Nonequilibrium state of electrochemically activated water and its biological activity]

Changes in the physicochemical parameters (pH, redox potential and electroconductivity) of catholyte and anolyte produced by membrane electrolysis of distilled water and dilute (c < 10(-3) M) sodium chloride solutions were studied. The relaxation of these parameters after electrolysis and the influence of catholyte and anolyte on the growth of roots of Tradescantia viridis grafts, the development of duckweed, and the motive activity of infusoria Spirostomum ambiguum were investigated. It was found that the anolyte of distilled water stimulated development of these biological objects. The direction of shift of physicochemical parameters of catholyte and anolyte from equilibrium values and the type of their biological activity (stimulation or inhibition) depend on salt concentration in initial solution. Barbotage of initial distilled water with argon or nitrogen leads to a greater decrease in the redox potential of catholyte during electrolysis. The physicochemical parameters relax to equilibrium values, and the biological activity of catholite and anolyte decreases with time and practically disappears by the end of the day. It was found that the oxidation of reducing agent by atmospheric oxygen is not the sole cause of the relaxation of catalyte redox potential. The increase in the ionic strength of catholite and anolyte by the addition of concentrated sodium chloride after electrolysis decreases the rate of redox potential relaxation several times. The redox potential can be maintained for long periods by freezing.     

Level of hydrogen peroxide in electrochemically activated solutions and study of its effect on growth of Escherichia coli

The formation of hydrogen peroxide in catholytes and anolytes of electrochemically activated solutions: bidistilled water and solutions of sodium chloride and nutrition medium M9 was studied. The concentration of hydrogen peroxide was determined by the method of enhanced chemiluminescence in a system peroxidase-luminol-p-iodophenol. It was shown that the concentration of hydrogen peroxide depends on the ionic content of the solution and varies from a few fractions of a micromole in catholytes of bidistilled water and sodium chloride solutions (10(-5) divided by 10(-2) M) to 20-25 microM in catholytes of medium M9. The concentration of H2O2 in anolytes of various solutions was 15-20 times lower than in the corresponding catholytes and was equal to a few nanomoles in bidistilled water and a few micromoles in medium M9. The biological activity of the catholyte of medium M9 was determined from changes in the growth of E. coli cells. It was found that this catholyte stimulates the cell growth. The stimulating effect was 20-25% and did not change after the decomposition of hydrogen peroxide in the catholyte by catalase. The addition of H2O2 at the corresponding concentration to the inactivated nutrient medium produced no stimulating effect. These data suggest that hydrogen peroxide formed in the catholyte of nutrient medium M9 does not affect its biological activity.     

Post-translational regulation of mercaptopyruvate sulfurtransferase via a low redox potential cysteine-sulfenate in the maintenance of redox homeostasis

3-Mercaptopyruvate sulfurtransferase (MST) (EC 2.8.1.2), a multifunctional enzyme, catalyzes a transsulfuration from mercaptopyruvate to pyruvate in the degradation process of cysteine. A stoichiometric concentration of hydrogen peroxide and of tetrathionate (S(4)O(6)(2-)) inhibited rat MST (k(i) = 3.3 min(-1), K(i) = 120.5 microM and k(i) = 2.5 min(-1), K(i) = 178.6 microM, respectively). The activity was completely restored by dithiothreitol or thioredoxin with a reducing system containing thioredoxin reductase and NADPH, but glutathione did not restore the activity. On the other hand, an excess molar ratio dose of hydrogen peroxide inactivated MST. Oxidation with a stoichiometric concentration of hydrogen peroxide protected the enzyme against reaction by iodoacetate, which modifies a catalytic Cys(247), suggesting that Cys(247) is a target of the oxidants. A matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis revealed that hydrogen peroxide- and tetrathionate-inhibited MSTs were increased in molecular mass consistent with the addition of atomic oxygen and with a thiosulfate (S(2)O(3)(-)), respectively. Treatment with dithiothreitol restored modified MST to the original mass. These findings suggested that there was no nearby cysteine with which to form a disulfide, and mild oxidation of MST resulted in formation of a sulfenate (SO(-)) at Cys(247), which exhibited exceptional stability and a lower redox potential than that of glutathione. Oxidative stress decreases MST activity so as to increase the amount of cysteine, a precursor of thioredoxin or glutathione, and furthermore, these cellular reductants restore the activity. Thus the redox state regulates MST activity at the enzymatic level, and on the other hand, MST controls redox to maintain cellular redox homeostasis.     

Digestive proteinase activity in corn earworm (Helicoverpa zea) after molting and in response to lowered redox potential

Insect digestive proteinases are often strongly influenced by ambient physicochemical conditions, such as pH, ionic strength, and oxidation-reduction potential. Although the effects of the former two parameters are well documented, the influence of redox potential on catalytic rates of digestive enzymes is not well understood. In this study, we manipulated the midgut redox potential of a generalist caterpillar (the corn earworm, Helicoverpa zea) by augmenting artificial diet with dithiothreitol, a powerful thiol reducing agent that lowers the redox potential in the lumen by 40-45 mV. Effects on total proteolytic activity, as well as on elastase, chymotrypsin, trypsin, leucine aminopeptidase, and carboxypeptidase A and B activities were measured using azocasein and nitroanilide model substrates. The profiles of proteinase activities in the epithelium and lumen were also monitored on days 1, 2, and 3 after the molt in penultimate instar larvae. Although the reducing agent strongly inhibited the activity of some proteinases in vitro, ingestion of the reducing diet failed to affect in vivo proteinase activities. There was also no effect on larval relative growth, consumption, or digestive efficiencies. We conclude that dietary reducing agents must lower midgut redox potential to below -40 mV to significantly impact digestive efficiency. Arch.     

Determination of the redox potential of deazariboflavin by equilibration with flavins.

The redox potential of deazariboflavin has been determined for pH values from 5.5 to 9.2 by equilibration with riboflavin and lumiflavin 3-acetate. The position of the equilibrium with riboflavin was measured spectrophotometrically and fluorimetrically; the equilibrium potential with lumiflavin 3-acetate was measured spectrophotometrically and potentiometrically. The Em7 for deazariboflavin was found to be--0.273 +/- 0.003 V against the standard hydrogen electrode. Equilibrium with flavodoxin at pH 9.5 and 10.0 was also used to determine the redox potential of deazariboflavin at high pH values. The pK of dihydrodeazariboflavin was found from the break in the potential vs. pH diagram and from spectrophotometric pH titration. The pK value obtained by both methods is 7.00 +/- 0.05. We found that borate, a product of the reducing agent borohydride, complexed with the ribityl sidechain of deazariboflavin, causing a shift in the pK for the reduced form to values of about 8.     

Effect of titanium (III) citrate as reducing agent on growth of rumen bacteria.

We compared the growth of 10 strains of rumen bacteria in an anaerobic medium reduced with cysteine hydrochloride, dithiothreitol, or titanium (III) citrate. The redox potential of medium reduced with cysteine hydrochloride was -167.8 mV; with dithiothreitol it was -175.8 mV; and with titanium(III) citrate it was -302.4 mV at a concentration of 5 X 10(-4) M titanium and -403.9 mV at 2 X 10(-3) M titanium. Maximum growth of the strains was generally lower with dithiothreitol or titanium(III) citrate than with cysteine hydrochloride, although growth was greater than in medium lacking an added reducing agent. Strains for which cysteine was required or markedly stimulatory grew only poorly with titanium(III) citrate. No strain grew in medium with sodium citrate as the energy source. Titanium(III) citrate could be used to reduce anaerobic media for some rumen bacteria if the exclusion of a sulfur-containing reducing agent is required.     

Mutation of conserved residues in Escherichia coli thioredoxin: effects on stability and function.

Mutations were made in three highly conserved residues in Escherichia coli thioredoxin. An internal charged residue, Asp-26, was changed to an alanine. The mutant protein was more stable than the wild type. It can function as a substrate for thioredoxin reductase with a 10-fold increase in the Km over the wild type. Although the redox potential was not substantially changed from that of the wild type, thioredoxin D26A was a poor reducing agent for ribonucleotide reductase. Asp-26 apparently serves to maintain an optimal charge distribution in the active site region for interaction with other proteins. Mutation of a surface Pro-34 in the active site disulfide ring to a serine had little effect on protein stability. A slight decrease in the redox potential (9 mV) made thioredoxin P34S a better reducing agent for ribonucleotide reductase. In contrast, mutation of the internal cis Pro-76 to an alanine destabilized the protein. The data indicate a change had also occurred in the charge distribution in the active site region. Thioredoxin P76A had a higher redox potential than the wild type protein and was not an effective reducing agent for ribonucleotide reductase. It was concluded that this residue is essential for maintaining the conformation of the active site and the redox potential of thioredoxin.     

Redox regulates mammalian target of rapamycin complex 1 (mTORC1) activity by modulating the TSC1/TSC2-Rheb GTPase pathway

Mammalian target of rapamycin (mTOR) is a kinase that plays a key role in a wide array of cellular processes and exists in two distinct functional complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although mTORC2 is primarily activated by growth factors, mTORC1 is regulated by numerous extracellular and intracellular signals such as nutrients, growth factors, and cellular redox. Previous study has shown that cysteine oxidants sufficiently activate mTORC1 activity under amino acid-depleted conditions and that a reducing agent effectively suppresses amino acid-induced mTORC1 activity, thereby raising the possibility that redox-sensitive mechanisms underlie amino acid-dependent mTORC1 regulation. However, the molecular mechanism by which redox regulates mTORC1 activity is not well understood. In this study, we show that the redox-sensitive regulation of mTORC1 occurs via Rheb but not the Rag small GTPase. Enhancing cellular redox potential with cysteine oxidants significantly increases Rheb GTP levels. Importantly, modulation of the cellular redox potential with a cysteine oxidant or reducing agent failed to alter mTORC1 activity in TSC1(-/-) or TSC2(-/-) mouse embryonic fibroblast cells. Furthermore, a cysteine oxidant has little effect on mTOR localization but sufficiently activates mTORC1 activity in both p18(-/-) and control mouse embryonic fibroblast cells, suggesting that the redox-sensitive regulation of mTORC1 occurs independent of the Ragulator·Rag complex. Taken together, our results suggest that the TSC complex plays an important role in redox-sensitive mTORC1 regulation and argues for the activation of mTORC1 in places other than the lysosome upon inhibition of the TSC complex.     

ON THE INFLUENCE OF AGGREGATES ON THE MEMBRANE POTENTIALS AND THE OSMOTIC PRESSURE OF PROTEIN SOLUTIONS

1. It is shown that when part of the gelatin in a solution of gelatin chloride is replaced by particles of powdered gelatin (without change of pH) the membrane potential of the solution is influenced comparatively little. 2. A measurement of the hydrogen ion concentration of the gelatin chloride solution and the outside aqueous solution with which the gelatin solution is in osmotic equilibrium, shows that the membrane potential can be calculated from this difference of hydrogen ion concentration with an accuracy of half a millivolt. This proves that the membrane potential is due to the establishment of a membrane equilibrium and that the powdered particles participate in this membrane equilibrium. 3. It is shown that a Donnan equilibrium is established between powdered particles of gelatin chloride and not too strong a solution of gelatin chloride. This is due to the fact that the powdered gelatin particles may be considered as a solid solution of gelatin with a higher concentration than that of the weak gelatin solution in which they are suspended. It follows from the theory of membrane equilibria that this difference in concentration of protein ions must give rise to potential differences between the solid particles and the weaker gelatin solution. 4. The writer had shown previously that when the gelatin in a solution of gelatin chloride is replaced by powdered gelatin (without a change in pH), the osmotic pressure of the solution is lowered the more the more dissolved gelatin is replaced by powdered gelatin. It is therefore obvious that the powdered particles of gelatin do not participate in the osmotic pressure of the solution in spite of the fact that they participate in the establishment of the Donnan equilibrium and in the membrane potentials. 5. This paradoxical phenomenon finds its explanation in the fact that as a consequence of the participation of each particle in the Donnan equilibrium, a special osmotic pressure is set up in each individual particle of powdered gelatin which leads to a swelling of that particle, and this osmotic pressure is measured by the increase in the cohesion pressure of the powdered particles required to balance the osmotic pressure inside each particle. 6. In a mixture of protein in solution and powdered protein (or protein micellæ) we have therefore two kinds of osmotic pressure, the hydrostatic pressure of the protein which is in true solution, and the cohesion pressure of the aggregates. Since only the former is noticeable in the hydrostatic pressure which serves as a measure of the osmotic pressure of a solution, it is clear why the osmotic pressure of a protein solution must be diminished when part of the protein in true solution is replaced by aggregates.     

Reductive dechlorination of tetrachloroethene by two compost samples with different maturity

This study sets up microcosms using two types of compost samples, bagasse/manure compost, and yard-trimming compost with different maturity, to evaluate their capacity for reductive dechlorination of tetrachloroethene (PCE). The experimental results show that less matured compost samples could reduce 300 μM of PCE to ethene within 180 days of incubation. Decreasing initial PCE concentration and removing dissolved oxygen from the solution could enhance reducing efficiency. The solution remains near neutral pH throughout the experiment, and ethene emerged when the redox potential dropped to below -150 mV. Different microbial inhibition agents, such as 2-bromoethanesulfonic acid and sodium molybdate 2-hydrate, exhibit different effects on the dechlorination efficiency. The potential advantages of using compost to remove PCE are discussed. Overall, due to their high carbon content, diverse microbial activity, high buffer capacity, and complex physical structure, compost samples could serve as suitable media for dechlorinating PCE.     

Leucine 41 is a gate for water entry in the reduction of Clostridium pasteurianum rubredoxin

Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large. It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions. The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the driving forces of the proteins involved. To determine the structural relaxation in a redox protein, we have developed methods to hold a redox protein in its final oxidation state during crystallization while maintaining the same pH and salt conditions of the crystallization of the protein in its initial oxidation state. Based on 1.5 A resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins (Rd) from Clostridium pasteurianum (Cp), the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated. First, expansion of the [Fe-S] cluster and concomitant contraction of the NH...S hydrogen bonds lead to greater electrostatic stabilization of the extra negative charge. Second, a gating mechanism caused by the conformational change of Leucine 41, a nonpolar side chain, allows transient penetration of water molecules, which greatly increases the polarity of the redox site environment and also provides a source of protons. Our method of producing crystals of Cp Rd from a reducing solution leads to a distribution of water molecules not observed in the crystal structure of the reduced Rd from Pyrococcus furiosus. How general this correlation is among redox proteins must be determined in future work. The combination of our high-resolution crystal structures and molecular dynamics simulations provides a molecular picture of the structural rearrangement that occurs upon reduction in Cp rubredoxin.     

Application of the redox potential for controling a sulfide oxidizing bioreactor

The investigations described show that the formation of elemental sulfur from the biological oxidation of sulfide can be optimized by controling the redox state of the solution. The nonsoluble sulfur can be removed by gravity sedimentation and re-used as a raw material, i.e., in bioleaching processes. It was shown that, by supplying an almost stoichiometrical amount of oxygen to the recirculated gas phase, the formation of sulfate is minimized. The redox potential is mainly determined by the sulfide concentration because this compound has a high standard exchange current density with the platinum electrode surface. By maintaining a particular redox setpoint value, in fact, the reactor becomes a "sulfide-stat." It was shown that in a sulfide-oxidizing bioreactor the measured redox potential, using a polished redox electrode, is kinetically determined rather than thermodynamically. The optimal redox value for sulfur formation is between -147 and -137 mV (H2 reference electrode, 30 degrees C, pH 8). The presented results are currently used for controling several full-scale installations, which desulfurize biogas and high-pressure natural gas. Copyright 1998 John Wiley & Sons, Inc.     

Redox potential affects the measured heat resistance of Escherichia coli O157:H7 independently of oxygen concentration

Cells of Escherichia coli O157:H7 were heat-treated at 59 °C and enumerated in (i) anaerobic medium with a low redox potential, (ii) anaerobic media with the oxidizing agents potassium ferricyanide or 2,6-dichloroindophenol (DPIP) added to raise the redox potential, (iii) aerobic medium with a high redox potential and (iv) aerobic medium with the reducing agent dithiothreitol added to lower the redox potential. The measured heat-resistance was greatest when the enumeration medium was highly anaerobic due to the absence of oxygen and the presence of hydrogen and cysteine HCl. Measured heat resistance was influenced by the redox potential of the enumeration medium independently of the chemical used to adjust it and therefore, independently of the presence of oxygen. Sub-lethally heat-damaged cells regained their ability to grow in media of high redox potential at a similar rate whether the redox potential was increased by the addition of potassium ferricyanide, DPIP or oxygen.     

Physiology of organotrophic and lithotrophic growth of the thermophilic iron-reducing bacteria Thermoterrabacterium ferrireducens and Thermoanaerobacter siderophilus

Growth physiology of the iron-reducing bacteria Thermoterrabacterium ferrireducens and Thermoanaerobacter siderophilus was investigated. The stimulation of the organotrophic growth of T. ferrireducens and T. siderophilus in the presence of Fe(III) was shown to be due to the utilization of ferric iron as an electron acceptor in catabolic processes and not to the effect exerted on the metabolism by Fe(II) or by changes in the redox potential. It was established that Fe(III) reduction in T. ferrireducens is not a detoxication strategy. In T. siderophilus, this process is carried out to relieve the inihibitory effect of hydrogen. T. ferrireducens was shown to be capable of lithoautotrophic growth with molecular hydrogen as electron donor and amorphous ferric oxide as electron acceptor, in the absence of any organic substances. The minimum threshold of H2 consumption was 3 x 10(-5) vol % of H2. The presence of CO dehydrogenase activity in T. ferrireducens suggests that CO2 fixation in this organism involves the anaerobic acetyl-CoA pathway. T. siderophilus failed to grow under lithoautotrophic conditions. The fact that T. ferrireducens contains c-type cytochromes and T. sidrophilus lacks them confirms the operation of different mechanisms of ferric iron reduction in these species.     

Temperature-induced structural changes in putidaredoxin: a circular dichroism and UV-VIS absorption study

Putidaredoxin (Pdx) is an 11,400-Da iron-sulfur protein that sequentially transfers two electrons to the cytochrome P450cam during the enzymatic cycle of the stereospecific camphor hydroxylation. We report two transitions in the Pdx UV-VIS absorption and circular dichroism (CD) temperature dependencies, occurring at 16.3+/-0.5 degrees C and 28.4+/-0.5 degrees C. The 16.3 degrees C transition is attributed to the disruption of the hydrogen bonding of the active center bridging sulfur atom with cysteine 45 and alanine 46. The transition at 28.4 degrees C occurs exclusively in the Pdx(ox) at very nearly the same temperature as the earlier reported biphasicity in the redox potential. The formal potential temperature slope constancy reflects the relative stability of the concentration ratio of both oxidation states. The lower temperature transition affects both Pdx(red) and Pdx(ox) to a comparable extent, and their concentration ratio remains constant. In contrast, the 28.4 degrees C transition preferentially destabilizes Pdx(ox) thereby accelerating the formal potential negative shift and lower redox reaction entropy. There is evidence to suggest that disrupting hydrogen bonding of the iron ligating cysteines 45, 39 with residues threonine 47, serine 44, glycine 41, and serine 42 causes the 28.4 degrees C transition. The sensitivity of the UV-VIS absorption and CD spectroscopy to subtle structural protein backbone transitions is demonstrated.     

Vasorelaxing activity of stable powder preparations of dinitrosyl iron complexes with cysteine or glutathione ligands.

Vasorelaxant activity of new stable powder preparations of dinitrosyl iron complexes (DNIC) with thiol-containing ligands was investigated on rat abdominal aorta rings. The preparations preserve their physicochemical characteristics (EPR and optical absorption) if stored for a long time in dry air (at least half-year). Three preparations of DNIC were tested: diamagnetic dimeric DNIC with glutathione (DNIC-GS 1:2) or cysteine (DNIC-cys 1:2) and paramagnetic monomeric DNIC with cysteine (DNIC-cys 1:20). Being dissolved in physiological solution the preparations induced relaxation of vessel similarly to that by earlier described non-stable DNICs which should be stored in liquid nitrogen. The amplitudes and kinetic characteristics of the relaxation were dependent on the incorporated thiolate ligands. Rapid transient relaxation followed by significant tone recovery to stationary level (plateau) was observed for DNIC-cys 1:2. DNIC-cys 1:20 also induced initial rapid relaxation followed by incomplete tone recovery. DNIC-GS 1:2 induced slow developing and long lasting relaxation. NO scavenger, hydroxocobalamin (2x10(-5)M) eliminated the rapid transitory relaxation induced by DNIC-cys 1:20 and did not influence significantly on the plateau level. SOD increased duration of the DNIC-cys 1:2 and DNIC-cys 1:20 induced relaxation. The addition of 5x10(-5)M DNIC-cys 1:2 or DNIC-cys 1:20 induced long lasting vasorelaxation within 20min and more. However the EPR measurements demonstrated full rapid disappearance (within 1-2min) of both type of DNIC-cys in Krebs medium bubbled with carbogen gas. This was not the case for DNIC-GS 1:2. We suggested that the long lasting vasorelaxation observed during the addition of DNICs-cys was induced by S-nitrosocysteine derived from DNICs-cys and stabilized by EDTA in Krebs medium. The suggestion is in line with the fact that strong ferrous chelator bathophenantroline disulfonate (BPDS) which is capable of rapid degradation of DNICs did not abrogate the vasorelaxtion induced by DNIC addition.     

Detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis

Regulation of protein function by reversible cysteine-targeted oxidation can be achieved by multiple mechanisms, such as S-glutathiolation, S-nitrosylation, sulfenic acid, sulfinic acid, and sulfenyl amide formation, as well as intramolecular disulfide bonding of vicinal thiols. Another cysteine oxidation state with regulatory potential involves the formation of intermolecular protein disulfides. We utilized two-dimensional sequential non-reducing/reducing SDS-PAGE (diagonal electrophoresis) to investigate intermolecular protein disulfide formation in adult cardiac myocytes subjected to a series of interventions (hydrogen peroxide, S-nitroso-N-acetylpenicillamine, doxorubicin, simulated ischemia, or metabolic inhibition) that alter the redox status of the cell. More detailed experiments were undertaken with the thiol-specific oxidant diamide (5 mm), a concentration that induces a mild non-injurious oxidative stress. This increase in cellular oxidation potential caused global intermolecular protein disulfide formation in cytosolic, membrane, and myofilament/cytoskeletal compartments. A large number of proteins that undergo these associations were identified using liquid chromatography-mass spectrometry/mass spectrometry. These associations, which involve metabolic and antioxidant enzymes, structural proteins, signaling molecules, and molecular chaperones, were confirmed by assessing "shifts" on non-reducing immunoblots. The observation of widespread protein-protein disulfides indicates that these oxidative associations are likely to be fundamental in how cells respond to redox changes.     

Thiol regulation of the thylakoid electron transport chain--a missing link in the regulation of photosynthesis?

Avoidance of over-reduction of the chloroplast ferredoxin pool is of paramount importance for plants in avoiding oxidative stress. The redox state of this pool can be controlled through regulation of the thylakoid electron transport chain. A model is presented for regulation of this chain via a thiol reduction mechanism, possibly involving a thioredoxin. It is shown in isolated thylakoids that electron transport is inhibited by the thiol reducing agent dithiothreitol. The kinetics of this reduction are rapid and readily reversible. The midpoint redox potential is -365 mV at pH 7.7, with a pH dependency of about -90 mV/pH. At physiological pH values, this places the potential of the species titrated between that of ferredoxin and NADPH and thus in the right potential range to be regulating the redox poise of the ferredoxin pool. This is also close to the potential of NADPH-malate dehydrogenase, an enzyme known to be regulated by thioredoxin. Regulation of electron transport by thioredoxin provides a mechanistic link between the regulation of photosynthesis and gene expression by sugars and the redox regulation of gene expression mediated through the plastoquinone pool.     

Thiol-linked peroxidase activity of human sensitive to apoptosis gene (SAG) protein

SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, which is redox responsive and protects mammalian cells from apoptosis, is a metal chelator and a potential reactive oxygen species (ROS) scavenger, but its antioxidant properties have not been completely defined. Here, we show that SAG possesses a potent peroxidase property to decompose hydrogen peroxide in the presence of dithiothreitol (DTT). However, without DTT as a reducing equivalent, SAG was not able to destroy hydrogen peroxide. The peroxidase activity was completely abolished by the reaction of SAG with N -ethylmaleimide (NEM), a chemical modification agent for the sulfhydryl of proteins. These observations suggested that the sulfhydryl of cysteines in SAG could function as strong nucleophiles to destroy hydrogen peroxide. In addition to the peroxidase activity used to remove hydrogen peroxide, SAG also showed t -butylhydroperoxide ( t -BOOH) and fatty acid hydroperoxide-selective peroxidase activity.     

STUDIES ON BIOLUMINESCENCE : XV. ELECTROREDUCTION OF OXYLUCIFERIN.

Oxyluciferin may be reduced to luciferin at cathodes, when an electric current is passed through the solution, or at cathodes formed by metal couples in solution, or at cathodes of oxidation-reduction cells of the NaCl - Pt - Pt - Na₂S type. It is also reduced at those metal surfaces (Al, Mn, Zn, and Cd) which liberate nascent hydrogen from water, although no visible hydrogen gas separates from the surface. Molecular hydrogen does not reduce oxyluciferin even though very finely divided but will reduce oxyluciferin in contact with palladium. Palladium has no reducing action except in presence of hydrogen, and apparently acts as a catalyst by virtue of some power of converting molecular into atomic hydrogen. Conditions are described under which a continuous luminescence of luciferin can be obtained. This luminescence may be used as a test for atomic hydrogen. It is suggested that the steady luminescence of bacteria is due to continuous oxidation of luciferin to oxyluciferin and reduction of oxyluciferin to luciferin in different parts of the bacterial cell.