Spectroscopic studies on the interaction of Au(III) with nucleosides,nucleotides, and dimethyl phosphate

A series of complexes of Au(III) with nucleosides and nucleotides and their methyl derivatives in different stoichiometry have been prepared. Ultraviolet, visible, ir, and nmr studies have been performed to determine the site of binding of these ligands with the metal ion. In (1:4) Au(III): guanosine complex, N₇ is the binding site, whereas at 1:1 complex, a bidentate type of chelation through C₆O and N₇ is observed. C₆-NH₂ is favored over N₁ as coordinating site at all stoichiometry in the adenosine complex. Inosine binds through N₁ at r = 1. In cytidine, N₁ is the binding site, whereas thymidine reacts only at high pH. In the case of nucleotides a bidentate type of chelation through the phosphate and the ring nitrogen occurs. The phosphate binding ability of Au(III) was further confirmed by studying the interaction of Au(III) with dimethyl phosphate—a conformational analog of the phosphate backbone in DNA chain.     

Purification and characterization of mouse DNA polymerase alpha devoid of primase activity

A simple method was developed for the isolation of primase-free DNA polymerase-alpha from the DNA polymerase-alpha-primase complex of mouse FM3A cells. The polymerase was separated from primase subunits by chromatography on a single-stranded DNA-cellulose column in the presence of 50% etylene glycol. The primase-free DNA polymerase-alpha contained two polypeptides with molecular masses of 180,000 and 68,000. Analysis of the DNA products with poly(dA)-oligo(dT)10 as template-primer revealed that both primase-free DNA polymerase-alpha and the DNA polymerase-alpha-primase complex predominantly synthesized short DNA with less than 30 nucleotides, but that the DNA polymerase-alpha-primase complex also synthesized some longer DNA with more than 300-400 nucleotides.     

Hemoglobin-mediated oxidation of the carcinogen 1,2-dimethylhydrazine to methyl radicals

Oxidation of 1,2-dimethylhydrazine (SDMH) catalyzed by hemoglobin is investigated by oxygen consumption studies, ESR spin-trapping experiments, and gas chromatography. Kinetic analysis and the study of the effects of superoxide dismutase, catalase, and azide on reaction rates indicate that SDMH oxidation is primarily dependent on ferric hemoglobin and autoxidatively formed H2O2. SDMH oxidation generates both methyl and hydroxyl radicals as ascertained by spin-trapping experiments with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, 5,5-dimethyl-1-pyrroline-N-oxide, and tert-nitrosobutane. Quantitative estimates indicate that the yield of the methyl radical trapped by alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone is about 8% of the consumed oxygen. Analysis of the gaseous products by gas chromatography shows methane formation at a yield 10 times lower than that obtained for the spin-trap methyl adduct. These results are discussed within the context of the spin-trapping technique. The relative efficiencies of oxyhemoglobin in catalyzing SDMH, 2-phenylethylhydrazine, and phenylhydrazine oxidation, defined as Vmax/KM, are estimated as 1, 13, and 386, respectively. The higher efficiency obtained for the monosubstituted derivatives leads us to suggest that hemoglobin-catalyzed oxidation could be a detoxification route for these compounds. By contrast, SDMH oxidation requires a peroxidase-like activity, a fact that may be related to the efficacy and specificity of this carcinogen.     

Reaction of iron(III) with theaflavin: complexation and oxidative products

Theaflavins are a family of compounds, whose chemistry has been sparsely investigated. They can comprise up to 40% the dry weight of black tea. They are known to chelate metals, however very little knowledge exists on the mechanisms involved. There is some correlation between both of these areas in that following degradation of the iron theaflavin complex, subsequent redox reactions may lead to the formation of similar products on both occasions. The interaction of iron(III) with theaflavin at pH < 3.0 is investigated by means of liquid chromatography mass spectroscopy (LC-MS), stopped flow spectroscopy and multivariate data analysis. Iron theaflavin complexes are formed which subsequently decay to form a number of oxidative species. The difficulties involved in the elucidation of the structure of polymeric phenolic compounds from black tea has been highlighted by numerous authors. The intermediates and major low molecular weight oxidised theaflavin products from the reaction of excess iron with theaflavin have been detected and identified using multivariate data analysis of diode array spectroscopic data. It is not possible to characterise the extremely polar high molecular weight oxidation products obtained from polyphenol oxidation. High performance liquid chromatography (HPLC) and electrospray mass spectroscopy (ES-MS) detected the low molecular weight oxidised theaflavin species present in the system. Enzymatic oxidation of theaflavin using peroxidase (POD) resulted in the formation of one major low molecular weight species oxidative product, which was fully characterised using nuclear magnetic resonance spectroscopy (NMR), high performance liquid chromatography (HPLC), electrospray mass spectroscopy (ES-MS), UV-visible (UV-Vis) and Fourier transform infra-red spectroscopy (FT-IR). The major objective of this work is to investigate the reaction of iron(III) with theaflavin and to add some insight into the mechanistic interaction of iron(III) with this family of compounds.     

A spectroscopic investigation of the binding interactions between 4,5-dihydroxyxanthone and heme

In order to investigate one possible mechanism by which xanthones inhibit growth of malaria-causing Plasmodium parasites, optical and NMR spectroscopic studies were performed on a prototypic xanthone, 4,5-dihydroxyxanthone (45X2), upon its complexation to heme. The 45X2 x heme complex stoichiometry in aqueous solution was found to be 1:2; this interaction was non-cooperative, and exhibited a very similar heme complex dissociation constant (K(d)=5.1 x 10(-6)) as observed for the common antimalarial agents, chloroquine and quinine. The 45X2 x heme(2) complex formation was found to be both pH- and solvent-dependent, with clear evidence of the xanthone carbonyl moiety coordinating with the iron of heme. Hydrogen bonding between the hydroxyl groups of 45X2 and the propionate side chains of heme, as well as pi-pi stacking between both aromatic systems appeared to contribute to the overall stability of the 45X2 x heme(2) complex, as judged by 1H NMR. It was concluded that 45X2 forms a complex with a heme dimer in aqueous solution, and that this interaction can be generalized to account for its in vivo detrimental effect of parasite growth through an effective inhibition of hemozoin aggregate formation.     

The binding of zinc (II) to a double-stranded oligodeoxyribonucleotide. A voltammetric study

Binding of zinc to a 19 mer double-stranded oligodeoxyribonucleotide was investigated by anodic stripping voltammetry and cyclic voltammetry in order to understand the roles of zinc in DNA cleavage catalyzed by mung bean nuclease. These methods rely on the direct monitoring of zinc oxidation current in the absence and in the presence of the oligo. Zinc titration curves with the ds-oligodeoxyribonucleotide were obtained in concentrations ranging from 3.62 x 10(-9) to 3.62 x 10(-8) M and 4.06 x 10(-10) to 5.25 x 10(-9) M. The acquired data were used to determine the dissociation constant, stoichiometry and zinc binding sites of the complex and to understand the specific changes of ds-oligodeoxyribonucleotide secondary structure by zinc binding. The oxidation-reduction process of zinc was also investigated by cyclic voltammetry through I (oxidation current) versus v(1/2) (square root of scan rate) curves in the absence and in the presence of the double-stranded oligodeoxyribonucleotide.     

Iron binding and oxidation kinetics in frataxin CyaY of Escherichia coli

Friedreich's ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein. Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (K(d) approximately 4 microM). Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone. The bound Fe(II) is oxidized slowly by O(2). However, oxidation occurs rapidly and completely with H(2)O(2) through a non-enzymatic process with a stoichiometry of two Fe(II)/H(2)O(2), indicating complete reduction of H(2)O(2) to H(2)O. In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemistry is greatly attenuated in the presence of CyaY. The Fe(III) produced from oxidation of Fe(II) by H(2)O(2) binds to the protein with a stoichiometry of six Fe(III)/CyaY monomer as independently measured by kinetic, UV-visible, fluorescence, iron analysis and pH-stat titrations. However, as many as 25-26 Fe(III)/monomer can bind to the protein, exhibiting UV absorption properties similar to those of hydrolyzed polynuclear Fe(III) species. Analytical ultracentrifugation measurements indicate that a tetramer is formed when Fe(II) is added anaerobically to the protein; multiple protein aggregates are formed upon oxidation of the bound Fe(II). The observed iron oxidation and binding properties of frataxin CyaY may afford the mitochondria protection against iron-induced oxidative damage.     

Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli

The genes for the protein (C5 protein) and RNA (M1 RNA) subunits of Escherichia coli RNase P have been subcloned and their products prepared in milligram quantities by rapid procedures. The interactions between the two subunits of the enzyme have been studied in vitro by a filter-binding technique. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant for the specific interactions of the subunits in the holoenzyme complex is approximately 4 x 10(-10) M. C5 protein also interacts with various RNA molecules in a non-specific manner with a dissociation constant of 2 x 10(-8) to 6 x 10(-8) M. Regions of M1 RNA required for interaction with C5 protein have been defined by deletion analysis and footprinting techniques. These interactions are localized primarily between nucleotides 82 to 96 and 170 to 270 of M1 RNA.     

Purification of ferrichrome synthetase from Aspergillus quadricinctus and characterisation as a phosphopantetheine containing multienzyme complex

Aspergillus quadricinctus was grown under iron limitation to induce the enzymes for ferrichrome biosynthesis. The mycelium was disintegrated by ultraturrax homogenization, and ferrichrome synthetase was purified by column chromatography on DEAE cellulose, hydroxyapatite and Bio-Gel A-5m. The enzyme was almost homogeneous in single fractions as shown in gel electrophoresis under non-denaturating conditions. By fast-protein liquid chromatography on Superose 6, the purified ferrichrome synthetase (molecular weight 9.6.10(5) dissociated partly into an enzyme complex with reduced ferrichrome synthetase activity of 8 x 10(5) Da, one acetylhydroxyornithine (AHO) activating protein of 5.5 x 10(5) Da and one glycine activating protein of 4 x 10(5) Da. After SDS treatment the AHO activating protein dissociated into subunits of 9 x 10(4) Da, while the glycine activating protein dissociated into subunits of 5 x 10(4) Da and 4 x 10(4) Da in a molar ratio of 6:1. No subunits were found after SDS treatment of the larger of the two ferrichrome synthetizing enzyme complexes. Pantetheine was detected in protein bands of defined molecular weights (4 x 10(4), 9 x 10(4) and greater than 3.4 x 10(5) after SDS polyacrylamide gel electrophoresis. Gel slices were cut out, and the growth factor activity for Lactobacillus plantarum ATCC 8014 was analyzed. The calculated content was 2 mol of pantetheine per mol of ferrichrome synthetase of 9.6 x 10(5) Da.     

Overproduction, purification, and characterization of chlorocatechol dioxygenase, a non-heme iron dioxygenase with broad substrate tolerance.

We show here that purified chlorocatechol dioxygenase from Pseudomonas putida is able to oxygenate a wide range of substituted catechols with turnover numbers ranging from 2 to 29 s-1. This enzyme efficiently cleaves substituted catechols bearing electron-donating or multiple electron-withdrawing groups in an intradiol manner with kcat/KM values between 0.2 x 10(7) and 1.4 x 10(7) M-1 s-1. These unique catalytic properties prompted a comparison with the related but highly specific enzymes catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase. The chlorocatechol dioxygenase gene (clcA) from the Pseudomonas plasmid pAC27 was subcloned into the expression vector pKK223-3, allowing production of chlorocatechol dioxygenase to approximately 7-8% of total cellular protein. An average of 4 mg of purified enzyme has been obtained per gram of wet cells. Protein and iron analyses indicate an iron stoichiometry of 1 iron/57.5-kDa homodimer, alpha 2Fe. The electronic absorption spectrum contains a broad tyrosinate to iron charge transfer transition centered at 430 nm (epsilon = 3095 M-1 cm-1 based on iron concentration) which shifts to 490 nm (epsilon = 3380 M-1 cm-1) upon catechol binding. The resonance Raman spectrum of the native enzyme exhibits characteristic tyrosine ring vibrations. Electron paramagnetic resonance data for the resting enzyme (g = 4.25, 9.83) is consistent with high-spin iron (III) in a rhombic environment. This similarity between the spectroscopic properties of the Fe(III) centers in chlorocatechol dioxygenase and the more specific dioxygenases suggests a highly conserved catalytic site. We infer that the unique catalytic properties of chlorocatechol dioxygenase are due to other characteristics of its substrate binding pocket.     

The kinetics and mechanisms of the complex formation and antioxidant behaviour of the polyphenols EGCg and ECG with iron(III)

(-)-Epigallocatechin-gallate ((-)-EGCg) and (-)-epicatechin-gallate ((-)-ECG) are important antioxidants which are found in green tea. The kinetics and mechanisms of the reactions of a pseudo-first order excess of iron(III) with EGCg and ECG have been investigated in aqueous solution at 25 degrees C and an ionic strength of 0.5M NaClO(4). Mechanisms have been proposed which account satisfactorily for the kinetic data. These are consistent with a mechanism in which the 2:1 metal:ligand complex initially formed on reaction of iron(III) with the ligand subsequently decomposes in an electron transfer step. Complex formation takes place at two separate binding sites via coupled reactions. Rate constants of 4.28(+/-0.06) x 10(6) M(-2) s(-1) and 2.83(+/-0.04) x 10(6) M(-2) s(-1) have been evaluated for the reaction of monohydroxy Fe(OH)2+ species with EGCg and ECG, respectively while rate constants for of 2.94(+/-0.4) x 10(4) M(-2) s(-1) and 2.41(+/-0.25) x 10(4) M(-2) s(-1) have been evaluated for the reaction of Fe3+ species with EGCg and ECG, respectively. The iron(III) assisted decomposition of the initial iron(III) complex formed was also investigated and the rate constants evaluated. Both the complex formation and subsequent electron transfer reactions of iron(III) with EGCg and ECG were monitored using UV-visible spectrophotometry. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time dependant spectra. The results obtained show that one molecule of either EGCg or EGC is capable of reducing up to four iron(III) species, a fact which is consistent with the powerful antioxidant properties of the ligands.     

Comparison of iron-catalyzed DNA and lipid oxidation

Lipid and DNA oxidation catalyzed by iron(II) were compared in HEPES and phosphate buffers. Lipid peroxidation was examined in a sensitive liposome system constructed with a fluorescent probe that allowed us to examine the effects of both low and high iron concentrations. With liposomes made from synthetic 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine or from rat liver microsomal lipid, lipid peroxidation increased with iron concentration up to the range of 10--20 microM iron(II), but then rates decreased with further increases in iron concentration. This may be due to the limited amount of lipid peroxides available in liposomes for oxidation of iron(II) to generate equimolar iron(III), which is thought to be important for the initation of lipid peroxidation. Addition of hydrogen peroxide to incubations with 1--10 microM iron(II) decreased rates of lipid peroxidation, whereas addition of hydrogen peroxide to incubations with higher iron concentrations increased rates of lipid peroxidation. Thus, in this liposome system, sufficient peroxide from either within the lipid or from exogenous sources must be present to generate equimolar iron(II) and iron(III). With iron-catalyzed DNA oxidation, hydrogen peroxide always stimulated product formation. Phosphate buffer, which chelates iron but still allows for generation of hydroxyl radicals, inhibited lipid peroxidation but not DNA oxidation. HEPES buffer, which scavenges hydroxyl radicals, inhibited DNA oxidation, whereas lipid peroxidation was unaffected since presumably iron(II) and iron(III) were still available for reaction with liposomes in HEPES buffer.     

A study of the interaction between cartilage proteoglycan and link protein.

The interaction between proteoglycan and link protein extracted from bovine articular cartilage (15-18-month-old animals) was investigated in 0.5 M-guanidinium chloride. The proteoglycans, radiolabelled as the aggregate (A1 fraction), were fractionated by two 'dissociative' density-gradient centrifugations (A1D1D1) followed by a rate-zonal centrifugation (S1) to yield an A1D1D1S1 preparation. At least 65% of these proteoglycans were able to bind to hyaluronate, but only 52% were able to bind to link protein as assessed by chromatography on Sepharose CL-2B. Over 80% of the [3H]link-protein preparation, radiolabelled as the aggregate, was able to interact with proteoglycan as assessed by chromatography on Sepharose CL-4B. Equilibrium-boundary-centrifugation studies performed at low link-protein concentrations (2.42 x 10(-9) M-5.93 x 10(-8) M) were analysed by Scatchard-type plots and indicated a Kd of 1.5 x 10(-8) M and a stoichiometry, n = 0.56, i.e. approx. 56% of those proteoglycans capable of binding to link protein had a strong site for link protein if a 1:1 stoichiometry were assumed. However, experiments performed at higher link-protein concentrations (3.5 x 10(-7) M and 8 x 10(-7) M) yielded stoichiometry values which were link-protein-concentration-dependent. Non-equilibrium binding studies using chromatography on Sepharose CL-2B and rate-zonal centrifugation yielded apparent stoichiometries between 0.6 and 7.5 link-protein molecules per proteoglycan monomer as a function of increasing link-protein concentration. It was concluded that a proportion of the proteoglycan molecules had a strong site for binding a single link protein (Kd 1.5 x 10(-8) M) and that at high link-protein concentrations a weaker, open-ended, process of link-protein self-association nucleated upon the strong link-protein-proteoglycan complex occurred. Hyaluronate oligosaccharides appeared to abolish a proportion of this self-association (as observed by Bonnet, Dunham & Hardingham [(1985) Biochem. J. 228, 77-85] in a study of link-protein-hyaluronate-oligosaccharide interactions) so as to leave a link protein:proteoglycan stoichiometry of 2. It is not clear whether this second link-protein molecule binds directly to the proteoglycan or to the first link protein.     

Electrochemical study of the interaction between cytochrome c and DNA at a modified gold electrode

The direct electron transfer of surface-confined horse heart cytochrome c (Cyt c) was achieved using COOH-terminated alkanethiolate-modified gold electrode. Later DNA was immobilized on the two-layer modified electrode. The quantitative determination of DNA was explored and the interaction between cytochrome c and DNA was studied. The binding site sizes were determined to be 15 bp per Cyt c molecule with double-stranded (ds) DNA and 30 nucleotides binding one Cyt c molecule with single-stranded (ss) DNA. At the dsDNA/Cyt c/MUA/Au electrode, the rate constant of oxidation electron transfer k(s,ox)=1.59x10(-3)cms-1 was obtained, at the ssDNA/Cyt c/MUA/Au electrode, the value was 2.43x10(-3)ms-1 when the scan rate was 1.0V/s. The different electrodes were characterized with electrochemical quartz crystal microbalance and atomic force microscope.     

Binding stoichiometry and structure of RecA-DNA complexes studied by flow linear dichroism and fluorescence spectroscopy. Evidence for multiple heterogeneous DNA co-ordination

The interaction between RecA and DNA (in the form of unmodified single-stranded DNA, fluorescent single-stranded DNA and double-stranded DNA) is studied with linear dichroism and fluorescence spectroscopy. RecA is found to form a complex with single-stranded DNA with a binding stoichiometry of about four nucleotides per RecA monomer, in which the DNA bases appear to have a random orientation. Addition of ATP gamma S (a non-hydrolyzable analog of ATP) reduces the stoichiometry to about three nucleotides per RecA and causes the DNA bases to adopt an orientation preferentially perpendicular to the fiber axis. This complex can incorporate an additional strand of single-stranded DNA or double-stranded DNA, yielding a total stoichiometry of six nucleotides or three nucleotides and three base-pairs, respectively, per RecA. RecA, in the presence of ATP gamma S, is also found to interact with double-stranded DNA, with a stoichiometry of about three base-pairs per RecA. In all studied complexes, the tryptophan residues in the RecA protein are oriented with their planes preferentially parallel to the fiber axis, whereas in complexes involving ATP gamma S the planes of the DNA bases are oriented preferentially perpendicular to the fiber. This virtually excludes the possibility that the tryptophan residues are intercalated in the DNA helix. On the basis of these results, a model for the research of homology in the RecA-mediated, strand-exchange reaction in the genetic recombination process is proposed.     

The unusual intersubunit ferroxidase center of Listeria innocua Dps is required for hydrogen peroxide detoxification but not for iron uptake. A study with site-specific mutants

The role of the ferroxidase center in iron uptake and hydrogen peroxide detoxification was investigated in Listeria innocua Dps by substituting the iron ligands His31, His43, and Asp58 with glycine or alanine residues either individually or in combination. The X-ray crystal structures of the variants reveal only small alterations in the ferroxidase center region compared to the native protein. Quenching of the protein fluorescence was exploited to assess stoichiometry and affinity of metal binding. Substitution of either His31 or His43 decreases Fe(II) affinity significantly with respect to wt L. innocua Dps (K approximately 10(5) vs approximately 10(7) M(-)(1)) but does not alter the binding stoichiometry [12 Fe(II)/dodecamer]. In the H31G-H43G and H31G-H43G-D58A variants, binding of Fe(II) does not take place with measurable affinity. Oxidation of protein-bound Fe(II) increases the binding stoichiometry to 24 Fe(III)/dodecamer. However, the extent of fluorescence quenching upon Fe(III) binding decreases, and the end point near 24 Fe(III)/dodecamer becomes less distinct with increase in the number of mutated residues. In the presence of dioxygen, the mutations have little or no effect on the kinetics of iron uptake and in the formation of micelles inside the protein shell. In contrast, in the presence of hydrogen peroxide, with increase in the number of substitutions the rate of iron oxidation and the capacity to inhibit Fenton chemistry, thereby protecting DNA from oxidative damage, appear increasingly compromised, a further indication of the role of ferroxidation in conferring peroxide tolerance to the bacterium.     

DNA-DNA cross-linking mediated by bifunctional [SalenAlIII]+ complex

The aluminum (III) complex [SalenAl(III)]Cl (1), (Salen=(R,R)-N,N'-bis[5-methyl-3-(4-methylpiperazinyl)-salicylidene]-1,2-diphenylethanediamine) has been synthesized and characterized by elemental analysis, FT-IR, (1)H and (13)C NMR measurements. The interaction of complex (1) with calf thymus (CT) DNA has been studied extensively by experimental techniques. Thermal denaturation study of DNA with (1) revealed the DeltaT(m) of 5+/-0.2 degrees C. Viscosity and steady-state fluorescence measurements showed that the complex cross-links DNA and the metal center is interacting with DNA during the cross-linking. Also, the phenyl ring in the complex may intercalate between the base pairs of the DNA during the cross-linking. Competitive binding study shows that the enhanced emission intensity of ethidium bromide (EB) in the presence of DNA was quenched by the addition of the metal complex indicating that it displaces EB from its binding site in DNA and the apparent binding constant has been estimated to be (2.8+/-0.2)x10(5) M(-1). Further, time-resolved fluorescence experiments confirm the binding of (1) with DNA and its cross-linking nature. Aluminum ions shown to precipitate DNA completely above the pH 6.0, but no such precipitation was observed with complex (1). The DNA-DNA cross-linking mediated by (1) is further confirmed by gel electrophoresis.     

Isolation and some properties of a deoxyribonuclease from Turbo cornutus.

1. Four DNases were found in the dried liver extract of a top shell, Turbo cornutus. The major one was purified 120-fold by phosphocellulose column chromatography, sulfoethylcellulose column chromatography and gel-filtration on Sephadex G-150. The yield was 2.7%. 2. The enzyme activity was not affected by Mg2+ (10(-3)--10(-2)M), EDTA (10(-3)--10(-2)M), or NaCl (10(-1)M). It showed a pH optimum of 4.7--4.8. Ionic strength was found to be critical for the maximal activity. The isoelectric point was 8.5--9.0. On heating at 50 degrees C C for 5 min the enzymic activity fell to half the initial value. 3. The enzyme preparation degraded native as well as heat-denatured DNA, but not RNA. It degraded heat-denatured DNA endonucleolytically to give oligonucleotides with 3'-phosphates. 4. The 3'-phosphate and 5'-hydroxy termini of oligonucleotides were investigated. At both the 3'- and 5'-terminal positions, purine nucleotides were predominant.     

Determination of non-protein-bound iron in rat tissue by ion chromatography with electrochemical detection

A new, rapid, sensitive, and specific method combining ion chromatography with electrochemical detection was developed for measuring non-protein-bound Fe(II) and Fe(III) in biological samples. The procedure was based on the separation of the iron-diethylenetriaminepentaacetic acid complex formed directly on the chromatographic column with anion-exchange resin followed by electrochemical detection. The method enabled more than 0.5 microM Fe(II) and Fe(III) to be determined for injection volumes of 10 microliters. This method was applicable for the determination of Fe(II) and Fe(III) in ultrafiltrates of the rat liver cytosolic fraction. It was found that release of iron from iron-bound proteins was pH dependent and that non-protein-bound iron in the tissues was determined in a ferrous state at low pH values.     

A Comparative study of Fe(II) and Fe(III) interactions with DNA duplex: major and minor grooves bindings

The involvement of the Fe cations in autoxidation in cells and tissues is well documented. DNA is a major target in such reaction, and can chelate Fe cation in many ways. The present study was designed to examine the interaction of calf-thymus DNA with Fe(II) and Fe(III), in aqueous solution at pH 6.5 with cation/DNA (P) (P = phosphate) molar ratios (r) of 1:160 to 1:2. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the cation binding site, the binding constant, helix stability and DNA conformation in Fe-DNA complexes. Structural analysis showed that at low cation concentration (r = 1/80 and 1/40), Fe(II) binds DNA through guanine N-7 and the backbone PO(2) group with specific binding constants of K(G) = 5.40 x 10(4) M(1) and K(P) = 2.40 x 10(4) M(1). At higher cation content, Fe(II) bindings to adenine N-7 and thymine O-2 are included. The Fe(III) cation shows stronger interaction with DNA bases and the backbone phosphate group. At low cation concentration (r = 1:80), Fe(III) binds mainly to the backbone phosphate group, while at higher metal ion content, cation binding to both guanine N-7 atom and the backbone phosphate group is prevailing with specific binding constants of K(G) = 1.36 x 10(5) M(-1) and K(P) = 5.50 x 10(4) M(-1). At r = 1:10, Fe(II) binding causes a minor helix destabilization, whereas Fe(III) induces DNA condensation. No major DNA conformational changes occurred upon iron complexation and DNA remains in the B-family structure.