Propane and n-Butane Oxidation by Pseudomonas putida GPo1

Propane and n-butane inhibit methyl tertiary butyl ether oxidation by n-alkane-grown Pseudomonas putida GPo1. Here we demonstrate that these gases are oxidized by this strain and support cell growth. Both gases induced alkane hydroxylase activity and appear to be oxidized by the same enzyme system used for the oxidation of n-octane.     

The oxidation of reduced nicotinamide nucleotides by hydrogen peroxide in the presence of lactoperoxidase and thiocyanate, iodide or bromide

Lactoperoxidase (EC 1.11.1.7) catalysed the oxidation of NADH by hydrogen peroxide in the presence of either thiocyanate, iodide or bromide. In the presence of thiocyanate, net oxidation of thiocyanate occurred simultaneously with the oxidation of NADH, but in the presence of iodide or bromide, only the oxidation of NADH occurred to a significant extent. In the presence of thiocyanate or bromide, NADH was oxidized to NAD(+) but in the presence of iodide, an oxidation product with spectral and chemical properties distinct from NAD(+) was formed. Thiocyanate, iodide and bromide appeared to function in the oxidation of NADH by themselves being oxidized to products which in turn oxidized NADH, rather than by activating the enzyme. Iodine, which oxidized NADH non-enzymically, appeared to be an intermediate in the oxidation of NADH in the presence of iodide. NADPH was oxidized similarly under the same conditions. An assessment was made of the rates of these oxidation reactions, together with the rates of other lactoperoxidase-catalysed reactions, at physiological concentrations of thiocyanate, iodide and bromide. The results indicated that in milk and saliva the oxidation of thiocyanate to a bacterial inhibitor was likely to predominate over the oxidation of NADH.     

Oxidation of phenol by Bacillus stearothermophilus strains]

The assimilation of phenol as a sole source of carbon and energy was studied with the thermophilic bacillus isolated from the geothermal zones in the South Ural. This ability was displayed by 11 strains of Bacillus stearothermophilus among 26 studied strains. The most active strains oxidized all phenol, when its content in the medium was 0.1--0.2%, during two days, at 56--58 degrees C, with aeration; a considerable amount of biomass was accumulated and the medium was acidified. The maximum concentration of phenol, which did not suppress the bacterial growth, was 0.3%. The majority of the strains of Bac. stearothermophilus capable of phenol oxidation were isolated from the regions heated with parothermal gases which contained phenols.     

Oxidized low density lipoprotein inhibits the hemolytic activity of Asp-hemolysin from Aspergillus fumigatus

We have examined the effect of chemically modified human low density lipoproteins (LDLs) , acetylated LDL and oxidized LDL, on the hemolytic activity of Asp-hemolysin. Oxidized LDL, but not acetylated LDL, inhibited the hemolytic activity of this toxin. The inhibitory effects of oxidized LDL increased with the time of Cu²⁺-induced LDL oxidation. Similar inhibition was observed in the filtrate which was separated from the incubation mixture of Asp-hemolysin with oxidized LDL (for 2 h of oxidation) following ultrafiltration through a membrane with a molecular mass cutoff of 100 000. However, at longer LDL oxidation times, the inhibition by the filtrates was less than the control mixture without ultrafiltration. We suggest that the inhibition by oxidized LDL was due to the binding of oxidized LDL to Asp-hemolysin at shorter LDL oxidation times .     

Reaction of peroxyacetyl nitrate with cysteine,cystine, methionine,lipoic acid,papain, and lysozyme

Peroxyacetyl nitrate was reactive with small molecular-weight sulfur-containing compounds The order of susceptibility was cysteine > reduced lipoic acid = reduced lipoamide > oxidized lipoic acid > oxidized lipoamide > methionine ? cystine. From thiols the predominant product was disulfide. In the early stages of oxidation methionine yielded methionine sulfoxide. Products of oxidation of oxidized lipoic acid and lipoamide were the respective sulfoxides. Cystine was resistant to oxidation, yielding cysteic acid when oxidation took place.Papain was readily inactivated by peroxyacetyl nitrate while lysozyme was resistant. The small amount of inactivation of lysozyme was correlated with methionine oxidation. Papain inactivation was correlated with thiol oxidation and could be reversed by thiol compounds. The oxidation product was judged to be a dimer by methods for determining molecular weight.     

Inhibition by serum components of oxidation and collagen-binding of low-density lipoprotein.

Low-density lipoprotein (LDL) is oxidized by cellular and noncellular mechanisms, both leading to an increased binding to collagen. We have investigated the effect of serum on lipid peroxidation, apoprotein oxidation and the binding of oxidized apoprotein to collagen. During noncellular oxidation, lipoprotein-deficient serum strongly inhibited all three processes. The serum fraction of M(r) > 100,000 was equally inhibitory; this effect was not due to alpha 1 or gamma globulins, alpha 2 macroglobulins, haptoglobins or ceruloplasmin. The serum fraction of M(r) 30,000-100,000 stimulated the binding of oxidized apoprotein but the albumin in this fraction inhibited lipid peroxidation and apoprotein oxidation. Serum ultrafiltrate (M(r) < 1000) inhibited lipid and protein oxidation, and binding; the inhibitory effect was abolished by deionization which removed histidine. The effects of lipoprotein-deficient serum and its fractions on cellular oxidation were similar but weaker than those on noncellular oxidation, HDL inhibited noncellular oxidation as well as binding of oxidized apoprotein. VLDL also inhibited oxidation; this could not be accounted for by its content of apo B. If present in vivo, these inhibitory effects would completely suppress both cellular and noncellular oxidation of LDL and its subsequent binding to collagen.     

The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL

For more than two decades, there has been continuing evidence of lipid oxidation playing a central role in atherogenesis. The oxidation hypothesis of atherogenesis has evolved to focus on specific proinflammatory oxidized phospholipids that result from the oxidation of LDL phospholipids containing arachidonic acid and that are recognized by the innate immune system in animals and humans. These oxidized phospholipids are largely generated by potent oxidants produced by the lipoxygenase and myeloperoxidase pathways. The failure of antioxidant vitamins to influence clinical outcomes may have many explanations, including the inability of vitamin E to prevent the formation of these oxidized phospholipids and other lipid oxidation products of the myeloperoxidase pathway. Preliminary data suggest that the oxidation hypothesis of atherogenesis and the reverse cholesterol transport hypothesis of atherogenesis may have a common biological basis. The levels of specific oxidized lipids in plasma and lipoproteins, the levels of antibodies to these lipids, and the inflammatory/anti-inflammatory properties of HDL may be useful markers of susceptibility to atherogenesis. Apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides may both promote a reduction in oxidized lipids and enhance reverse cholesterol transport and therefore may have therapeutic potential.     

Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO).

Production and consumption processes in soils contribute to the global cycles of many trace gases (CH4, CO, OCS, H2, N2O, and NO) that are relevant for atmospheric chemistry and climate. Soil microbial processes contribute substantially to the budgets of atmospheric trace gases. The flux of trace gases between soil and atmosphere is usually the result of simultaneously operating production and consumption processes in soil: The relevant processes are not yet proven with absolute certainty, but the following are likely for trace gas consumption: H2 oxidation by abiontic soil enzymes; CO cooxidation by the ammonium monooxygenase of nitrifying bacteria; CH4 oxidation by unknown methanotrophic bacteria that utilize CH4 for growth; OCS hydrolysis by bacteria containing carbonic anhydrase; N2O reduction to N2 by denitrifying bacteria; NO consumption by either reduction to N2O in denitrifiers or oxidation to nitrate in heterotrophic bacteria. Wetland soils, in contrast to upland soils are generally anoxic and thus support the production of trace gases (H2, CO, CH4, N2O, and NO) by anaerobic bacteria such as fermenters, methanogens, acetogens, sulfate reducers, and denitrifiers. Methane is the dominant gaseous product of anaerobic degradation of organic matter and is released into the atmosphere, whereas the other trace gases are only intermediates, which are mostly cycled within the anoxic habitat. A significant percentage of the produced methane is oxidized by methanotrophic bacteria at anoxic-oxic interfaces such as the soil surface and the root surface of aquatic plants that serve as conduits for O2 transport into and CH4 transport out of the wetland soils. The dominant production processes in upland soils are different from those in wetland soils and include H2 production by biological N2 fixation, CO production by chemical decomposition of soil organic matter, and NO and N2O production by nitrification and denitrification. The processes responsible for CH4 production in upland soils are completely unclear, as are the OCS production processes in general. A problem for future research is the attribution of trace gas metabolic processes not only to functional groups of microorganisms but also to particular taxa. Thus, it is completely unclear how important microbial diversity is for the control of trace gas flux at the ecosystem level. However, different microbial communities may be part of the reason for differences in trace gas metabolism, e.g., effects of nitrogen fertilizers on CH4 uptake by soil; decrease of CH4 production with decreasing temperature; or different rates and modes of NO and N2O production in different soils and under different conditions.     

Cometabolism of methyl tertiary butyl ether and gaseous n-alkanes by Pseudomonas mendocina KR-1 grown on C5 to C8 n-alkanes

Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C5 to C8), and 1 degrees alcohols (C2 to C8) but not on other aromatics, gaseous n-alkanes (C1 to C4), isoalkanes (C4 to C6), 2 degrees alcohols (C3 to C8), methyl tertiary butyl ether (MTBE), or tertiary butyl alcohol (TBA). Cells grown under carbon-limited conditions on n-alkanes in the presence of MTBE (42 micromoles) oxidized up to 94% of the added MTBE to TBA. Less than 3% of the added MTBE was oxidized to TBA when cells were grown on either 1 degrees alcohols, toluene, or dextrose in the presence of MTBE. Concentrated n-pentane-grown cells oxidized MTBE to TBA without a lag phase and without generating tertiary butyl formate (TBF) as an intermediate. Neither TBF nor TBA was consumed by n-pentane-grown cells, while formaldehyde, the expected C1 product of MTBE dealkylation, was rapidly consumed. Similar Ks values for MTBE were observed for cells grown on C5 to C8 n-alkanes (12.95 +/- 2.04 mM), suggesting that the same enzyme oxidizes MTBE in cells grown on each n-alkane. All growth-supporting n-alkanes (C5 to C8) inhibited MTBE oxidation by resting n-pentane-grown cells. Propane (Ki = 53 micromoles) and n-butane (Ki = 16 micromoles) also inhibited MTBE oxidation, and both gases were also consumed by cells during growth on n-pentane. Cultures grown on C5 to C8 n-alkanes also exhibited up to twofold-higher levels of growth in the presence of propane or n-butane, whereas no growth stimulation was observed with methane, ethane, MTBE, TBA, or formaldehyde. The results are discussed in terms of their impacts on our understanding of MTBE biodegradation and cometabolism.     

Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants

Human serum paraoxonase (PON1) can protect low density lipoprotein (LDL) from oxidation induced by either copper ion or by the free radical generator azo bis amidinopropane hydrochloride (AAPH). During LDL oxidation in both of these systems, a time-dependent inactivation of PON arylesterase activity was observed. Oxidized LDL (Ox-LDL) produced by lipoprotein incubation with either copper ion or with AAPH, indeed inactivated PON arylesterase activity by up to 47% or 58%, respectively. Three possible mechanisms for PON inactivation during LDL oxidation were considered and investigated: copper ion binding to PON, free radical attack on PON, and/or the effect of lipoprotein-associated peroxides on the enzyme. As both residual copper ion and AAPH are present in the Ox-LDL preparations and could independently inactivate the enzyme, the effect of minimally oxidized (Ox-LDL produced by LDL storage in the air) on PON activity was also examined. Oxidized LDL, as well as oxidized palmitoyl arachidonoyl phosphatidylcholine (PAPC), lysophosphatidylcholine (LPC, which is produced during LDL oxidation by phospholipase A2-like activity), and oxidized cholesteryl arachidonate (Ox-CA), were all potent inactivators of PON arylesterase activity (PON activity was inhibited by 35%-61%). PON treatment with Ox-LDL (but not with native LDL), or with oxidized lipids, inhibited its arylesterase activity and also reduced the ability of the enzyme to protect LDL against oxidation. PON Arylesterase activity however was not inhibited when PON was pretreated with the sulfhydryl blocking agent, p-hydroxymercurybenzoate (PHMB). Similarly, on using recombinant PON in which the enzyme's only free sulfhydryl group at the position of cysteine-284 was mutated, no inactivation of the enzyme arylesterase activity by Ox-LDL could be shown. These results suggest that Ox-LDL inactivation of PON involves the interaction of oxidized lipids in Ox-LDL with the PON's free sulfhydryl group. Antioxidants such as the flavonoids glabridin or quercetin, when present during LDL oxidation in the presence of PON, reduced the amount of lipoprotein-associated lipid peroxides and preserved PON activities, including its ability to hydrolyze Ox-LDL cholesteryl linoleate hydroperoxides. We conclude that PON's ability to protect LDL against oxidation is accompanied by inactivation of the enzyme. PON inactivation results from an interaction between the enzyme free sulfhydryl group and oxidized lipids such as oxidized phospholipids, oxidized cholesteryl ester or lysophosphatidylcholine, which are formed during LDL oxidation. The action of antioxidants and PON on LDL during its oxidation can be of special benefit against atherosclerosis since these agents reduce the accumulation of Ox-LDL by a dual effect: i.e. prevention of its formation, and removal of Ox-LDL associated oxidized lipids which are generated during LDL oxidation.     

Oxidation of low-density lipoprotein by iron at lysosomal pH: implications for atherosclerosis

Low-density lipoprotein (LDL) has recently been shown to be oxidized by iron within the lysosomes of macrophages, and this is a novel potential mechanism for LDL oxidation in atherosclerosis. Our aim was to characterize the chemical and physical changes induced in LDL by iron at lysosomal pH and to investigate the effects of iron chelators and α-tocopherol on this process. LDL was oxidized by iron at pH 4.5 and 37 °C and its oxidation monitored by spectrophotometry and high-performance liquid chromatography. LDL was oxidized effectively by FeSO(4) (5-50 μM) and became highly aggregated at pH 4.5, but not at pH 7.4. The level of cholesteryl esters decreased, and after a pronounced lag, the level of 7-ketocholesterol increased greatly. The total level of hydroperoxides (measured by the triiodide assay) increased up to 24 h and then decreased only slowly. The lipid composition after 12 h at pH 4.5 and 37 °C was similar to that of LDL oxidized by copper at pH 7.4 and 4 °C, i.e., rich in hydroperoxides but low in oxysterols. Previously oxidized LDL aggregated rapidly and spontaneously at pH 4.5, but not at pH 7.4. Ferrous iron was much more effective than ferric iron at oxidizing LDL when added after the oxidation was already underway. The iron chelators diethylenetriaminepentaacetic acid and, to a lesser extent, desferrioxamine inhibited LDL oxidation when added during its initial stages but were unable to prevent aggregation of LDL after it had been partially oxidized. Surprisingly, desferrioxamine increased the rate of LDL modification when added late in the oxidation process. α-Tocopherol enrichment of LDL initially increased the rate of oxidation of LDL but decreased it later. The presence of oxidized and highly aggregated lipid within lysosomes has the potential to perturb the function of these organelles and to promote atherosclerosis.     

The bacterial metabolism of 2,4-xylenol

1. Measurements of the rates of oxidation of various compounds by a fluorescent Pseudomonas indicated that metabolism of 2,4-xylenol was initiated by oxidation of the methyl group para to the hydroxyl group. 2. 4-Hydroxy-3-methylbenzoic acid was isolated as the product of oxidation of 2,4-xylenol by cells inhibited with alphaalpha'-bipyridyl. 3. 4-Hydroxyisophthalic acid accumulated at low oxygen concentrations when either 2,4-xylenol or 4-hydroxy-3-methylbenzoic acid was oxidized by cells grown with 2,4-xylenol. 4. When supplemented with NADH, but not with NADPH, cell extracts oxidized 4-hydroxy-3-methylbenzoic acid readily. 2-Hydroxy-5-methylbenzoic acid was not oxidized. 5. Both 4-hydroxyisophthalic acid and p-hydroxybenzoic acid were oxidized to beta-oxoadipic acid by cell extracts supplemented with either NADH or NADPH. 4,5-Dihydroxyisophthalic acid was not oxidized. 6. From measurements of oxygen consumed and carbon dioxide evolved it was concluded that protocatechuic acid is an intermediate in the conversion of 4-hydroxyisophthalic acid into beta-oxoadipic acid.     

Oxidation rates of some desialylated glycoproteins by galactose oxidase

Patterns of oxidation of dilute solutions of desialylated fetuin and submaxillary mucin by galactose oxidase have been examined. A significant portion (20-40%) of the terminal galactosyls exposed on the glycoproteins, which theoretically were expected to be accessible to the enzyme, was not oxidized. In comparison, galactosyls in oligosaccharides released from completely desialylated glycoproteins were oxidized more effectively with an apparently lower degree of crypticity to the enzyme. Partial desialylation usually resulted in a reduction of both the rate and the final level of substrate oxidation. A second cycle of oxidation of a desialylated substrate earlier oxidized by galactose oxidase and then reduced by NaB3H4 revealed a selectivity in the pattern of galactosyl oxidation. The same galactosyl residues oxidized in the first cycle were again the most susceptible to oxidation in the second cycle, leaving unmodified the same fraction of galactosyls throughout both cycles. The relevance of these results to the application of the galactose oxidase-NaBH4 procedure for detecting and measuring desialylated glycoconjugates in solution and in biological membranes is discussed.     

Protein oxidation and aging

Organisms are constantly exposed to various forms of reactive oxygen species (ROS) that lead to oxidation of proteins, nucleic acids, and lipids. Protein oxidation can involve cleavage of the polypeptide chain, modification of amino acid side chains, and conversion of the protein to derivatives that are highly sensitive to proteolytic degradation. Unlike other types of modification (except cysteine oxidation), oxidation of methionine residues to methionine sulfoxide is reversible; thus, cyclic oxidation and reduction of methionine residues leads to consumption of ROS and thereby increases the resistance of proteins to oxidation. The importance of protein oxidation in aging is supported by the observation that levels of oxidized proteins increase with animal age. The age-related accumulation of oxidized proteins may reflect age-related increases in rates of ROS generation, decreases in antioxidant activities, or losses in the capacity to degrade oxidized proteins.     

Protein oxidation and aging

Organisms are constantly exposed to various forms of reactive oxygen species (ROS) that lead to oxidation of proteins, nucleic acids, and lipids. Protein oxidation can involve cleavage of the polypeptide chain, modification of amino acid side chains, and conversion of the protein to derivatives that are highly sensitive to proteolytic degradation. Unlike other types of modification (except cysteine oxidation), oxidation of methionine residues to methionine sulfoxide is reversible; thus, cyclic oxidation and reduction of methionine residues leads to consumption of ROS and thereby increases the resistance of proteins to oxidation. The importance of protein oxidation in aging is supported by the observation that levels of oxidized proteins increase with animal age. The age-related accumulation of oxidized proteins may reflect age-related increases in rates of ROS generation, decreases in antioxidant activities, or losses in the capacity to degrade oxidized proteins.     

Studies of Auxin Protectors VIII. Evidence that Auxin Protectors Act as Cellular Poisers

Auxin protectors completely inhibit the peroxidase-catalyzed oxidation of indoleacetic acid (IAA). Presumably only when the protector substance itself has been oxidized, does IAA oxidation begin. Reduced nicotinamide-adenine dinucleotide (NADH) mimics the native auxin protectors: In the presence of NADH, the peroxidase-catalyzed oxidation of IAA does not begin until almost all the NADH has been oxidized. Auxin protectors slow the oxidation of NADH in the presence of the peroxidase complex (enzyme plus manganese). However, in the absence of the peroxidase complex, protectors actually accelerate the spontaneous oxidation of NADH. Protectors can also accelerate the oxidation of the dye 2,6-dichlorophenol-indophenol, especially in the presence of manganese. Protector oxidized by boiling with traces of hydrogen peroxide will act as an electron acceptor in the peroxidase-catalyzed oxidation of NADH. The reversible redox role of auxin protectors implies that they can act as cellular poisers.     

Transamination and oxidation of leucine and valine in rat adipose tissue

Leucine was oxidized by rat adipose tissue at a rate which was not limited by the activity of branched chain amino acid transaminase since high concentrations (10 mM) of [1-14C]leucine and its transamination product, alpha-keto[1-14C]isocaproate, were oxidized at similar rates. Despite the apparent abundance of transaminase activity, however, [1-14C]valine was oxidized at only 10 to 25% of the rate of its transamination product, alpha-keto[1-14C]isovalerate. The net rate at which [1-14C] valine was transaminated by intact tissues was estimated as the sum of the rates of 14CO2 production and alpha-ketoiso[1-14C]valerate release into the medium. Transamination did not limit the rate of valine oxidation since valine was transaminated 3 times as fast as it was oxidized. The rate of valine transamination increased 18-fold when its concentration was raised 100-fold, but the fraction of [1-14C]valine oxidized to 14CO2 remained constant over the range of incubation conditions studied. The oxidation/transamination ratio for leucine was also constant and exceeded the oxidation/transamination ratio for valine unless valine oxidation was stimulated, either by the addition of glucose or leucine. Stimulation of valine oxidation did not increase its transamination but reduced the rate at which alpha-ketoisovalerate was released from the tissue. The faster oxidation of alpha-ketoisocaproate than of alpha-ketoisovalerate may be due to the activation of branched chain alpha-keto acid dehydrogenase by alpha-ketoisocaproate, but the alpha-keto acid oxidation rates do not fully account for the faster transamination of leucine than of valine.     

Abiotic synthesis of organic molecules from minerals containing traces of dissolved H2O,CO2 and N2 part II: Experimental data

Even though a given mineral, for instance olivine, may contain only traces of dissolved H₂O, CO₂ and N₂ the gases which evolve from its surface during heating comprise (a) highly reduced molecules such as H₂, CH₄, C_mH_n and more complex hydrocarbons, HCN and other N-bearing compounds (b) oxidized species in various degrees of oxidation from formaldehyde and CO to oxygen. These gases evolve sequentially besides H₂O, CO₂ and possibly N₂, their relative amounts being controlled by experimental parameters such as the rate of heating. Preliminary indications of amino acids have been obtained by liquid extraction. The chemical complexity is a consequence of radical reactions between different solute species in the surface and the bulk of the mineral grains. Data for synthetic MgO and for mantle-derived olivine are presented.     

Degradation of oxidized extracellular proteins by microglia

In living organisms a permanent oxidation of protein oxidation occurs. The degradation of intracellular oxidized proteins is intensively studied, but knowledge about the fate of oxidatively modified extracellular proteins is still limited. We studied the fate of exogenously added oxidized proteins in microglial cells. Both primary microglial cells and RAW cells are able to remove added oxidized laminin and myelin basic protein from the extracellular environment. Moderately oxidized proteins are degraded most efficiently, whereas strongly oxidized proteins are taken up by the microglial cells without an efficient degradation. Activation of microglial cells enhances the selective recognition and degradation of moderately oxidized protein substrates by proteases. Inhibitor studies also revealed an involvement of the lysosomal and the proteasomal system in the degradation of extracellular proteins. These studies let us conclude that microglial cells are able to remove oxidized proteins from the extracellular environment in the brain.     

Binding of anti-band 3 autoantibody to oxidatively damaged erythrocytes. Formation of senescent antigen on erythrocyte surface by an oxidative mechanism

Incubation of human erythrocytes oxidized by iron catalysts, ADP/Fe3+ or xanthine/xanthine oxidase/Fe3+, with autologous IgG resulted in IgG binding as detected by enzyme immunoassay using protein A-beta-galactosidase conjugate. The binding of autologous IgG to ADP/Fe3(+)-treated erythrocytes maximized when the cells were treated with 1.8:0.1 mM ADP/Fe3+, and declined when treated above this concentration, suggesting that autologous IgG binds to moderately but not to excessively oxidized erythrocytes. The antibody involved in the binding was anti-Band 3, the autoantibody known to bind to aged erythrocytes, because isolated anti-Band 3 bound to the oxidized cells, but anti-Band 3-depleted autologous IgG did not. In addition, purified Band 3 inhibited the autologous IgG binding. Anti-alpha-galactosyl IgG, another natural antibody which has been reported to bind to aged erythrocytes, did not bind to the oxidized cells. Oxidation of membrane lipids, SH-groups of membrane proteins, and Hb of these cells was slight, but the cells contained an increased amount of membrane-bound native Hb, indicating that the oxidized cell membrane has an altered property. alpha-Tocopherol prevented the lipid oxidation and the subsequent IgG binding. Reduction of the oxidized erythrocytes with dithiothreitol resulted in a loss of the IgG binding. These results suggest that anti-Band 3 binding sites (Band 3 senescent antigen) are formed on moderately oxidized erythrocytes as a result of oxidation of membrane protein SH-groups which can be mediated by the membrane lipid oxidation and that formation of the anti-Band 3 binding sites on the oxidized cells is an essentially reversible membrane event which is linked to oxidation and restoration of the protein SH-groups.