Stereospecific oxidation of ascorbic acid by chiral association complexes between polypeptides and iron(III) chelate ions

2,2,′,2″,2?-Tetrapyridineiron(III) complex ions anchored to poly(L -glutamate) (FeL) or poly(D -glutamate) (FeD) were used as catalysts for the H₂O₂ oxidation of L (+)-ascorbic acid at pH 7 and varying complex:polymer-residue molar ratios [C]/[P]. Evidence is produced that the reaction is a composite process reflecting contributions from parallel pathways, one of which corresponds to a catalytic route and is [H₂O₂]-independent and the other to an uncatalyzed electron-transfer process between the ascorbate anion and hydrogen peroxide. Stereospecific effects in the catalysis are observed on increasing the complex:polymer ratio, which corresponds to an increase of the amount of α-helical fraction in the polypeptide supports (x_a). Thus, at [C]/[P] = 0.01 (x_a < 0.05), k_FeD/k_FeL = 1.0; but at [C]/[P] = 0.20 (x_a ≈ 0.70), k_FeD/k_FeL = 4.0 ± 0.5, k_FeD and k_FeL being the second-order rate constants of the electron-transfer reaction between the FeD or FeL isomer of the asymmetric catalyst and the L -ascorbate anion. The activation energies were found to increase markedly on going from the former to the latter complex:polymer ratio but, at the same time, to exhibit equal values with both enantiomeric catalysts. Stereoselectivity therefore appears to be an entropy-controlled process, arising from the conformational rigidity of the precursor complex, which very likely sees the substrate molecules bound to the chiral residues of the ordered polymer surrounding the active sites. The implications of the stereochemical features of the substrate–catalyst adduct on the mechanism of electron transfer are also discussed. Evidence is presented that the asymmetric [Fe(tetpy)(OH)₂]⁺–polyelectrolyte systems play the additional role of environmental controller of the uncatalyzed oxidation of the L -ascorbate anion.     

Saccharose complexes of manganese in different oxidation states

The interaction between saccharose and manganese in different oxidation states was studied in alkaline media by polarographic, potentiometric, ESR spectroscopic and UV-Vis spectrophotometric methods. The results showed that stable manganese(II) and manganese(III) complexes and a complex of manganese(II,III) in a mixed oxidation state were formed with the composition [Mn^IIL(OH)₂], [Mn₂^IIIL₂(OH)₈]²⁻ and [Mn^IIMn^IIIL₂(OH)₆]⁻, respectively. The manganese(II)-saccharose complex was shown to dimerize in alkaline media. The stability constants of the Mn(II,III) and Mn(III) complexes were determined. The oxidation of the manganese(II)-saccharose complex by a stoichiometric amount of K₃ [FeCN]₆ resulted in the formation of the manganese(III) and manganese(IV) complexes. However, oxidation by molecular oxygen only yielded the manganese(III) complex which reduced spontaneously in inert atmosphere to the mixed valence Mn(II,III) complex. The latter was able to be oxidized again by oxygen to the Mn(III) complex. This process proved to be reversible and could be repeated several times.     

Effects of scavengers of oxygen free radicals on the anaerobic oxidation of 6-hydroxydopamine by H2O2

H₂O₂, a product of the aerobic autoxidation of 6-hydroxydopamine, is also consumed as a reactant, contributing progressively more to the oxidation as the concentration of O₂ becomes limiting H₂O₂ is a less effective oxidant than O₂, since the anaerobic peroxidatic oxidation of 6-hydrodopamine is slower than the aerobic oxidation by three orders of magnitude. The anaerobic peroxidation was inhibited by the hydroxyl scavengers mannitol (13–40%), glucose (41–62%) and benzoate (15–100%), implying a catalytic role for ^.OH. -9e strongly inhibitory action of desferrioxamine (76–91%), regardless of which other scavengers were present, suggests a specific role for iron in the reaction, despite the use of Chelex 100-treated buffers. Further addition of diethylenetriaminepentaacetate (DTPA), benzoate or formate to the desferrioxamine-treated reactions resulted in complete inhibition. In contrast, the presence of DTPA alone, accelerated the reaction by 160%. This acceleration is in part due to stimulation by DTPA of production of ^.OH (by Fenton-type reactions), since it was partially prevented by the hydroxyl scavengers benzoate (32% inhibition) and glucose (41%). Thus, DTPA inhibits the participation of metals other than iron, but potentiates the catalytic role of iron, in the reduction of hydrogen peroxide. The semidehydromannitol radical can reduce the DTPA-Fe³⁺ chelate directly, since mannitol further accelerated the DTPA-stimulated peroxidation (by 55%). Superoxide dismutase also accelerated the reaction (by 57–84%). This activation was seen regardless of which other scavengers were present. These effects are explained in terms of potentiating or moderating interactions among the reactive intermediates which propagate the overall reaction.     

Stereoselective electron transfer between chiral substrates and metal chelates anchored to polypeptides

Electron transfer from ortho-dihydroxy substrates, such as L(+)-ascorbic acid, L-adrenaline, and L-dopa, to iron(III) in [Fe(tetpy)(OH)2]+ ions anchored to sodium poly(L-glutamate) (FeTL) or poly(D-glutamate) (FeTD) was found to proceed stereoselectively when structurally ordered and partially shielded active sites prevent easy approach for redox partner. Oxidant-reductant interactions are then mediated by the polypeptide, whose conformational asymmetry ensures an efficient sterically discriminating environment. Evidence is produced that stereoselectivity chiefly arises from transition state effects, while thermodynamic discrimination is of minor importance. Theoretical models of the diastereomeric electron-transfer complexes were constructed by conformational energy calculations based on Coulombic, nonbonded, and hydrogen-bonded energy terms. The molecular parameters of the models enabled "differential" thermodynamic functions of the diastereomeric pairs and stereoselectivity to be evaluated and satisfactorily compared with those experimentally determined. The models give good insight into the observed topochemical phenomena and support the idea that stereoselectivity is coupled with a remote attack mechanism on the central metal ion where the peripheral tetpy ligand of the active sites acts as an electron-transfer agent.     

Pulse-radiolytic investigations of catechols and catecholamines II. Reactions of Tiron with oxygen radical species

Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (°OH), superoxide anions (O₂^?) or a combination of both oxygen radicals. The rate constant with °OH radicals was determined at 1.0·10⁹ M^?1·s^?1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11–16), the rate constant with O₂^? of 1.0·10⁷ M^?1·s^?1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with °OH and O₂^? of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of °OH radicals, leading to the same products with catechol compounds.     

Copper(II), nickel(II) and zinc(II) complexes of N-acetyl-His-Pro-His-His-NH2: equilibria, solution structure and enzyme mimicking

The copper(II), nickel(II) and zinc(II) binding ability of the multi-histidine peptide N-acetyl-His-Pro-His-His-NH₂ has been studied by combined pH-potentiometry and visible, CD and EPR spectroscopies. The internal proline residue, preventing the metal ion induced successive amide deprotonations, resulted in the shift of this process toward higher pH values as compared to other peptides. The metal ions in the parent [ML]²⁺ complexes are exclusively bound by the three imidazole side chains. In [CuH₋₁L]⁺, formed between pH 6-8, the side chains of the two adjacent histidines and the peptide nitrogen between them are involved in metal ion binding. The next deprotonation results in the proton loss of the coordinated water molecule (CuH₋₁L(OH)). The latter two species exert polyfunctional catalytic activity, since they possess superoxide dismutase-, catecholase- (the oxidation of 3,5-di-tert-butylcatechol) and phosphatase-like (transesterification of the activated phosphoester 2-hydroxypropyl-4-nitrophenyl phosphate) properties. On further increase of the pH rearrangement of the coordination sphere takes place leading to the [CuH₋₃L]⁻ species, the deprotonated amide nitrogen displaces a coordinated imidazole nitrogen from the equatorial position of the metal ion. The shapes of the visible and CD spectra reflect a distorted arrangement of the donor atoms around the metal ion. In presence of zinc(II) the species [ZnL]²⁺ forms only above pH 6, which is shortly followed by precipitation. On the other hand, the [NiL]²⁺ complex is stable over a wide pH range, its deprotonation takes place only above pH 8. At pH 10 an octahedral NiH₋₂L species is present at first, which transforms slowly to a yellow square planar complex.     

Use of Fenton's Reagent for the Degradation of TCE in Aqueous Systems and Soil Slurries

Fenton's reaction is comprised of hydrogen peroxide (H₂O₂) catalyzed by iron, producing the hydroxyl radical (·OH), a strong oxidant. ·OH in turn may react with H₂O₂ and iron and is capable of destroying a wide range of organic contaminants. In this laboratory study, Fenton's reaction was observed in aqueous and soil slurry systems using trichloroethylene (TCE) as the target contaminant, with the goal of maximizing TCE degradation while minimizing H₂O₂ degradation. Fenton's reaction triggers a complex matrix of reactions involving ·OH, H₂O₂, iron, TCE, and soil organics. In soil slurries with a high fraction of organic carbon (f_OC), iron tends to sorb to soil organics and/or particles. In aqueous systems the optimal ratio of H₂O₂:Fe²⁺:TCE to degrade TCE in a timely fashion, minimize costs, and minimize H₂O₂ degradation is 300?mg/L: 25?mg/L: 60?mg/L (19:1:1 molar ratio), while soil slurries with a f_OC up to approximately 1% and a soil:water ratio of 1:5 (weight ratio) require about ten times the amount of H₂O₂, the optimal ratio being 3000?mg/L: 5?mg/L: 60?mg/L (190:0.2:1 molar ratio). TCE degradation rates were observed to decrease in soil slurries with higher f_OC because of competition by soil organic matter, which appears to act as a sink for ·OH. H₂O₂ degradation rates tended to increase in soil slurries with higher f_OC, most likely due to increased demand for ·OH by soil organics, increased available iron and other oxidation processes.     

Macrocyclic dinuclear Zn(II) complexes: synthesis, structure and hydrolysis of tris(p-nitrophenyl) phosphate

A series of mono- and dinuclear zinc complexes of 3,6,9,17,20,23-hexaaza-29,30-dihydroxy-13,27-dimethyl-tricyclo[23,3,1^11,15]triaconta-1(28),11,13,15(30),25,26-hexaene (H₂L or BDBPH) have been defined in solution by potentiometry. The crystal structure of [Zn₂C₂₆H₄₀N₆O₂(CH₃OH)₂]·Br₂ has been determined by X-ray. Each zinc ion is coordinated to three nitrogen atoms, a bridged-phenolic oxygen atom, and a methanolic oxygen atom, which define a six-coordinated octahedron. Bond lengths of ZnN are in the range of 2.104(3)-2.120(3) Å and distances between Zn and O (bridged-phenolic oxygen) are 2.052(2), 2.062(2) Å, respectively. The dinuclear complexes: [Zn₂L]²⁺ and [Zn₂L(OH)]⁺ play crucial roles in hydrolytic reaction of tris(4-nitrophenyl)phosphate. A possible mechanism showed that [Zn₂L(OH)]⁺ acts as a nucleophile and [Zn₂L]²⁺ stabilizes the formation of the intermediate: [Zn₂L-BNP].     

Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis

Nitrous oxide (N₂O), a greenhouse gas, is emitted during autotrophic and heterotrophic ammonia oxidation. This emission may result from either coupling to aerobic denitrification, or it may be formed in the oxidation of hydroxylamine (NH₂OH) to nitrite (NO₂ ⁻). Therefore, the N₂O production during NH₂OH oxidation was studied with Alcaligenes faecalis strain TUD. Continuous cultures of A. faecalis showed increased N₂O production when supplemented with increasing NH₂OH concentrations. ¹⁵N-labeling experiments showed that this N₂O production was not due to aerobic denitrification of NO₂ ⁻. Addition of ¹⁵N-labeled NH₂OH indicated that N₂O was a direct by-product of NH₂OH oxidation, which was subsequently reduced to N₂. These observations are sustained by the fact that NO₂ ⁻ production was low (0.23 mM maximum) and did not increase significantly with increasing NH₂OH concentration in the feed. The NH₂OH-oxidizing capacity increased with increasing NH₂OH concentrations. The apparent V _max and K _m were 31 nmol min⁻¹ mg dry weight⁻¹ and 1.5 mM respectively. The culture did not increase its growth yield and was not able to use NH₂OH as the sole N source. A non-haem hydroxylamine oxidoreductase was partially purified from A. faecalis strain TUD. The enzyme could only use K₃Fe(CN)₆ as an electron acceptor and reacted with antibodies raised against the hydroxylamine oxidoreductase of Thiosphaera pantotropha. Received: 1 September 1998 / Received revision: 5 November 1998 / Accepted: 7 November 1998     

In Vitro Effect of H2O2, Some Transition Metals and Hydroxyl Radical Produced Via Fenton and Fenton-Like Reactions,on the Catalytic Activity of AChE and the Hydrolysis of ACh

It is well known that the principal biomolecules involved in Alzheimer’s disease (AD) are acetylcholinesterase (AChE), acetylcholine (ACh) and the amyloid beta peptide of 42 amino acid residues (Aβ42). ACh plays an important role in human memory and learning, but it is susceptible to hydrolysis by AChE, while the aggregation of Aβ42 forms oligomers and fibrils, which form senile plaques in the brain. The Aβ42 oligomers are able to produce hydrogen peroxide (H₂O₂), which reacts with metals (Fe²⁺, Cu²⁺, Cr³⁺, Zn²⁺, and Cd²⁺) present at high concentrations in the brain of AD patients, generating the hydroxyl radical (^·OH) via Fenton (FR) and Fenton-like (FLR) reactions. This mechanism generates high levels of free radicals and, hence, oxidative stress, which has been correlated with the generation and progression of AD. Therefore, we have studied in vitro how AChE catalytic activity and ACh levels are affected by the presence of metals (Fe³⁺, Cu²⁺, Cr³⁺, Zn²⁺, and Cd²⁺), H₂O₂ (without Aβ42), and ^· OH radicals produced from FR and FLR. The results showed that the H₂O₂ and the metals do not modify the AChE catalytic activity, but the ^·OH radical causes a decrease in it. On the other hand, metals, H₂O₂ and ^·OH radicals, increase the ACh hydrolysis. This finding suggests that when H₂O₂, the metals and the ^·OH radicals are present, both, the AChE catalytic activity and ACh levels diminish. Furthermore, in the future it may be interesting to study whether these effects are observed when H₂O₂ is produced directly from Aβ42.     

Atmospheric Oxidation of Hydrocarbons

Hydrocarbon oxidation in the atmosphere proceeds generally by the following sequence of reactions: hydrocarbon + OH → alkyl radical + H₂O, alkyl radical + O,(³I) → alkylperoxy radical, alkylperoxy radical + NO → alkoxy radical + NO₂, alkoxy radical + O₂(³X) -→ aldehyde + HO,. The atmospheric lifetimes of hydrocarbons are determined by their reactivity towards OH as well as by the average OH concentration level. They are compound specific and vary from several hours to several years. Hydrocarbon oxidation chains couple with other trace gases (O_v, HO_x, and NO_v). For the conditions of the average continental atmosphere an increase of the oxidative potential (HO_v, O_x) is predicted through hydrocarbon oxidation.     

Synthesis,Crystal Structure,and Biological Activities of Two Chiral Mononuclear Mn(III) Complexes

Two new chiral mononuclear Mn^(III) complexes, [Mn L ^{( R )}Cl (C₂H₅OH)]?C₂H₅OH ( 1 ) and [Mn L ^{( S )} (CH₃OH)₂]Cl?CH₃OH ( 2 ), {H₂ L = (R,R)‐or (S,S)‐N,N’‐bis‐(2‐hydroxy‐1‐naphthalidehydene)‐cyclohexanediamine} were synthesized and characterized by various physicochemical techniques. Bond valence sum (BVS) calculations and the Jahn‐Teller effect indicate that the Mn centers are in a +3 oxidation state. The statuses of the two complexes in the solution were confirmed as a pair of enantiomers by electrospray ionization, mass spectrometry (ESI‐MS) spectrum. The binding ability of the complexes with calf thymus CT‐DNA was investigated by spectroscopic and viscosity measurements. Both of the complexes could interact with CT‐DNA via an intercalative mode with the order of 1 ( R ‐enantiomer) > 2 ( S ‐enantiomer). Under the physiological conditions, the two compounds exhibit efficient DNA cleavage activities without any external agent, which also follows the order of R ‐enantiomer > S ‐enantiomer. Interestingly, the concentration‐dependent DNA cleavage experiments indicate an optimal concentration of 17.5 μM. In addition, the interaction of the compounds with bovine serum albumin (BSA) was also investigated, which indicated that the complexes could quench the intrinsic fluorescence of BSA by a static quenching mechanism. Chirality 27:142‐150, 2015. © 2014 Wiley Periodicals, Inc.     

Laser-excited raman spectroscopy of biomolecules. XII. Thermally induced conformational changes in poly(L-glutamic acid)

The α-helical from of poly(L -glutamic acid) [α-poly(Glu)] gives rise to the same amide I and III lines as α-poly(γ-benzyl-L -glutamate) at 1652 and 1296 cm^?1, respectively. The latter is a superposition of the amide III line near 1290 cm^?1 and a line deu to vibrational made of CH₂ groups of the side chain near 1300 cm^?1. A line at 924 cm^?1 is tentatively identified as characteristics of α-poly(Glu). Both the β₁- and β₂- forms of poly(Glu) give rise to characteristic of β-amide. III frequencies that are similar because of their similar backbone structures. Differences in the conformations of their side chains and in the environments of the backbone are reflected in the region 800–1200 cm^?1 and in the amide I. A line at 1042 cm^?1 and a pair at 1021 and 1059 cm^?1 are tentatively assigned as characteristic of β₁-poly(Glu) and β₂-poly(Glu), respectively. The α-β₂ transition in poly(L -Glu⁷⁸L -Val²²) is shown by the appearance of all the β₂-characteristic lines in the thermally transformed sample. The same features observed in poly(L -Glu⁹⁵L -Val⁵) also indicate that the α-β₂ transition of poly(Glu) is facilitated by the presence of L -valine and that the content of L -valine is not critical for this purpose. Investigation of the Raman spectra of the calcium, strontium, barium and sodium slats of poly(Glu) shows that these salts, under the conditions of preparation used, all the have random-coil conformations.     

Potent methyl oxidation of 5-methyl-2′-deoxycytidine by halogenated quinoid carcinogens and hydrogen peroxide via a metal-independent mechanism

Halogenated quinones are a class of carcinogenic intermediates and are newly identified chlorination disinfection by-products in drinking water. We found recently that the highly reactive and biologically important hydroxyl radical (^•OH) can be produced by halogenated quinones and H₂O₂ independent of transition metal ions. However, it is not clear whether these quinoid carcinogens and H₂O₂ can oxidize the nucleoside 5-methyl-2′-deoxycytidine (5mdC) to its methyl oxidation products and, if so, what the underlying molecular mechanism is. Here we show that three methyl oxidation products, 5-(hydroperoxymethyl)-, 5-(hydroxymethyl)-, and 5-formyl-2′-deoxycytidine, could be produced when 5mdC was treated with tetrachloro-1,4-benzoquinone (TCBQ) and H₂O₂. The formation of the oxidation products was markedly inhibited by typical ^•OH scavengers and under anaerobic conditions. Analogous effects were observed with other halogenated quinones and the classic Fenton system. Based on these data, we propose that the oxidation of 5mdC by TCBQ/H₂O₂ might be through the following mechanism: ^•OH produced by TCBQ/H₂O₂ may first abstract hydrogen from the methyl group of 5mdC, leading to the formation of 5-(2′-deoxycytidylyl)methyl radical, which may combine with O₂ to form the peroxyl radical. The unstable peroxyl radical transforms into the corresponding hydroperoxide 5-(hydroperoxymethyl)-2′-deoxycytidine, which reacts with TCBQ and results in the formation of 5-(hydroxymethyl)-2′-deoxycytidine and 5-formyl-2′-deoxycytidine. This is the first report that halogenated quinoid carcinogens and H₂O₂ can induce potent methyl oxidation of 5mdC via a metal-independent mechanism, which may partly explain their potential carcinogenicity.     

Unusual Contribution of 2-Aminoadenine to the Thermostability of DNA

Abstract

The poly(dA-dU) and poly(dl-dC) duplexes have very similar thermostabilities (T_m). This similarity extends also to the pyrimidine 5-methyl group-containing poly(dA-dT) and poly(dI-m⁵dC). The differences between chemical structures of the A:U and I:C or the A:T and I:m⁵C base-pairs seem to be unimportant for the thermostability of the DNA. However, on the insertion of an amino group into position 2 of the purines the similarities disappear. Thermostabilities of poly(n²dA-dU) and poly(dG-dC) as well as the poly(n²dA-dT) and poly(dG-m⁵dC) are radically different. This is also the case with their other 5-substituted pyrimidine-containing derivatives, the 5-ethyl, 5-n-butyl and 5-bromo analogues. The G:C-based polynucleotides are more stable by an average of 40°C than the n²A.U-based ones. Poly(dA,n²dA-dT)-s containing various proportions of A and n²A as well as the natural DNA of S-2L cyanophage that contains n²A bases instead of A were also studied. It was found that dependence of T_m on the n²A-content was non-linear and that the lower T_m is not the consequence of a particular nucleotide sequence. The possible structural reasons for the lower thermostabilization of these B-DNAs by the n²A:T base-pair as compared to the G:C are discussed.     

Interactions of poly-N6-methyladenylic Acid and N6-substituted-9-methyladenines with poly-5-bromouridylic Acid: A Novel Base-pairing

Abstract

The interaction of poly-N⁶-methyladenylic acid (poly(m⁶A)) with poly-5-bromouridylic acid (poly(BU)) was studied by the mixing curve method. A 1 m⁶A: 2 BU stoichiometry was clearly indicated over a wide range of ionic strengths at neutral pH, while the binding of poly(m⁶A) to poly(U) is known to occur with 1 m⁶A:1 U. Digestion by nuclease S1 confirmed this stoichiometry, indicating the absence of single strands in a 1:2 mixture. Heating profile analysis and hydroxyapatite column chromatography provided further confirmation of this finding. To determine whether 1:2 stoichiometry holds in a monomer-polymer system, the interaction of N⁶-methyl-9-methyladenine (m⁶m⁹A), a corresponding monomer of poly(m⁶A), with poly(BU) was investigated.

Equilibrium dialysis experiments showed the stoichiometry of the interaction to be 1 m⁶A: 2 BU. Thus, we would describe some structural studies of the above complexes using c.d. and i. r. spectroscopy. Poly (m⁶A)·2poly(BU) and m⁶m⁹A·2poly(BU) are helical and analogous to each other in structure, and the bases in the complexes are all bound by hydrogen-bonding. N⁶-(Δ²-isopentenyl)- and N⁶-allyl-9-methyladenine were also found to form complexes with poly(BU), giving similar c.d. spectra with that of m⁶m⁹A·2poly(BU). The melting experiments indicated the T_ms to be substantially decreased, compared to the parent unmodified complexes, even though the T_m dependence of the polymer complex on salt concentration conforms to the typical triple strand. In the following, the biological significance of this novel pairing will be discussed.     

施用甲醇刺激矮牵牛的生长效应与其代谢关系初探

大量研究表明施用甲醇能够促进多种植物的生长,在甲醇刺激植物生长的机理中,支持碳源假说的证据最多。该研究通过考察矮牵牛甲醇代谢与甲醇刺激其生长的相关性,对碳源假说进行验证。结果表明:(1)在MS固体培养基上添加2和6mmol/L CH3OH均可促进矮牵牛的生长和叶绿素含量增加,但2mmol/L CH3OH(低浓度)效果好于6mmol/L(高浓度),而且添加6mmol/L CH3OH会诱发较强的氧化胁迫。(2)进一步用13 C-NMR分析矮牵牛对不同浓度13 CH3OH的代谢作用发现,6mmol/L 13 CH3OH处理矮牵牛中[U-13 C]Fruc和[U-13 C]Gluc的生成量显著大于2mmol/L 13 CH3OH处理,即来自甲醇的碳源在代谢过程中虽被卡尔文循环同化为糖类物质,但这部分碳源对甲醇刺激矮牵牛的生长贡献不大。这些证据表明CH3OH代谢与其刺激矮牵牛生长的效果没有关联性,该实验结果不支持碳源假说。     

Effect of growth conditions on enzyme activities,intracellular kinetics and biomass yields of a new RuMP-type methylotroph (T15)

Summary A new methylotrophic strain (T15), which employs the ribulose monophosphate (RuMP) cycle of formaldehyde assimilation, was isolated on the basis of high in vitro activities of formaldehyde and formate dehydrogenases (19 and 678 mU per mg protein, respectively). Serial subculturing of the strain in batch cultures, on 4 g/l CH₃OH for 6 months, led to loss of substantial percentages of the NAD-linked formaldehyde (25%) and formate (98%) dehydrogenases. The activities of these two enzymes were partially recovered when cells were grown continuously at very low dilution rate (0.03 h^–1). We found large variations (40 to 1000%) in the activities of other key enzymes of carbon-substrate oxidation (both linear and cyclic) and assimilation, in batch cultures with pure and mixed substrates, and in continuous cultures of different dilution rates. Key intracellular reaction rates, including those of the cyclic and linear substrate oxidation, were measured in vivo using a ¹⁴C-tracer technique in both continuous and batch cultures. The results indicate significant variations in these reaction rates, particularly those of linear and cyclic carbon oxidation. Overall, the cyclic oxidation appears to be employed to a larger (although not predominant) extent in strain T15 compared with another RuMP strain (L3) we have previously examined. T15 exhibits high biomass yields (up to 0.63 g cells per g CH₃OH) and growth rates (up to 0.46 h^–1) on CH₃OH in batch cultures. CH₃NH₂ can also be utilized as a substrate. In continuous culture, T15 could be grown at dilution rates up to 0.36 h^–1 with a corresponding biomass yield of 0.4. Examination of a large number of data on the biomass yields of strains T15 and L3 reveals that the large variations in yields derive from the variable branching of carbon flow between linear and cyclic oxidation and assimilation, rather than changes in the biosynthetic efficiency of carbon incorporation into biomass.     

Spectral and kinetic investigation on oxidation and reduction of water soluble porphyrin-manganese(III) complex

In this work the oxidation and reduction reactions of Mn^III-Coproporphyrin-I (Mn^III-CPI) have been studied and four forms of manganese-CPI complexes have been characterized. This complex was observed to be highly reactive (at basic pH) towards Mn(II), hypochlorite, hydrogen peroxide and oxone, forming [Mn^IV(O)CPI(OH)]⁻ that was unstable and, after a short time, formed again [Mn^IIICPI(OH)₂]⁻. With an excess of NaClO, a further oxidation of the complex [Mn^IV(O)CPI(OH)]⁻, provoked a significant spectral change for the [Mn^V(O)CPI(OH)] formation that showed, in the time, a partial polymerization. [Mn^IIICPI(OH)₂]⁻ was reduced by sodium dithionite to form the very unstable complex of [Mn^IICPI(OH)]⁻ that successively degraded with Mn(II) release.