EDTA Molecules Research Articles

DNA cleavage mediated by copper superoxide dismutase via two pathways

The known action of Cu, Zn superoxide dismutase (Cu 2Zn 2SOD) that converts O 2 - to O 2 and H 2O 2 plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of Cu 2Zn 2SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much Cu 2Zn 2SOD may be injurious to the cells. The present study examined the DNA cleavage activity mediated by a Cu n SOD that contains 1–4 copper ions, in order to obtain an insight into the aberrant copper-mediated oxidative chemistry in the enzyme. A high SOD activity was observed upon metallation of the apo-form of Cu 2Zn 2SOD with Cu(II), indicating that nearly all of the Cu(II) in the Cu n SOD is as active as the Cu(II) in the copper site of fully active Cu 2Zn 2SOD. Using a supercoiled DNA as substrate, significant DNA cleavage was observed with the Cu n SOD in the presence of hydrogen peroxide or mercaptoethanol, whereas DNA cleavage with free Cu(II) ions can occur only <5% under the same conditions. Comparison with other proteins shows that the DNA cleavage activity is specific to some proteins including the Cu n SOD. The steady state study suggests that a cooperative action between the SOD protein and the Cu(II)may appear in the DNA cleavage activity, which is independent of the number of Cu(II) in the Cu n SOD. The kinetic study shows that a two-stage reaction was involved in DNA cleavage. The effects of various factors including EDTA, radical scavengers, bicarbonate anion, and carbon dioxide gas molecules on the Cu n SOD-mediated DNA cleavage activity were also investigated. It is proposed that DNA cleavage occurs via both hydroxyl radical oxidation and hydroxide ion hydrolysis pathways. This work implies that any form of the copper-containing SOD enzymes (including Cu 2Zn 2SOD and its mutants) might have the DNA cleavage activity.

Crystal Structure of the Hypoxia-inducible Form of 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3): A POSSIBLE NEW TARGET FOR CANCER THERAPY

The hypoxia-inducible form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) plays a crucial role in the progression of cancerous cells by enabling their glycolytic pathways even under severe hypoxic conditions. To understand its structural architecture and to provide a molecular scaffold for the design of new cancer therapeutics, the crystal structure of the human form was determined. The structure at 2.1 A resolution shows that the overall folding and functional dimerization are very similar to those of the liver (PFKFB1) and testis (PFKFB4) forms, as expected from sequence homology. However, in this structure, the N-terminal regulatory domain is revealed for the first time among the PFKFB isoforms. With a beta-hairpin structure, the N terminus interacts with the 2-Pase domain to secure binding of fructose-6-phosphate to the active pocket, slowing down the release of fructose-6-phosphate from the phosphoenzyme intermediate product complex. The C-terminal regulatory domain is mostly disordered, leaving the active pocket of the fructose-2,6-bisphosphatase domain wide open. The active pocket of the 6-phosphofructo-2-kinase domain has a more rigid conformation, allowing independent bindings of substrates, fructose-6-phosphate and ATP, with higher affinities than other isoforms. Intriguingly, the structure shows an EDTA molecule bound to the fructose-6-phosphate site of the 6-phosphofructo-2-kinase active pocket despite its unfavorable liganding concentration, suggesting a high affinity. EDTA is not removable from the site with fructose-6-P alone but is with both ATP and fructose-6-P or with fructose-2,6-bisphosphate. This finding suggests that a molecule in which EDTA is covalently linked to ADP is a good starting molecule for the development of new cancer-therapeutic molecules.

Crystal structures and spectroscopic properties of palladium complexes isolated from Pd–EDTA solutions

Two complexes were isolated from aqueous Pd(NO 3) 2–Na 2H 2EDTA solutions and studied by single crystal X-ray diffraction, IR/Raman spectroscopy, and photoelectron spectroscopy. The first complex, Pd(Hkpda) 2 (kpda = ketopiperazinediacetate dianion, C 8H 10N 2O 5), forms yellow parallelepipeds during slow evaporation of Pd(NO 3) 2–Na 2H 2EDTA solution at 25 °C, and is a result of EDTA oxidation. The second one, [Pd(μ-H 2EDTA)] 2 · 2NaNO 3 · 7.5H 2O, with two EDTA molecules acting as bridges between two palladium atoms, forms yellow bipyramides during fast evaporation at 60 °C. In both complexes, the palladium atoms adopt a planar trans-geometry by bonding to two nitrogen and two oxygen atoms of two ligand molecules.

Degradation of EDTA by a chemostat culture of bacterial strain DSM 9103

Characteristics of growth and EDTA degradation by chemostat culture of bacterial strain DSM 9103 were investigated. The effect of specific growth rate ( μ) on mass cell yield ( Y X/S), specific rate of EDTA uptake ( q S) and the rate of Mg–EDTA degradation by a cell suspension ( q S rest) was studied. Based on the experimental data, kinetic constants of dependencies q S( S) and μ( S) were determined. The rates of substrate and energy expenditures for cell maintenance, m S and m e, were 0.02 and 0.014 h −1, respectively. High efficiency of DSM 9103 growth on EDTA in a chemostat was established; the maximum mass cell yield ( Y X/S m) and the maximum energy cell yield ( η X/S m) were as high as 35.3 and 50.5%, respectively. Results indicated that oxidation of side chains in the EDTA molecule by monooxygenase is coupled with energy utilization by the cells. Cells harvested from a chemostat (resting cells) retain the capability of EDTA degradation, although at a rate about four to five times lower than growing cells.

A novel two-dimensional 4f-3d coordination polymer Ce2Co3(EDTA)3(H2O)11·12H2O

A novel two-dimensional 4f-3d heterometallic coordination polymer Ce2Co3(EDTA)3(H2O)11·12H2O (EDTA = ethylenediamine-N,N,N′,N′-tetraacetate) has been synthesized using complex Na[Ce(EDTA)] as ligand. The complex crystallines in the space group C2, with lattice parameters a = 15.974(3) A, b = 14.676(3) A, c = 14.773(3) A, β = 116.39(3)°,V = 3102.4(11) A3, Dx = 1.858 g/m3, Z = 2. It consists of a two-dimensional sheet in which the Ce(III) is ten-coordinated by six carboxylic O atoms from three EDTA molecules and four O atoms of water molecules, forming a distorted bicapped square antiprism, and the Co(II) atom is seven-coordinated by two N, four O atoms from one EDTA molecule and one O atom from the water molecule, giving rise to a monocapped trigonal prism. Each chelated Co(II) atom is connected to two hydrated Ce(III) atoms through tridendate bridging carboxylate groups, while each Ce(III) atom has three Co(II) atoms as neighbors. These sheets are linked into a three-dimensional network by the water molecules between layers through rich hydrogen bonds.

Experimental study of the effect of EDTA on Cd adsorption by Bacillus subtilis: a test of the chemical equilibrium approach

The interactions between aqueous metals, organic acids, and bacteria can significantly affect mass transport in many geologic settings. This study examines the Cd–EDTA– Bacillus subtilis system to investigate these interactions, and to test the applicability of a chemical equilibrium approach to quantify aqueous and surface complexation reactions that occur in the system. The experiments indicate that fully-protonated EDTA adsorbs onto the bacterial surface through hydrophobic interaction, and that adsorption of the deprotonated EDTA molecule is negligible. Aqueous EDTA can strongly compete with the bacterial surface for aqueous Cd, and the presence of aqueous EDTA significantly diminishes Cd adsorption onto B. subtilis. Independent of the experimental measurements, we use chemical equilibrium modeling to estimate the extent of Cd adsorption in the Cd–EDTA–bacteria systems. The observed adsorption behavior is in excellent agreement with the estimations, suggesting that chemical equilibrium modeling can successfully account for the distribution of mass in a system in which both aqueous and surface metal–organic complexation occurs.

Energetics of Ca 2+–EDTA interactions: calorimetric study

The interaction between Ca 2+ and EDTA has been studied using isothermal titration calorimetry to elucidate the detailed mechanism of complex formation and to relate the apparent thermodynamic parameters of calcium binding to the intrinsic effects of ionization. It has been shown that Ca 2+ binding to EDTA is an exothermic process in the temperature range 5–48°C and is highly dependent on the buffer in which the reaction occurs. Calorimetric measurements along with pH-titration of EDTA under different solvent conditions shows that the apparent enthalpy effect of the binding is predominantly from the protonation of buffer. Subtraction of the ionization effect of buffer from the total enthalpy shows that the enthalpy of binding Ca 2+ to EDTA is −0.56 kcal mol −1 at pH 7.5. The Δ H value strongly depends on solvent conditions as a result of the degree of ionization of the two amino groups in the EDTA molecule, but depends little on temperature, indicating that the heat capacity increment for metal binding is close to zero. At physiological pH values where the amino groups of EDTA with p K a=6.16 and p K a=10.26 are differently ionized, the coordination of the Ca 2+ ion into the complex leads to release of one proton due to deprotonation of the amino group having p K a=10.26. Increasing the pH up to 11.2, where little or no ionization occurs, leads to elimination of the enthalpy component due to ionization, while its decrease to pH 2 leads to its increase, due to protonation of the two amino groups. The heat effect of Ca 2+/EDTA interactions, excluding the deprotonation enthalpy of the amino groups, i.e. that associated with the intrinsic enthalpy of binding, is higher in value (Δ b H o=−5.4 kcal mol −1) than the apparent enthalpy of binding. Thus, the large Δ G value for Ca 2+ binding to EDTA arises not only from favorable entropic but also enthalpic changes, depending on the ionization state of the amino groups involved in coordination of the calcium. This explains the great variability in Δ H obtained in previous studies. The ionization enthalpy is always unfavorable, and therefore dramatically decreases Ca 2+ affinity by reduction of the enthalpy term of the stability function. The origin of the enthalpy and entropy terms in the stability of the Ca 2+–EDTA complex is discussed.

Mineral precipitation and dissolution in aqueous solution: in-situ microscopic observations on barite (001) with atomic force microscopy

Crystal growth and dissolution mechanisms on the barite (001) surface have been observed in-situ in aqueous solution as a function of the saturation state at molecular scales using Atomic Force Microscopy (AFM). On freshly cleaved barite (001) surfaces, cleavage steps with a step height of 7 Å occur, representing the height of a unit cell in the c direction. Growth and dissolution occurs via the advance and retreat of steps with a step height of half a unit cell (3.5 Å). In supersaturated BaSO 4 solution, monolayer step growth increases linearly with the BaSO 4 concentration. Step velocities of one BaSO 4 half-unit cell layer are faster in [100] relative to [−100], whereas in the underlying BaSO 4 half-unit cell layer, step velocities are faster in [−100] than in [100]. A 2 1 screw axis parallel to [001] causes the directional growth in opposite directions in different half-unit cell layers. At low to moderate supersaturations, up to about 80 μM BaSO 4, spiral growth is the dominant growth mechanism, whereas at higher BaSO 4 concentrations, surface nucleation dominates the growth process. In the presence of a crystal growth inhibitor (NTMP, nitrilotri(methylenephosphonic) acid), the nucleation rate as well as step growth is retarded depending on the inhibitor concentration. The morphology of monolayer step edges becomes irregularly curved and jagged, thus suggesting that NTMP molecules attach preferentially to step edges. In pure water, shallow etch pits form with a morphology defined by monolayer steps parallel to [010] and [110]. All etch pits within one BaSO 4 layer point in the same direction, whereas etch pits in the underlying BaSO 4 layer point in the opposite direction, as a result of the 2 1 screw axis. Chelating agents, such as EDTA, ethylenediaminetetraacetic acid, dissolve barite effectively by the formation of Ba-EDTA surface complexes and their desorption resulting in an increased etch pit formation rate. Different types of etch pits could be distinguished: (i) shallow etch pits are defined by steps parallel to [010] and [110], (ii) whereas deep etch pits are defined by steps parallel to [100] and [010]. Step velocities of retreating monolayer steps parallel to [110] retreat faster than steps parallel to [010] resulting in an elongated etch pit morphology. With increasing EDTA concentration from 0.1 mM to 100 mM, the velocity of steps parallel to [110] is reduced by one order of magnitude. Etch pit morphology as well as monolayer step kinetics suggest that EDTA molecules attach to the barite (001) surface and detach a BaEDTA 2− complex preferentially from steps parallel to [110].

Study of coordination and ligand structure in cobalt-EDTA complexes with vibrational microspectroscopy

Micro -Raman scattering experiments and micro-infrared absorption studies have been conducted on the cobalt-EDTA complexes [Co(H 2O)H 2EDTA] · 2H 2O ( I) and [Co(H 2O) 6] 2+[Co(H 2O) HEDTA] 2 2− · 2H 2O ( II) (where HEDTA is the mono-protonated EDTA 4− ligand anion, or HEDTA 3−, and EDTAH 2 is the di-protonated anion, or H 2EDTA 2−, of the neutrally charged parent EDTA acid) that previously have been documented with respect to their structural chemistry. The two complexes have been found to exhibit vibrational spectra that allow one to differentiate them from one another. The vibrational data for the two compounds are discussed in relation to their structures, particularly with respect to subtle bonding differences of both the EDTA ligands and the coordinated and un-coordinated water molecules. Several vibrational assignments are proposed for both the EDTA and water molecules in the complexes for the variably protonated EDTA molecule.

Effect of configuration of viologen-linked porphyrin on photocurrent generation and on photoinduced hydrogen evolution

Two types of viologen-linked porphyrin with different configuration between the porphyrin and the bonded viologen have been synthesized and Langmuir–Blodgett (LB) films prepared. The effects of the different configurations on the photoelectric conversion by indium tin oxide (ITO) electrodes modified with the LB films and on photoinduced hydrogen evolution, using the LB films and H 2 PtCl 6 as a catalyst in the presence of ethylenediaminetetraacetic acid (EDTA) as a sacrificial electron donor, have been investigated. No effect was detected for the photocurrent generation but photoinduced hydrogen evolution was dependent upon the configuration. These results show that the rate of diffusion of small electrolyte ions is the same in both LB films but diffusion of large EDTA molecules is affected by the configuration. Hence, the molecular structure of viologen-linked porphyrin must be taken into account in order to make diffusion of reactant in the LB film easy and thus to increase the reaction yield.

Crystal structure of aquo (hydroxo) (ethylenediaminetetraacetato) sodium(I) tin(IV), NaSn(OH)(edta)(H2O)

NaSn(OH)(edta)(H2O) is monoclinic, space groupP21/c, witha=9.747(3)A,b=9.121(3)A,c=16.430(6)A, β=98.69(4)°, A3, andZ=4. The coordination environment of Sn(IV) is a capped octahedron. Sn−O distances range from 1.990(6)A to 2.351(7)A. Na(I) is five coordinated to three different edta molecules. Na−O distances range from 2.283(9)A to 2.414(7)A. The edta ligand presents the E, G/R conformation. The crystal structure is composed of sheets parallel to (001): inside a sheet Sn(OH)(edta) molecules are connected to each other by the Na(I) interactions.

Synthesis and Crystal Structure of MCu(edta).cntdot.4H2O: Molecular Precursors for MCuO2 (M = Ca, Sr, Ba)

The compounds MCu(edta){center_dot}4H{sub 2}O (edta = ethylenediaminetetraacetic acid; M = Ca, Sr, Ba) have been synthesized and characterized by analytical and spectroscopic techniques. The structures of the strontium and barium complexes have been determined by X-ray diffraction methods. Both compounds are isostructural and crystallize in the monoclinic system, space group P2{sub 1}/c, with four formula weights in a cell measuring a = 8.717(1), b = 10.568(3), c = 19.374(3) {angstrom}, {beta} = 100.39(1){degrees} for [SrCu(edta)(H{sub 2}O){sub 3}]{center_dot}H{sub 2}O and a = 8.818(1), b = 10.200(2), c = 19.737(2) {angstrom}, {beta} = 102.40(1){degrees} for [BaCu(edta)(H{sub 2}O){sub 3}]{center_dot}H{sub 2}O. The structures were refined to R = 0.046 (R{sub w}=0.051) and R = 0.034 (R{sub w}=0.037) for strontium and barium compounds, respectively. The geometry of the complexes consists of an intricate network of ligand-bridged coordination polyhedra, where the arrangement of the copper(II) ion exhibits a distorted octahedral topology. The geometry of the alkaline-earth metal cations is near to the bicapped trigonal prism, being bound to three water molecules and five oxygen atoms from different edta molecules. In comparison with the structural results of the [CaCu(edta)(H{sub 2}O){sub 2}]{center_dot}2H{sub 2}O, a change in the coordination polyhedron of calcium ion has been established. Thermal treatment ofmore » the complexes using TGA involves three consecutive steps: dehydration, ligand pyrolysis and the formation of inorganic residue. Taking these results into account, further thermal treatments have been performed in order to obtain MCuO{sub 2} phases (M = Ba, Sr, Ca). 33 refs., 6 figs., 4 tabs.« less

Leaching Behavior of Edta In A Silica Sol-Gel Matrix

ABSTRACTEthylene diamine tetra-acetic acid (EDTA) doped silica gels were made by the sol-gel process for the potential application as a filter for heavy metal ions in wastewater. The behavior of the organic molecule in the matrix was studied by investigating the percentage of EDTA leached out with a variation in the timing of addition of the EDTA molecule into the starting silica gel solution. Leach tests using water as the medium were performed for 2, 12 and 24 hours in order to determine the amount of EDTA trapped in the pores or in the matrix. A minimum amount of EDTA leached from the gels was detected in the samples to which EDTA was added 4 hours after initial hydrolysis of TEOS. This result was correlated to having a greater percentage of pore volume in the range of 15–20 Å, which was further substantiated with density measurements. The physical changes in the silica matrix altering the pore volume distribution were attributed to the addition of the water into which the EDTA molecule was initially dissolved.

Preparation, IR, 13C and 119 mSn Mössbauer spectral studies of organotin(IV) derivatives of ethylenediaminetetraacetic acid

New diorganotin(IV) and triorganotin(IV) derivatives (R 2Sn) n EDTA (R  Me, Et, Pr, nBu, nOct; n = 1, 2); (R 2Sn) 3(EDTAH) 2 (R  nBu, nOct); and (R 3Sn) 3EDTAH (R  ME, nBu, Ph, Cyh) have been prepared by reacting diorganotindichloride and triorganothinchloride with di-, tri- and tetra-silver salts of ethylenediaminetetraacetic acid (EDTAH 4) in 1:1, 2:1, 3:2 and 3:1 (metal/ligand) molar ratio, respectively. The 3:2 diorganotin(IV) compounds possess an octahedral geometry around the Sn atom whereas the diorganotin (1:1, 2:1) and triorganotin (3:1) have five coordinate trigonal bipyramidal geometry around the Sn atom. One of the organotin moieties in (R 2Sn) 3(EDTAH) 2 acts as a bridge between two EDTA molecules, resulting in a novel type of complex.

Implications of heavy metal toxicity related to EDTA exposure

Owing to its poor degradability, EDTA can be found in relatively high concentrations in many drinking waters produced from surface waters and river bank filtrates. In its toxicological assessment it has to be taken into account that heavy metals found in these waters are present mainly in the form of their EDTA complexes whose chronic low‐dose toxicity has not yet been adequately investigated. Moreover, the biochemistry, pharmacodynamics and pharmacokinetics of the EDTA molecule itself preclude at present a definitive answer as to its toxicological profile. Some recent biochemical data show that EDTA could intensify the renal toxicity of cadmium due to the higher binding affinity of this complexing agent for zinc. Consequently, there is a need for a toxicological re‐evaluation of EDTA whose admission as a food additive in some European countries is based on data obtained at least 14 years ago.

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The mass spectra of EDTA salts and complexes were measured by means of FABMS. Three main quasi-molecular ions, (M-A+H+H)+, (M+H)+, and (M+A)+ were detected, in which M and A represent EDTA molecule and alkali metal element, respectively. In the case of EDTA containing only alkali metal element, six characteristic ions {(H5-n AnY)+ n=0 to 5, and Y represents (-OOCCH2)2 NCH2CH2N (CH2COO-)2} were observed. Comparing the intensities of three characteristic ions of EDTA containing alkali earth metal elements (Mg, Ca, and Sr) with (2G+H)+ ion of glycerin, we found that the intensities of characteristic ions were in proportion to the order of the inverse of the ionic radii of the elements. However, in the cases of the EDTA complexes containing transition metal elements, the order of the intensities of characteristic ions are turned out to be as follows,Mn2+ > Co2+ > Ni2+ > Cu2+ < Zn2+.In the case of EDTA containing Fe(III), both oxidation and reduction reactions were recognized. Moreover, in the case of non-metal EDTA compound, both (M+H)+ and (M+NH4)+ quasi-molecular ion were detected. In a few EDTA complexes, dimer quasi-molecular ions were also detected.

Mechanism of electron transfer to coordinated dioxygen of oxyhemoglobins to yield peroxide and methemoglobin. Protein control of electron donation by aquopentacyanoferrate(II).

Reactions of human oxyhemoglobin A with iron(II) compounds have been investigated. Human oxyhemoglobin (HbO2) reacts with aquopentacyanoferrate(II), Fe(II)(CN)5H2O3-, to yield hydrogen peroxide, aquomethemoglobin and Fe(III)(CN)5H2O2-. The reaction follows a second order rate law, first order in the pentacyanide and in HbO2. Since reaction rates are lower in the presence of catalase, the H2O2 produced must promote metHb formation in reactions independent of pentacyanide. Changes in concentrations of effectors (e.g. H+, inositol hexaphosphate, Cl-, and Zn2+), alkylation of beta-93 cysteine with N-ethylmaleimide, and substitution at distal histidine (as in Hb Zurich with beta-63 His----Arg) in each case can markedly affect pentacyanide reaction rates demonstrating a fine control of rates by protein structure. Hexacyanoferrate(II) (ferrocyanide) reacts with HbO2 to produce cyano-metHb as well as aquo-metHb but the reaction with the hexacyanide is much slower than with the aquopentacyanide. Iron(II) EDTA converts HbO2 to deoxy-Hb with no evidence for formation of metHb as an intermediate. These findings support a mechanism in which the pentacyanide anion reacts directly with coordinated dioxygen. One-electron transfers to O2 from both pentacyanide iron(II) and heme iron(II) result in the formation of a mu-peroxo intermediate, HbFe(III)-O-O-Fe(III) (CN)5(3-). Hydrolysis of this intermediate yields metHb . H2O, H2O2, and FeIII(CN)5H2O2-. The reaction of HbO2 with Fe(CN)6(4-) must follow an outer sphere electron transfer mechanism. However, the very slow rate that is seen with Fe(CN)6(4-) could arise entirely from the pentacyanide produced from loss of one cyanide ligand from the hexacyanide. Fe(II)EDTA reacts rapidly with free O2 in solution but can not interact directly with the heme-bound O2 of HbAO2. The dynamic character of the O2 binding sites apparently permits access of the Fe2+ of the pentacyanide to coordinated dioxygen but the protein structure is not sufficiently flexible to allow the larger Fe2+EDTA molecule to react with bound O2. It is necessary for maintenance of the oxygen transport function of the red cell for reductants such as the methemoglobin reductase system, glutathione, and ascorbate to be able to reduce metHb to deoxy-Hb. It is also important for these reductants to be unable to donate an electron to HbO2 to yield H2O2 and metHb. Thus, a mechanistic requirement for the delivery of one-electron directly to the dioxygen ligand, if peroxide is to be produced, enables the protein to protect the oxygenated species from those electron donors normally present in the cell by denying these reductants steric access to coordinated O2.(ABSTRACT TRUNCATED AT 400 WORDS)

Surface analysis of dealuminated Y zeolites by ESCA

A series of dealuminated NaY zeolites was studied by ESCA. NaY zeolite was dealuminated using an aqueous solution of H4EDTA. Changes in surface compositions and chemical states as a result of dealumination are reported. ESCA data indicated that aluminum and sodium are preferentially removed from the external surface of the zeolite during dealumination. This is presumably due to diffusional limitations of EDTA molecules into the internal cavities of the zeolite particles. It was found also that Al atoms in the zeolite crystal termination positions are either neutralized by protons or tri-coordinated.

Surface analysis of dealuminated Y zeolites by ESCA

A series of dealuminated NaY zeolites was studied by ESCA. NaY zeolite was dealuminated using an aqueous solution of H 4EDTA. Changes in surface compositions and chemical states as a result of dealumination are reported. ESCA data indicated that aluminum and sodium are preferentially removed from the external surface of the zeolite during dealumination. This is presumably due to diffusional limitations of EDTA molecules into the internal cavities of the zeolite particles. It was found also that Al atoms in the zeolite crystal termination positions are either neutralized by protons or tri-coordinated.

A critical evaluation of Bernhard's EDTA regressive staining technique for RNA.

The EDTA regressive staining procedure to detect RNA (Bernhard's technique) is based on the proposition that after staining ultrathin sections with uranyl the stain is preferentially removed from DNA rather than RNA by the action of the chelating agent EDTA. Whilst attempting to use the EDTA regressive staining procedure to detect the presence of RNA in the large granule complexes of chicken erythrocyte nuclei, certain anomalous staining patterns were observed in the chromatin of these nuclei. Essentially, these were that the edges of condensed chromatin bodies stained positively for RNA even though this molecule is known not to be present there in significant quantities. The staining patterns suggested that chromatin was retaining its stain in a manner expected of RNA but not DNA as a consequence of EDTA-containing species failing to pass freely through the section. This hypothesis was tested by carrying out the EDTA procedure on embedded specimens of a DNA-containing virus, simian virus 40 (SV40), small enough not to be exposed at the surface of the section. In this way it has been shown that virus particles completely surrounded by resin destain so much more slowly than chromatin, which is accessible at the surface of the section, that without any other information it would be concluded that the viruses contained RNA, not DNA. This apparently anomalous result arises because the difficulties encountered by stain or EDTA molecules in passing through a plastic section were not appreciated at the time of the initial publication of the technique. The observations are discussed in the light of recent knowledge that has been gained on the kinetics of staining by measuring the electron-scattering densities of stained sections, similar measurements having been made on sections stained by Bernhard's technique. A model for the mechanism of the EDTA regressive staining technique consistent with the experimental observations is proposed and the conditions under which Bernhard's staining procedure retains its specificity are defined. Briefly, these conditions are that: (a) the sections be stained for only a short period with uranyl before treating them with EDTA even though such brief staining is undesirable for quantitative measurements of stain uptake into biological material; and (b) that sections stained in lead only be compared as controls with sections stained by Bernhard's technique so that any specificity of lead for sub-cellular components is not confused with a positive indication of the presence of RNA. Unless these conditions are fulfilled, results obtained by the use of the regressive staining technique may be highly misleading.