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At least 19 recordsLinked to original sources
PubMed1991

The influence of reducing agent and 1,10-phenanthroline concentration on DNA cleavage by phenanthroline + copper.

Copper in the presence of excess 1,10-phenanthroline, a reducing agent, and molecular oxygen causes cleavage of DNA with a preference for T-3',5'-A-steps, particularly in TAT triplets. The active molecular species is commonly thought to be the bis-(1,10-phenanthroline)Cu(I) complex, (Phen)2Cu(I), regardless of the reducing agent type. We have found that (Phen)2Cu(I) is not the predominant copper complex when 3-mercaptopropionic acid (MPA) or 2-mercaptoethanol are used as the reducing agents, but (Phen)2Cu(I) predominates when ascorbate is used as the reducing agent. Substitution of ascorbate for thiol significantly enhances the rate of DNA cleavage by 1,10-phenanthroline + copper, without altering the sequence selectivity. We show that (Phen)2Cu(I) is the complex responsible for DNA cleavage, regardless of reducing agent, and that 1,10-phenanthroline and MPA compete for copper coordination sites. DNA cleavage in the presence of ascorbate also occurs under conditions where the mono-(1,10-phenanthroline)Cu(I) complex predominates (1:1 phenanthroline:copper ratio), but preferential cleavage was observed at a CCGG sequence and not at TAT sequences. The second phenanthroline ring of the (Phen)2Cu(I) complex appears essential for determining the T-3',5'-A sequence preferences of phenanthroline + copper when phenanthroline is in excess.

3-Mercaptopropionic Acid↗
PubMed1978

Effects of 1,10-phenanthroline and a zinc complex of 1,10-phenanthroline on nucleic acid synthesis in mouse liver and spleen.

The effects of 1,10-phenanthroline and a zinc complex of 1,10-phenanthroline on nucleic acid synthesis were examined by noting the capcity of these agents to incorporate [methyl-3H]thymidine, or [5-3H]uridine into hepatic and splenic DNA and RNA. Within 32 hours after the intraperitoneal injection of 1,10-phenanthroline in mice, a decrease of [methyl-3H]thymidine incorporation into hepatic DNA was observed. The administration of the zinc complex of 1,10-phenanthroline decreased the incorporation of [5-3H] uridine into hepatic RNA within 24 hours and decreased the formation of [3H]DNA from [methyl-3H]thymidine in the liver within 24 and 32 hours. An increase of the isotopic incorporation into hepatic RNA and of the pool size of ATP within 2 hours after the administration of the complex was also noted. In the splenic studies, the zinc complex significantly inhibited the formation of labeled DNA and RNA at 2, 8, 24, and 32 and 2, 8 and 24 hours postinjection, respectively. A relationship exists between the results obtained from sequential treatment (zinc chloride after 1,10-phenanthroline) and those derived from the zinc complex of 1,10-phenanthroline pertaining to an inhibition of isotopic incorporation into hepatic and splenic nucleic acid.

Adenine Nucleotides↗
PubMed1977

Teratogenicity of zinc chloride, 1,10-phenanthroline, and a zinc-1,10-phenanthroline complex in mice.

Zinc chloride, in single doses of 12.5, 20.5 and 25 mg/kg ip on Day 8,9,10, or 11 of gestation in CF-1 albino mice, produced skeletal anomalies without accompanying soft tissue defects. Ripple ribs, the most unusual anomaly, first appeared when the zinc salt was given on Day 9 of gestation in a dose of 20.5 mg/kg, becoming more prevalent when 25 mg/kg of the drug was administered on Day 11.1,10-Phenanthroline, in single doses of 30 mg/kg ip on Day 8,9,10, or 11 of gestation elicited skeletal defects comparable to those caused by zinc chloride as well as soft tissue anomalies, but a significant incidence of the former occurred with this agent only following its injection on Day 8 of gestation. A zinc-1,10-phenanthroline complex in single doses of 50 mg/kg on Day 8,9,10, or 11 of gestation yielded significant incidences of skeletal and soft tissue anomalies only when the complex was administered on Day 8 or 9 of gestation. This dosage level was toxic to both the mother and fetus when given on Day 10 of gestation. However, when the complex was given on Day 8,9,10, or 11 in a dose of 25 mg/kg, neither toxic nor teratogenic effects were observed in the mother or fetus, respectively.

Animals↗
PubMed1988

Structures of chloro(glycinato)(1,10-phenanthroline)copper(II) monohydrate (I) and aqua(1,10-phenanthroline)(L-phenylalaninato)copper(II) nitrate monohydrate (II).

(I) [CuCl(C2H4NO2)(C12H8N2)].H2O, Mr = 371.28, orthorhombic, P2(1)2(1)2(1), a = 6.795 (3), b = 12.496 (4), c = 17.273 (5) A, V = 1467 (1) A3, Dx = 1.680 Mg m-3, Z = 4, F(000) = 756, lambda(Mo K alpha) = 0.71069 A, mu(Mo K alpha) = 1.742mm-1. Room temperature. Final R = 0.046 for 1302 unique observed reflections. (II) [Cu(C9H10NO2)(C12H8N2)(H2O)]NO3.-H2O, Mr = 505.98, monoclinic, P2(1), a = 5.782 (2), b = 20.700 (6), c = 9.355 (3) A, beta = 97.58 (2)degrees, V = 1110 (1) A3, Dx = 1.514 Mg m-3, Z = 2, F(000) = 522, lambda(Mo K alpha) = 0.71069 A, mu(Mo K alpha) = 1.076 mm-1. Room temperature. Final R = 0.069 for 1929 unique observed reflections. The Cu ion displays distorted square-pyramidal coordination in both (I) and (II), with the chlorine atom (I) or the water molecule (II) in the apical position. The Cu-N bond lengths alter according to the electronegative character of the trans atom. The conformations of the five-membered chelate rings appear to depend on H bonding and van der Waals interactions.

Hydrogen Bonding↗
PubMed1991

Noncovalent DNA binding of bis(1,10-phenanthroline)copper(I) and related compounds.

The noncovalent DNA binding of the bis(1,10-phenanthroline)copper(I) complex [(Phen)2CuI] was examined under anaerobic conditions by absorption and circular dichroism spectroscopy, and viscometry, as a function of phenanthroline concentration. Analyses according to the McGhee-von Hippel method indicated that binding exhibited both neighbor-exclusion and positive cooperativity effects, with a neighbor-exclusion parameter n approximately 2 and a cooperativity parameter omega approximately 4. The association constant for (Phen)2CuI binding decreased with increasing concentration of phenanthroline in excess over that required to stoichiometrically generate (Phen)2CuI, indicating that free phenanthroline was a weak competitive inhibitor of (Phen)2CuI binding. The maximal association constant for DNA binding of (Phen)2CuI in 0.2 M NaCl and 9.8% ethanol, extrapolated to zero concentration of excess phenanthroline, was 4.7 x 10(4) M-1 (DNA base pairs). The magnitude of the neighbor-exclusion parameter, the changes in spectral properties of (Phen)2CuI induced by DNA binding, and the increase in DNA solution viscosity upon (Phen)2CuI addition are consistent with a model for DNA binding by (Phen)2CuI involving partial intercalation of one phenanthroline ring of the complex between DNA base pairs in the minor groove as suggested previously [Veal & Rill (1989) Biochemistry 28, 3243-3250]. Viscosity measurements indicated that the mono(phenanthroline)copper(I) complex also binds to DNA by intercalation; however, no spectroscopic or viscometric evidence was found for DNA binding of free phenanthroline or the bis(2,9-dimethyl-1,10-phenanthroline)copper(I) complex. DNA binding of free phenanthroline may be cooperative and induced by prior binding of (Phen)2CuI.

3-Mercaptopropionic Acid↗
PubMed1977

Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by 1,10-phenanthroline.

In confluent cultures of 3T3 fibroblasts, incubated for 24 h with 1,10-phenanthroline at 10(-5)--10(-9) M, the activity of prolyl hydroxylase was significantly increased. 1,10-Phenanthroline was inhibitory at concentrations greater than 10(-4) M. The stimulatory effect of 1,10-phenanthroline manifests itself after 6 h incubation and increased with time up to 48 h. 2,2'-dipyridyl and 5,6-dimethyl-1,10-phenanthroline were also stimulatory; a nonchelating analog, 1,7-phenanthroline had no effect. Cycloheximide did not modify the 1,10-phenanthroline effect. The stimulatory effect does not seem to depend on the shift of an inactive precursor of prolyl hydroxylase to an active form because 1,10-phenanthroline was shown to be ineffective in logarithmically growing cells. While dialysis of washed and homogenized cells significantly increased prolyl hydroxylase activity in cell extracts, undialyzed 1,10-phenanthroline treated samples exhibited higher prolyl hydroxylase activity than dialyzed controls. These data suggested to us that 1,10-phenanthroline and other chelating agents may be forming complexes with certain metal ions or protein-metal ions which are inhibitory towards prolyl hydroxylase.

2,2'-Dipyridyl↗
PubMed1988

The binding of 1,10-phenanthroline to specifically active-site cobalt(II)-substituted horse-liver alcohol dehydrogenase. A probe for the open-enzyme conformation.

We have studied the binding of 1,10-phenanthroline to specifically active-site cobalt(II)-substituted horse-liver alcohol dehydrogenase [Co(II)-LADH]. The dissociation constant is a factor of 6500 smaller than in the native enzyme. Spectral evidence is given which shows that 1,10-phenanthroline does not remove the catalytic Co(II) ion and that binding of 1,10-phenanthroline renders the catalytic metal ion pentacoordinate. The maximum limiting rate constant for the association of 1,10-phenanthroline to Co(II)-LADH is about 60 s-1. This is about a third of the value (169 s-1) determined for native horse-liver alcohol dehydrogenase, Zn(II)LADH [Frolich et al. (1978) Arch. Biochem. Biophys. 189, 471-480]. For cadmium(II)-substituted horse-liver alcohol dehydrogenase, [Cd(II)LADH] the maximum limiting rate constant for association of 1,10-phenanthroline increased to 590 s-1. These findings demonstrate that the rate-limiting step is strongly dependent on the chemical nature of the catalytic metal ion and its immediate environment. 1,10-Phenanthroline is shown to bind to the Co(II)-LADH.NAD+ complex in the open conformation. The maximum limiting rate constant remains unchanged in the presence of NAD+. The data have been used to derive a kinetic scheme for the formation of ternary complexes including NAD+ that involves a slow intermediary step.

Alcohol Dehydrogenase↗
PubMed1988

Conformational stability of ferrocytochrome c. Electrostatic aspects of the oxidation by tris(1,10-phenanthroline)cobalt(III) at low ionic strength.

At ionic strengths below 0.1 M the oxidation of horse ferrocytochrome c by tris(1,10-phenanthroline)cobalt (III) and tris(2,2'-bipyridine)cobalt(III) proceeds by a pathway which is independent of the transition metal complex concentration. Formation of an activated form of the protein appears to be rate limiting. The rate of oxidation decreases as the ionic strength increases. This dependence of the reaction rate on inert electrolyte concentration indicates that electrostatic association of anions under physiological ionic strength confers stability to the protein. The activated form of the protein, which reacts at least 10(4) times as fast as the predominant form, is thought to be a conformation of the reduced protein with an open heme crevice. Binding of the open form of ferrocytochrome c with the redox-inactive cationic transition metal complexes hexamminecobalt(III) and tris(1,10-phenanthroline)chromium(III) inhibits the oxidation by tris(1,10-phenanthroline)cobalt(III). Reactions of tris(1,10-phenanthroline)cobalt(III) with 4-carboxy-2,5-dinitrophenyllysine 13 and 72 ferrocytochromes c show no dependence on ionic strength. NMR studies at pH 7 demonstrate that ferricytochrome c is partly (15%) in the open conformation at low ionic strength. Furthermore, the interaction of redox-inert tris (1,10-phenanthroline)chromium(III) with ferricytochrome c under conditions identical to those of the kinetic studies demonstrates that the transition metal complex binds only to the open form of the protein. Titration with increasing amounts of tris(1,10-phenanthroline) chromium(III) shows changes in the NMR spectrum that are inconsistent with a single binding site.

Algorithms↗
PubMed1988

[Artificial nucleases: specific cleavage of the double helix of DNA by oligonucleotides linked to copper-phenanthroline complex].

A homopyrimidine oligonucleotide d(TTTCCTCCTCT) was covalently linked to 1,10-phenanthroline via a 5'-thiophosphate group. In the presence of copper ions and a reducing agent the copper-phenanthroline complex induced cleavage reactions in duplex DNA. The oligonucleotide binds to the major groove of DNA at a homopurine.homopyrimidine sequence, forming a local triple helix. It is oriented parallel to the homopurine strand. Watson-Crick A.T and G.C base pairs are recognized via Hoogsteen-type hydrogen bonding by thymine and protonated cytosine, respectively. The cleavage patterns on opposite strands of duplex DNA at the homopurine.homopyrimidine sequence are asymmetric. They are shifted toward the 3'-side indicating that cleavage takes place from the minor groove even though the oligonucleotide is bound to the major groove. It is therefore suggested that the phenanthroline ring attached to the oligonucleotide intercalates into DNA at the junction between the triple and the double helix and that the copper complex forms in the minor groove where radical reactions leading to strand cleavage occur. The homopyrimidine oligodeoxynucleotide d(TTTCCTCCTCT) tethered to phenanthroline binds to a single site on SV 40 DNA. It cleaves circular and linear SV 40 DNA at this single binding site. Cleavage requires both copper ions and a reducing agent. The unsubstituted oligonucleotide competes with the oligonucleotide-phenanthroline conjugate and prevents site-specific cleavage. These results demonstrate that oligonucleotide-phenanthroline conjugates can be used to induce sequence-specific cleavage of duplex DNA. Such artificial endonucleases could be used, among other things, to map genes on long DNA fragments, to induce site-specific mutations or to block gene expression at the transcriptional level.

Base Composition↗
PubMed1992

Kinetics of the course of inactivation of aminoacylase by 1,10-phenanthroline.

The kinetic theory of the substrate reaction during modification of enzyme activity previously described [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381-436] has been applied to a study on the kinetics of the course of inactivation of aminoacylase by 1,10-phenanthroline. Upon dilution of the enzyme that had been incubated with 1,10-phenanthroline into the reaction mixture, the activity of the inhibited enzyme gradually increased, indicating dissociation of a reversible enzyme--1,10-phenanthroline complex. The kinetics of the substrate reaction with different concentrations of the substrate chloroacetyl-L-alanine and the inactivator suggest a complexing mechanism for inactivation by, and substrate competition with, 1,10-phenanthroline at the active site. The inactivation kinetics are single phasic, showing that the initial formation of an enzyme-Zn(2+)-1,10-phenanthroline complex is a relatively rapid reaction, followed by a slow inactivation step that probably involves a conformational change of the enzyme. The presence of Zn2+ apparently stabilizes an active-site conformation required for enzyme activity.

Amidohydrolases↗
PubMed1989

Sequence-specific recognition and cleavage of duplex DNA via triple-helix formation by oligonucleotides covalently linked to a phenanthroline-copper chelate.

Homopyrimidine oligodeoxynucleotides recognize the major groove of the DNA double helix at homopurine.homopyrimidine sequences by forming local triple helices. Phenanthroline was covalently attached to the 5' end of an 11-mer homopyrimidine oligonucleotide of sequence d(TTTCCTCCTCT). Simian virus 40 DNA, which contains a single target site for this oligonucleotide, was used as a substrate for the phenanthroline-oligonucleotide conjugate. In the presence of copper ions and a reducing agent, a single specific double-strand cleavage site was observed at 20 degrees C by agarose gel electrophoresis. The efficiency of double-strand cleavage was greater than 70% at 20 degrees C and pH 7.4. Secondary cleavage sites were observed when binding of the oligonucleotide to mismatched sequences was allowed to take place at low temperature. The exact location of the cleavage sites was determined by polyacrylamide gel electrophoresis of denatured fragments by using both simian virus 40 DNA and a synthetic DNA fragment containing the target sequence. The asymmetric distribution of the cleavage sites on the two strands revealed that the cleavage reaction took place in the minor groove even though the phenanthroline linker was located in the major groove. Linkers of different lengths were used to tether phenanthroline to the oligonucleotide and their relative efficacies of DNA cleavage were compared. Based on these comparative studies and on model building, it is proposed that the phenanthroline ring carried by the oligonucleotide intercalates from the major groove and that copper chelation locks the complex in place from within the minor groove where the cleavage reaction occurs.

Base Sequence↗
PubMed1986

Effects of o-phenanthroline, 2,2'-dipyridyl and neocuproine on the activities of bleomycin to inhibit DNA synthesis and growth of cultured cells.

Effects of o-phenanthroline, 2,2'-dipyridyl and neocuproine, which form stable complexes preferentially with Fe(II), Fe(II) and specifically with Cu(I), respectively, on the inhibitory activity of bleomycin against DNA synthesis of rat ascites hepatoma AH66 cells were examined. The inhibitory activity of metal-free bleomycin was suppressed in the presence of o-phenanthroline or 2,2'-dipyridyl, but not by neocuproine, though these chelating agents also showed the inhibitory activity against the DNA synthesis of the cells by themselves alone. The activity of bleomycin-Cu(II) was also suppressed by o-phenanthroline, but bleomycin-Fe(II) and bleomycin-Fe(III) exhibited some activities in the presence of o-phenanthroline. The growth inhibitory activity of bleomycin against HeLa cells was also suppressed by o-phenanthroline. From these results, bleomycin-iron complexes were suggested to be responsible to the bleomycin action in cells.

2,2'-Dipyridyl↗
PubMed1977

Action of 1,10-phenanthroline transition metal chelates on P388 mouse lymphocyte leukaemic cells.

(1)Fully coordinated 1,10-phenanthroline and 2,2'-bipyridine chelates of Ru(II) are lethal in vitro to cultured and ascites P388 mouse lymphocytic leukaemic cells; 1,10-phenanthroline chelates are generally more potent than corresponding 2,2'-bipyridine compounds, and mixed-ligand (acetylacetonato) monovalent chelates of both series are more active than the corresponding identical-ligand divalent chelates. Lethal potency is greatest for Ru(II) chelates containing highly alkylated ligands. (2) Within two series of tetramethyl-1,10-phenanthroline chelates, the inert Ru(II) and Ni(II) members are less active against P388 cultured and ascites cells than the corresponding more labile chelates of Cu(II), Cd(II), Zn(II), Fe(II), and Co(II); for the ascites cells, the rank order of lethal potency of the chelates correlates reasonably well with their anticipated rank order of kinetic reactivity. (3) Repeated subculture of P388 cells in the presence of a mixed-ligand Ru(II) chelate has produced a cell line that shows a stable 10-fold resistance to the chelate; the resistant cell line is selectively cross-resistant to certain Ru(II) identical and mixed-ligand chelates. (4) The presence of a fluorescent Ru(II) chelate has been demonstrated at the surface and within the cytoplasm and nucleus of P388 ascites cells exposed to it either in vitro or in the mouse. (5) Ru(II) and Cu(II) chelates of tetramethyl-1,10-phenanthroline do not appear to be chemotherapeutically active against P388 ascites cells in the mouse.

Animals↗
PubMed1988

Sequence-targeted cleavage of nucleic acids by oligo-alpha-thymidylate-phenanthroline conjugates: parallel and antiparallel double helices are formed with DNA and RNA, respectively.

Oligodeoxynucleotides can be synthesized by using the alpha anomers of nucleoside units. Oligo-alpha-deoxynucleotides are resistant to nucleases and could be used to regulate gene expression in vivo. Theoretical calculations were carried out to determine the conformational energy of an oligomeric alpha-beta duplex (dA)5.(dT)5 where the adenosine strand contains natural beta-deoxyribonucleotides and the thymidine strand contains synthetic alpha-deoxyribonucleotides. These calculations predict that in the more stable B-like conformation the two strands of the double helix should run parallel to each other whereas in the more stable A-like conformation the two strands should adopt an antiparallel orientation. In order to test these predictions 1,10-phenanthroline was covalently attached to the 5'-end of an alpha-octathymidylate. In the presence of copper ions and a reducing agent (beta-mercaptopropionic acid), the (phenanthroline)2-copper complex generates OH. radicals that cleave phosphodiester bonds in the complementary sequence to which the alpha-octathymidylate is bound. By use of a 27mer oligo-beta-deoxynucleotide containing an octadeoxyadenylate sequence as a target for the phenanthroline-substituted alpha-(dT)8, cleavage was observed on the 5'-side of the (dA)8 sequence, demonstrating that the alpha-beta DNA-DNA hybrid formed a double helix with parallel orientation of the two strands. The same result was obtained when alpha-(dT)8 was bound to beta-(dA)n with n = 8 or 10. When a beta-oligoriboadenylate was used as a target, cleavage occurred exclusively on the 3'-side of the (rA)8 or (rA)10 sequence, indicating that the alpha-beta DNA-RNA hybrid formed a double helix with an antiparallel orientation of the two strands. When a phenanthroline-substituted beta-octathymidylate was used instead of the alpha-octathymidylate, an antiparallel double helix was formed independently of whether the target beta sequence was a DNA or an RNA.

Base Sequence↗
PubMed1986

Nuclease activity of 1,10-phenanthroline-copper: sequence-specific targeting.

The nuclease activity of 1,10-phenanthroline-copper ion can be targeted to specific DNA sequences by attachment of the ligand to the 5' end of complementary deoxyoligonucleotides via a phosphoramidate linkage. To synthesize the adduct, the phosphorimidazolide of the deoxyoligonucleotide is prepared using a water-soluble carbodiimide and is then coupled to 5-glycylamido-1,10-phenanthroline. After hybridization to the target DNA, sequence-specific cleavage is observed upon the addition of cupric ion and 3-mercaptopropionic acid. Two methods of assaying the cutting of the operator sequence of the lac operon have been employed using the oligonucleotide 5'-AATTGTTATCCGCTCACAATT-3' representing sequence positions 21-1 of the template strand. In the first, the single-stranded DNA of the phage M13mp8 was the target, and cuts were detected using a primer-extension assay. In the second, the substrate was an EcoRI fragment 3' labeled in the nontemplate strand. After denaturation and reannealing to the oligonucleotide-1,10-phenanthroline adduct, cupric ion and 3-mercaptopropionic acid were added, and the products were analyzed directly on a sequencing gel. With the phenanthroline moiety attached to position 21 of the oligonucleotide carrier, cutting was observed at positions 20-25 using both assays.

Base Sequence↗
PubMed1992

Interactions of 1,10-phenanthroline and its copper complex with Ehrlich cells.

Mechanistic details of the interaction of 1,10-phenanthroline and its copper complex with Ehrlich ascites tumor cells were examined, using inhibition of cell proliferation, DNA breakage, and increased membrane permeability as indices of cellular damage. The metal chelating agent, 1,10-phenanthroline (OP), the 1:0.5 complex of 1,10-phenanthroline and CuCl2 [(OP)2Cu], and CuCl2 inhibited growth of Ehrlich ascites tumor cell monolayers during 48-h treatments by 50% at about 3.5, 2, and 70 nmol/10(5) cells/mL, respectively. (OP)2Cu at 10 nmol/10(5) cells also enhanced uptake of trypan blue dye during 6 h of treatment, while dye uptake in OP- and CuCl2-treated cells remained similar to controls. DNA breakage, measured by DNA alkaline elution, was produced during 1-h treatments with (OP)2Cu at drug/cell ratios similar to those producing growth inhibition. Copper uptake was similar for both (OP)2Cu and CuCl2. Electron spin resonance (ESR) spectroscopy suggested that cellular ligands bind copper added as (OP)2Cu or CuCl2 and then undergo time-dependent reductions of Cu(II) to Cu(I) for both forms. Inhibition of (OP)2Cu-induced single-strand scission and trypan blue uptake by scavengers of activated oxygen is consistent with participation of superoxide and H2O2 in both processes. In contrast, superoxide dismutase (SOD) did not reduce the magnitude of the fraction of cellular DNA appearing in lysis fractions prior to alkaline elution of (OP)2Cu-treated cells. Dimethyl sulfoxide (DMSO) inhibited uptake of trypan blue dye but did not inhibit DNA strand scission produced by (OP)2Cu. Thus, multiple mechanisms for generation of oxidative damage occur in (OP)2Cu-treated cells. Growth inhibition produced by OP or (OP)2Cu, as well as the low levels of strand scission produced by OP, was not reversed by scavengers.

Animals↗