Samvel G. Haroutiunian

Theoretical and Experimental Study of DNA Helix-Coil Transition in Acidic and Alkaline Medium

The theoretical approach to the calculation of the influence of selective binding of small ligands on DNA helix-coil transition has been described in the previous paper (Lando D. Yu., J. Biomol. Struct. Dyn., (1994)). In the present paper that method is used for the study of DNA protonation and deprotonation in acidic and alkaline medium by theoretical analysis of pH effect on DNA heat denaturation. The mechanism of DNA protonation in acidic medium and pK values of nucleotides are well known. It gave us an opportunity to check the theory without any fitting of pK values. A good agreement between experimental and calculated functions Tm(pH) and delta T(pH) (melting temperature and melting range width) obtained for acidic medium proved the validity of the theory. However, for alkaline medium there was not even qualitative agreement when the agreed-upon mechanism of deprotonation was considered. Looking into the cause of the discrepancy, we have studied the DNA melting for different mechanisms of deprotonation by calculation of Tm(pH) and delta T(pH). As a result, it has been established that the discrepancy is due to deprotonation of bonded GC base pairs of helical DNA regions (pK = 11). It was shown that the early known protonation and newly found deprotonation of helical DNA essentially stabilised double helix in alkaline and acidic medium.

Interactions of Meso-tetra-(4-N-oxyethylpyridyl) Porphyrin, Its 3-N Analog and Their Metallocomplexes with Duplex DNA

Abstract Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.

Effects of two representative antitumor ruthenium(III) complexes on thermal denaturation profiles of DNA

The effects of two representative antitumor ruthenium(III) complexes — namely (dichloro-1,2-propylenediamine tetraacetate ruthenium(III)) Ru(III)) RAP and (sodium trans-(dimethylsulfoxide)(imidazole)tetrachloro ruthenate(III)) NAMI — on thermal denaturation profiles of calf thymus DNA were analyzed. It is shown that both complexes slightly stabilize calf thymus DNA when tested in the concentration range 0.01<r<0.1; minor effects on the melting interval ΔT are also observed. Differential melting curves permit monitoring of the effects of ruthenium complexes, on the different regions of calf thymus DNA depending on their GC content. Preferential binding of NAMI to GC-rich tracts of DNA is observed. Notably both complexes favor renaturation of calf thymus DNA although to a lesser extent than cisplatin does. CD spectra reveal only very modest conformational changes of DNA upon addition of either ruthenium complex in the concentration range used in the melting studies. Implications of the present results for the mechanism of action of antitumor ruthenium(III) complexes are discussed.

Microcalorimetric investigation of DNA, poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] melting in the presence of water soluble (meso tetra (4 N oxyethylpyridyl) porphyrin) and its Zn complex.

Abstract Thermodynamic parameters of melting process (δHm, Tm, δTm) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C))·poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased Tm of satellite fraction by 24°C, but ZnTOEpyp(4), on the contrary, predominately bound with AT-rich sites and increased DNA main stage Tm by 18°C, and Tm of poly(dA)poly(dT) increased by 40 °C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes—strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode—strong binding—took place. The weak binding is characterized with shifting of Tm by some grades, and for the strong binding Tm shifts by ∼ 30–40°C. Invariability of ΔHm of DNA and poly(dA)poly(dT), and sharp increase of Tm in the range of r from 0.08 to 0.25 for thymus DNA and 0.01–0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H2O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width ΔTm caused by increase of added ZnTO- Epyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which ΔT∼TGC-TAT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193–205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.

Melting of complexes of DNA-cis-DDP in acidic environment

The peculiarities of helix-coil transition of DNA at complex-formation with cis-diamminedichlorplatinum (II) (cis-DDP) have been investigated in acidic environment by UV absorbance technique. It is shown that observed features of behavior ΔT (pH) and Tm (pH) DNA at pH 2.8-3.0 are possible to explain by formation in DNA of pseudo-ring structures at covalent linking of cis-DDP with DNA.

Short-Range Interactions and Size of Ligands Bound to DNA Strongly Influence Adsorptive Phase Transition Caused by Long-Range Interactions

Abstract Long-range interaction between all the ligands bound to DNA molecule may give rise to adsorption with the character of phase transition of the first kind (D. Y. Lando, V. B. Teif, J. Biomol. Struct & Dynam. 18, 903–911 (2000)). In this case, the binding curve, c(c o), is characterized by a sudden change of the relative concentration of bound ligands (c) at a critical concentration of free (unbound) ligands, c o=cocr , from a low c value to a high one where c o is molar concentration of free ligands. Such a transition might be caused by some types of DNA condensation or changes in DNA topology. For the study of the conditions necessary for adsorption with the character of phase transition, a calculation procedure based on the method of the free energy minimum is developed. The ligand size and two types of interactions between ligands adsorbed on DNA molecule are taken into consideration: long-range interaction between all the ligands bound to DNA and contact interactions between neighboring ligands. It was found that a) Stronger long-range interaction is required for longer ligands to induce phase transition that is occurred at greater cocr values; b) Pure contact interaction between neighboring ligands can not itself initiate phase transition. However contact cooperativity strongly decreases the threshold value of energy of long-range interaction necessary to give rise to the transition.

Adsorption of Ligands on Macromolecules in the Fluctuating Medium

Abstract In the present work fluctuations of number of ligands adsorbed on macromolecule are investigated. We have taken into account the adsorption and desorption of ligands under the circumstance of some adsorption centers fluctuations affected by medium fluctuation. The correlation function and spectral density of number of ligands adsorbed on macromolecule are calculated. The properties of these fluctuations which allow identifying a noisemaker are determined. It has been shown, that “fast” and “sluggish” adsorption can be distinguished by properties of dispersion and spectral density. It has been also shown, that comparison of experimental and theoretical correlation functions (or spectral densities) allows to calculate constants of ligand—adsorption center binding and unbinding.

The dependence of cis-diamminedichloroplatinum(II) binding to DNA upon the GC content: a thermal and spectrophotometrical investigation

Abstract The binding of cis -diamminedichloroplatinum(II) ( cis -DDP) to DNA is investigated as a function of the GC content and of the cis -DDP concentration. Analysis of the melting curves and of the UV and CD spectra shows that binding of cis -DDP to DNA is highly sequence specific, GC rich DNAs being destabilized more effectively. At low levels of platination, stabilization of GC- and destabilization of AT-base pairs can be noted. In AT rich DNA, cis - DDP binding favours the B→ A conformational transition. It is also shown that, in addition to specific interactions between cis -DDP and DNA, several non-specific processes take place.

Compensation of DNA Stabilization and Destabilization Effects Caused by Cisplatin is Partially Disturbed in Alkaline Medium

Abstract Binding of the antitumor compound cisplatin to DNA locally distorts the double helix. These distortions correlate with a decrease in DNA melting temperature (Tm). However, the influence of cisplatin on DNA stability is more complex because it decreases the DNA charge density. In this way, cisplatin increases the melting temperature and partially compensates for the destabilizing influence of structural distortions. The stabilization is stronger at low Na+ ion concentration. Due to this compensation, the total decrease in the DNA melting temperature after cisplatin binding is much lower than the decrease caused by the distortions themselves, especially at low [Na+]. It is shown in this study that, besides Na+ concentration, pH also strongly influences the value of a change in the melting temperature caused by cisplatin. In alkaline medium (pH=10.5–10.8), a fall in the melting temperature caused by platination is enhanced several times with respect to neutral medium. Such a stronger drop in Tm is explained by a decrease in pK values of base pairs caused by lowering the charge density under platination that facilitates proton release. At neutral pH, the proton release is low for both control and platinated DNA and does not influence the melting behavior. Therefore, lowering in the charge density under platination, besides stabilization, gives additional destabilization just in alkaline medium. Destabilization caused by structural distortions due to this pH induced compensation of stabilizing effect is more pronounced. In the presence of carbonate ion, destabilization caused by high pH value is strengthened. As a decrease in DNA charge density, interstrand crosslinking caused by cisplatin also increases the DNA stability due to loss in the entropy of the melted state. However, computer modeling of DNA stability demonstrates that interstrand crosslinks formed by cisplatin do not stabilize long DNA. It is shown that the increase in Tm caused by interstrand crosslinking itself is compensated for by a local destabilization of the double helix at the sites of location of interstrand crosslinks formed by cisplatin.

In Vitro Testing of Cyto- and Genotoxicity of New Porphyrin Water-Soluble Metal Derivatives

Porphyrins and porphyrin derivatives have an outstanding potential for discovery of novel pharmacological agents due to their ability for numerous chemical modifications and a variety of mechanisms of biological effects. New water-soluble Ag and Zn derivatives of tetrachloride meso-tetra (4-N-oxiethylpyridyl) porphyne were synthesized. Cyto- and genotoxicity of these substances were tested in vitro by the vital dye (trypan blue) exclusion and the micronucleus tests, respectively. Both metalloporphyrins were shown to be cytotoxic for Cos-7 (fibroblast-like African green monkey kidney cells transformed by simian virus 40 [SV40]), DU 145 (epithelial-like cells of human prostate carcinoma), and K-562 (human chronic myeloid leukemia cells) cell lines. At the same time they did not cause chromosome fragmentation in K-562 cell line at as high concentrations as IC50 (20 μmol/L for Ag and 70 μmol/L for Zn derivative). Thus, the metalloporphyrins tested meet at least two important demands to potential anticancer drugs as they combine the cytotoxicity with low genotoxicity. The three in vitro tumor models used are relevant to further in vitro and in vivo pre-clinical investigation of the studied metalloporphyrins as potential chemotherapeutics.