatment led to decline of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives. Short-term exposure of tomato seedlings to CAN lowered the protein nitration level. Activity of peroxidase, polyamine oxidase and NADPH oxidase, enzymes acting as modulators of H2O2 concentration and governing root architecture and growth were determined. Activities of all enzymes were stimulated by CAN, but no strict CAN concentration dependence was observed. We conclude, that although CAN treatment led to a decline in the nitric oxide level, PTMs observed in roots of plants exposed to CAN are linked rather to the formation of carbonyl groups than to nitration, and are detected particularly after 24 h. Thus, oxidative stress and oxidative modifications of proteins seems to be of significant importance in the rapid response of plants to CAN.

Protection of Clitoria ternatea flower petal extract against free radical-induced hemolysis and oxidative damage in canine erythrocytes

The present study assessed the antioxidant activity and protective ability of Clitoria ternatea flower petal extract (CTE) against in vitro 2,2′-azobis-2-methyl-propanimidamide dihydrochloride (AAPH)-induced hemolysis and oxidative damage of canine erythrocytes. From the phytochemical analysis, CTE contained phenolic compounds, flavonoids, and anthocyanins. In addition, CTE showed antioxidant activity as measured by oxygen radical absorbance capacity (ORAC) method and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. CTE (400 µg/ml) remarkably protected erythrocytes against AAPH-induced hemolysis at 4 h of incubation. Moreover, CTE (400 µg/ml) reduced membrane lipid peroxidation and protein carbonyl group formation and prevented the reduction of glutathione concentration in AAPH-induced oxidation of erythrocytes. The AAPH-induced morphological alteration of erythrocytes from a smooth discoid to an echinocytic form was effectively protected by CTE. The present results contribute important insights that CTE may have the potential to act as a natural antioxidant to prevent free radical-induced hemolysis, protein oxidation and lipid peroxidation in erythrocytes.

Vicenin 2 isolated from Artemisia capillaris exhibited potent anti-glycation properties

Vicenin 2, isolated from a traditionally used medicinal plant Artemisia capillaris, is a 6,8-di-C-glucoside of apigenin which has been previously reported to possess a wide variety of pharmacological activities including antioxidant, anti-inflammatory, anti-cancer, and hepatoprotective. However, there have not been any reports concerning its anti-diabetic potential until now. Therefore, in the present study, we evaluated the anti-diabetic potential of vicenin 2 via α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), rat lens aldose reductase (RLAR), and advanced glycation end products (AGE) formation inhibitory assays. Vicenin 2 strongly inhibited α-glucosidase, PTP1B, and RLAR in the corresponding assays. In addition, vicenin 2 inhibited the formation of both fluorescent AGE and nonfluorescent AGE, e.g., CML, as well as the level of fructosamine in glucose–fructose-induced bovine serum albumin (BSA) glycation. In the test system, vicenin 2 suppressed glycation-induced protein oxidation by attenuating the formation of protein carbonyl groups as well as by inhibiting the modification of protein thiol groups. Moreover, vicenin 2 was found to be a potent inhibitor of glycation-induced formation of amyloid cross-β structures in BSA. Taken together, vicenin 2 might be a useful lead for the development of multiple target-oriented therapeutic modalities for the treatment of diabetes and diabetes-associated complications.

Chemical Modification of Lysozyme, Glucose 6-Phosphate Dehydrogenase, and Bovine Eye Lens Proteins Induced by Peroxyl Radicals: Role of Oxidizable Amino Acid Residues

Chemical and structural alterations to lysozyme (LYSO), glucose 6-phosphate dehydrogenase (G6PD), and bovine eye lens proteins (BLP) promoted by peroxyl radicals generated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions were investigated. SDS-PAGE analysis of the AAPH-treated proteins revealed the occurrence of protein aggregation, cross-linking, and fragmentation; BLP, which are naturally organized in globular assemblies, were the most affected proteins. Transmission electron microscopy (TEM) analysis of BLP shows the formation of complex protein aggregates after treatment with AAPH. These structural modifications were accompanied by the formation of protein carbonyl groups and protein hydroperoxides. The yield of carbonyls was lower than that for protein hydroperoxide generation and was unrelated to protein fragmentation. The oxidized proteins were also characterized by significant oxidation of Met, Trp, and Tyr (but not other) residues, and low levels of dityrosine. As the dityrosine yield is too low to account for the observed cross-linking, we propose that aggregation is associated with tryptophan oxidation and Trp-derived cross-links. It is also proposed that Trp oxidation products play a fundamental role in nonrandom fragmentation and carbonyl group formation particularly for LYSO and G6PD. These data point to a complex mechanism of peroxyl-radical mediated modification of proteins with monomeric (LYSO), dimeric (G6PD), and multimeric (BLP) structural organization, which not only results in oxidation of protein side chains but also gives rise to radical-mediated protein cross-links and fragmentation, with Trp species being critical intermediates.

Oversynthesis of Riboflavin in the Yeast Pichia guilliermondii is Accompanied by Reduced Catalase and Superoxide Dismutases Activities

Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. Under iron deprivation conditions, Pichia guilliermondii cells increase production of riboflavin and malondialdehyde and the formation of protein carbonyl groups, which reflect increased intracellular content of reactive oxygen species. In this study, we found that P. guilliermondii iron deprived cells showed dramatically decreased catalase and superoxide dismutase activities. Previously reported mutations rib80, rib81, and hit1, which affect repression of riboflavin synthesis and iron accumulation by iron ions, caused similar drops in activities of the mentioned enzymes. These findings could explain the previously described development of oxidative stress in iron deprived or mutated P. guilliermondii cells that overproduce riboflavin. Similar decrease in superoxide dismutase activities was observed in iron deprived cells in the non-flavinogenic yeast Saccharomyces cerevisiae.

Striatal Neuroprotection from Neonatal Hypoxia-Ischemia in Piglets by Antioxidant Treatment with EUK-134 or Edaravone

Striatal neurons are highly vulnerable to hypoxia-ischemia (HI) in term newborns. In a piglet model of HI, striatal neurons develop oxidative stress and organelle disruption by 3–6 h of recovery and ischemic cytopathology over 6–24 h of recovery. We tested the hypothesis that early treatment with the antioxidants EUK-134 (a manganese-salen derivative that acts as a scavenger of superoxide, hydrogen peroxide, nitric oxide or NO and peroxynitrite) or edaravone (MCI-186, a scavenger of hydroxyl radical and NO) protects striatal neurons from HI. Anesthetized newborn piglets were subjected to 40 min of hypoxia and 7 min of airway occlusion. At 30 min after resuscitation, the piglets received vehicle, EUK-134 or edaravone. Drug treatment did not affect arterial blood pressure, blood gases, blood glucose or rectal temperature. At 4 days of recovery, the density of viable neurons in the putamen of vehicle-treated piglets was 12 ± 6% (±SD) of sham-operated control density. Treatment with EUK-134 increased viability to 41 ± 17%, and treatment with edaravone increased viability to 39 ± 19%. In the caudate nucleus, neuronal viability was increased from 54 ± 11% in the vehicle group to 78 ± 15% in the EUK-134 group and to 73 ± 13% in the edaravone group. Antioxidant drug treatment accelerated recovery from neurologic deficits and decreased oxidative and nitrative damage to nucleic acids. Treatment with EUK-134 reduced the HI-induced formation of protein carbonyl groups and tyrosine nitration at 3 h of recovery. We conclude that systemic administration of antioxidant agents by 30 min after resuscitation from HI can reduce oxidative stress and salvage neurons in the highly vulnerable striatum in a large-animal model of neonatal HI. Therefore, oxidative stress is an important mechanism for this injury, and antioxidant therapy is a rational, mechanism-based approach to neuroprotection in the newborn brain.

Oxidative stress induced by the Fe/ascorbic acid system or model ischemia in vitro: effect of carvedilol and pyridoindole antioxidant SMe1EC2 in young and adult rat brain tissue.

New effective strategies and new highly effective neuroprotective agents are being searched for the therapy of human stroke and cerebral ischemia. The compound SMe1EC2 is a new derivative of stobadine, with enhanced antioxidant properties compared to the maternal drug. Carvedilol, a non-selective beta-blocker, possesses besides its cardioprotective and vasculoprotective properties also an antioxidant effect. We compared the effect of carvedilol and SMe1EC2, antioxidants with a similar chemical structure, in two experimental models of oxidative stress in young and adult rat brain tissue. SMe1EC2 was found to improve the resistance of hippocampal neurons to ischemia in vitro in young and even in 18-month-old rats and inhibited formation of protein carbonyl groups induced by the Fe2+/ascorbic acid pro-oxidative system in brain cortex homogenates of young rats. Carvedilol exerted a protective effect only in the hippocampus of 2-month-old rats and that at the concentration 10-times higher than did SMe1EC2. The inhibitory effect of carvedilol on protein carbonyl formation induced by the pro-oxidative system was not proved in the cortex of either young or adult rats. An increased baseline level of the content of protein carbonyl groups in the adult versus young rat brain cortex confirmed age-related changes in neuronal tissue and may be due to increased production of reactive oxygen species and low antioxidant defense mechanisms in the adult rat brain. The results revealed the new pyridoindole SMe1EC2 to be more effective than carvedilol in neuroprotection of rat brain tissue in both experimental models involving oxidative stress.

Protection of wheat bran feruloyl oligosaccharides against free radical-induced oxidative damage in normal human erythrocytes

The present work assessed the protective effect of water-soluble feruloyl oligosaccharides (FSH), ferulic acid ester of oligosaccharides from wheat bran, against in vitro oxidative damage of normal human erythrocytes induced by a water-soluble free radical initiator, 2,2′-azobis-2-amidinopropane dihydrochloride (AAPH). In the whole process of AAPH-initiated oxidation, hemolysis occurred quickly after the lag time. The rate of hemolysis is correlated dose-dependently with AAPH concentration. Significant decrease in reduced glutathione (GSH) levels of erythrocyte with concomitant enhancement in oxidized gluthione (GSSG) levels was noticed. It was also observed that lipid and protein peroxidation of erythrocytes induced by AAPH was significantly increased, and scanning electron microscopy observations showed that AAPH induced obvious morphological alteration in the erythrocytes from a smooth discoid to an echinocytic form. FSH suppressed depletion of GSH, lipid peroxidation, and methaemoglobin and protein carbonyl group formation of erythrocytes in concentration- and time-dependent manners, remarkably delayed AAPH-induced hemolysis. Morphological changes to erythrocyte caused by AAPH were effectively protected by FSH. It was also observed that FSH could work synergistically with endogenous antioxidants in erythrocytes. These results indicated that FSH efficiently protected normal human erythrocytes against oxidative stress, and they could be used as a potential source of natural antioxidants.

Would Carnosine or a Carnivorous Diet Help Suppress Aging and Associated Pathologies?

Carnosine (beta-alanyl-L-histidine) is found exclusively in animal tissues. Carnosine has the potential to suppress many of the biochemical changes (e.g., protein oxidation, glycation, AGE formation, and cross-linking) that accompany aging and associated pathologies. Glycation, generation of advanced glycosylation end-products (AGEs), and formation of protein carbonyl groups play important roles in aging, diabetes, its secondary complications, and neurodegenerative conditions. Due to carnosine's antiglycating activity, reactivity toward deleterious carbonyls, zinc- and copper-chelating activity and low toxicity, carnosine and related structures could be effective against age-related protein carbonyl stress. It is suggested that carnivorous diets could be beneficial because of their carnosine content, as the dipeptide has been shown to suppress some diabetic complications in mice. It is also suggested that carnosine's therapeutic potential should be explored with respect to neurodegeneration. Olfactory tissue is normally enriched in carnosine, but olfactory dysfunction is frequently associated with neurodegeneration. Olfactory administration of carnosine could provide a direct route to compromised tissue, avoiding serum carnosinases.

Regulation of antioxidant enzyme gene expression in response to oxidative stress and during differentiation of mouse skeletal muscle

Various properties of skeletal muscle, including high metabolic activity and high levels of heme-containing proteins, render it particularly susceptible to free radical injury. Indeed, cellular injury from reactive oxygen species (ROS) has been implicated in many muscle disorders. Thus muscle cell survival is critically dependent on the ability of the cell to respond to periods of oxidative stress. To investigate this important homeostatic response, we studied the effect of oxidative challenges on the expression of genes encoding the antioxidant enzymes Cu,Zn-superoxide dismutase (CuZnSOD), Mn-superoxide dismutase (MnSOD), glutathione peroxidase (GPx), and catalase (CAT) in myotube cultures. Using Northern blot analysis, we found that treatment with the pro-oxidant paraquat resulted in time- and dose-dependent increases of transcript levels that were greatest for GPx and CAT (∼4–5 fold). CuZnSOD and MnSOD transcripts were also increased, albeit more modestly (∼2–3 fold). Transcript levels were also induced by treatment of the cells with two other pro-oxidants, menadione and H 2O 2, and correlated with the level of oxidative injury to the cells, measured as protein carbonyl group formation. Activities of all of the enzymes increased in response to the oxidative challenges, although the magnitudes of the increases were less robust than the increases of the respective transcript levels. In studying the effect of cellular differentiation on antioxidant gene expression and susceptibility to oxidative stress, we found that pro-oxidant treatment resulted in greater oxidative injury to differentiated myotubes than to undifferentiated myoblasts. Furthermore, the increased susceptibility of myotubes correlated with decreased antioxidant defenses—as muscle cells differentiated, both transcript and activity levels of antioxidant enzymes decreased. These data suggest that muscle cells regulate antioxidant defenses in response to oxidative stress and cellular differentiation.

Posttranslational modifications of cardiac and skeletal muscle proteins by reactive oxygen species after burn injury in the rat.

To determine the involvement of oxidative damage in muscle wasting after burn injury. Burn injury damages tissue at the site of the burn and also affects peripheral tissue. There is evidence to suggest that reactive oxygen species may be generated in increased amounts after burn, and these may contribute to wound healing and to posttranslational modifications of tissue constituents distant from the wound site. The oxidation of muscle proteins was assessed, using the dinitrophenylhydrazine assay for carbonyl content, in muscles of rats after a full-thickness skin scald burn covering 20% of the total body surface area, over a 6-week period. In this model, rats failed to incur normal body weight or muscle weight gain. Soleus, extensor digitorum longus, diaphragm, and heart ventricle proteins were oxidatively damaged after injury. The extent of tissue protein oxidation, however, differed depending on the time points studied. In general, higher levels of protein carbonyl group formation, an indicator of oxidative damage, were found to occur within 1 to 5 days after injury, and the oxidized protein content of the various tissues decreased during the later stages. Both sarcoplasmic and myofibrillar carbonyl-containing proteins accumulated in diaphragm 3 days after burn injury and were rapidly removed from the tissue during a 2-hour in vitro incubation. This coincided with increased proteolytic activity in diaphragm. These observations suggest that the loss of proteins modified by reactive oxygen species may contribute to the burn-induced protein wasting in respiratory and other muscles by a proteolytically driven mechanism.

ROLE OF MITOCHONDRIA DYSFUNCTION IN THE DEVELOPMENT OF INHERITED PREDISPOSITION TO THE DISEASES INVOLVING ENHANCED OXYGEN RADICAL FORMATION IN THE RATS WISTAR/S

By selecting and inbreeding Wistar rats which are sensitive (S) or resistant (R) to the cataractogenic effect of galactose the S and R inbreed rat strains were created. Intense generation of oxygen radicals and enhanced lipid peroxidation were revealed in the liver and myocardium of the S rats. DNA rearrangements were found in the genome of S rats. Decreased oxidative phosphorilation and the lower respiratory control ratio were found in liver mitochondria of S rats. The formation of protein carbonyl groups and membrane fluidity were increased. SOD and catalase activity were decreased. Tumors, premature aging (low fecundity, growth retardation and short life-span), cataracts, cardiomyopathy-like changes in the myocardium, scoliosis are characteristics of the S rats. It was found that dysfunction of rat liver mitochondria is increased with age. Moreover, the increasing of mitochondria dysfunction is in good correlation with oxygen radical formation and oxidative damage of lipids and proteins. Thus, we suppose that mitochondria dysfunction plays an important role in the development of inherited predisposition to the enhancement of oxygen radical generation in the rats Wistar/S resulting in multipathological states.

The prooxidant effect of 5-aminolevulinic acid in the brain tissue of rats: Implications in neuropsychiatric manifestations in porphyrias

5-Aminolevulinic acid (ALA), a heme precursor accumulated during the clinical expression of acute intermittent porphyria, lead poisoning, and tyrosinosis, has been hypothesized to act as an endogenous source of oxyradicals. We now report oxidative effects on brain tissue of rats submitted to ALA treatment. Upon acute treatment (40 mg/kg body weight) increased total nonheme iron in the cortex (20%) was observed. After prolonged ALA administration (40 mg/kg body weight on alternate days during 2 weeks), the following indicators of oxidative stress were found to be significantly increased: CuZnSOD activity (67%) in total brain homogenate, total iron (68%) and ferritin (71%) in the cortex, ferritin in striatum (44%), protein carbonyls in homogenate of cerebral cortex (threefold) and 45Ca 2+ uptake by cortical synaptosomes (45%). In addition, synaptic membranes prepared from whole brain assayed with the radioligand 3H-muscimol, revealed increased K d values (twofold) of the high-affinity GABAergic receptor binding and formation of protein carbonyl groups, thiobarbituric acid reactive products, and conjugated dienes. In vitro, ALA produced similar effects upon the high affinity 3H-muscimol binding. No apparent alteration of either dopaminergic or serotonergic [ 3h]-ligand binding was observed. These results argue in favor of ALA-triggered oxidative stress in brain accompanied by iron metabolism alterations and GABAergic receptor damage, which may be implicated in the neuropsychiatric manifestations of the aforementioned porphyrias.

Inhibition of protein carbonyl formation and lipid peroxidation by glutathione in rat liver microsomes

The peroxidation of rat liver microsomal lipids is stimulated in the presence of iron by the addition of NADPH or ascorbate and is inhibited by the addition of glutathione (GSH). The fate of GSH and the oxidative modification of proteins under these conditions have not been well studied. Rat liver microsomes were incubated at 37 °C under 95% O 2:5% CO 2 in the presence of 10 μ m ferric chloride, 400 μ m ADP, and either 450 μ m ascorbic acid or 400 μ m NADPH. Lipid peroxidation was assessed in the presence 0, 0.2, 0.5, 1, or 5 m m GSH by measuring thiobarbituric acid reactive substance (TBARS) and oxidative modification of proteins by measuring protein thiol and carbonyl groups. GSH inhibited TBARS and protein carbonyl group formation in both ascorbate and NADPH systems in a dose-dependent manner. Heat denaturing of microsomes or treatment with trypsin resulted in the loss of this protection. The formation of protein carbonyl groups could be duplicated by incubating microsomes with 4-hydroxynonenal. Ascorbate-dependent peroxidation caused a loss of protein thiol groups which was diminished by GSH only in fresh microsomes. Both boiling and trypsin treatment significantly decreased the basal protein thiol content of microsomes and enhanced ascorbate-stimulated lipid peroxidation. Protection against protein carbonyl group formation by GSH correlated with the inhibition of lipid peroxidation and appeared not to be due to the formation of the GSH conjugate of 4-hydroxynonenal as only trace amounts of this conjugate were detected. Ninety percent of the GSH lost after 60 min of peroxidation was recoverable as borohydride reducible material in the supernatant fraction. The remaining 10% could be accounted for as GSH-bound protein mixed disulfides. However, only 75% of the GSH lost during peroxidation appeared as glutathione disulfide, suggesting that some was converted to other soluble borohydride reducible forms. These data support a role for protein thiol groups in the GSH-mediated protection of microsomes against lipid peroxidation.