AXanthomonas maltophilia isolate tolerating up to 1% sodium azide in Tris/HCl buffer

A bacterium tolerating up to 1% NaN₃ found as a contaminant of Sephadex colums being run with Tris/HCl buffer, was identified asXanthomonas maltophilia. It had low nutrient requirements, was strongly proteolytic and interfered with Sephadex columns run with Tris/HCl buffers.The authors are with the Department of Plant Pathology, Swedish University of Agricultural Sciences, Box 7044, S-750 07 Uppsala, Sweden;     

Oxidation of Good's buffers by hydrogen peroxide

Good's zwitterionic buffers are widely used in biological and biochemical research in which hydrogen peroxide is a solution component. This study was undertaken to determine whether Good's buffers exhibit reactivity toward H(2)O(2). It is found that H(2)O(2) oxidizes both morpholine ring-containing buffers (e.g., Mops, Mes) and piperazine ring-containing zwitterionic buffers (e.g., Pipes, Hepes, and Epps) to produce their corresponding N-oxide forms. The percentage of oxidized buffer increases as the concentration of H(2)O(2) increases. However, the rate of oxidation is relatively slow. For example, no oxidized Mops was detected 2h after adding 0.1M H(2)O(2) to 0.1M Mops (pH 7.0), and only 5.7% was oxidized after 24h exposure to H(2)O(2). Thus, although all of these buffers can be oxidized by H(2)O(2), their slow reaction does not significantly perturb levels of H(2)O(2) in the time frame and at the concentrations of most biochemical studies. Therefore, the previously reported rapid loss of H(2)O(2) produced from the ferroxidase reaction of ferritin is unlikely due to reaction of H(2)O(2) with buffer, a conclusion supported by the fact that H(2)O(2) is also lost rapidly when the solution pH of the ferroxidase reaction is controlled by a pH stat apparatus in the absence of buffer.     

Baroresistant buffer mixtures for biochemical analyses

Hydrostatic pressure is a useful tool in the study of varied fields such as protein aggregation, association, folding, ligand binding, and allostery. Application of pressure can have a significant effect on the pK(a) values of buffers commonly used for biochemical analysis. Consequently, cationic buffers, rather than neutral ones, are generally used to minimize pH effects; however, even with these buffers, the change in pH over 3 kbar may be consequential in highly pH-sensitive biochemical systems. Using fluorescence-based assays, we have systematically examined the effects of pressure on various buffers in the neutral pH range. We show that many commonly used cationic and Good's buffers increase in pH with pressure on the order of 0.1 to 0.3 pH units/kbar, in agreement with other published values. Carboxylates and phosphate decrease in pH to a similar extent. Buffer mixtures, composed of both cationic and carboxylate or phosphate components, are shown to be an order of magnitude less pressure sensitive than the individual component buffers. Using various relative concentrations of Tris and either phosphate, tricarballylate (1,2,3-propanetricarboxylate), or CDA (1,1-cyclohexane diacetate) at pH values between 7 and 8 yields baroresistant buffer mixtures. Buffer mixtures can be optimized for a specific pH, and a list of mixtures is presented for general laboratory use.     

The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals

Hydroxyl radicals, generated by reaction of an iron-EDTA complex with H2O2 in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added hydroxyl radical "scavengers" compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. For a wide range of compounds, rate constants obtained in this way are similar to those determined by pulse radiolysis. It is suggested that the deoxyribose assay is a simple and cheap alternative to pulse radiolysis for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of ATP, ADP, and Good's buffers with hydroxyl radicals have been determined by this method.     

Chromatographic behavior of Bothrops erythromelas phospholipase and other venom constituents on Superdex 75

In a chromatographic method modification intended to preserve protease activity in Bothrops erythromelas venom, 2 mM CaCl2 was added to the gel filtration buffer [50mM Tris/HCl/150mM NaCl (pH 8.0)], in lieu of an equimolar portion of NaCl. This minor compositional change induced significant differences in the venom elution profile on Superdex 200. For this reason, the influence of buffer composition on chromatographic behavior was investigated using an analytical Superdex 75 HR 10/30 column. Phospholipase (PLA) was used as a marker because Naja atra PLA had previously been observed to interact hydrophobically with this resin. PLA elution volumes generally increased as buffer pH decreased. Addition of 20% acetonitrile to the Tris buffer with CaCl2, reduced hydrophobic interaction of the PLA so significantly that its elution was non-overlapping in the two buffers. Other venom constituents, including bradykinin-potentiating peptides and probable hemorrhagic metalloproteases, were similarly affected. Buffer calcium, bound by vicinal dextran hydroxyl groups, appears to retard elution of this acidic PLA.     

Oxidation of Tris to One-Carbon Compounds in a Radical-Producing Model System,in Microsomes,in Hepatocytes and in Rats

The buffer substance tris(hydroxymethyl)aminomethane (Tris) is converted to formaldehyde in an hydroxyl radical producing model system and in rat liver microsomes, and to CO₂ in rat hepatocytes and in the intact rat. In microsomes, formaldehyde formation from Tris is inhibited by catalase, by the antioxidant propylgallate and by the iron chelator deferoxamine, formaldehyde formation is stimulated by the addition of Fe (II) EDTA. In hepatocytes, the formation of [¹⁴C] CO₂ from [¹⁴C] Tris is inhibited by propylgallate and by the iron chelator o-phenanthroline and is stimulated by the presence of a xanthine oxidase system plus Fe (II) EDTA in the medium. In the intact rat, the administration of [¹⁴C] Tris results in the exhalation of [¹⁴C] CO². The results indicate that an oxidant formed via a Fenton-type reaction, possibly the hydroxyl radical, may be involved in the formation of one-carbon compounds from Tris.     

Model Studies of the Iron-Catalysed Haber-Weiss Cycle and the Ascorbate-Driven Fenton Reaction

Complementary hydroxylation assays and stopped-flow e.s.r. techniques have been employed in the investigation of the effect of various iron chelators (of chemical, biological and clinical importance) on hydroxyl-radical generation via the Haber-Weiss cycle and the ascorbate-driven Fenton reaction.

Chelators have been identified which selectively promote or inhibit various reactions involved in hydroxyl-radical generation (for example, NTA and EDTA promote all the reactions of both the Haber-Weiss cycle and the ascorbate-driven Fenton reaction, whereas DTPA and phytate inhibit the recycling of iron in these reactions). The biological chelators succinate and citrate are shown to be relatively poor catalysts of the Haber-Weiss cycle, whereas they are found to be effective catalysts of ·OH generation in the ascorbate-driven Fenton reaction.

It is also suggested that continuous redox-cycling reactions between iron, oxygen and ascorbate may represent an important mechanism of cell death in biological systems.     

A selective lignin-degrading fungus,Ceriporiopsis subvermispora,produces alkylitaconates that inhibit the production of a cellulolytic active oxygen species,hydroxyl radical in the presence of iron and H(2)O(2)

A cellulolytic active oxygen species, hydroxyl radicals (.OH), play a leading role in the erosion of wood cell walls by brown-rot and non-selective white-rot fungi. In contrast, selective white-rot fungi have been considered to possess unknown systems for the suppression of .OH production due to their wood decay pattern with a minimum loss of cellulose. In the present paper, we first report that 1-nonadecene-2,3-dicarboxylic acid, an alkylitaconic acid (ceriporic acid B) produced by the selective white-rot fungus Ceriporiopsis subvermispora intensively inhibited .OH production by the Fenton reaction by direct interaction with Fe ions, while non-substituted itaconic acid promoted the Fenton reaction. Suppression of the Fenton reaction by the alkylitaconic acid was observed even in the presence of the Fe(3+) reductants, cysteine and hydroquinone. The inhibition of .OH production by the diffusible fungal metabolite accounts for the extracellular system of the fungus that attenuates the formation of .OH in the presence of iron, molecular oxygen, and free radicals produced during lignin biodegradation.     

Aromatic hydroxylation of phenylalanine as an assay for hydroxyl radicals: application to activated human neutrophils and to the heme protein leghemoglobin

Attack of hydroxyl radical (.OH), generated by a Fenton system at physiological pH, upon L-phenylalanine produces three isomeric tyrosines, o-tyrosine (2-hydroxyphenylalanine), m-tyrosine (3-hydroxyphenylalanine), and p-tyrosine (4-hydroxyphenylalanine). These may be separated by high-performance liquid chromatography and measured using an electrochemical detector. Since L-phenylalanine is relatively nontoxic, it is proposed that generation of these three tyrosines from phenylalanine can be used as an assay for .OH in biological systems. The use of the assay to measure .OH production by leghemoglobin (plus H2O2) and by activated human neutrophils is described. No .OH production by activated human neutrophils was observed unless a source of iron ions was added to the reaction mixture, which suggests that these cells do not release an iron "promoter" of .OH generation from superoxide and hydrogen peroxide.     

Hemoglobin: A mechanism for the generation of hydroxyl radicals

Oxyhemoglobin (HbO₂) reduces Fe(III) NTA aerobically to become methemoglobin (metHb) and Fe(II)NTA. These conditions are favorable for the generation via Fenton chemistry of the hydroxyl radical that was measured by HPLC using salicylate as a probe. The levels of hydroxyl radicals generated are a function of both the percent metHb formed and the chemical nature of the buffer. The rates of formation of both metHb and hydroxyl radicals were dependent upon the concentration of Fe(III)NTA. Of the buffers tested, HEPES was the most effective scavenger of hydroxyl radicals while the other buffers scavenged in the order: HEPES > Tris > MOPS > NaCl ≈ unbuffered. The addition of catalase to remove H₂0₂ or bathophenanthroline to chelate Fe(II) inhibited virtually all hydroxyl radical formation. Carbonyl formation from free radical oxidation of amino acids was found to be 0.1 mol/mol of hemoglobin. These experiments demonstrate the ability of hemoglobin to participate directly in the generation of hydroxyl radicals mediated by redox metals, and provide insight into potential oxidative damage from metals released into the blood during some pathologic disorders including iron overload.     

Induction of polymerization of purified tubulin by sulfonate buffers. Marked differences between 4-morpholineethanesulfonate (Mes) and 1,4-piperazineethanesulfonate (Pipes)

Interactions of both purified tubulin and microtubule protein (tubulin plus associated proteins) with two commonly used sulfonate buffers were examined. 1,4-Piperazineethanesulfonate (Pipes) and 4-morpholineethanesulfonate (Mes) at high concentrations induce the polymerization of purified tubulin in reactions requiring only buffer, tubulin and GTP. While both reactions were temperature-dependent, cold-reversible and inhibited by GDP, colchicine or Ca2+, there were significant differences between them. Substantially lower tubulin and buffer concentrations were required for Pipes-induced polymerization; and turbidity was much more intense in the Pipes-induced than in the Mes-induced reaction at the same protein concentration. Electron microscopy demonstrated that for the most part typical smooth-walled microtubules were formed in Mes, while aberrant forms were the predominant structures formed in Pipes. When the polymerization of microtubule protein was examined as a function of buffer concentration, biphasic patterns were observed with both Pipes and Mes: polymerization occurred at both low and high, but not intermediate, buffer concentrations. The turbidity observed at high concentrations of Pipes greatly exceeded that at low concentrations. With Mes, equivalent turbidity developed at both high and low buffer concentrations. Although associated proteins copolymerized with tubulin at low buffer concentrations, they were excluded from the polymerized material at high buffer concentrations. Pipes and Mes were compared to sodium phosphate, Tris/HCl and imidazole/HCl buffers at 0.1 M in several polymerization systems using both purified tubulin and microtubule protein. The sulfonate buffers were invariably associated with more vigorous reactions than the other buffers.     

Light-dependent inhibitory action of p-nitrothiophenol on Photosystem II in relation to the redox state of electron carriers

The photosystem-II activity of chloroplasts was inhibited by the treatment with p-nitrothiophenol (NphSH) in the light, and the inhibition was accompanied by a change of the fluorescence spectrum. Aromatic mercaptans examined were active in causing this inhibition and fluorescence change. These effects of p-nitrothiophenol were highly accelerated by blocking the electron transport on the oxidation side of photosystem II by carbonyl cyanide-m-chlorophenylhydrazone (CCCP) or Tris · HCl or heat pre-treatment, whereas these were suppressed by blocking the transport on the reduction side by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). It was deduced that the site of NphSH action in the electron transport chain is closer to the reaction center of photosystem II that the blocking site of CCCP or Tris · HCl or heat, and that such a site in photosystem II is exposed to be modified with NphSH when electron carriers on the oxidation side of photosystem II are oxidized by illumination.     

Essential Oil Phenyl Propanoids. Useful as OH Scavengers?

In order to search for radical scavengers which could be used as raw materials for cosmetics, phenyl propanoids (eugenol, isoeugenol, dehydrodieugenol, dehydrodieugenol B and coniferyl aldehyde) were examined for their hydroxyl radical (· OH) scavenging ability. A Fenton system was used to produce -OH. In order to see scavenging by these phenyl propanoids, competition reactions between a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and these phenyl propanoids for -OH were studied. The relative yield of the spin adduct of -OH (DMPO-OH) was measured by electron spin resonance spectroscopy. The approximate rate constants of the reactions between these phenyl propanoids and -OH estimated by measuring the reduced height of the ESR signals of DMPO-OH were found to be at least in the order of 10⁹ M^-1 s^-1 (diffusion-controlled). Also, using the TBA tests, the reactions between ·OH and several compounds reactive with ·OH were investigated in the presence of the phenyl propanoids and it was found that the phenyl propanoids compete with such reactive compounds for ·OH. These results indicate that these phenyl propanoids can be used as antioxidants for skin damage perhaps caused by -OH generated by UV-light.     

Resurveying the Tris buffer solution: the specific interaction between tris(hydroxymethyl)aminomethane and lysozyme

An unusual phenomenon, the specific interaction between tris(hydroxymethyl)aminomethane (Tris) and lysozyme (LZM), was demonstrated for the first time by rapid screen analysis of interactions using a quartz crystal microbalance (QCM) biosensor. This phenomenon was also observed in a surface plasmon resonance (SPR) system. Further study using high-performance affinity chromatography (HPAC) confirmed this specific interaction between LZM and immobilized Tris with an apparent dissociation constant (K_D) of 6.7 × 10⁻⁵ M. Molecular docking was carried out to identify possible modes of binding between LZM and Tris linked to a binding arm. The estimated binding free energy was −6.34 kcal mol⁻¹, corresponding to a K_D of 2.3 × 10⁻⁵ M, which correlated well with the experimental value. Based on the docking model, the three hydroxyl groups of Tris form intermolecular H bonds with Asp52, Glu35, and Ala107 in LZM. This study reinforces the importance of buffer selection in quantitative biochemical investigations. For a lysozyme ligand binding study, it is better to avoid using Tris when the ligands under study are weak binders.     

Divalent metal ion effect on helix-coil transition of high molecular weight DNA in neutral and alkaline solutions

Effect of Mg(2+), Ca(2+), Ni(2+) and Cd(2+) ions on parameters of DNA helix-coil transition in sodium cacodylate (pH 6.5), Tris (pH 8.5) and sodium tetraborate (pH 9.0) buffers have been studied by differential UV-visible spectroscopy and by thermal denaturation. Anomalous behavior of the melting temperature T(m) and the melting interval ΔT in the presence of MgCl(2) was observed in Tris, but not in cacodylate or tetraborate buffers. Changes in the buffer type and pH did not influence T(m) and ΔT dependence on Ca(2+) and Cd(2+) concentrations. Decrease of the T(m) and ΔT of DNA in the presence of Ni(2+) and Cd(2+) was caused by preferential ion interaction with N7 of guanine. This type of interaction was also found for Mg(2+) in Tris buffer. The anomalous decrease in the T(m) and ΔT values was connected to formation of complexes between metal ions and Tris molecules. Transition of DNA single-stranded regions into a compact form with the effective radius of the particles of 300±100 ? was induced by Mg(2+) ions in Tris buffer.     

ADP-iron as a Fenton reactant: radical reactions detected by spin trapping, hydrogen abstraction, and aromatic hydroxylation

A mixture of ADP, ferrous ions, and hydrogen peroxide (H2O2) generates hydroxyl radicals (OH) that attack the spin trap DMPO (5,5-dimethyl-pyrollidine-N-oxide) to yield the hydroxyl free radical spin-adduct, degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, and hydroxylate benzoate to give fluorescent products. Inhibition studies, with scavengers of the OH radical, suggest that the behavior of iron-ADP in the reaction is complicated by the formation of ternary complexes with certain scavengers and detector molecules. In addition, iron-ADP reacting with H2O2 appears to release a substantial number of OH radicals free into solution. During the generation of OH radicals the ADP molecule was, as expected, damaged by the iron bound to it. Damage to the iron ligand in this way is not normally monitored in reaction systems that use specific detector molecules for OH radical damage. Under certain reaction conditions the ligand may be the major recipient of OH radical damage thereby leading to the incorrect assumption that the iron ligand is a poor Fenton reactant.     

Quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid enhance the Fenton reaction in phosphate buffer.

Quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid enhanced the Fenton reaction in phosphate buffer, respectively. The enhancement by quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid of the Fenton reaction may be partly related to their respective actions in the biological systems such as a neurotoxic effect (quinolinic acid), a marked growth-inhibitory action on rice seeding (alpha-picolinic acid and fusaric acid), and an antiseptic (2,6-pyridinedicarboxylic acid). The ultraviolet-visible absorption spectrum of the mixture of alpha-picolinic acid with ferrous ion showed a characteristic visible absorbance band with a lambda(max) at 443 nm, suggesting that alpha-picolinic acid chelate of Fe2+ ion forms in the solution. Similar characteristic visible absorbance band was also observed for the mixture of Fe2+ ion with quinolinic acid (or fusaric acid, or 2,6-pyridinedicarboxylic acid). The chelation seems to be related to the enhancement by quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid of the Fenton reaction. alpha-Picolinic acid was reported to be a toxic substance isolated from the culture liquids of blast mould (Piricularia oryzae CAVARA). On the other hand, it has also been known that chlorogenic acid protects rice plants from the blast disease. The chlorogenic acid inhibited the formation of the hydroxyl radical in the reaction mixture of alpha-picolinic acid, FeSO4(NH4)2SO4, and H2O2. Thus the inhibition may be a possible mechanism of the protective action of the chlorogenic acid against the blast disease.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.     

Radicals from "Good's" buffers

Oxidative deposition of iron in ferritin or the autoxidation of iron in the absence of protein produces radicals from Good's buffers. Radical species are formed from the piperazine ring-based buffers Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Epps 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, and Pipes 1,4-piperazinediethanesulfonic acid, but not from Mes (4-morpholineethanesulfonic acid) which contains a morpholine ring. The radicals all have half-lives around 10 min and display very similar electron paramagnetic resonance spectra consisting of at least 30 lines. The Hepes radical can be formed by the addition of potassium superoxide directly to the buffer and its production during iron(II) autoxidation is inhibited by superoxide dismutase (EC 1.15.1.1). Catalase (EC 1.11.1.6) accelerates the decay of the EPR spectrum. Thus the buffer radicals are secondary radical species produced from oxygen radicals formed during the iron catalyzed Haber-Weiss process. The deoxyribose/thiobarbituric acid assay for hydroxyl radical production shows that Hepes is an effective hydroxyl radical scavenging agent. The Hepes radical can also be formed electrolytically at a potential of +0.8 V (vs standard hydrogen electrode). Oxidation of Hepes at pH 10 during the autoxidation of iron(II) or by the addition of hydrogen peroxide produces a nitroxide radical. These results indicate that piperazine ring Good buffers should be avoided in studies of redox processes in biochemistry.