The buoyant and potentiometric titrations of human immuno-gamma globulin.

The buoyant density and potentiometric hydrogen ion titration curves of human immuno-gamma globulin in 3M cesium chloride have been recorded. In addition, the amino acid analysis of the IgG employed has been completed. The hydration of the protein and the variation of the hydration with pH have been calculated from the buoyant density data. The potentiomtric hydrogen ion titration curve has been employed to estimate the intrinsic pK′s of the acidic and histidyl residues of the molecule, and to confirm the hypothesis that it does in fact conform to the oil drop model of protein conformation. Correlations have been drawn between the three sets of data in the following manner. The results of the potentiometric hydrogen ion titration have been checked against the amino acid analysis to determine whether the numbers of groups observed to titrate and the numbers of groups observed in the amino acid analysis do correspond. Second, previous hypotheses as to the direct correlation between potentiometric hydrogen ion titration behaviour and buoyant density titration behaviour have been investigated and substantially confirmed.     

Protective mechanisms against peptide and protein peroxides generated by singlet oxygen

Reaction of certain amino acids, peptides, and proteins with singlet oxygen yields substrate-derived peroxides. Recent studies have shown that these species are formed within intact cells and can inactivate key cellular enzymes. This study examines potential mechanisms by which cells might remove or detoxify such peroxides. It is shown that catalase, horseradish peroxidase, and Cu/Zn superoxide dismutase do not react rapidly with these peroxides. Oxymyoglobin and oxyhemoglobin, but not the met (Fe3+) forms of these proteins, react with peptide but not protein, peroxides with oxidation of the heme iron. Glutathione peroxidase, in the presence of reduced glutathione (GSH) rapidly removes peptide, but not protein, peroxides, consistent with substrate size being a key factor. Protein thiols, GSH, other low-molecular-weight thiols, and the seleno-compound ebselen react, in a nonstoichiometric manner, with both peptide and protein peroxides. Cell lysate studies show that thiol consumption and peroxide removal occur in parallel; the stoichiometry of these reactions suggests that thiol groups are the major direct, or indirect, reductants for these species. Ascorbic acid and some derivatives can remove both the parent peroxides and radicals derived from them, whereas methionine and the synthetic phenolic antioxidants Probucol and BHT show little activity. These studies show that cells do not have efficient enzymatic defenses against protein peroxides, with only thiols and ascorbic acid able to remove these materials; the slow removal of these species is consistent with protein peroxides playing a role in cellular dysfunction resulting from oxidative stress.     

A nonenzymatic method for determination of hydrogen peroxide and organic peroxides

Reduction of hydrogen peroxide and organic peroxides (t-butyl hydroperoxide and linoleic acid hydroperoxide) was achieved with homovanillic acid as hydrogen donor in the presence of the triethylenetetramine-Fe3+ complex. By the catalytic action of this complex, homovanillic acid is oxidized to its fluorescent dimer. Based on this reaction a fluorometric method for the measurement of the hydroperoxides mentioned above is described. The method can be extended to the determination of substrate-enzyme systems that produce hydrogen peroxide, e.g., glucose-glucose oxidase. The method allows the determination of substances such as hydrogen peroxide and t-butyl hydroperoxide with an accuracy and precision of less than 3%. Glucose can be determined with similar precision and an accuracy of 4.7%.     

The effectiveness of a lipid peroxide in oxidizing protein and non-protein thiols

1. Thiol oxidation by a lipid peroxide or hydrogen peroxide was as efficient in denatured non-haem proteins as in small thiols. Both peroxides were relatively ineffective in oxidizing haemoprotein thiols, especially at low pH. Increased amounts of haematin decreased greatly the efficiency of GSH oxidation by peroxides especially at low pH. 2. Other than the haematin ring, the thiol group was found to be probably the group in proteins most sensitive to modification by peroxides. 3. At low concentrations, the fatty acid moiety of a lipid peroxide appeared to impede thiol oxidation in proteins, probably by hydrophobic bonding to the protein, rather than to stimulate thiol oxidation by denaturing the protein and thereby increasing the exposure and reactivity of the thiol group. 4. The relative rates of thiol oxidation by peroxides in the different thiols were: haemoprotein thiols>small thiols>other protein thiols. In all cases, thiol oxidation was much more rapid by the lipid peroxide than by hydrogen peroxide.     

Production of Yeast Cells from Methanol

Using immunochemical technique thermal denaturation of soybean 11S globulin, dissolved in different ionic strength solutions (µ=0~4.0) and heated at 100°C for 5 min, has been quantitatively studied. The curves of the percentage of antigenicity remaining were obtained as a function of salt concentration. The 11S globulin became strongly resistant to thermal denaturation with increasing both KCl and potassium phosphate. The stabilizing effect (in terms of percent antigenicity) was separated into three regions. At ionic strength below 0.7, potassium phosphate had no stabilizing effect while KCl had aslightly effect. The rise in stabilizing effect up to about 50%, near 1.0~1.5 µ, represented a second transition to a different denatured state which retains undissociated molecule. At rises up to 75~95%, near 2.5~3.5µ, a different conformational state resulted in which thermally denatured 11S globulin maintained almost intact native conformation after heating. The selection of an adequate ionic strength of protein solution has enabled preparation of thermally denatured 11S globulins which have desired-residual amounts of structured regions.     

Photo-oxidation of cells generates long-lived intracellular protein peroxides

Singlet oxygen is generated by several cellular, enzymatic, and chemical reactions as well as by exposure to UV or visible light in the presence of a sensitizer. Consequently, this oxidant has been proposed to be a damaging agent many pathologies. Proteins are major targets for singlet oxygen as a result of their abundance and high rate constants for reaction. In this study, we show that illumination of viable rose bengal-loaded THP-1 (human monocyte-like) cells with visible light gives rise to intracellular protein-derived peroxides. The peroxide yield increases with illumination time, requires the presence of rose bengal, is enhanced in D(2)O, and is decreased by azide, consistent with the mediation of singlet oxygen. The concentration of peroxides detected, which is not affected by glucose or ascorbate loading of the cells, corresponds to about 1.5 nmoles peroxide per 10(6) cells, or 10 nmoles/mg cell protein, and account for up to approximately 15% of the O(2) consumed by the cells. Similar peroxides have been detected on isolated cellular proteins exposed to light in the presence of rose bengal and oxygen. After cessation of illumination, cellular protein peroxide levels decrease with t(1/2) about 4 h at 37 degrees C. Decomposition of protein peroxides formed within cells, or on isolated cellular proteins, by metal ions gives rise to radicals as detected by EPR spin trapping. These studies demonstrate that exposure of intact cells to visible light in the presence of a sensitizer leads to novel long-lived, but reactive, intracellular protein peroxides via singlet oxygen-mediated reactions.     

Striatal dopamine-NMDA receptor interactions in the modulation of glutamate release in the substantia nigra pars reticulata in vivo: opposite role for D1 and D2 receptors

To investigate the antioxidative capacities of oligodendrocytes, rat brain cultures enriched for oligodendroglial cells were prepared and characterized. These cultures contained predominantly oligodendroglial cells as determined by immunocytochemical staining for the markers galactocerebroside and myelin basic protein. If oligodendroglial cultures were exposed to exogenous hydrogen peroxide (100 micro m), the peroxide disappeared from the incubation medium following first order kinetics with a half-time of approximately 18 min. Normalization of the disposal rate to the protein content of the cultures by calculation of the specific hydrogen peroxide detoxification rate constant revealed that the cells in oligodendroglial cultures have a 60% to 120% higher specific capacity to dispose of hydrogen peroxide than cultures enriched for astroglial cells, microglial cells or neurones. Oligodendroglial cultures contained specific activities of 133.5 +/- 30.4 nmol x min(-1) x mg protein(-1) and 27.5 +/- 5.4 nmol x min(-1) x mg protein(-1) of glutathione peroxidase and glutathione reductase, respectively. The specific rate constant of catalase in these cultures was 1.61 +/- 0.54 min(-1) x mg protein(-1). Comparison with data obtained by identical methods for cultures of astroglial cells, microglial cells and neurones revealed that all three of the enzymes which are involved in hydrogen peroxide disposal were present in oligodendroglial cultures in the highest specific activities. These results demonstrate that oligodendroglial cells in culture have a prominent machinery for the disposal of hydrogen peroxide, which is likely to support the protection of these cells in brain against peroxides when produced by these or by surrounding brain cells.     

Characterization of a stellate cell activation-associated protein (STAP) with peroxidase activity found in rat hepatic stellate cells

A proteome approach for the molecular analysis of the activation of rat stellate cell, a liver-specific pericyte, led to the discovery of a novel protein named STAP (stellate cell activation-associated protein). We cloned STAP cDNA. STAP is a cytoplasmic protein with molecular weight of 21,496 and shows about 40% amino acid sequence homology with myoglobin. STAP was dramatically induced in in vivo activated stellate cells isolated from fibrotic liver and in stellate cells undergoing in vitro activation during primary culture. This induction was seen together with that of other activation-associated molecules, such as smooth muscle alpha-actin, PDGF receptor-beta, and neural cell adhesion molecule. The expression of STAP protein and mRNA was augmented time dependently in thioacetamide-induced fibrotic liver. Immunoelectron microscopy and proteome analysis detected STAP in stellate cells but not in other hepatic constituent cells. Biochemical characterization of recombinant rat STAP revealed that STAP is a heme protein exhibiting peroxidase activity toward hydrogen peroxide and linoleic acid hydroperoxide. These results indicate that STAP is a novel endogenous peroxidase catabolizing hydrogen peroxide and lipid hydroperoxides, both of which have been reported to trigger stellate cell activation and consequently promote progression of liver fibrosis. STAP could thus play a role as an antifibrotic scavenger of peroxides in the liver.     

Oxidative stress regulates the interaction of p16 with Cdk4

Oxidative stress can have a myriad of effects on many different cell types. The mechanisms by which these effects occur are not completely known. Chimeric proteins of the GAL4 DNA binding domain and Cdk4, or the GAL4 activation domain with p16, were expressed in the yeast two-hybrid system. Cells expressing these chimeric proteins were cultured with hydrogen peroxide and decreases in beta-galactosidase activity were observed when compared to cells incubated without hydrogen peroxide. When cells, which expressed the intact GAL4 binding protein, were cultured in the presence of hydrogen peroxide the opposite was observed. Incubation of cells with buthionine sulfoximine augmented these responses to hydrogen peroxide. These data suggest that one of the mechanisms by which oxidative stress acts is via the modulation of protein-protein interactions and demonstrate that the yeast two-hybrid system may be a model by which to study protein interactions due to oxidative stress.     

Spin trapping investigation of peroxide- and isoniazid-induced radicals in Mycobacterium tuberculosis catalase-peroxidase

A new approach, the immuno-spin trapping assay, used a novel rabbit polyclonal anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antiserum to detect protein radical-derived DMPO nitrone adducts in the hemoprotein Mycobacterium tuberculosis catalase-peroxidase (KatG). This work demonstrates that the formation of protein nitrone adducts is dependent on the concentrations of tert-BuOOH and DMPO as shown by Western blotting and an enzyme-linked immunosorbent assay (ELISA). We have also detected protein-protein cross-links formed during turnover of Mtb KatG driven by tert-butyl peroxide ( tert-BuOOH) or enzymatic generation of hydrogen peroxide. DMPO inhibits this dimerization due to its ability to trap the amino acid radicals responsible for the cross-linkage. Chemical modifications by tyrosine and tryptophan blockage suggest that tyrosine residues are one site of formation of the dimers. The presence of the tuberculosis drug isoniazid (INH) also prevented cross-linking as a result of its competition for the protein radical. Protein-DMPO nitrone adducts were also generated by a continuous flux of hydrogen peroxide. These findings demonstrated that the protein-based radicals were formed not only during Mtb KatG turnover with alkyl peroxides but also in the presence of hydrogen peroxide. Furthermore, the formation of protein-DMPO nitrone adducts was accelerated in the presence of isoniazid.     

Involvement of polyamines in evening primrose extract-induced apoptosis in Ehrlich ascites tumor cells

Summary. We previously demonstrated that evening primrose extract (EPE) induced apoptosis and inhibited the DNA synthesis in Ehrlich ascites tumor cells (EATC) and suggested that EPE-induced inhibition of the growth of EATC are via at least two pathway differentially modulated by reactive oxygen species, notably intracellular peroxides. These are (a) the EPE-induced apoptosis pathway which is dependent on increases in hydrogen peroxide and (b) the EPE-induced inhibition of cell proliferation which is hydrogen peroxide independent. In this study, EPE brought about a significant decrease in intracellular polyamine levels. Furthermore, the addition of polyamines reversed the EPE-induced decrease in cell viability and suppressed the EPE-induced increase in intracellular hydrogen peroxides. However, the addition of polyamines did not reverse EPE-induced decrease in DNA synthesis and phosphorylation of Rb protein, and EPE-induced translocation of AIF. These results suggest the involvement of polyamines in the EPE-induced apoptosis pathway which is dependent on increase in hydrogen peroxide.     

Cross reactivity and enzyme sensitivity of immunoaffinity purified Sm/RNP antigens

Immunoaffinity purified Sm/RNP antigens from buffalo and goat liver were studied to determine the role of RNA and proteins towards the antigenicity of Sm and RNP antigens. A more direct approach using enzyme-linked immunosorbent assay on nylon beads has been utilized to look into the problem. The effect of enzyme treatment and the role of RNA and protein fractions in influencing antigenicity have been described. RNA seems to be involved in the maintenance of RNP specific polypeptides in suitable conformation so as to keep them in solution. Removal of RNA leads to insolubilization of RNP specific polypeptides. Antibodies to Sm and RNP antigens have been shown to cross react with poly A containing heterogeneous nuclear ribonucleoprotein with no cross reactivity with thymus RNA or DNA.     

Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein.

A simple and sensitive method for the direct measurement of lipid peroxides in lipoprotein and liposomes is described. The method is based on the principle of the rapid peroxide-mediated oxidation of Fe2+ to Fe3+ under acidic conditions. The latter, in the presence of xylenol orange, forms a Fe(3+)-xylenol orange complex which can be measured spectrophotometrically at 560 nm. Calibration with standard peroxides, such as hydrogen peroxide, linoleic hydroperoxide, t-butyl hydroperoxide, and cumene hydroperoxide gives a mean apparent extinction coefficient of 4.52 x 10(4) M-1 cm-1 consistent with a chain length of approximately 3 for ferrous ion oxidation by hydroperoxides. Endoperoxides are less reactive or unreactive in the assay. The assay has been validated in the study of lipid peroxidation of low density lipoprotein and phosphatidyl choline liposomes. By pretreatment with enzymes known to metabolize peroxides, we have shown that the assay measures lipid hydroperoxides specifically. Other methods for measuring peroxidation, such as the assessment of conjugated diene, thiobarbituric acid reactive substances and an iodometric assay have been compared with the ferrous oxidation-xylenol orange assay.     

The capacity of glutathione reductase in cell protection from the toxic effect of heated oils

This empirical study tries to focus on the evidence that the wrong use of oil in food cooking leads to health problems. High temperatures associated with the repeated use of the same oil lead to the breakdown of some fatty acids, forming numerous toxic polymer compounds and peroxides. The obtained data have showed that the ratios of polymer compounds reached 11.3% in oil heated continuously for 10 h at 220 degrees C, and 37.8% in frying oil (FO). Moreover, the polar compound ratios reached 25.6% and 47.6% in continuously heated oil (CHO) and FO, respectively. However, the peroxide concentrations were 157.1 and 133.6 mM/kg in CHOs and FOs, respectively. The observed results have allowed the study of the role of the glutathione redox system in the detoxification and elimination of different toxic peroxides resulting from heated oils. On a diet of 10% of CHO and FO, a significant increase in glutathione peroxidase (GPx) and glutathione reductase (GR) activities appears. This combined relationship between the decreased glutathione content and the increased GPx and GR activities in rats fed on CHO and FO confirms the participation of the glutathione redox system in the detoxifying reactions of continuously accumulated peroxides.     

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and protein peroxides generated by singlet oxygen attack.

Reaction of certain peptides and proteins with singlet oxygen (generated by visible light in the presence of rose bengal dye) yields long-lived peptide and protein peroxides. Incubation of these peroxides with glyceraldehyde-3-phosphate dehydrogenase, in the absence of added metal ions, results in loss of enzymatic activity. Comparative studies with a range of peroxides have shown that this inhibition is concentration, peroxide, and time dependent, with H2O2 less efficient than some peptide peroxides. Enzyme inhibition correlates with loss of both the peroxide and enzyme thiol residues, with a stoichiometry of two thiols lost per peroxide consumed. Blocking the thiol residues prevents reaction with the peroxide. This stoichiometry, the lack of metal-ion dependence, and the absence of electron paramagnetic resonance (EPR)-detectable species, is consistent with a molecular (nonradical) reaction between the active-site thiol of the enzyme and the peroxide. A number of low-molecular-mass compounds including thiols and ascorbate, but not Trolox C, can prevent inhibition by removing the initial peroxide, or species derived from it. In contrast, glutathione reductase and lactate dehydrogenase are poorly inhibited by these peroxides in the absence of added Fe2+-EDTA. The presence of this metal-ion complex enhanced the inhibition observed with these enzymes consistent with the occurrence of radical-mediated reactions. Overall, these studies demonstrate that singlet oxygen-mediated damage to an initial target protein can result in selective subsequent damage to other proteins, as evidenced by loss of enzymatic activity, via the formation and subsequent reactions of protein peroxides. These reactions may be important in the development of cellular dysfunction as a result of photo-oxidation.     

Superoxide-dependent lipid peroxidation. Problems with the use of catalase as a specific probe for fenton-derived hydroxyl radicals

Hydroxyl radicals (OH.) can initiate lipid oxidation by hydrogen abstraction. Transition metals however, particularly iron and copper, stimulate lipid oxidation by reacting with lipid peroxides to form new radical species. The haem-iron protein catalase can react non-specifically with lipid peroxides in this way resulting in loss of their conjugated diene structures. When a superoxide-generating system is used to stimulate lipid autoxidation, catalase can conceivably inhibit the reaction in two ways (A) by decomposing lipid peroxides as they are formed (B) through the removal of hydrogen peroxide preventing OH. radical formation. Results presented here suggest that the latter interpretation, although commonly presented, cannot be automatically assumed.     

The reaction of horseradish peroxidase with hydroperoxides derived from Triton X-100

All of the commercially available Triton X-100 examined gave Compound I upon reaction with horseradish peroxidase, followed by its gradual transition into Compound II. Titration of horseradish peroxidase with Triton X-100 to form Compound I indicated that 1% (v/v) aqueous solutions of the detergent contained 0.4 to 3.2 microM equivalent peroxide but iodometric titration revealed 1.1 to 5.0 microM peroxide, suggesting the occurrence of different types of peroxides, reactive and unreactive with the peroxidase. The rate constant for Compound I formation was 1.5 X 10(7) M-1 S-1 at pH 7.4 at 25 degrees C, and for conversion into Compound II apparent first-order rate constants were 5.2 X 10(-3) to 1.7 X 10(-2) S-1. These results indicate that the Triton peroxides are as highly reactive as hydrogen peroxide. The amount of Triton peroxides increased as aqueous solutions of the detergent were allowed to stand, but the peroxides were destroyed by treatment with sodium borohydride. Although freshly prepared aqueous solutions of sodium cholate, sodium dodecyl sulfate, Tween 20 (polyoxyethylene sorbitan monolaurate), and Emasol 1130 (an equivalent of Tween 20) did not contain any detectable amount of peroxide, aged solutions of sodium dodecyl sulfate and Emasol 1130 contained peroxides. These observations suggest the need for appropriate precautions when biologically active substances vulnerable to attack by peroxides are incubated with Triton X-100 either for their solubilization from biomembranes or for other processing.     

Effects of titanium compounds on a D-glucose-D-glucose oxidase assay system.

Titanium compounds affect the measurement of D-glucose oxidase (and therefore D-glucose) by the D-glucose-D-glucose oxidase-peroxidase-2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) assay system. The validity of measurement of the activity of D-glucose oxidase immobilised on supports based on titanium oxides is affected by complexation of the intermediate hydrogen peroxide with the support, and such supports may prove to be unsuitable for the immobilisation of D-glucose oxidase. The formation of titanic peroxides is among the reasons discussed for the various interactions encountered. The use of the assay system for the determination of D-glucose oxidase contaminated with catalase and for the determination of hydrogen peroxide is also described.     

A High-Throughput Microtiter Plate Based Method for the Determination of Peracetic Acid and Hydrogen Peroxide

Peracetic acid is gaining usage in numerous industries who have found a myriad of uses for its antimicrobial activity. However, rapid high throughput quantitation methods for peracetic acid and hydrogen peroxide are lacking. Herein, we describe the development of a high-throughput microtiter plate based assay based upon the well known and trusted titration chemical reactions. The adaptation of these titration chemistries to rapid plate based absorbance methods for the sequential determination of hydrogen peroxide specifically and the total amount of peroxides present in solution are described. The results of these methods were compared to those of a standard titration and found to be in good agreement. Additionally, the utility of the developed method is demonstrated through the generation of degradation curves of both peracetic acid and hydrogen peroxide in a mixed solution.     

Formation of lipid peroxides in isolated rat liver microsomes by singlet molecular oxygen.

Rat liver microsomes were incubated in neutral aqueous solution of potassium peroxychromate, a system which generates singlet molecular oxygen. Such incubation resulted both in a rapid decline in NADPH-cytochrome c reductase activity, and in an increase in formation of lipid peroxides. These reactions were not inhibited by either superoxide dismutase (SOD) or mannitol, nor were they entirely duplicated by incubating microsomes with hydrogen peroxide. However, a high concentration of 1,4-diazabicyclo-[2,2,2]octane (DABCO), a known scavenger of singlet oxygen, prevented both decline in reductase activity and formation of lipid peroxides. These results suggest that the observed effects are, in fact, attributable to singlet oxygen, and not to hydrogen peroxide, superoxide radical, or hydroxyl radical.