Building reactive copper centers in human carbonic anhydrase II

Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu²⁺ ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu²⁺-binding events have different affinities (K _a approximately 5 × 10¹² and 1 × 10¹⁰), and both are enthalpically driven processes. Reconstituting these Cu²⁺ sites under a range of conditions has allowed us to assign the Cu²⁺-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu²⁺ sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.     

Electron paramagnetic resonance evidence of metal-ion binding sites in Micrococcus lysodeikticus (M. luteus) F1-ATPase

Evidence is presented for the presence of divalent cation binding sites in purified F₁-ATPase from Micrococcus lysodeikticus (Micrococcus luteus). Electron paramagnetic resonance studies of native F₁-ATPase indicate that the enzyme binds Mn²⁺ and Cu²⁺. Scatchard-type plot for Mn²⁺ binding to the enzyme indicates the presence of 3–4 independent and identical sites with a dissociation constant of 18.3 · 10⁻⁶ M. Cu²⁺ binds to the enzyme at only one kind of site(s). This Cu²⁺ binding site(s) is characterized by a moderately ionic ligand field provided by the protein and by a tetragonal symmetry of nitrogen and/or oxigen ligands. Competition studies indicate that Mg²⁺ binds at these Mn²⁺ and Cu²⁺ binding sites.     

Biological processes involved in the cycling of elements between soil or sediments and the aqueous environment

The biochemical basis for resistance to toxicity is complicated by the great variety of reactions at the molecular and cellular levels even in closely related organisms and tissues. Several strategies for resistance to intoxication have been identified. Metal ion interactions in biology can be divided into three classes representing fast, intermediate and slow exchange with biological ligands. Examples of those elements in fast exchange include the alkali metals Na⁺ and K⁺, the alkali earth metals Ca²⁺ and Mg²⁺, and, of course, H⁺. Those which can sometimes be in intermediary exchange are Fe²⁺ and Mn²⁺. Examples of those in slow exchange are generally in the active sites of metalloenzymes, e.g., Fe³⁺, Zn²⁺, Ni²⁺, Cu²⁺. In the presented paper, the cycling of one essential element (nickel) and one non-essential element (mercury) are reviewed with special emphasis on their mobilities in the event of in situ sediment contamination.     

The identity of a new copper(II) electron paramagnetic resonance signal in cytochrome c oxidase

In reoxidation experiments with cytochrome c oxidase (EC 1.9.3.1) in the presence of both reducing substrate and molecular oxygen, a new EPR signal from Cu²⁺ has been observed. The new signal corresponds to 0.45 Cu per functional unit. It is concluded that the new EPR signal originates from Cu²⁺_B, the copper which is EPR-nondetectable in the resting enzyme.Optical absorption changes in the 500–700 nm region accompanies the decay of the new Cu²⁺ EPR signal.Based on the results in this investigation a catalytic cycle for cytochrome oxidase is proposed.     

The YHS-Domain of an Adenylyl Cyclase from Mycobacterium phlei Is a Probable Copper-Sensor Module

YHS-domains are small protein modules which have been proposed to bind transition-metal ions like the related TRASH-domains. They are found in a variety of enzymes including copper-transporting ATPases and adenylyl cyclases. Here we investigate a class IIIc adenylyl cyclase from Mycobacterium phlei which contains a C-terminal YHS-domain linked to the catalytic domain by a peptide of 8 amino acids. We expressed the isolated catalytic domain and the full-length enzyme in E. coli. The catalytic domain requires millimolar Mn²⁺ as a cofactor for efficient production of cAMP, is unaffected by low micromolar concentrations of Cu²⁺ and inhibited by concentrations higher than 10 μM. The full-length enzyme also requires Mn²⁺ in the absence of an activator. However, 1–10 μM Cu²⁺ stimulate the M. phlei adenylyl cyclase sixfold when assayed with Mn²⁺. With Mg²⁺ as the probable physiological cofactor of the adenylyl cyclase Cu²⁺ specifically switches the enzyme from an inactive to an active state. Other transition-metal ions do not elicit activity with Mg²⁺. We favor the view that the YHS-domain of M. phlei adenylyl cyclase acts as a sensor for copper ions and signals elevated levels of the transition-metal via cAMP. By analogy to TRASH-domains binding of Cu²⁺ probably occurs via one conserved aspartate and three conserved cysteine-residues in the YHS-domain.     

Ligand Trapping by Cytochrome c Oxidase: IMPLICATIONS FOR GATING AT THE CATALYTIC CENTER*

Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe_a3³⁺–Cu_B²⁺) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme. Here we show that cytochrome oxidase pretreated with a low concentration of azide exhibits a significant increase in the apparent rate of cyanide binding relative to that of free enzyme. However, this increase in rate does not reflect a conformational change enhancing the rapid formation of a Fe_a3³⁺–CN–Cu_B²⁺ complex. Instead the cyanide-induced transition of a preformed Fe_a3³⁺–N₃–Cu_B²⁺ to the ternary complex of Fe_a3³⁺–N₃ Cu_B²⁺–CN is the most likely reason for the observed acceleration. Significantly, the slow rate of azide release from the ternary complex indicates that cyanide ligated to Cu_B blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at Cu_B and that, during catalysis, ligands present at this copper center control access to the iron of heme a₃ from the bulk medium.     

Copper(II)-induced secondary structure changes and reduced folding stability of the prion protein

The cellular isoform of the prion protein PrP^C is a Cu²⁺-binding cell surface glycoprotein that, when misfolded, is responsible for a range of transmissible spongiform encephalopathies. As changes in PrP^C conformation are intimately linked with disease pathogenesis, the effect of Cu²⁺ ions on the structure and stability of the protein has been investigated. Urea unfolding studies indicate that Cu²⁺ ions destabilise the native fold of PrP^C. The midpoint of the unfolding transition is reduced by 0.73 ± 0.07 M urea in the presence of 1 mol equiv of Cu²⁺. This equates to an appreciable difference in free energy of unfolding (2.02 ± 0.05 kJ mol^− 1 at the midpoint of unfolding). We relate Cu²⁺-induced changes in secondary structure for full-length PrP(23-231) to smaller Cu²⁺ binding fragments. In particular, Cu²⁺-induced structural changes can directly be attributed to Cu²⁺ binding to the octarepeat region of PrP^C. Furthermore, a β-sheet-like transition that is observed when Cu ions are bound to the amyloidogenic fragment of PrP (residues 90-126) is due only to local Cu²⁺ coordination to the individual binding sites centred at His95 and His110. Cu²⁺ binding does not directly generate a β-sheet conformation within PrP^C; however, Cu²⁺ ions do destabilise the native fold of PrP^C and may make the transition to a misfolded state more favourable.     

Metal ion reconstitution studies of yeast copper-zinc superoxide dismutase: the "phantom" subunit and the possible role of Lys7p

 Using a corrected molar extinction coefficient for yeast apo copper-zinc superoxide dismutase (CuZnSOD), we have confirmed that the metal binding properties of this protein in vitro differ greatly from those of the bovine and human CuZnSOD enzymes. Thus yeast apo CuZnSOD was found to bind only one Co²⁺ per protein dimer under the conditions in which the bovine and human CuZnSOD apoenzymes readily bind two per dimer. The spectroscopic properties characteristic of the two Cu²⁺ plus two Co²⁺ per dimer or four Cu²⁺ per dimer metal-substituted bovine apo CuZnSOD derivatives were obtained for the yeast apoprotein but by the addition of only half of the appropriate metals, i.e., one Cu²⁺ plus one Co²⁺ per dimer or two Cu²⁺ per dimer. This half-metallated yeast CuZnSOD has been characterized by UV-visible and EPR spectroscopy as well as by native polyacrylamide gel electrophoresis. We conclude that yeast apo CuZnSOD, unlike the bovine and human apoproteins, cannot be reconstituted fully with metal ions under the same conditions. Instead, only one subunit of the homodimer, the "normal" subunit, can be remetalled in a fashion reminiscent of the well-characterized bovine protein. The other "phantom" subunit is not competent to bind metals in this fashion. Furthermore, we have shown that CuZnSOD protein isolated from Saccharomyces cerevisiae that lacks the gene coding for the copper chaperone, Lys7p, contains only one metal ion, Zn²⁺, per protein dimer. The possibility that yeast CuZnSOD can exist in multiple conformational states may represent an increased propensity of the yeast protein to undergo changes that can occur in all CuZnSODs, and may have implications for amyotrophic lateral sclerosis. Received: 8 June 1998 / Accepted: 9 September 1998     

The metal function in the reactions of bovine serum amine oxidase with substrates and hydrazine inhibitors

 Bovine serum amine oxidase (BSAO) reacts with 2-hydrazinopyridine, which binds the organic cofactor 2,4,5-trihydroxyphenylalanine quinone, forming a band at 435 nm. The band shifts to 526 nm around 60  °C, to 415 nm upon denaturation, but only shifts to 429 nm upon Cu²⁺ depletion. Its wavelength and intensity suggest that the adduct has the azo conformation, whilst the same adduct of crystallineEscherichia coli amine oxidase (ECAO) shows the hydrazone conformation in the X-ray structure. The steady state kinetics of aminomethyl- and aminoethylpyridines confirm that the formation of the product Schiff base, analogous to the azo form of the 2-hydrazinopyridine adduct, is not hindered in solution. The structural stability of the adduct in the absence of Cu²⁺ is taken to imply hydrogen bonding of the pyridyl nitrogen to a conserved aspartate, as in the ECAO adduct. Thus the ECAO adduct provides a good model for a transient intermediate leading to formation of the BSAO azo adduct. On the basis of this model and of the catalytic competence of Co²⁺-substituted BSAO, confirmed by the present data, a catalytic reaction scheme is proposed. Received: 2 December 1998 / Accepted: 22 March 1999     

Influence of divalent copper, manganese and zinc ions on fibril nucleation and elongation of the amyloid-like yeast prion determinant Sup35p-NM

There is a large body of evidence that divalent metal ions, particularly copper, might play a role in several protein folding pathologies like Alzheimer’s disease, Parkinson’s disease or the prion diseases. However, contribution of metal ions on pathogenesis and their molecular influence on the formation of amyloid structures is not clear. Therefore, the general influence of metals on the formation of amyloids is still controversially discussed. We have utilized the well established system of yeast Sup35p-NM to investigate the role of three different metal ions, Cu²⁺, Mn²⁺ and Zn²⁺, on amyloidogenesis. Recently, it has been shown that the prion determining region NM of the Saccharomyces cerevisiae prion protein Sup35p, which is responsible for the yeast prion phenotype [PSI⁺], specifically binds Cu²⁺ ions. We further characterized the affinity of NM for Cu²⁺, which were found to be comparable to that of other amyloidogenic proteins like the mammalian prion protein PrP. The specific binding sites could be located in the aminoterminal N-region which is known to initiate formation of amyloidogenic nuclei. In the presence of Cu²⁺, fibril nucleation was significantly delayed, probably due to influences of copper on the oligomeric ensemble of soluble Sup35p-NM, since Cu²⁺ altered the tertiary structure of soluble Sup35p-NM, while no influences on fibril elongation could be detected. The secondary structure of soluble or fibrous protein and the morphology of the fibrils were apparently not altered when assembled in presence of Cu²⁺. In contrast, Mn²⁺ and Zn²⁺ did not bind to Sup35p-NM and did not exhibit significant effects on the formation of NM amyloid fibrils.     

Copper bioreduction and nanoparticle synthesis by an enrichment culture from a former copper mine

Microorganisms can facilitate the reduction of Cu²⁺, altering its speciation and mobility in environmental systems and producing Cu-based nanoparticles with useful catalytic properties. However, only a few model organisms have been studied in relation to Cu²⁺ bioreduction and little work has been carried out on microbes from Cu-contaminated environments. This study aimed to enrich for Cu-resistant microbes from a Cu-contaminated soil and explore their potential to facilitate Cu²⁺ reduction and biomineralisation from solution. We show that an enrichment grown in a Cu-amended medium, dominated by species closely related to Geothrix fermentans, Azospira restricta and Cellulomonas oligotrophica, can reduce Cu²⁺ with subsequent precipitation of Cu nanoparticles. Characterisation of the nanoparticles with (scanning) transmission electron microscopy, energy-dispersive x-ray spectroscopy and electron energy loss spectroscopy supports the presence of both metallic Cu(0) and S-rich Cu(I) nanoparticles. This study provides new insights into the diversity of microorganisms capable of facilitating copper reduction and highlights the potential for the formation of distinct nanoparticle phases resulting from bioreduction or biomineralisation reactions. The implications of these findings for the biogeochemical cycling of copper and the potential biotechnological synthesis of commercially useful copper nanoparticles are discussed.     

Differential copper binding to alpha-synuclein and its disease-associated mutants affect the aggregation and amyloid formation

BackgroundCopper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson’s Disease. Hence, residue specific interaction of Cu²⁺ to α-Syn along with the familial mutants H50Q and G51D needs to be studied in detail.MethodsWe investigated the residue specific mapping of Cu²⁺ binding sites and binding strength using solution-state NMR and ITC respectively. The aggregation kinetics, secondary structural changes, and morphology of the formed fibrils in the presence and absence of Cu²⁺ were studied using fluorescence, CD, and AFM respectively.ResultsCopper binding to α-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q, the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu²⁺ showed an enhanced rate of fibril formation with a pronounced effect for G51D.ConclusionCu²⁺ binding results in the destabilization of long-range tertiary interactions in α-Syn leading to the exposure of highly amyloidogenic NAC region which results in the increased rate of fibril formation. Although the residues 48-53 have a stronger affinity for Cu²⁺ in case of WT and G51D, the binding is not responsible for enhancing the rate of fibril formation in case of H50Q.General SignificanceThese findings will help in the better understanding of Cu²⁺ catalyzed aggregation of synucleins.     

Mechanism of oxidation of oxymyoglobin by copper ions: comparison of sperm whale, horse, and pig myoglobins

The influence of Cu²⁺ concentration, pH, and ionic strength of the solution as well as redox-inactive zinc ions on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins (oxy-Mb) by copper ions has been studied. These myoglobins have homologous spatial structures and equal redox potentials but differ in the number of histidines located on the surface of the proteins. It was shown that oxy-Mb can be oxidized in the presence of Cu²⁺ through two distinct pathways depending on which histidine binds the reagent and how stable the complex is. A slow pH-dependent catalytic process is observed in the presence of equimolar Cu²⁺ concentration for sperm whale and horse oxymyoglobins. The curves of pH dependence in both cases are sigmoid with pK _eff corresponding to the ionization. The process is caused by the strong binding of Cu²⁺ to His113 and His116, an analogous His residue being absent in pig Mb. In contrast, rapid oxidation of 10-15% of pig oxy-Mb is observed under the same conditions (fast phase), which is not accompanied by catalysis because the reduced copper is apparently not reoxidized. The complexing of Cu²⁺ with His97 situated near the heme is probably responsible for the fast phase of the reaction. The affinity of His97 for Cu²⁺ must be significantly lower than those of the catalytic His residues since the fast phase does not contribute markedly to the rate of sperm whale and horse oxy-Mb oxidation. Increasing copper concentration does not produce a proportional growth in the oxidation rate of sperm whale and horse oxy-Mbs. Which Cu²⁺ binding sites of Mb make main contributions to the His reaction rate at different Cu²⁺/Mb ratios from 0.25 to 10 is discussed.     

Investigations on the effects of Cu2+ on the structure and function of human serum albumin

Human serum albumin (HSA) is the most prominent protein in blood plasma with important physiological functions. Although copper is an essential metal for all organisms, the massive utilization of copper has led to concerns regarding its potential health impact. To better understand the potential toxicity and toxic mechanisms of Cu²⁺, it is of vital importance to characterize the interaction of Cu²⁺ with HSA. The effect of Cu²⁺ on the structure and function of HSA in vitro were investigated by biophysical methods including fluorescence techniques, circular dichroism (CD), time‐resolved measurements, isothermal titration calorimetry (ITC), molecular simulations and esterase activity assay. Multi‐spectroscopic measurements proved that Cu²⁺ quenched the intrinsic fluorescence of HSA in a dynamic process accompanied by the formation of complex and alteration of secondary structure. But the Cu²⁺ had minimal effect on the backbone and secondary structure of HSA at relatively low concentrations. The ITC results indicated Cu²⁺ interacted with HSA spontaneously through hydrophobic forces with approximately 1 thermodynamic identical binding sites at 298 K. The esterase activity of HSA was inhibited obviously at the concentration of 8 × 10^‐5 M. However, molecular simulation showed that Cu²⁺ mainly interacted with the amino acid residues Asp (451) by the electrostatic force. Thus, we speculated the interaction between Cu²⁺ and HSA might induce microenvironment of the active site (Arg 410). This study has provided a novel idea to explore the biological toxicity of Cu²⁺ at the molecular level. Copyright © 2015 John Wiley & Sons, Ltd.     

Sorption of Copper and Zinc to the Plasma Membrane of Wheat Root

Sorption of Cu²⁺ and Zn²⁺ to the plasma membrane (PM) of wheat root (Triticum aestivum Lcv. Scout 66) vesicles was measured at different pH values and in the presence of organic acids and other metals. The results were analyzed using a Gouy-Chapman-Stem model for competitive sorption (binding and electrostatic attraction) to a negative binding site. The binding constants for the two investigated cations as evaluated from the sorption experiments were 5 M^–1 for Zn²⁺ and 400 M^–1 for Cu²⁺. Thus, the sorption affinity of Cu²⁺ to the PM is considerably larger than that of Ca²⁺, Mg²⁺ or Zn²⁺. The greater binding affinity of Cu²⁺ was confirmed by experiments in which competition with La³⁺ for sorption sites was followed. The amount of sorbed Cu²⁺ decreased with increasing K⁺, Ca²⁺, or La³⁺ concentrations, suggesting that all these cations competed with Cu²⁺ for sorption at the PM binding sites, albeit with considerable differences among these cations in effectiveness as competitors with Cu²⁺. The sorption of Cu²⁺ and Zn²⁺ to the PM decreased in the presence of citric acid or malic acid. Citric acid (as well as pH) affected the sorption of Cu²⁺ or Zn²⁺ to PM more strongly then did malic acid.     

Cu2+ probe of metal-ion binding sites in melanin using electron paramagnetic resonance spectroscopy. I. Synthetic melanins

The isotope ⁶³Cu²⁺ has been used to probe the metal-ion binding sites of synthetic (autoxidized) catechol and 3,4-dihydroxyphenylalanine melanins using electron paramagnetic resonance spectroscopy. Samples were in aqueous media over a wide range of pH values. Assignments of the structures of the melanin-copper complexes are based in part on model studies of the complexes formed with melanin precursors, catechol and 3,4-dihydroxyphenylalanine, and with phenanthroline. Nearly all complexes involve just one or two ligands from melanin. In catechol melanin below pH 5.0, complexes with carboxyl groups are formed; above 6.0, Cu²⁺ forms complexes with phenolic hydroxyl groups. These same complexes were found in 3,4-dihydroxyphenylalanine melanin and binding of Cu²⁺ at amino acid type sites also was detected. After partial reduction of copper ions bound to 3,4-dihydroxyphenylalanine melanin, a weak signal of copper with four melanin ligands (oxygen and nitrogen in various combinations) was observed.     

The cupric ion as an inhibitor of photosynthetic electron transport in isolated chloroplasts

Strong inhibition of uncoupled photosynthetic electron transport by Cu²⁺ in isolated spinach chloroplasts was observed by measuring changes in O₂ concentration in the reaction medium. Inhibition was dependent not only on the concentration of the inhibitor, but also on the ratio of chlorophyll to inhibitor. Binding of Cu²⁺ to the chloroplast membranes resulted in removal of Cu²⁺ from solution. When chloroplasts were exposed to preincubation in light, there was increased inhibition as a result of Cu²⁺ binding to inhibitory sites. Preincubation in the dark resulted in Cu²⁺ binding to noninhibitory sites and decreased inhibition. The degree of inhibition was lower at low light intensities than at high light intensities.     

Capturing a Reactive State of Amyloid Aggregates: NMR-BASED CHARACTERIZATION OF COPPER-BOUND ALZHEIMER DISEASE AMYLOID β-FIBRILS IN A REDOX CYCLE*

The interaction of redox-active copper ions with misfolded amyloid β (Aβ) is linked to production of reactive oxygen species (ROS), which has been associated with oxidative stress and neuronal damages in Alzheimer disease. Despite intensive studies, it is still not conclusive how the interaction of Cu⁺/Cu²⁺ with Aβ aggregates leads to ROS production even at the in vitro level. In this study, we examined the interaction between Cu⁺/Cu²⁺ and Aβ fibrils by solid-state NMR (SSNMR) and other spectroscopic methods. Our photometric studies confirmed the production of ∼60 μm hydrogen peroxide (H₂O₂) from a solution of 20 μm Cu²⁺ ions in complex with Aβ(1–40) in fibrils ([Cu²⁺]/[Aβ] = 0.4) within 2 h of incubation after addition of biological reducing agent ascorbate at the physiological concentration (∼1 mm). Furthermore, SSNMR ¹H T₁ measurements demonstrated that during ROS production the conversion of paramagnetic Cu²⁺ into diamagnetic Cu⁺ occurs while the reactive Cu⁺ ions remain bound to the amyloid fibrils. The results also suggest that O₂ is required for rapid recycling of Cu⁺ bound to Aβ back to Cu²⁺, which allows for continuous production of H₂O₂. Both ¹³C and ¹⁵N SSNMR results show that Cu⁺ coordinates to Aβ(1–40) fibrils primarily through the side chain N_δ of both His-13 and His-14, suggesting major rearrangements from the Cu²⁺ coordination via N_ϵ in the redox cycle. ¹³C SSNMR chemical shift analysis suggests that the overall Aβ conformations are largely unaffected by Cu⁺ binding. These results present crucial site-specific evidence of how the full-length Aβ in amyloid fibrils offers catalytic Cu⁺ centers.     

On the magnetic properties of cobalt substituted bovine superoxide dismutase derivatives.

We have measured the magnetic susceptibility in the temperature range 1.4–77°K of three derivatives of bovine superoxide dismutase in which Co²⁺ was substituted for Zn²⁺: (1) 2Co²⁺ — in which Co²⁺ binds to the normal Zn²⁺ site and the Cu²⁺ site is unoccupied, (2) 2Co²⁺2Cu²⁺ — in which the Zn²⁺ site is occupied by Co²⁺ and the copper sites contains Cu²⁺ and (3) 2Co²⁺2Cu⁺ — which is the reduced form of the second derivative. The 2Co²⁺ protein exhibits Curie paramagnetism indicating $\text{S′}\text{=}\text{1}\text{2}$ and the zero-field splitting must be greater than ?20 cm^?1. The same propeties have been observed with the 2Co²⁺2Cu⁺-protein. By contrast, the 2Co²⁺2Cu²⁺-derivative exhibits relatively little paramagnetism, some of which arises from non-specifically associated metal ions. The lower susceptibility is due to antiferromagnetic coupling between Co²⁺ and Cu²⁺, and the magnitude of the coupling constant is probably ?5 cm^?1.     

The influence of pH on the inhibition of DNA cleavages induced by pyrogallol

Abstract

Induction of DNA damage by pyrogallol has been shown at physiological pH, but mutagenesis data also suggest there is inhibition in acidic media. In the present work, the plasmid pBSK was incubated with pyrogallol, under aerobic conditions at 37°C, at pH 7.4, 4.5 or 3.5, for 1, 3 or 5 h, in the absence or presence of Cu²⁺. Cleavage of the supercoiled DNA form was analyzed through topology modifications by agarose gel electrophoresis and quantified by densitometry. Independently of the presence of Cu²⁺ , DNA cleavage at pH 7.4 was significantly (P < 0.001) induced and occurred extensively after 1-h incubation. At pH 4.5, the cleavage was significantly (P < 0.05) induced only after 5 h incubation in the absence of Cu²⁺ , but was extensive (P < 0.001) after 1-h incubation when the metal ion was present. At pH 3.5, DNA cleavage was inhibited (P > 0.05), after 5-h incubation, even in the presence of Cu²⁺. Our results provide evidence that DNA cleavage by pyrogallol is pH-dependent, catalyzed by Cu²⁺ , and extensively decreased in acidic pH. Due to the abundant presence of the pyrogallate ion in physiological media, we suggest that this conjugate base form is responsible for DNA cleavage.