Ethyl Hydrogen Peroxide Research Articles

Abstract 4510: Real-time imaging of tumor cells after exposure to NDC-1308 identifies the temporal order of cellular events leading to cancer cell death

Abstract Recent evidence suggests that the growth of cancer cells is influenced by the estrogen receptor (ER) activity. We previously reported the discovery of Endece's family of small molecule ER-β agonists that will be used to treat human diseases, like cancer. Our lead candidate, NDC-1308, is cytotoxic to a broad range of human tumor cell lines (EC50 5-20 µM) and inhibits human tumor growth in mouse xenograft models. NDC-1308 binds with similar affinity to both ER-α and ER-β (70-100 nM), but activates ER-β nearly 5-fold more than ER-α. This finding is consistent with recent studies reported in the literature that show ER-β activation correlates with cell growth inhibition and ultimately cell death. Microarray analysis of three human tumor cell lines (A549-lung, SKOV-3-ovary, and Panc-1-pancreatic) treated with NDC-1308 (10-50 µM) for 24 hours caused significant effects on the regulation of genes associated with growth arrest and cell death pathways. For growth arrest, this includes the up-regulation of genes impacting the S and G2/M cell cycle checkpoints such as GADD45A (10-fold), the down-regulation of 21 key genes critical for chromosome replication (3-8 fold), and the up-regulation of nucleases such as GzmK (16-fold) and AEN (2-fold). For cell death, the up-regulation of genes within pro-apoptotic pathways includes BBC3 (4-fold) and GDF15 (12-fold), while the death receptor pathway gene TNFRSF10B (DR5) is up-regulated 5-fold. Since NDC-1308 treatment can lead to cancer cell death by simultaneously impacting cell cycle checkpoints, DNA damage and the apoptotic pathways, it holds great promise as an anticancer therapeutic. However, these studies have not identified the primary trigger event leading to NDC-1308 mediated cancer cell death. We therefore utilized real-time, live cell imaging with green fluorescent protein (GFP) reporters to better understand the initial mechanism of tumor cell death upon exposure to the drug. The tumor cells’ (A549 and Panc-1) response to NDC-1308 was compared to known DNA damaging agents, like ethyl methanesulfonate and hydrogen peroxide, which allowed for identification of well-characterized components of the pathways noted above. Utilizing a variety of different reporters provided information about the level of RNAs and intracellular localization of proteins known to be active in the proliferation and survival cellular pathways. As the measurements are non-destructive, a dense temporal profile of cellular elements that control the cells’ response to NDC-1308 and the other DNA damaging agents was produced. This profile will be presented along with our understanding of how NDC-1308 binding to estrogen receptors impacts cancer cell proliferation. Future studies will lead to the development of potential biomarkers for use in Phase 1 human clinical trials with NDC-1308. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4510. doi:10.1158/1538-7445.AM2011-4510

Oxyferryl Heme and Not Tyrosyl Radical Is the Likely Culprit in Prostaglandin H Synthase-1 Peroxidase Inactivation

Prostaglandin H synthase-1 (PGHS-1) is a bifunctional heme protein catalyzing both a peroxidase reaction, in which peroxides are converted to alcohols, and a cyclooxygenase reaction, in which arachidonic acid is converted into prostaglandin G2. Reaction of PGHS-1 with peroxide forms Intermediate I, which has an oxyferryl heme and a porphyrin radical. An intramolecular electron transfer from Tyr385 to Intermediate I forms Intermediate II, which contains two oxidants: an oxyferryl heme and the Tyr385 radical required for cyclooxygenase catalysis. Self-inactivation of the peroxidase begins with Intermediate II, but it has been unclear which of the two oxidants is involved. The kinetics of tyrosyl radical, oxyferryl heme, and peroxidase inactivation were examined in reactions of PGHS-1 reconstituted with heme or mangano protoporphyrin IX with a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and ethyl hydrogen peroxide (EtOOH). Tyrosyl radical formation was significantly faster with 15-HPETE than with EtOOH and roughly paralleled oxyferryl heme formation at low peroxide levels. However, the oxyferryl heme intensity decayed much more rapidly than the tyrosyl radical intensity at high peroxide levels. The rates of reactions for PGHS-1 reconstituted with MnPPIX were approximately an order of magnitude slower, and the initial species formed displayed a wide singlet (WS) radical, rather than the wide doublet radical observed with PGHS-1 reconstituted with heme. Inactivation of the peroxidase activity during the reaction of PGHS-1 with EtOOH or 15-HPETE correlated with oxyferryl heme decay, but not with changes in tyrosyl radical intensity or EPR line shape, indicating that the oxyferryl heme, and not the tyrosyl radical, is responsible for the self-destructive peroxidase side reactions. Computer modeling to a minimal mechanism was consistent with oxyferryl heme being the source of peroxidase inactivation.

High-field EPR study of tyrosyl radicals in prostaglandin H(2) synthase-1.

Various tyrosyl radicals generated by reaction of both native and indomethacin-inhibited ovine prostaglandin H synthase-1 with ethyl hydrogen peroxide were examined by using high-field/high-frequency EPR spectroscopy. The spectra for the initially formed tyrosyl radical commonly referred to as the "wide-doublet" species and the subsequent "wide-singlet" species as well as the indomethacin-inhibited "narrow-singlet" species were recorded at several frequencies and analyzed. For all three species, the g-values were distributed. In the case of the wide doublet, the high-field EPR spectra indicated that dominant hyperfine coupling was likely to be also distributed. The g(x)-values for all three radicals were found to be consistent with a hydrogen-bonded tyrosyl radical. In the case of the wide-doublet species, this finding is consistent with the known position of the radical and the crystallographic structure and is in contradiction with recent ENDOR measurements. The high-field EPR observations are consistent with the model in which the tyrosyl phenyl ring rotates with respect to both the protein backbone and the putative hydrogen bond donor during evolution from the wide-doublet to the wide-singlet species. The high-field spectra also indicated that the g-values of two types of narrow-singlet species, self-inactivated and indomethacin-inhibited, were likely to be different, raising the possibility that the site of the radical is different or that the binding of the inhibitor perturbs the electrostatic environment of the radical. The 130 GHz pulsed EPR experiments performed on the wide-doublet species indicated that the possible interaction between the radical and the oxoferryl heme species was very weak.

Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1.

The tyrosyl radicals generated in reactions of ethyl hydrogen peroxide with both native and indomethacin-pretreated prostaglandin H synthase 1 (PGHS-1) were examined by low-temperature electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. In the reaction of peroxide with the native enzyme at 0 degrees C, the tyrosyl radical EPR signal underwent a continuous reduction in line width and lost intensity as the incubation time increased, changing from an initial, 35-G wide doublet to a wide singlet of slightly smaller line width and finally to a 25-G narrow singlet. The 25-G narrow singlet produced by self-inactivation was distinctly broader than the 22-G narrow singlet obtained by indomethacin treatment. Analysis of the narrow singlet EPR spectra of self-inactivated and indomethacin-pretreated enzymes suggests that they reflect conformationally distinct tyrosyl radicals. ENDOR spectroscopy allowed more detailed characterization by providing hyperfine couplings for ring and methylene protons. These results establish that the wide doublet and the 22-G narrow singlet EPR signals arise from tyrosyl radicals with different side-chain conformations. The wide-singlet ENDOR spectrum, however, is best accounted for as a mixture of native wide-doublet and self-inactivated 25-G narrow-singlet species, consistent with an earlier EPR study [DeGray et al. (1992) J. Biol. Chem. 267, 23583-23588]. We conclude that a tyrosyl residue other than the catalytically essential Y385 species is most likely responsible for the indomethacin-inhibited, narrow-singlet spectrum. Thus, this inhibitor may function by redirecting radical formation to a catalytically inactive side chain. Either radical migration or conformational relaxation at Y385 produces the 25-G narrow singlet during self-inactivation. Our ENDOR data also indicate that the catalytically active, wide-doublet species is not hydrogen bonded, which may enhance its reactivity toward the fatty-acid substrate bound nearby.

Rapid kinetics of tyrosyl radical formation and heme redox state changes in prostaglandin H synthase-1 and -2

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34–35 G) was formed by 4 ms, followed by transition to a wide singlet (33–34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25–26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.

Rapid Kinetics of Tyrosyl Radical Formation and Heme Redox State Changes in Prostaglandin H Synthase-1 and -2

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34-35 G) was formed by 4 ms, followed by transition to a wide singlet (33-34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25-26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.

Homolytic ethoxycarbonylation of 3-nitropyridines

The homolytic ethoxycarbonylation of alkyl-3-nitropyridines has been accomplished for the first time by the action of ethyl pyruvate and hydrogen peroxide in the presence of ferrous sulfate.

Detection of an Fe2+-protoporphyrin-IX intermediate during aspirin-treated prostaglandin H2 synthase II catalysis of arachidonic acid to 15-HETE.

Spectral intermediates associated with the dioxygenase and peroxidase activities of prostaglandin H2 (PGH2) synthase I and II were monitored by stopped-flow spectrometry. During reactions of PGH2 synthase I with arachidonic acid (AA) and ethyl hydrogen peroxide (EtOOH), compound I (Fe5+; formally (protoporphyrin-IX) x +Fe4+=O) and compound II (Fe4+; formally (protoporphyrin-IX)Fe4+=O) were detected. These intermediates were observed sooner with EtOOH (within 50 ms) than with AA (within 200 ms). Compound I and compound II were found to be kinetically competent with respect to AA-dependent O2 uptake. These findings are consistent with a mechanism in which peroxidative cleavage precedes AA dioxygenation. During reactions with PGH2 synthase II with AA, compound I and compound II were again observed within 200 ms and were kinetically competent to participate in dioxygenation. However, during reactions of PGH2 synthase II with EtOOH, compound I and compound II were detected much later (after 10 s). These findings would be inconsistent with a mechanism in which peroxidative cleavage precedes AA dioxygenation. When aspirin-treated PGH2 synthase II was reacted with EtOOH, a normal peroxidase cycle occurred with compound I and compound II formation occurring over 10 s. However, when aspirin-treated PGH2 synthase II was reacted with AA, a unique spectral intermediate with lambda(max) at 446 nm was detected within 3 ms and was strikingly similar to ferrous (Fe2+) protoporphyrin-IX. Aspirin-treated PGH2 synthase II was found to produce 15-HETE, and the appearance of the Fe2+ intermediate (within 3 ms) indicated that it was kinetically competent to participate in the 15-dioxygenation event. The detection of this Fe2+ intermediate and the slow formation of compound I and compound II observed with EtOOH in PGH2 synthase II suggest that peroxidative cleavage is not the initiating event in dioxygenation. Instead, it is proposed that the reduction of Fe3+ in heme to Fe2+ oxidizes a peroxide to yield an initiating peroxy radical. Since it is unlikely that 11- and 15-dioxygenation occurs via different mechanisms, our findings question mechanisms of catalysis in both PGH2 synthases.

Prostaglandin H synthase. Kinetics of tyrosyl radical formation and of cyclooxygenase catalysis.

Hydroperoxides are known to induce the formation of tyrosyl free radicals in prostaglandin (PG) H synthase. To evaluate the role of these radicals in cyclooxygenase catalysis we have analyzed the temporal correlation between radical formation and substrate conversion during reaction of the synthase with arachidonic acid. PGH synthase reacted with equimolar levels of arachidonic acid generated sequentially the wide doublet (34 G peak-to-trough) and wide singlet (32 G peak-to-trough) tyrosyl radical signals previously reported for reaction with hydroperoxide. The kinetics of formation and decay of the doublet signal corresponded reasonably well with those of cyclooxygenase activity. However, the wide singlet free radical signal accumulated only after prostaglandin formation had ceased, indicating that the wide singlet is not likely to be an intermediate in cyclooxygenase catalysis. When PGH synthase was reacted with 25 equivalents of arachidonic acid, the wide doublet and wide singlet radical signals were not observed. Instead, a narrower singlet (24 G peak-to-trough) tyrosyl radical was generated, similar to that found upon reaction of indomethacin-treated synthase with hydroperoxide. Only about 11 mol of prostaglandin were formed per mol of synthase before complete self-inactivation of the cyclooxygenase, far less than the 170 mol/mol synthase produced under standard assay conditions. Phenol (0.5 mM) increased the extent of cyclooxygenase reaction by only about 50%, in contrast to the 460% stimulation seen under standard assay conditions. These results indicate that the narrow singlet tyrosyl radical observed in the reaction with high levels of arachidonate in this study and by Lassmann et al. (Lassmann, G., Odenwaller, R., Curtis, J.F., DeGray, J.A., Mason, R.P., Marnett, L.J., and Eling, T.E. (1991) J. Biol. Chem. 266, 20045-20055) is associated with abnormal cyclooxygenase activity and is probably nonphysiological. In titrations of the synthase with arachidonate or with hydroperoxide, the loss of enzyme activity and destruction of heme were linear functions of the amount of titrant added. Complete inactivation of cyclooxygenase activity was found at about 10 mol of arachidonate, ethyl hydrogen peroxide, or hydrogen peroxide per mol of synthase heme; maximal bleaching of the heme Soret absorbance peak was found with 10 mol of ethyl hydroperoxide or 20 mol of either arachidonate or hydrogen peroxide per mol of synthase heme. The peak concentration of the wide doublet tyrosyl radical did not change appreciably with increased levels of ethyl hydroperoxide. In contrast, higher levels of hydroperoxide gave higher levels of the wide singlet radical species, in parallel with enzyme inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Removal of hydroperoxides by immobilized borohydride: A good method for purification of biochemical materials

Borohydride was immobilized on a quaternary ammonium type anion exchange resin, Amberlite IRA-400, by an exchange reaction in N,N-dimethylformamide. The reducing ability of borohydride on the polymer beads was examined; 0.1 g resin was applied for about 30 min to 3 ml solutions of hydrogen peroxide, ethyl hydrogen peroxide, and peracetic acid, at a concentration of ∼40 m m, m-chloroperbenzoic acid (3.13 m m), and 5-phenyl-4-pentenyl-1-hydroperoxide (1 m m), respectively. The solutions were then assayed for remaining hydroperoxide by use of horseradish peroxidase or prostaglandin H synthase. In addition, the effect of treatment on the ability of 5-phenyl-4-pentenyl-1-hydroperoxide to initiate the cyclooxygenase activity of prostaglandin H synthase was investigated. Results indicated that immobilized borohydride is very efficient in removing hydroperoxides. It can be used in either organic or aqueous media. It is convenient for both large and small scales, particularly important for purification of biochemical materials.

Purification, partial characterization, and possible role of catalase in the bacterium Vitreoscilla

Vitreoscilla is a gram-negative bacterium that contains a unique bacterial hemoglobin that is relatively autoxidizable. It also contains a catalase whose primary function may be to remove hydrogen peroxide produced by this autoxidation. This enzyme was purified and partially characterized. It is a protein of 272,000 Da with a probable A 2B 2 subunit structure, in which the estimated molecular size of A is 68,000 Da and that of B, 64,000 Da, and an average of 1.6 molecules of protoheme IX per tetramer. The turnover number for its catalase activity was 27,000 s −1 and the K m for hydrogen peroxide was 16 m m. The peroxidase activity measured using o-dianisidine was 0.6% that of the catalase activity. Cyanide, which inhibited both catalase and peroxidase activities, bound the heme in a noncooperative manner. Azide inhibited the catalase activity but stimulated the peroxidase activity. An apparent compound II was formed by the reaction of the enzyme with ethyl hydrogen peroxide. The enzyme was reducible by dithionite, and the ferrous enzyme reacted with CO. The cellular content of Vitreoscilla hemoglobin varies during the growth cycle and in cells grown under different conditions, but the ratio of hemoglobin to catalase activity remained relatively constant, indicating possible coordinated biosynthesis and supporting the putative role of Vitreoscilla catalase as a scavenger of peroxide generated by Vitreoscilla hemoglobin.

In vivo thiyl free radical formation from hemoglobin following administration of hydroperoxides

Although free radical formation due to the reaction between red blood cells and organic hydroperoxides in vitro has been well documented, the analogous in vivo ESR spectroscopic evidence for free radical formation has yet to be reported. We successfully employed ESR to detect the formation of the 5,5-dimethyl-1-pyrroline- N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats dosed with DMPO and tert-butyl hydroperoxide, cumene hydroperoxide, ethyl hydrogen peroxide, 2-butanone hydroperoxide, 15( S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid, or hydrogen peroxide. We found that pretreating the rats with either buthionine sulfoximine or diethylmaleate prior to dosing with tert-butyl hydroperoxide decreased the concentration of nonprotein thiols within the red blood cells and significantly enhanced the DMPO/hemoglobin thiyl radical adduct concentration. Finally, we found that pretreating rats with the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea prior to dosing with tert-butyl hydroperoxide enhanced the DMPO/hemoglobin thiyl radical adduct concentration and induced the greatest decrease in nonprotein thiol concentration within the red blood cells.

The Heme Environment of Ovoperoxidase as Determined by Optical Spectroscopy

Native ovoperoxidase exhibited an optical absorption spectrum with certain similarities to lactoperoxidase, but not horseradish peroxidase, over the pH range 4.5-11.5. Ovoperoxidase had three distinct spectral forms dependent on pH, with transitions at apparent pKa values of 6.6 and 3.0. Complexes of ovoperoxidase with CN-, N3-, F-, or when reduced and ligated to carbon monoxide, CN-, or pyridine, were distinct from other peroxidases. Ovoperoxidase formed two specific and different spectral derivatives at pH 6.0 and 8.0, either in the native state, or when combined with CN-, when reduced, or when reduced and ligated to CN-. The position of the Soret band when mixed with near-stoichiometric amounts of H2O2. This cycling was inhibited by phenylhydrazine, 3-amino-1,2,4-triazole, or low pH (less than or equal to 6). Compound II was formed when ovoperoxidase was mixed with ethyl hydrogen peroxide in a 1:3 ratio, but not with H2O2. With a great excess of H2O2, Compound III was formed at pH 8.0; at pH 6.0 or below, the Soret band shifted slightly with excess of H2O2, but Compound III was never formed. Even when ovoperoxidase was bound to proteoliaisin (Weidman, P. J., and Shapiro, B. M. (1987) J. Cell Biol. 105, 561-567), ovoperoxidase exhibited spectral characteristics of the free enzyme.

Lack of antibacterial activity after intravenous hydrogen peroxide infusion in experimental Escherichia coli sepsis

The intravenous administration of hydrogen peroxide has been reported to benefit patients with pneumonia and to reduce Plasmodium parasitemia in experimentally infected mice. We assessed the antibacterial activity of intravenously infused hydrogen peroxide against hydrogen peroxide-susceptible Escherichia coli (MBC of hydrogen peroxide, 0.23 mM) in experimentally infected rabbits. No decrease in the level of bacteremia was detected at the maximum intravenous infusion rate of hydrogen peroxide physiologically tolerated by the rabbits (2.0 mumol/h). Moreover, the addition ex vivo of greater amounts of hydrogen peroxide to human or murine blood containing E. coli resulted in no detectable antibacterial action. In contrast, ethyl hydrogen peroxide, which is not affected by catalase, was bactericidal when added ex vivo to human blood containing E. coli. These results suggest that extracellular hydrogen peroxide, whether of exogenous or endogenous origin, does not have antibacterial activity in the blood of animals having even low levels of catalase.

Multiple structures and functions of cytochrome oxidase

X-ray absorption studies have been used to investigate the structure of the four redox centers (2Fe, 2Cu) of the terminal enzyme in the respiratory chain, cytochrome c oxidase in the resting oxidized form as well as in the functional intermediates that are freeze-trapped. Methods of x-ray fluorescence detection for these low-concentration samples together with low temperature cryostats and simultaneous optical monitoring were developed to ensure good signal-to-noise data and sample integrity. The resting oxidized form contains a sulfur bridge between the copper and iron of the active site which are separated by ~ 3.8 Å. This separation of the active site metal atoms was uniquely identified by comparison of both the iron and copper EXAFS data and iron EXAFS of the copper-depleted enzyme. In the reduced state, the CO or O 2 is bound to the active site iron having a structure identical to CO or oxy hemoglobin while the sulfur remains with the active site copper. Little change in structure is observed for the other iron and copper. It is the sulfur bridged active site form that is isolated by the Yonetani and Caughy methods with ⩾ 85% homogeneity but not the Hartzell-Beinert or similar methods. Another form observed in the redox cycle is also fully oxidized but lacks the sulfur bridged active site with the iron of the active site having a structure identical to that of the peroxidases. This form exhibits peroxidase as well as oxidase activity, and a stable intermediate is formed with hydrogen and ethylhydrogen peroxide in which the iron of the active site is structurally similar to that of the peroxidase intermediate. The active site copper, however, does not participate in the peroxidatic role and the structures of the other iron and copper are identical to those of the sulfur bridged resting oxidized form. Thus this unique enzyme has peroxidase activity which may serve to safeguard its main oxidase function.

Reaction of catalase with ethylhydrogen peroxide

C2H5OOH reacts with catalse in a basically irreversible reaction in the course of which the species called compound (I) is formed and decomposed. The formation of compound (I) is preceded by the formation of a precursor complex which is able to react with a further molecule of C2H5OOH to yield an inactive biperoxy complex. The biperoxy complex causes a diminution of the extent of formation of compound (I) at high [C2H5OOH]. As a consequence, compound (I) can never be formed quantitatively. Some of its physical constants can, nevertheless, be evaluated. Compound (I) with C2H5OOH appears to retain C2H5OH in its structure.

Fermentation of tea in aqueous suspension. Influence of tea peroxidase

AbstractThe natural peroxidase and catalase of tea leaf were shown to be active under the conditions employed for the fermentation of minced tea leaf in aqueous suspension. Fermentations carried out under nitrogen with ethyl hydrogen peroxide, which is not decomposed by catalase, yielded predominantly thearubigins, a high proportion of which were polymeric in nature. Fermentations using an excess of hydrogen peroxide gave similar results. Fermentations under conditions of controlled dissolved oxygen using both air and hydrogen peroxide resulted in a pigment distribution intermediate between those observed using air alone and using ethyl hydroperoxide under nitrogen. Adjustment of the amount of hydrogen peroxide used to around 120 μmol g−1 leaf resulted in a pigment distribution closely similar to that of a good quality black tea. A mixture of theaflavins was stable in the presence of air and tea polyphenoloxidase but decomposed rapidly to yield polymeric materials under the action of hydrogen peroxide and peroxidase derived from tea or horseradish. Kinetic measurements suggest that tea flavanols are generally poorer substrates for peroxidase than for catechol oxidase. However, the level of peroxidase is high and, furthermore, in view of the respective pH optima of the two enzymes, its action would be favoured as the pH falls during fermentation. It is postulated that the nature and distribution of the pigments formed during the fermentation of tea is governed in part by the relative actions of catechol oxidase and peroxidase. These actions are, in turn, influenced by the availability of oxygen and the action of catalase.

N-Demethylation reactions catalyzed by chloroperoxidase.

The peroxidase-supported N-demethylations catalyzed by chloroperoxidase, a heme protein isolated from Caldariomyces fumago, have been investigated as models for cytochrome P-450-catalyzed N-dealkylations. The turnover number for the ethyl hydrogen peroxide-supported dealkylation of N,N-dimethylaniline by chloroperoxidase (1476) was much greater than that for cytochrome P-450-catalyzed dealkylations. The dealkylations of N,N-dimethylaniline by chloroperoxidase yielded N-methylaniline and formaldehyde in equimolar amounts with no other products detectable by high pressure liquid chromatography analysis of the reaction mixture. Ethyl hydrogen peroxidase could be replaced by other hydroperoxides, peroxides, or peracids. Chloride ions stimulated the reaction at low pH. The dealkylation reaction exhibited normal Michaelis-Menten saturation kinetics with respect to N,N-dimethylaniline (Km = 0.08 mM) and ethyl hydrogen peroxide (Km = 0.8 mM) at low substrate concentrations. However, substrate inhibition occurred at higher concentrations of N,N-dimethylaniline. The chloroperoxidase-catalyzed demethylations were inhibited by inhibitors of cytochrome P-450 such as azide or n-propyl gallate, but not by metyrapone, SKF-525A, or piperonyl butoxide. Although tiron and DL-epinephrine, trapping agents for the superoxide anion, inhibited the demethylation reactions, superoxide dismutase had no effect. There was no significant inhibition by alpha-phenyl-t-butyl-nitrone or 5,5-dimethyl-pyrroline-N-oxide, which react with free radicals. Diphenylfuran and DL-histidine, which react with singlet oxygen, did not inhibit the reaction. Substitution of D2O for H2O resulted in a marked inhibition with a solvent isotope effect (VH/VD) of 3.6. Chloroperoxidase did not catalyze the demethylation of N,N-dimethylaniline-N-oxide, indicating that the reaction does not proceed via an N-oxide intermediate.

Determination of structure of polyunsaturated hydrocarbons and polymers by a new method of ozonolysis in volatile solvents

A new, efficient method has been devised for determining the structure of polyunsaturated hydrocarbons and polymers by ozonizing them in a volatile solvent with subsequent contact between the peroxide products and Lindlar's catalyst in a hydrogen atmosphere and preoxidation of the aldehydes formed with an ozone-oxygen mixture in ethyl acetate or hydrogen peroxide in the presence of SeO2 in tert-butanol.

Optical measurements of intracellular oxygen concentration of rat heart in vitro

The optical characteristics of hemoglobin-free perfused rat heart have been examined in detail. Ethyl hydrogen peroxide is found to convert myoglobin into “ferryl compound” in the perfused heart, as is also seen in vitro. After pretreatment with ethyl hydrogen peroxide, a typical mitochondrial absorption spectrum, similar to that of isolated rat heart mitochondria, is obtained in perfused heart. The overall absorption spectrum of the heart obtained by the aerobic to anaerobic transition is a superposition of the mitochondrial spectrum on that of myoglobin. By comparing these spectra, it is found that measurement of cytochrome a + a 3 at 605–620 nm is possible in spite of the absorbance change due to the oxygenation-deoxygenation of myoglobin, whereas the wavelength pairs for cytochrome c at 550-540 nm, cytochrome b at 562–575 nm and cytochrome a + a 3 at 445–450 nm can not be used in the heart because of interference from the absorption change of myoglobin. The partial pressure of O 2 (P 50) which is required for half maximal deoxygenation (or oxygenation) of myoglobin in perfused heart is found to be 2.4 mm Hg at room temperature and the Hill constant, n, is 1.1; these values are similar to those of myoglobin purified from rat heart. The steady-state O 2 titration has been performed by using absorbancy changes of myoglobin and cytochrome a + a 3 as intracellular O 2 indicators. In the perfused heart, the percentage change of oxygenation-deoxygenation of myoglobin parallels the oxidation-reduction of cytochrome a + a 3, while the mixture of purified myoglobin and isolated mitochondria shows a deviation, reflecting the difference of O 2 affinities between myoglobin and cytochrome a + a 3. The results indicate that there may be an O 2 gradient between cytosolic and mitochondrial compartments in the hemoglobin-free perfused heart. The absorption changes of myoglobin and of cytochrome a + a 3 can be measured in a single contraction-relaxation cycle. A triple beam method was introduced to eliminate the effect of light scattering changes in these measurements. The results demonstrated that myoglobin is more oxygenated during the systolic and diastolic periods and deoxygenated in the resting period, whereas cytochrome a + a 3 is more reduced in systole and diastole and oxidized in the resting state. Changing the perfusion conditions greatly alters the time course of the events which occur during the contraction-relaxation cycle of the perfused heart.