Oxidation Of Protein Sulfhydryl Groups Research Articles

Antioxidant Activity of Quercetin in a H2O2-Induced Oxidative Stress Model in Red Blood Cells: Functional Role of Band 3 Protein.

During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H2O2. Markers of OS, including % hemolysis, reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, CD47 and B3p expression, methemoglobin formation (% MetHb), as well as the anion exchange capability through Band 3 protein (B3p) have been analyzed in RBCs treated for 1 h with 20 mM H2O2 with or without pre-treatment for 1 h with 10 μM Q, or in RBCs pre-treated with 20 mM H2O2 and then exposed to 10 µM Q. The results show that pre-treatment with Q is more effective than post-treatment to counteract OS in RBCs. In particular, pre-exposure to Q avoided morphological alterations (formation of acanthocytes), prevented H2O2-induced OS damage, and restored the abnormal distribution of B3p and CD47 expression. Moreover, H2O2 exposure was associated with a decreased rate constant of SO42− uptake via B3p, as well as an increased MetHb formation. Both alterations have been attenuated by pre-treatment with 10 μM Q. These results contribute (1) to elucidate OS-related events in human RBCs, (2) propose Q as natural antioxidant to counteract OS-related alterations, and (3) identify B3p as a possible target for the treatment and prevention of OS-related disease conditions or aging-related complications impacting on RBCs physiology.

Açaì (Euterpe oleracea) Extract Protects Human Erythrocytes from Age-Related Oxidative Stress.

Aging is a process characterised by a general decline in physiological functions. The high bioavailability of reactive oxygen species (ROS) plays an important role in the aging rate. Due to the close relationship between aging and oxidative stress (OS), functional foods rich in flavonoids are excellent candidates to counteract age-related changes. This study aimed to verify the protective role of Açaì extract in a d-Galactose (d-Gal)-induced model of aging in human erythrocytes. Markers of OS, including ROS production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, as well as the anion exchange capability through Band 3 protein (B3p) and glycated haemoglobin (A1c) have been analysed in erythrocytes treated with d-Gal for 24 h, with or without pre-incubation for 1 h with 0.5–10 µg/mL Açaì extract. Our results show that the extract avoided the formation of acanthocytes and leptocytes observed after exposure to 50 and 100 mM d-Gal, respectively, prevented d-Gal-induced OS damage, and restored alterations in the distribution of B3p and CD47 proteins. Interestingly, d-Gal exposure was associated with an acceleration of the rate constant of SO42− uptake through B3p, as well as A1c formation. Both alterations have been attenuated by pre-treatment with the Açaì extract. These findings contribute to clarify the aging mechanisms in human erythrocytes and propose functional foods rich in flavonoids as natural antioxidants for the treatment and prevention of OS-related disease conditions.

Role of Non-prolamin Proteins and Low Molecular Weight Redox Agents in Protein Folding and Polymerization in Wheat Grains and Influence on Baking Quality Parameters

The various enzyme systems and low molecular weight (LMW) redox agents are related to the folding and polymerization of prolamins in the ripening wheat grains and the formation of baking quality. Protein disulfide isomerases (PDIs) and cyclophylins accelerate "correct" folding of prolamins, which is most likely necessary for the subsequent formation of the macromolecular structure of the gluten protein matrix. PDIs are also involved in the polymerization of prolamins, catalyzing the oxidation of protein sulfhydryl groups. Molecular chaperone binding BiP protein facilitates folding of prolamins, with its role increasing in the stressful conditions. Reducing systems of thioredoxin and glutaredoxin, LMW redox pairs GSH/GSSG and Asc/DHAsc, thiol oxidases, and lipoxygenases (LOXs) regulate redox balance and the rate of polymerization of prolamins at the different stages of grain ripening. Additionally, LOX is probably involved in the protein-starch-lipid interactions between the starch granule and the protein matrix, mediated by puroindolines, determining the formation of grain texture. It is assumed that the high variability of baking quality in different environmental conditions is due to the interaction of labile enzyme systems with the storage proteins in the developing wheat caryopsis.

Protein-bound and free UV filters in cataract lenses. The concentration of UV filters is much lower than in normal lenses

In human cataract lenses the UV filters, 3-hydroxykynurenine glucoside (3OHKG) and kynurenine (Kyn) were found to be covalently bound to proteins and the levels in the nucleus were much higher than in the cortex. The levels of the bound UV filters in cataract nuclei were much lower than those in age-matched normal lenses. 3-Hydroxykynurenine could not be detected in cataract lenses. As with normal lenses, protein-bound 3OHKG in cataract lenses was found at the highest levels followed by Kyn. Free UV filter concentrations were also markedly reduced in cataract lenses. This feature may well contribute to the lower protein-bound levels; however, there was no clear relationship between free and bound UV filter contents when individual lenses were examined. We propose that since cysteine is a major site for UV filter binding, the well-documented oxidation of protein sulfhydryl groups during the progression of nuclear cataract may account, in part, for the pronounced decrease in bound UV filters in cataract lenses.

Activation of microsomal glutathione s-transferase by peroxynitrite.

Peroxynitrite (ONOO-) toxicity is associated with protein oxidation and/or tyrosine nitration, usually resulting in inhibition of enzyme activity. We examined the effect of ONOO- on the activity of purified rat liver microsomal glutathione S-transferase (GST) and found that the activity of reduced glutathione (GSH)-free enzyme was increased 4- to 5-fold by 2 mM ONOO-; only 15% of this increased activity was reversed by dithiothreitol. Exposure of the microsomal GST to ONOO- resulted in concentration-dependent oxidation of protein sulfhydryl groups, dimer and trimer formation, protein fragmentation, and tyrosine nitration. With the exception of sulfhydryl oxidation, these modifications of the enzyme correlated well with the increase in enzyme activity. Nitration or acetylation of tyrosine residues of the enzyme using tetranitromethane and N-acetylimidazole, respectively, also resulted in increased enzyme activity, providing additional evidence that modification of tyrosine residues can alter catalytic activity. Addition of ONOO--treated microsomal GST to microsomal membrane preparations caused a marked reduction in iron-induced lipid peroxidation, which raises the possibility that this enzyme may act to lessen the degree of membrane damage that would otherwise occur under pathophysiological conditions of increased ONOO- formation.

Kinetics of oxidation of the lipids and proteins of cod sarcoplasmic reticulum.

Lipid and protein oxidation in an NADH-Fe enzymic and an ascorbate-Fe nonenzymic system were determined simultaneously. The nonenzymic iron-reducing system gave rapid oxidation of lipid that leveled off at values much lower than those achieved in the enzymic system, which showed a continuous increase over the 1 or 2 h incubation times used. Protein sulfhydryl oxidation was more rapid in the nonenzymic system for total and accessible sulfhydryl groups, but the enzymic system oxidized the inaccessible sulfhydryl groups more rapidly. Both lipid and protein oxidations appeared to begin simultaneously. In the enzymic system, more lipid oxidation was achieved on a molar basis than oxidation of protein sulfhydryl groups, while in the nonenzymic system this was reversed. These data probably reflect the site specificity of the production of oxidizing elements in the two systems. The greater lipid oxidation in the enzymic system suggests that this may be the more important ferric iron-reducing system during storage of fish muscle.

Peroxynitrite Inactivates Tryptophan Hydroxylase via Sulfhydryl Oxidation: COINCIDENT NITRATION OF ENZYME TYROSYL RESIDUES HAS MINIMAL IMPACT ON CATALYTIC ACTIVITY

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me(2)SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25 degrees C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.

Inhibition of Glutathione Reductase by Isoproterenol Oxidation Products

Oxidative stress induced by catecholamines is a well recognized toxic event. This effect has been extensively observed in the heart, where high levels of catecholamines cause enzyme inhibition, lipid peroxidation, energy depletion and myocardial necrosis. Catecholamines can be converted into o-quinones and undergo cyclization into aminochromes. This process can occur enzymatically or through autoxidation and involves the formation of free radicals. Aminochromes are highly reactive molecules that can cause oxidation of protein sulfhydryl groups and deamination catalysis, among other deleterious effects; in addition, inhibition of some enzymes has been also reported. We have studied the effects of isoproterenol oxidation products (IOP) on glutathione reductase (GR) activity in vitro. Isoproterenol (ISO) autoxidation was conducted at 37 degrees C in the dark, for 4 h at pH 7.0 and this process was monitored by UV spectrophotometry at both 340 and 490nm. Addition of the autoxidized solution to GR in the presence of oxidized glutathione (GSSG) and NADPH showed that IOP inhibits GR in a competitive mode and that this effect increases during the 4 h incubation period. This inhibitory effect of IOP was partially prevented by the addition of reduced glutathione (GSH), L-cysteine and ascorbic acid to the reaction mixtures.

Priming of human neutrophils by peroxynitrite: potential role in enhancement of the local inflammatory response.

Peroxynitrite is a potent oxidant generated from the reaction of nitric oxide (NO) and superoxide anion (O2-), both of which can be produced in inflammatory tissues. In these studies, we analyzed what direct effect peroxynitrite had on neutrophil (PMN) function. We found that peroxynitrite was an effective priming agent for PMNs, as demonstrated by enhanced O2- production on subsequent activation with low doses of PMA or N-formyl-methionine-leucine-phenylalanine (fMLF), changes in the expression of PMN surface markers (L-selectin, Mac-1, flavocytochrome b, and fMLF receptor), and increased intracellular calcium levels. Analysis of the mechanism of PMN priming by peroxynitrite demonstrated that peroxynitrite resulted in minimal oxidation of protein sulfhydryl groups and subsequent protein cross-linking. In contrast, treatment of PMNs with peroxynitrite resulted in significant nitration of tyrosine residues on neutrophil proteins. In addition, inhibition of tyrosine nitration with a pyrrolopyrimidine antioxidant blocked the majority of peroxynitrite-induced priming effects, further suggesting that PMN priming was mediated primarily by nitration of tyrosine residues on PMN proteins. The ability of peroxynitrite to serve as an effective priming agent for PMNs at sites of inflammation may play a key role in modulating the host-defense process.

Inhibitory effects of ascorbic acid on dopamine uptake by rat striatal synaptosomes: relationship to lipid peroxidation and oxidation of protein sulfhydryl groups

Ascorbic acid is frequently added in the incubation medium to prevent oxidation of dopamine (DA) during uptake assays. However, a preliminary study showed that the presence of ascorbic acid induced a decrease of DA uptake after prolonged incubation. The purpose of this study was to determine the mechanism underlying ascorbic acid-induced alterations of DA uptake in rat striatal synaptosomes. In this context, the effects of physiological concentrations of ascorbic acid (100–500 μM) on DA uptake and Na +/K + ATPase activity (which is essential for DA transporter function) were assessed in synaptosomes before and after incubation at 37°C. The capacity of synaptosomes to take up DA was significantly decreased after incubation owing to a reduction in DA transporters (but with no modification of their affinity for DA). This partial inhibition was associated with a decrease of Na +/K + ATPase activity, a production of thiobarbituric acid reactive substances (TBARS) and malonaldehyde (MDA), and a loss of sulfhydryl group content. Addition of Trolox C to the medium prevented the reduction of DA uptake, the inhibition of Na +/K + ATPase activity, the decrease in sulfhydryl group content and the production of TBARS and MDA. These results suggest that ascorbic acid in the presence of contaminant ferrous ions induced a decrease in functional DA transporters, probably through a lipid peroxidation process involving oxidation of sulfhydryl groups and at least in part through a decrease of Na +/K + ATPase activity.

Increase in the Intramolecular Disulfide Bonding of Alpha-A Crystallin During Aging of the Human Lens

Aging of the normal human lens is accompanied by oxidation of protein sulfhydryl groups to disulfide groups. Although this has been known for many years, very little is known about the exact amino acid residues involved. In addition, almost nothing is known concerning the temporal sequence of this oxidative process over the lifetime of the individual. To address these two concerns for alpha-A crystallin, the polypeptide was purified from total proteins of the human lens, followed by digestion with lys-C endoprotease. Mass spectral analysis of the resulting fragments demonstrated that the two cysteine residues (cysteine-131 and cysteine-142) are present as a mixture of an intramolecular disulfide bond and free sulfhydryl groups. Reverse phase chromatography was used to resolve and quantitate the relative amounts of the two forms present in alpha-A crystallin from normal lenses of different age. Even in very young lenses (4 months and 5 months of age) there is significant oxidation of the two cysteine residues. However, the oxidative state of these two residues does not significantly change during the next ∼27 years of age, after which there is an increase in the relative amount of intramolecular disulfide bonding. Together, these results have identified and quantitated the relative change in the oxidative state of two specific cysteine residues of alpha-A crystallin in human lenses of different age, and have established that age-dependent oxidation of these two residues occurs primarily during the later part of life.

Production of Rat Protein Disulfide Isomerase in Saccharomyces cerevisiae

Protein disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes the oxidation of protein sulfhydryl groups and the isomerization and reduction of protein disulfide bonds. Saccharomyces cerevisiae cells lacking PDI are inviable. PDI is a component of many different protein processing complexes, and the actual activity of PDI that is required for cell viability is unclear. A cDNA that codes for rat PDI fused to the α-factor pre-pro segment was expressed in a protease-deficient strain of S. cerevisiae under the control of an ADH2-GAPDH hybrid promoter. The cells processed the resulting protein and secreted it into the medium as a monomer, despite having a KDEL or HDEL sequence at its C-terminus. The typical yield of isolated protein was 2 mg per liter of culture. The catalytic activity of the PDI from S. cerevisiae was indistinguishable hom that of PDI isolated from bovine liver, This expression system is unique in allowing the same plasmid to be used both to complement pdi1Δ S. cerevisiae and to produce PDI for detailed in vitro analyses. Correlations of the in vivo behavior and in vitro properties of PDI are likely to reveal structure-function relationships of biological importance.

Oxidant stress decreases Na+/H+ antiport activity in bovine pulmonary artery endothelial cells.

We determined the effects of oxidant stress by the use of tert-butyl hydroperoxide (t-BOOH) on Na+/H+ exchange in pulmonary artery endothelial cells. Monolayers were exposed to the hydroperoxide, followed by measurement of intracellular pH and the rate of recovery from acidosis by utilizing the pH-sensitive probe 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein. t-BOOH (0.4 mM) decreased the rate of acid recovery after a 2-h exposure without evidence of overt cytotoxicity (51Cr-release assay). Glutathione repletion (N-acetyl-L-cysteine) abolished the effect of the hydroperoxide. Lowering intracellular glutathione with buthionine sulfoximine decreased the acid recovery rate at a dose of t-BOOH (0.04 mM) that was not normally associated with a change in this parameter. Preincubation with vitamin E had no protective effect. Dithiothreitol abolished the effect of the hydroperoxide, suggesting oxidation of protein sulfhydryl groups as a mechanism for the altered kinetics of acid recovery. There was no difference in cell buffering capacity between control and treated monolayers. The findings suggest that the decrease in Na+/H+ antiport activity in this model of oxidant stress represents an early perturbation of membrane function and illustrate the role of the glutathione redox system in maintaining the functional integrity of the Na+/H+ antiport in these cells.

Oxidative Damages and Repair in <italic>Euglena gracilis</italic> Exposed to Ozone II. Membrane Permeability and Uptake of Metabolites

We have shown that exposure to O 3 resulted in the oxidation of protein sulfhydryl groups and fatty acid lipids and reduced growth. In our effort to elucidate the nature of these damages and their effect on the plasma membrane function, we examined the effect of O 3 exposure on two specific transport systems associated with the functional integrity of the plasma membrane, the transport of vitamine B 12 and acetate

Myocardial ischemia and reperfusion: The role of oxygen radicals in tissue injury

Thrombolytic therapy has gained widespread acceptance as a means of treating coronary artery thrombosis in patients with acute myocardial infarction. Although experimental data have demonstrated that timely reperfusion limits the extent of infarction caused by regional ischemia, there is growing evidence that reperfusion is associated with an inflammatory response to ischemia that exacerbates the tissue injury. Ischemic myocardium releases archidonate and complement-derived chemotactic factors, e.g., leukotriene B4 and C5a, which attract and activate neutrophils. Reperfusion of ischemic myocardium accelerates the influx of neutrophils, which release reactive oxygen products, such as superoxide anion and hydrogen peroxide, resulting in the formation of a hydroxyl radical and hypochlorous acid. The latter two species may damage viable endothelial cells and myocytes via the peroxidation of lipids and oxidation of protein sulfhydryl groups, leading to perturbations of membrane permeability and enzyme function. Neutrophil depletion by antiserum and inhibition of neutrophil function by drugs, e.g., ibuprofen, prostaglandins (prostacyclin and PGE1), or a monoclonal antibody, to the adherence-promoting glycoprotein Mo-1 receptor, have been shown to limit the extent of canine myocardial injury due to coronary artery occlusion/reperfusion. Recent studies have challenged the hypothesis that xanthine-oxidase-derived oxygen radicals are a cause of reperfusion injury. Treatment with allopurinol or oxypurinol may exert beneficial effects on ischemic myocardium that are unrelated to the inhibition of xanthine oxidase. Furthermore, the human heart may lack xanthine oxidase activity. Further basic research is needed, therefore, to clarify the importance of xanthine oxidase in the pathophysiology of reperfusion injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Cataracts induced by microwave and ionizing radiation

Microwaves most commonly cause anterior and/or posterior subcapsular lenticular opacities in experimental animals and, as shown in epidemiologic studies and case reports, in human subjects. The formation of cataracts seems to be related directly to the power of the microwave and the duration of exposure. The mechanism of cataractogenesis includes deformation of heat-labile enzymes, such as glutathione peroxide, that ordinarily protect lens cell proteins and membrane lipids from oxidative damage. Oxidation of protein sulfhydryl groups and the formation of high-molecular-weight aggregates cause local variations in the orderly structure of the lens cells. An alternative mechanisms is thermoelastic expansion through which pressure waves in the aqueous humor cause direct physical damage to the lens cells. Cataracts induced by ionizing radiation (e.g., X-rays and gamma rays) usually are observed in the posterior region of the lens, often in the form of a posterior subcapsular cataract. Increasing the dose of ionizing radiation causes increasing opacification of the lens, which appears after a decreasing latency period. Like cataract formation by microwaves, cataractogenesis induced by ionizing radiation is associated with damage to the lens cell membrane. Another possible mechanism is damage to lens cell DNA, with decreases in the production of protective enzymes and in sulfur-sulfur bond formation, and with altered protein concentrations. Until further definitive conclusions about the mechanisms of microwaves and ionizing radiation induced cataracts are reached, and alternative protective measures are found, one can only recommend mechanical shielding from these radiations to minimize the possibility of development of radiation-induced cataracts.

Considerations in the isolation of rat liver nuclear matrix, nuclear envelope, and pore complex lamina

A number of recent studies have demonstrated a salt-, nuclease, and detergent-resistant subnuclear structure termed the nuclear protein matrix which consists of a fibrogranular intranuclear network, residual components of the nucleolus, and a peripheral lamina. Other workers, however, have shown that somewhat similar methods result in the isolation of the peripheral lamina devoid of the intranuclear components. In this report we demonstrate that seemingly slight changes in the isolation procedure cause major changes in the morphology of the residual structures obtained. When freshly purified rat liver nuclei were digested with DNase I and RNase A and then extracted with buffers of low magnesium ion concentration (LS buffer) and high ionic strength (HS buffer), the resulting structures isolated prior to or after Triton X-100 extraction lacked the extensive intranuclear network and the easily identifiable residual nucleoli present in the nuclear protein matrix. Systematic modification of this extraction procedure revealed that morphologically identifiable residual nucleoli were present when digestion with RNase A followed extraction with HS buffer but were absent when the order of these steps was reversed. The removal of the nucleolus by RNase A and HS buffer correlated with the removal of nuclear RNA by the same treatments. These coordinate events could not be prevented by treatment with protease inhibitors but were prevented by treatment of the RNase A with diethylpyrocarbonate, an RNase inhibitor. The extensive intranuclear network seen in the nuclear protein matrix was sparse or absent when residual structures were prepared from DNase- and RNase-treated nuclei under conditions which minimized the oxidation of protein sulfhydryl groups. In contrast, an extensive non-chromatin intranuclear network was seen if the formation of intermolecular protein disulfide bonds was promoted by extraction of nuclei with cationic detergents, by overnight incubation, or by treatment with oxidizing agents like sodium tetrathionate prior to nuclease digestion and subsequent extraction. By varying the order of extraction steps and the extent of disulfide cross-linking, it is possible to isolate from a single batch of nuclei residual structures with a wide range of morphologies and compositions.

Comparison of the effects of bacteriophage T4 infection and N-ethylmaleimide on the translational specificity of Escherichia coli ribosomes

Treatment of ribosomes with N-ethylmaleimide (NEM) specifically inhibited the translation of f2 RNA by reacting with components of the high-salt wash fraction as well as the high-salt washed ribosomes themselves. The NEM-treated ribosomes were unable to efficiently bind fMet-tRNA at low concentrations of f2 RNA, but at higher concentrations the binding of initiator tRNA approached that of control ribosomes. The fMet-tRNA bound under the latter conditions could be completely released by puromycin. In spite of this, the NEM ribosomes could not efficiently translate even high concentrations of f2 RNA. Because the effects of NEM treatment mimic almost exactly those of bacteriophage T4 infection, in vitro translational specificity associated with phage infection may result from the oxidation of protein sulfhydryl groups either during infection or during subsequent isolation of the ribosomes.

Fatty Acid Synthetase from Lactating Rat Mammary Gland: III. DISSOCIATION AND REASSOCIATION

Abstract Fatty acid synthetase from lactating rat mammary gland is shown to be a cold-labile multienzyme complex. The native (13 S) form slowly dissociates into half-molecular weight (9 S) subunits on aging in the cold. Fatty acid synthetase activity observed on addition of the 9 S subunits to the assay system between 20 and 30°, is shown to result from rapid reassociation of the subunits to the 13 S form. Dissociation of the enzyme into subunits is accompanied by a change in the number of protein sulfhydryl groups accessible to 5,5'-dithiobis(2-nitrobenzoate), but reassociation to the parent form is not dependent on the presence of a reduced thiol. The dissociation does not involve oxidation of protein sulfhydryl groups, as the total number of sulfhydryl groups titratable with 5,5'-dithiobis(2-nitrobenzoate) in the presence of 6 m urea was the same in the native enzyme and its subunits. Blocking more than two of the 28 subunit sulfhydryl groups with p-chloromercuribenzoate restricted the ability of the subunits to undergo heat-induced reassociation. Evidence is presented which suggests that although protein sulfhydryl groups may be involved in the dissociation-reassociation phenomenon, hydrophobic bonding is probably a more critical factor. Similar studies carried out with the fatty acid synthetase purified from rat liver indicate that this enzyme too exhibits cold lability.

Sulfhydryl Groups in Radiation Damage

Several lines of evidence indicate that radiation-induced oxidation of protein sulfhydryl groups play a role in the development of radiation damage. In all -SH enzymes so far studied the radiation inactivation in solution can largely be accounted for by destruction of sulfhydryl groups. Since sulfhydryl groups have different functions in different enzymes, the inactivation may be brought about by different mechanisms. The over-all yield of inactivation of sulfhydryl enzymes shows a wide range of variation, depending on the number of sulfhydryl groups in the enzyme, their function and their radiation sensitivity. The radiation sensitivity of protein -SH groups is parallelled by their reactivity towards chemical agents. The most reactive sulfhydryl groups which are capable of interacting with disulfides, are by and large oxidized by ionizing radiation with high G-values (0.7–1.8), while the less reactive sulfhydryl groups are generally oxidized at low yields. In certain cases the products of radiation oxida...