F. Quadrifoglio

The interaction of adriamycin and its β anomer with DNA

Abstract The results of thermal denaturation, fluorescence, calorimetric and viscosimetric studies on the interaction of adriamycin and its β anomer with DNA are reported. Whereas all equilibrium, hydrodynamic and thermodynamic measurements are consistent with the proposed intercalative binding model for the adriamycin · DNA complex, the binding mechanism for the reaction of the β anomer with DNA remains uncertain. All DNA binding properties of this stereoisomer are substantially different from those of the parent compound. The results suggest that the amino sugar residue of the natural antibiotic may interact stereo-specifically with the DNA helix, thus dictating the orientation of the tetracyclic chromophore within the intercalation site. The alteration in the DNA binding capacity and the changes in interactions with DNA following the inversion of configuration at C-1′, parallel a lack of biological activity observed for the β anomer.

On the binding of actinomycin and of daunomycin to DNA: A calorimetric and spectroscopic investigation

Abstract The “strong” binding of two antibiotics, actinomycin D and daunomycin, to native DNA (calf-thymus) in dilute aqueous solution has been studied by means of calorimetric and spectroscopic measurements. In essence our results show: (1) Daunomycin interaction with DNA is an exothermic process, all features of which depend in a discontinuous way on the fraction of DNA binding sites engaged by the drug. Fluorescence data indicate that such a discontinuous trend should be independent of the GC content of DNA. (2) Actinomycin binding to DNA is, on the contrary, characterized by a positive enthalpy. For such binding, no discontinuity appears discernible with increasing the molar ratio of drug to DNA (phosphorous) on the basis of calorimetric and fluorescence data. (3) Both antibiotics can be bound simultaneously to DNA: our results would suggest that their binding sites on the biopolymer are independent. Discussion is focussed on the possible information derivable from our data on whether or not intercalation may indeed be the main process through which each antibiotic considered “strongly” interacts with DNA.

Thermodynamic behaviour of the heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG) forming B and Z hairpins in aqueous solution.

UV and CD data of the partially self-complementary heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG), obtained as a function of temperature, salt and strand concentration, show that: at low NaCl and strand concentration the oligomer exhibits, on increasing the temperature, a biphasic thermal profile which is indicative of two structural transitions, from dimeric duplex to hairpin and from hairpin to coil; the loop stabilizes enthalpically both B and Z hairpin structures with respect to the corresponding unconstrained hexamer d(CGCGCG) by a few Kcal/mol; the oligomer undergoes a B-Z transition which appears to be complete, at 0 degree C, when induced by NaClO4; by contrast the B-Z transition induced by NaCl does not reach completeness even at salt saturation. The independence of the denaturation temperature, at high salt conditions, on the oligomer concentration indicates that the Z structure is present also in the hairpin.

Short oligodeoxynucleotides with d(G-C)n sequence do not assume left-handed conformation in high salt conditions.

d(G-C)n oligodeoxynucleotides (with n varying from 3 to 7) were studied by the circular dichroism technique in 5 M-NaCl. Contrary to what was previously found with the d(C-G)n series in the same solvent, the left-handed double-stranded Z-conformation appears less stable than the B-form for low n values. The influence of base sequence on the relative stability of B- and Z-conformations for the two series is discussed.

Fluorescence of buried tyrosine residues in proteins

Histone H1 contains only one tyrosine and no tryptophan. The intrinsic fluorescence of the tyrosine rises by about 400% as the protein folds from a random coil to a globular structure (Giancotti, V., Fonda, M. and Crane-Robinson, C. (1977) Biophys. Chem. 6, 379-383). Measurements of external quenching by a large variety of quenchers shows very much reduced quenching in the folded state as compared to the disordered. It is concluded that the tyrosine is a buried residue. This is supported by the observation that the fluorescence of modified amino-tyrosyl H1 is similar to that of buried tyrosines in ribonuclease. The classification of tyrosine fluorescence in tryptophan-free proteins (Cowgill, R.W. (1976) in Biochemical Fluorescence Concepts, Vol. 2 to include the case of residues buried in a hydrophobic environment and having a relative quantum yield RTyr, greater than unity.

The interaction of natural tetra-azacyclopentazulene dyes with DNA and their effects on the DNA AND RNA polymerase reactions

The interaction of two natural tetra-azacyclopentazulene dyes with native calf thymus DNA was studied by means of microcalorimetric, viscosimetric, and spectroscopic measurements. The results are consistent with the hypothesis of an intercalative-binding. However, comparison of calorimetric studies shows that the changes in enthalpy associated with the interaction of these compounds with DNA are, in absolute value, significantly lower than those found with known intercalating agnets (daunomycin, ethidium bromide). The influence of these dyes on the template capacity of DNA in the in vitro synthesis of nucleic acids was also determined. Under the conditions used, these compounds selectively inhibited DNA synthesis. No appreciable inhibitory effect upon E. coli RNA polymerase was observed. Both compounds had greater inhibitory effect on rat liver high molecular weight DNA polymerase than E. coli DNA polymerase I. Zoanthoxanthin was a more effective inhibitor than 3-norzoanthoxanthin.

On the binding of ethidium bromide in synthetic polyelectrolyte solutions

Abstract Results are reported for spectroscopic and calorimetric studies of ethidium bromide (EBr) in water and in aqueous solutions of three alternating maleic acid copolymers. The dimerization constant, dimerization enthalpy and the dimer spectrum of EBr are derived. The interaction of EBr with the polycarboxylates is characterized on the basis of spectral and microcalorimetric data according to two limiting mechanisms: at low dye-to-polymer concentration ratios, EBr would be bound essentially in monomeric form with small exothermic effects: at high concentration ratios, the observed comparatively large exothermic heat is attributed to bound dye stacking.

Influence of some chromophore substituents on the intercalation of anthracycline antibiotics into DNA

Abstract The interaction between some chromopore-modified daunorubicin derivatives and calf thymus DNA was studied using a number of physical techniques in order to investigate the effect substituents on the aromatic ring system have on the capacity to intercalate into DNA and on the DNA binding affinity. The modifications examined include methylation of the hydroxyl groups at the 6 and 11 positions of the B ring and removal of the 11-hydroxyl group. The studies showed that only 11-deoxydaunorubicin retains the ability to bind to DNA by the intercalation mechanism typical of the parent compound, although the structural modification leads to an appreciably weaker binding. In contrast, methylation of any hydroxyl group dramatically reduces the affinity of the drug for DNA. At physiological ionic strength both methyl ether derivatives showed no evidence of intercalation. Structure activity correlations for the intercalation reaction deduced from these studies are in agreement with earlier findings and hypotheses relating to antitumour activity.

Biochemical and biological activity of the anthracycline analog, 4-demethyl-6-0-methyl-doxorubicin

SummaryThe chromophore-modified derivative of doxorubicin, 4-demethyl-6-0-methyl-doxorubicin, has been tested for antitumor activity in a range of experimental murine tumor systems. In contrast to the inactive 6-0-methyl derivative of daunorubicin, 4-demethyl-6-0-methyl-doxorubicin provided antitumor effects comparable to that of the parent compound. In addition, detailed DNA-interaction studies showed that the doxorubicin derivative retains the ability to bind DNA by the intercalation mechanism. However, the binding affinity was appreciably reduced following structural modification in the anthraquinone chromophore. On the basis of the proposed models of intercalation, these results could be rationalized in terms of steric influence of the bulky methoxy group. The results of this study are in agreement with the correlation already observed between DNA binding and relative antitumor activity of anthracyclines.

Separation and properties of an H2B histone variant from the sperm chromatin of the sea urchin Sphaerechinus granularis

Abstract The purification and the physico-chemical characterization of one of the two H2B histone variants from the sperm of the sea urchin Sphaerechinus granularis are reported. The molecule shows, in addition to a distinctive molecular weight value, an amino acid composition different both from that of calf thymus H2B histone and from those of H2B histones from chromatin of sperm and embryos of other sea urchins. Circular dichroism and fluorescence data are discussed in comparison to those of calf thymus H2B.