Production of superoxide radicals with pulse radiolysis of water with high linear energy transfer

The radiolysis of water with heavy ions of high linear energy transfer (LET) (-dE/dx) is characterized, in deaerated medium, by the production of superoxide anions, the radiolytic yields of which increase with the LET. Radiobiological interest in such radical species comes from the oxidative stress which may be generated by their dismutation in O2 and H2O2 in anoxic medium (radiotherapy with heavy ions). A brief review of the measurements of superoxide free radicals in aqueous solution by indirect or direct methods is presented. Moreover, some experimental results obtained by pulse radiolysis with Ar18+ ions (TEL = 290 keV x microm(-1)), are described. The interpretation of the kinetics takes into account the superoxide absorbance and that of hydrogen peroxide, which is present at the millisecond time scale.     

Dose-response of Fricke- and PAGAT-dosimetry gels in kilovoltage and megavoltage photon beams: Impact of LET on sensitivity

Purpose: Dosimetry of ionizing radiation quantifies the energy deposited by an incident beam to the medium. This study presents the relative response of two types of gel dosimeters describing their differences by estimating radiation chemical yields produced in water radiolysis.Methods: Two types of gel dosimeter were used, namely an acid ferrous ion solution infused with xylenol orange known as Fricke gel and a polymer gel based on acrylamide and N,N’-methylenebis(acrylamide) known as PAGAT. Samples were irradiated using two photon beam energies, one from a conventional X-ray tube operated at 44 kV and the other one from a LINAC operated at 6 MV. The dosimeters were analyzed by optical absorbance and magnetic resonance imaging. Additionally, the linear energy transfer of each beam was calculated using Monte Carlo simulations for further estimation of the radiation chemical yields produced during water radiolysis.Results: Obtained results for both gel dosimeters indicate that their response at 44 kV and 6 MV are different, regardless of the read-out technique. On average, the sensitivity at 44 kV was found to be 65 % of the response at 6 MV. The calculated radiation chemical yields are in agreement with the observed experimental results.Conclusions: The main reason for the difference in the response of the dosimeters may be related to the linear energy transfer of each photon beam, which varies the production of primary chemical species during water radiolysis.     

Monte Carlo calculation of the primary radical and molecular yields of liquid water radiolysis in the linear energy transfer range 0.3-6.5 keV/micrometer: application to 137Cs gamma rays

Monte Carlo simulations of the radiolysis of neutral liquid water and 0.4 M H(2)SO(4) aqueous solutions at ambient temperature are used to calculate the variations of the primary radical and molecular yields (at 10(-6)s) as a function of linear energy transfer (LET) in the range approximately 0.3 to 6.5 keV/micrometer. The early energy deposition is approximated by considering short (approximately 20-100 micrometer) high-energy (approximately 300-6.6 MeV) proton track segments, over which the LET remains essentially constant. The subsequent nonhomogeneous chemical evolution of the reactive species formed in these tracks is simulated by using the independent reaction times approximation, which has previously been used successfully to model the radiolysis of water under various conditions. The results obtained are in good general agreement with available experimental data over the whole LET range studied. After normalization of our computed yields relative to the standard radical and molecular yields for (60)Co gamma radiation (average LET approximately 0.3 keV/micrometer), we obtain empirical relationships of the primary radiolytic yields as a function of LET over the LET range studied. Such relationships are of practical interest since they allow us to predict a priori values of the radical and molecular yields for any radiation from the knowledge of the average LET of this radiation only. As an application, we determine the corresponding yields for the case of (137)Cs gamma radiation. For this purpose, we use the value of approximately 0.91 keV/micrometer for the average LET of (137)Cs gamma rays, chosen so that our calculated yield G(Fe(3+)) for ferrous-ion oxidation in air-saturated 0.4 M sulfuric acid reproduces the value of 15.3 molecules/100 eV for this radiation recommended by the International Commission on Radiation Units and Measurements. The uncertainty range on those primary radical and molecular yields are also determined knowing the experimental error (approximately 2%) for the measured G(Fe(3+)) value. The following values (expressed in molecules/100 eV) are obtained: (1) for neutral water: G(e(-)(aq)) = 2.50 +/- 0.16, G(H(.)) = 0.621 +/- 0.019, G(H(2)) = 0.474 +/- 0.025, G((.)OH) = 2.67 +/- 0.14, G(H(2)O(2)) = 0.713 +/- 0.031, and G(-H(2)O) = 4.08 +/- 0.22; and (2) for 0.4 M H(2)SO(4) aqueous solutions: G(H(.)) = 3.61 +/- 0.09, G(H(2)) = 0.420 +/- 0.019, G((.)OH) = 2.78 +/- 0.12, G(H(2)O(2)) = 0.839 +/- 0.037, and G(-H(2)O) = 4.46 +/- 0.16. These computed values are found to differ from the standard yields for (60)Co gamma rays by up to approximately 6%.     

The hydroxylation of the salicylate anion by a Fenton reaction and T-radiolysis: a consideration of the respective mechanisms

The yield of 2,3- and 2,5-dihydroxybenzoates (dHB's) from the reaction of .OH radicals with salicylate (SA) ions has been measured as a function of pH and in the presence of oxidants. Under steady-state radiolysis conditions, the production of these products occurs via the reactions .OH + SA----HO-SA. (radical adduct) HO-SA. H+.OH+----2-carboxyphenoxyl radical (SA.) + H2O HO-SA. + SA.----2,3-/2,5-dHB + SA The addition of the oxidants O2, Fe3+ edta, or Fe(CN)63- increases the relative yield of 2,5-dHB/2,3-dHB from about 0.2 to 1. A model to account for this effect is presented. Steady-state radiolyses of 3- and 4-hydroxybenzoate give dihydroxybenzoate products consistent with the phenol group being an ortho-para director in the electrophilic attack of the hydroxyl radical on the aromatic ring. A comparison of product distributions from the reaction of ferrous edta with hydrogen peroxide using salicylate as a scavenger strongly suggests that the same hydroxyl radical adducts are formed as in the radiation experiments.     

High-LET ion radiolysis of water: visualization of the formation and evolution of ion tracks and relevance to the radiation-induced bystander effect

Ionizing radiation-induced bystander effects, commonly observed in cell populations exposed to high-linear energy transfer (LET) radiations, are initiated by damage to a cellular molecule which then gives rise to a toxic signal exported to neighboring cells not directly hit by radiation. A major goal in studies of this phenomenon is the identification of this initial radiation-induced lesion. Liquid water being the main constituent of biological matter, reactive species produced by water radiolysis in the cellular environment are likely to be major contributors to the induction of this lesion. In this context, the radiation track structure is of crucial importance in specifying the precise location and identity of all the radiolytic species and their subsequent signaling or damaging effects. We report here Monte Carlo track structure simulations of the radiolysis of liquid water by four different impacting ions 1H+, 4He2+, 12C6+ and 20Ne10+, with the same LET ( approximately 70 keV/ microm). The initial radial distribution profiles of the various water decomposition products (eaq(-), *OH, H*, H2 and H2O2) for the different ions considered are presented and discussed briefly in the context of track structure theory. As an example, the formation and temporal evolution of simulated 24 MeV 4He2+ ion tracks (LET approximately 26 keV/microm) are reported for each radiolytic species from 1 ps to 10 micros. The calculations reveal that the ion track structure is completely lost by approximately 1 micros.     

Antioxidant and radical scavenging properties of curcumin

Curcumin (diferuoyl methane) is a phenolic compound and a major component of Curcuma longa L. In the present paper, we determined the antioxidant activity of curcumin by employing various in vitro antioxidant assays such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH*) scavenging, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) radical scavenging activity, total antioxidant activity determination by ferric thiocyanate, total reducing ability determination by the Fe(3+)-Fe(2+) transformation method, superoxide anion radical scavenging by the riboflavin/methionine/illuminate system, hydrogen peroxide scavenging and ferrous ions (Fe(2+)) chelating activities. Curcumin inhibited 97.3% lipid peroxidation of linoleic acid emulsion at 15 microg/mL concentration (20 mM). On the other hand, butylated hydroxyanisole (BHA, 123 mM), butylated hydroxytoluene (BHT, 102 mM), alpha-tocopherol (51 mM) and trolox (90 mM) as standard antioxidants indicated inhibition of 95.4, 99.7, 84.6 and 95.6% on peroxidation of linoleic acid emulsion at 45 microg/mL concentration, respectively. In addition, curcumin had an effective DPPH* scavenging, ABTS*(+) scavenging, DMPD*(+) scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, ferric ions (Fe(3+)) reducing power and ferrous ions (Fe(2+)) chelating activities. Also, BHA, BHT, alpha-tocopherol and trolox, were used as the reference antioxidant and radical scavenger compounds. According to the present study, curcumin can be used in the pharmacological and food industry because of these properties.     

Oxidative damage to fibronectin. II. The effect of H2O2 and the hydroxyl radical.

The effect of H2O2 and the hydroxyl radical (.OH) on fibronectin was investigated. .OH was generated in three ways: (i) by radiolysis with 60Co under N2O, or by the Fenton system using either (ii) equimolar Fe(2+)-EDTA and H2O2 or (iii) H2O2 and catalytic amounts of Fe(2+)-EDTA recycled with ascorbate. Each system had a different effect. H2O2 alone caused no changes, even at an 800-fold molar excess. Radiolytic .OH caused a rapid loss of tryptophan fluorescence, an increase in bityrosine fluorescence, and extensive crosslinking. The Fenton system using Fe-EDTA, H2O2, and ascorbate caused a loss in tryptophan fluorescence, a smaller increase in bityrosine than was seen with radiolytic .OH, and a threefold increase in carbonyl groups. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis fragmentation of fibronectin was seen. In contrast, when .OH was generated with equimolar Fe-EDTA and H2O2, the only change was a small increase in bityrosine fluorescence at the highest dose of oxidant. None of the systems used affected cysteine. All the changes except the loss of tryptophan by radiolytic .OH were completely inhibited with mannitol. The differences seen with radiolytic .OH and the Fe-EDTA, H2O2, ascorbate system were not solely due to O2 in the latter system since similar results were obtained under N2. The differences between radiolytic .OH and the Fenton systems could be partly due to the components of the latter systems reacting with .OH and thus competing with fibronectin. Our results demonstrate that the extent and type of fibronectin damage by .OH is dependent on the mode of radical generation.     

A Monte-Carlo step-by-step simulation code of the non-homogeneous chemistry of the radiolysis of water and aqueous solutions—Part II: calculation of radiolytic yields under different conditions of LET,pH, and temperature

The importance of the radiolysis of water in the initial events following irradiation of biological systems has motivated considerable theoretical and experimental work in the field of radiation chemistry of water and aqueous systems. These studies include Monte-Carlo simulations of the radiation track structure and of the non-homogeneous chemical stage, which have been successfully used to calculate the yields of radiolytic species (H^·, ^·OH, H₂, H₂O₂, e_aq⁻, …). Most techniques used for the simulation of the non-homogeneous chemical stage such as the independent reaction time (IRT) technique and diffusion kinetics methods do not calculate the time evolution of the positions of the radiolytic species. This is a major limitation to their extension to the simulation of the irradiation of radiobiological systems. Step-by-step (SBS) simulation programs provide such information, but they are very demanding in term of computer power and storage capacity. Recent improvements in computer performance now allow the regular use of the SBS method in radiation chemistry simulations. In the first of a series of two papers, the SBS method has been reviewed in details and the implementation of a SBS code has been discussed. In this second paper, the results of several studies are presented: (1) the time evolution of the radiolytic yields from the formation of the radiation track to 10⁻⁶ s; (2) the effect of pH on yields (pH ~ 0.4–7.0); (3) the effect of proton energy (and LET) on yields (300 MeV-0.1 MeV), and iv) the effect of the ion type (¹H⁺, ⁴He²⁺, ¹²C⁶⁺) on yields. Nonbiological applications, i.e., the study of the temperature on the yields (about 25–300°C) and the simulation of the time evolution of G(Fe³⁺) in the Fricke dosimeter are also discussed.     

Methodology in the radiolysis of biochemical compounds with cyclotron beams at low flux densities.

A modification of the standard beam-optics of the LBL 88-Inch Cyclotron now makes it practicable to use cyclotron radiations in the detailed study of the effects of linear energy transfer (LET) in the radiation chemistry of organic compounds in the solid state. Dosages and dose-rates are comparable to those employed in conventional γ-ray studies. The modification involves passing the focused beam through a pair of “beam-sweeping” electromagnets so that a circular target area up to 10 cm in diameter can be uniformly irradiated with beams in the nanoamp range. Yield data are given for the radiolysis of the Fricke dosimeter with beams of H⁺, H⁺², Be⁺⁴, C⁺⁶ and Ne⁺¹⁰ at ～10 MeV per nucleon. Preliminary data on the effects of LET in the radiolysis of solid glycine are reported.     

On (3)H beta-particle and (60)Co gamma irradiation of aqueous systems

The chemistry of water and aqueous solutions is very different after irradiation with (3)H beta particles and high-energy electrons or (60)Co gamma rays. The greater the linear energy transfer (LET) of the medium for (3)H beta particles compared to high-energy electrons or (60)Co gamma rays leads to an increased local concentration of reactants. There is an increased amount of intratrack chemistry, which reduces the escape yield of and OH by about 50%, but increases the yield of H(2) by about 50% and of H(2)O(2) by about 35%. Analysis of stochastic-diffusion kinetic calculations employing simulated track structures reveals that the yield of H(2) produced by diffusion-kinetic processes increases significantly for (3)H beta particles compared to (60)Co gamma radiation, while production of H(2) by sub-picosecond processes is essentially the same. In both (3)H beta-particle and (60)Co gamma radiolysis, the reactions + and are equally important in the production of H(2). In the former case, each reaction has a yield of approximately 0.18, and in the latter a yield of approximately 0.08. In neutral water, the reaction (H + H) is negligible. The yield of Fe(III) in (3)H beta-particle radiolysis of the Fricke dosimeter is much smaller than in radiolysis with more energetic electrons. Simulations show that this change is primarily due to the reduced escape yield of H, formed from the scavenging of by the bulk H(3)O(+) of the acid. The chemical differences observed in experiments, and in calculations, reflect the underlying structure of the electron tracks: Examination of the track structure simulations demonstrates that primary events are considerably more well-separated in high-energy electron tracks compared to (3)H beta-particle tracks.     

Effects of glutaraldehyde polymerization on oxygen transport and redox properties of bovine hemoglobin.

Crosslinking of bovine Hb (HbBv) with glutaraldehyde produces a mixture of low oxygen affinity (P(50)) tetrameric and polymeric Hb species (PolyHbBv). Under physiological conditions the P(50) of HbBv and PolyHbBv were 27 and 35 mmHg, respectively. The dependence of the P(50) on pH and chloride ions and the cooperativity (n(50)) of the protein were diminished as a result of glutaraldehyde modification. Rapid kinetic studies showed greater overall rates of oxygen dissociation (k(off)) with little or no change in the association of CO (k(on)) to the modified protein. The rate of nitric oxide (NO)-induced oxidation of the PolyHbBv was slightly lower than that of HbBv. Autoxidation rate of PolyHbBv was 1.4 times faster than that of HbBv. The reaction of hydrogen peroxide (H2O2) with the ferrous (Fe(2+)) and ferric (Fe(3+)) forms of the proteins led to the formation of a more stable ferrylHb (Fe(4+)) in the case of PolyHbBv. Glutaraldehyde polymerization of HbBv alters its normal allosteric mechanisms, autoxidation kinetics and other related redox properties, which may compromise its function and cause greater toxicity when used as an oxygen transport fluid.     

Apoptotic, antioxidant and antiradical effects of majdine and isomajdine from Vinca herbacea Waldst. and kit

In the present study, apoptotic, antioxidant and antiradical effects of majdine and isomajdine from Vinca herbacea Waldst. and Kit were studied. For testing the possible apoptotic effects of majdine and isomajdine from V. herbacea, DNA fragmentation assay was conducted on the rat brain cortical tissue homogenates, in vitro. Also their possible effects on mitochondrial activity were tested by using the same tissue samples of rats. In addition, the antioxidant activity of isomajdine and majdine was determined using various in vitro antioxidant assays, including 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS(?+)) radical scavenging and N,N-dimethyl-p-phenylenediamine (DMPD(?+)) radical scavenging, ferric ions (Fe(3+)) and cupric ions (Cu(2+)) reducing abilities and ferrous ions (Fe(2+)) chelating activity. On the other hand, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were used as reference antioxidants.     

Pulse radiolysis of aromatic amino acids — State of the art

Summary The pulse radiolysis method as well as the primary processes of water radiolysis and the spectroscopic characteristics of H, OH, HO₂/O₂ ^– and e _aq ^- are briefly presented. Subsequently, kinetic and spectroscopic data of the transients resulting from the resolved multi site attack on aromatic amino acids are discussed. The reactivity of H and e _aq ^- with the same substrates, as well as the effect of oxygen on the major radiolytic processes are reviewed. Finally, the formation of tryptophan radical cation is mentioned shortly. The presented radiation mechanisms are the fundamentals for radiolytic processes occurring in proteins, enzymes and hormones in the living cells.     

Reactivity of chalcones with 1-hydroxymethyl radicals

Reactivity of chalcones with reactive species issued from methanol radiolysis was investigated in the absence or presence of dioxygen. Chalcones are natural antioxidants that are present in fruit and vegetables. Their degradation in the radiolysed solutions was followed by HPLC, NMR, FAB-LSIMS mass spectroscopy and analytical TLC in deaerated solution. Among the 18 identified radiolytic compounds, 16 were new. The formation of the radiolytic products was not influenced by A- and B-ring substitutions. To explain the degradation process, we thus suggested that the primary step was an attack of the alpha,beta-double bond by the 1-hydroxymethyl radical, either at C(alpha) or at C(beta). This step was followed by addition, cyclization or bond dissociations. Different chemical pathways were discussed that implicate the reactive species issued from methanol radiolysis. This paper highlights the relative importance of the different radical species, especially the carbon-centered radical, 1-hydroxymethyl (HMR) and the corresponding oxygen-centered isomer. In addition, an interesting unusual role of dioxygen should be noted; indeed, in the presence of dioxygen, degradation of chalcones was inhibited.     

Kinetic behavior of the monodehydroascorbate radical studied by pulse radiolysis

The reactions of the monodehydroascorbate radical (As.-) with various biological molecules were investigated by pulse radiolysis. As.- reacted with both fully reduced and semiquinone forms of hepatic NADH-cytochrome b5 reductase with second-order rate constants of 4.3 x 10(6) and 3.7 x 10(5) M-1 s-1, respectively, at pH 7.0. In contrast, no reaction of As.- with ferrous cytochrome b5 could be detected by pulse radiolysis, whereas the oxidation of cytochrome b5 by As.- was observed by ascorbate-ascorbate oxidase method. This suggests that the rate constant of As.- with the ferrous cytochrome b5 must be several orders in magnitude smaller than that of the disproportionation of As.-. On the other hand, As.- reduced Fe3+EDTA with a second-order rate constant of 4.0 x 10(6) M-1 s-1 but did not reduce ferric hemoproteins such as metmyoglobin, methemoglobin, and cytochrome b5 by either the pulse radiolysis or the ascorbate-ascorbate oxidase method.     

Microbial leaching of metals from sulfide minerals

Microorganisms are important in metal recovery from ores, particularly sulfide ores. Copper, zinc, gold, etc. can be recovered from sulfide ores by microbial leaching. Mineral solubilization is achieved both by 'direct (contact) leaching' by bacteria and by 'indirect leaching' by ferric iron (Fe(3+)) that is regenerated from ferrous iron (Fe(2+)) by bacterial oxidation. Thiobacillus ferrooxidans is the most studied organism in microbial leaching, but other iron- or sulfide/sulfur-oxidizing bacteria as well as archaea are potential microbial agents for metal leaching at high temperature or low pH environment. Oxidation of iron or sulfur can be selectively controlled leading to solubilization of desired metals leaving undesired metals (e.g., Fe) behind. Microbial contribution is obvious even in electrochemistry of galvanic interactions between minerals.     

Reactions of the NAD radical with higher oxidation states of horseradish peroxidase

The reactions of the NAD radical (NAD.) with ferric horseradish peroxidase and with compounds I and II were investigated by pulse radiolysis. NAD. reacted with the ferric enzyme and with compound I to form the ferrous enzyme and compound II with second-order rate constants of 8 X 10(8) and 1.5 X 10(8) M-1 s-1, respectively, at pH 7.0. In contrast, no reaction of NAD. with native compound II at pH 10.0 nor with diacetyldeutero-compound II at pH 5.0-8.0 could be detected. Other reducing species generated by pulse radiolysis, such as hydrated electron (eaq-), superoxide anion (O2-), and benzoate anion radical, could not reduce compound II of the enzyme to the ferric state, although the methylviologen radical reduced it. The results are discussed in relation to the mechanism of catalysis of the one-electron oxidation of substrates by peroxidase.     

Antioxidant activity of L-adrenaline: a structure-activity insight

L-adrenaline belongs to a group of the compounds known as catecholamines, which play an important role in the regulation of physiological process in living organisms. The antioxidant activity and antioxidant mechanism of L-adrenaline was clarified using various in vitro antioxidant assays including 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), N,N-dimethyl-p-phenylenediamine (DMPD(+)), and superoxide anion radicals (O(2)(-)) scavenging activity, hydrogen peroxide (H(2)O(2)), total antioxidant activity, ferric ions (Fe(3+)) and cupric ions (Cu(2+)) reducing ability, ferrous ions (Fe(2+)) chelating activity. L-adrenaline inhibited 74.2% lipid peroxidation of a linoleic acid emulsion at 30 microg/mL concentration. On the other hand, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol and trolox displayed 83.3, 82.1, 68.1 and 81.3% inhibition on the peroxidation of linoleic acid emulsion at the same concentration, respectively. BHA, BHT, alpha-tocopherol and trolox were used as reference antioxidants and radical scavenger compounds. Moreover, this study will bring an innovation for further studies related to antioxidant properties of L-adrenaline. According to present study, L-adrenaline had effective in vitro antioxidant and radical scavenging activity.     

Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2'-deoxyguanosine in solutions of free 2'-deoxyguanosine and calf thymus DNA

Formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in solutions of free 2'-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+). The oxidation of Fe2+ to Fe3+ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe2+ >or=phosphate concentration, the rate of Fe2+ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe2+ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe2+ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)-HCl.     

Balance of macroergic compounds during the growth of Thiobacillus ferrooxidans

The balance of energy-rich compounds (ERC) was drawn up for the growth of Thiobacillus ferrooxidans in a medium with ferrous ions as an energy source. The balance items and the phosphorylating efficiency of oxidation (P/2e-) were calculated basing on the experimental yield values using the ERC balance equation. At a specific growth rate of 0.1 h-1, 55% of ferrous ions are used for the synthesis of cell biomass, 7.5% for maintainance, 4% of the ions are oxidized to reduce NAD+, and 34% are used to produce ERC necessary for the reduction. Here, 24% of ERC are used for the synthesis of monomers from CO2, 42% for the production of NADH, 24% for the biomass synthesis from monomers, and 10% for maintaining cell activity. The P/2e- for the oxidation of ferrous ions is 0.19 mole of ERC per 2e-. This is possible only when the [Fe3+]/[Fe2+] ratio in the cell periplasm is 1 X 10(3)-1 X 10(4).