Interactions of selected gold(III) complexes with calf thymus DNA

DNA represents the primary target for platinum antitumor metal complexes and is the probable target for newly developed cytotoxic gold(III) complexes. To test this hypothesis the reactions with calf thymus DNA of five representative gold(III) complexes--namely [Au(en)(2)]Cl(3), [Au(dien)Cl]Cl(2), [Au(cyclam)](ClO(4))(2)Cl, [Au(terpy)Cl]Cl(2) and [Au(phen)Cl(2)]Cl--were analyzed in vitro through various physicochemical techniques including circular dichroism, absorption spectroscopy, DNA melting, and ultradialysis. It is shown that all tested complexes interact with DNA and modify significantly its solution behavior. The solution conformation of DNA is affected to variable extents by the individual complexes as shown by CD titration experiments. Notably, in all cases, the gold(III) chromophore is not largely perturbed by addition of calf thymus DNA ruling out occurrence of gold(III) reduction. Ultradialysis experiments point out that the binding affinity of the various complexes for the DNA double helix is relatively low; in most cases the gold(III)/DNA interaction is electrostatic in nature and reversible. The implications of these findings for the mechanism of action of antitumor gold(III) complexes are discussed.     

A circular dichroism study uncovers a two-step interaction of antitumor azolato-bridged dinuclear platinum(II) complexes with calf thymus DNA

The interactions of four antitumor azolato-bridged dinuclear platinum(II) complexes, [{cis-Pt(NH(3))(2)}(2)(μ-OH)(μ-azolato)](2+), with calf thymus DNA were monitored dose- and time-dependently, by using circular dichroism. Complexes 1-4 reacted with DNA via a two-step interaction that comprised a prompt diffusion-controlled reaction, which induced a B- to C-form transition, and a relatively slow temperature-dependent reaction.     

Synthesis of cholesteryl polyamine carbamates: pK(a) studies and condensation of calf thymus DNA

Novel polyamine carbamates have been designed and prepared from cholesterol. Our synthesis uses an orthogonal protection strategy based upon trifluoroacetyl and Boc-protecting groups. These unsymmetrical polyamine carbamates have been prepared from symmetrical (e.g., spermine and thermine) polyamines. Detailed interpretations of (1)H and (13)C NMR spectroscopic data led to the unambiguous assignment of these polyamine carbamates. These target conjugates contain a variety of positive charges distributed along methylene chains. Their pK(a)s have been determined potentiometrically for conjugates substituted with up to five amino functional groups. Condensation of calf thymus DNA into particles was monitored using light scattering at 320 nm. Salt-dependent binding affinity for calf thymus DNA was determined using an ethidium bromide fluorescence quenching assay. These cholesteryl polyamine carbamates are models for lipoplex formation with respect to gene delivery (lipofection), a key first step in gene therapy.     

Spectroscopic studies on the interaction of quercetin-terbium(III) complex with calf thymus DNA

The interaction of native calf thymus DNA (CT-DNA) with quercetin-terbium(III) [Q-Tb(III)] complex at physiological pH was monitored by UV absorption spectrophotometry, circular dichroism, fluorescence spectroscopy, and viscosimetric techniques. The complex displays binding properties to the CT-DNA and was found to interact with CT-DNA through outside binding, demonstrated by a hypochromic effect of Q-Tb(III) on the UV spectra of CT-DNA and the calculated association constants (K). Also, decrease in the specific viscosity of CT-DNA, decrease in the fluorescence intensity of Q-Tb(III) solutions in the presence of increasing amounts of CT-DNA, and detectable changes in the circular dichroism spectrum of CT-DNA are other evidences to indicate that Q-Tb(III) complex interact with CT-DNA through outside binding.     

Influence of Cr(III) and Cr(VI) on the interaction between sparfloxacin and calf thymus DNA

Cr(III) and Cr(VI) have different binding capacity with sparfloxacin, and have different combination modes with calf thymus DNA. Selecting these two different metal ions, the influence of them on the binding constants between sparfloxacin (SPFX) and calf thymus DNA, as well as the related mechanism has been studied by using absorption and fluorescence spectroscopy. The result shows that Cr(III) has weaker binding capacity to SPFX in the SPFX-Cr(III) binary system, but influences the binding between SPFX and DNA obviously in SPFX-DNA-Cr(III) ternary system. However, although Cr(VI) has a stronger binding capacity to SPFX, it has no effect on the binding between SPFX and DNA. Referring to the different modes of Cr(III) and Cr(VI) binding to DNA, the mechanism of the influence of metal ions on the binding between SPFX and DNA has been proposed. SPFX can directly bind to DNA by chelating DNA base sites. If a metal ion at certain concentration binds mainly to DNA bases, it can decrease the binding constants between SPFX and DNA through competing with SPFX. While if a metal ion at certain concentration mainly binds to phosphate groups of DNA, it can increase the binding constants by building a bridge between SPFX and DNA. If a metal ion at certain concentrations binds neither to bases nor phosphate groups in DNA, it will have no effect on the binding constant between SPFX and DNA. Our result supports Palumbo's conclusion that the binding between SPFX and the phosphata groups is the precondition for the combination between SPFX and DNA, which is stabilized through stacking interactions between the condensed rings of SPFX and DNA bases.     

Efficient calf thymus DNA condensation upon binding with novel bile acid polyamine amides

Polyamine amides have been prepared from lithocholic and cholic acids (5beta-colanes) by acylation of tri-Boc-protected tetraamines spermine and thermine. These designed ligands for DNA are polyammonium ions at physiological pH. In NMR spectra, they display 14N-1H 1J = 51 Hz, 1:1:1 triplets, due to the symmetry of the R14NH(3)+ cations. The binding affinities of these conjugates for calf thymus DNA were determined using an ethidium bromide fluorescence quenching assay and compared with spermine and polylysine. DNA-binding affinities were dependent upon both salt concentration and the hydrophobicity or intermolecular bonding (facial effects) of the lipid moieties in these conjugates. Light scattering at 320 nm was used to determine DNA condensation and particle formation. The observed self-assembly phenomena are discussed with respect to DNA charge neutralization and DNA bending with loss of ethidium cation intercalation sites, ultimately leading to DNA condensation. These polyamine amides are models for lipoplex formation with respect to gene delivery (lipofection), a key first step in gene therapy.     

Poly(ADP-ribosylation) of DNA topoisomerase I from calf thymus

We demonstrate that the activity of the major DNA topoisomerase I from calf thymus is severely inhibited after modification by purified poly(ADP-ribose) synthetase. Polymeric chains of poly(ADP-ribose) are covalently attached to DNA topoisomerase I. These observations with highly purified enzymes suggest that poly(ADP-ribosylation) may be a cellular mechanism for modulating DNA topoisomerase I activity in response to the state of DNA in the nucleus. Although extensive poly(ADP-ribosylation) of the Mr = 100,000 DNA topoisomerase I from calf thymus resulted in greater than 90% enzyme inhibition, exogenous poly(ADP-ribose) does not, by itself, inhibit topoisomerase activity. After modification, the apparent molecular weight of both the topoisomerase enzyme protein and of the topoisomerase enzyme activity was increased. In vitro, the extent of modification of DNA topoisomerase I could be controlled either by changing the ratio of topoisomerase to the synthetase or by varying the reaction time. More than 40 residues of ADP ribose per topoisomerase molecule could be added by the synthetase. Analysis of a poly(ADP-ribosylated) topoisomerase preparation that was about 50% inhibited revealed an average polymer chain length of 7.4, with 1-2 chains per enzyme molecule.     

Interaction between ellagic acid and calf thymus DNA studied with flow linear dichroism UV-VIS spectroscopy

The interaction between ellagic acid and DNA has been characterized with respect to the geometry of the ellagic acid-DNA complex, and the active form of ellagic acid has been identified. Optical spectroscopic methods have been employed to examine the interaction between double-stranded calf thymus DNA and ellagic acid in low-ionic-strength aqueous solutions at pH values of 5.5, 7.0, and 8. 8. Based on normal absorption titration and flow linear dichroism experiments, it is confirmed that the neutral form of ellagic acid present at pH 5.5 binds to double-stranded DNA. It is found that the plane of the ellagic acid chromophore is positioned at an angle relative to the DNA helix axis, which is in accordance with intercalation of ellagic acid in DNA. It is concluded that at higher values of pH no or a very limited amount of ellagic acid binds to DNA. These results prove that the direct interaction between ellagic acid and DNA must be taken into account when evaluating the mechanism underlying the observed biological effects of this plant phenol.     

Fe(III) and Cu(II) conalbumin visible circular dichroism spectra.

The single polypeptide chain of conalbumin strongly binds two Fe(III) or two Cu(II) ions to yield intense absorption in the visible region similar to that shown by the related protein transferrin. Comparison of the metal-ion-binding sites in the two proteins is made by exploiting the sensitivity to ligand geometry of circular dichroism (CD). For the Fe(III) proteins strong similarities of the CD spectra outweigh marginal differences. For Cu(II) conalbumin an additional negative extremum near 506 nm appears between two positive ones at 634 and 410 nm suggesting greater subtraction of oppositely signed CD components leading to lesser magnitudes for the two positive peaks than are found in Cu(II)-transferrin. The two Fe(III)-binding sites within conalbumin are compared by noting the strong similarities of the CD and MCD of proteins with Fe(III) in one site and Ga(III) in the other site, and vice versa, with the protein containing Fe(III) in both sites. Due to features of the amino acid sequences of the single protein chains, the four strong metal ion binding sites in conalbumin and transferrin cannot be identical in all particulars, yet CD spectra of their metal ion complexes are closely similar. From a study of model phenolate complexes and the wavelength maxima of visible absorption in the Fe(III), Cu(II), and Co(III) proteins near 465, 440, and 405 nm, respectively, these strong absorption bands are identified as ligand to metal ion electron-transfer transitions. It is suggested that tyrosyl residues are the donors in the electron transfer transitions and that they lock in the metal ions after being keyed into position by binding of bicarbonate or other anions.     

Mercury-induced DNA polymorphism: probing the conformation of Hg(II)-DNA via staphylococcal nuclease digestion and circular dichroism measurements

Exposing native calf thymus DNA to increasing concentrations of Hg(ClO4)2 not only produces dramatic changes in its circular dichroism (CD) but results also in the decrease, and ultimate cessation, of endonucleolytic DNA cleavage by staphylococcal nuclease. Let r = [moles of added Hg(II)]/[mole of DNA base]: the conservative CD spectrum of the DNA B-form becomes nonconservative in appearance at 0.01 less than r less than 0.12 (resembling DNA in C-form geometry) and assumes the spectral characteristics of a left-handed DNA double helix at 0.12 less than r less than or equal to 1.0. DNA cleavage proceeds at or near the rates exhibited by untreated DNA at 0 less than r less than 0.08. At Hg(II) levels of 0.08 less than r less than 0.5, the rate of DNA hydrolysis decreases monotonically with increasing Hg(II) concentrations, and at r greater than 0.4, DNA cleavage ceases. Both the CD changes and the changes in the rate of DNA digestion are totally reversible upon the removal of Hg(II), at least up to r = 1.0, demonstrating that Hg(II) keeps all base pairs in register. For comparison purposes, native calf thymus DNA was also treated with methylmercury [CH3Hg(II)], an agent known to disrupt the secondary structure of DNA. The treatment yielded single-stranded methylmercurated DNA with preserved right-handed helix screwness. In addition, this DNA is digested by staphylococcal nuclease much more rapidly than double-stranded control DNA. Lastly, neither the CD nor the cleavage rate changes are reversible upon the removal of methylmercury.(ABSTRACT TRUNCATED AT 250 WORDS)     

The condensation of DNA by chromium (III) ions

Cr(III), one of the most potent inorganic carcinogens, induces condensation of DNA into a very compact product at 37 degrees, as shown by electron microscopy. The condensation begins with the appearing of some supercoil structures and complete condensation occurs at relatively low Cr(III) concentrations; for 3 and 30 mM ionic strength they are 4.5 and 45 microM, respectively. Under these conditions, Cr(III) inhibits the interaction between ethidium and DNA as shown by absorption and fluorescence spectra.     

Interactions of two cytotoxic organotin(IV) compounds with calf thymus DNA

The reactions with DNA of two antitumor active organotin(IV) compounds, the dimer of bis[(di-n-butyl 3,6-dioxaheptanoato)tin] (C(52)H(108)Sn(4)O(1) x 2H(2)O), compound 1, and tri-n-butyltin 3,6,9-trioxodecanoate (C(19)H(40)SnO(5) x 1/2H(2)O), compound 2, were analysed by circular dichroism, DNA melting experiments and gel mobility shift assays. It is found that both complexes modify only slightly the B-type circular dichroism spectroscopy (CD) spectrum of calf thymus DNA. On the other hand, both complexes were found to affect significantly the parameters of the thermally induced helix-to-coil transition. Addition of 1 or 2 to calf thymus DNA samples does not favor DNA renaturation after melting ruling out formation of interstrand crosslinks. Moreover, the effects of both compounds on plasmid DNA gel mobility were investigated. From the analysis of the present results it is inferred that both organotin(IV) compounds do interact with DNA, probably at the level of the phosphate groups.     

Purification of DNA ligase II from calf thymus and preparation of rabbit antibody against calf thymus DNA ligase II

DNA ligase II has been purified about 4,000-fold to apparent homogeneity from a calf thymus extract. The ligase consists of a single polypeptide with a molecular weight of 68,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On fluorography after electrophoresis, a DNA ligase-[3H]AMP complex gave a single band corresponding to a molecular weight of 68,000. The Km values of the ligase for ATP and nicked DNA (5'-phosphoryl ends) were obtained to be 40 and 0.04 microM, respectively. Antibody against calf thymus DNA ligase II was prepared by injecting the purified enzyme into a rabbit. The antibody cross-reacted with DNA ligase II but not with calf thymus DNA ligase I. DNA ligase II was not affected by antibody against calf thymus DNA ligase I with a molecular weight of 130,000 (Teraoka, H. and Tsukada, K. (1982) J. Biol. Chem. 257, 4758-4763). These results indicate that DNA ligase II (Mr = 68,000) is immunologically distinct from DNA ligase I (Mr = 130,000).     

Conformation and circular dichroism of DNA.

CD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum-uv region to about 168 nm for the A-, B-, and C-forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double-stranded helix. The A-form spectra confirm that these bands are indeed sensitive to secondary structure. In the near-uv, the CD of B-form DNA is well analyzed as a linear combination of 27% A-form and 78% C-form. However, an analysis of the extended spectrum demonstrates that the near-uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B- and C-forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.