E. Fredericq

The association of insulin molecular units in aqueous solutions

Abstract The size and shape of insulin molecules at pHs 2.7 and 10 were determined from sedimentation, diffusion, and viscosity measurements. Molecular weights close to 7500 were found at low concentrations, indicating a dissociation into the chemical units of Sanger. Above pH 7, the composition and the size of zinc-insulin molecules were studied. The nature of the energies involved in the association process has been discussed.

Terbium(3+) as a probe of nucleic acids structure. Does it alter the DNA conformation in solution?

At low ionic strength, Tb3+ binding strongly alters the secondary structure of DNA. Circular dichroism and electro-optical techniques are more sensitive than fluorescence to study these alterations in double-stranded DNA, at low Tb3+/DNA phosphate (I/P) ratios. Both techniques yield the following conclusion: as I/P is increased, native and sonicated DNA undergo a transition from the B- to psi-form, the latter being a compact structure characteristic of aggregated DNA. Our study of alkylated DNA establishes that the accessibility of N-7 guanine to Tb3+ is clearly required for structural alterations in an aggregated state to occur. The chelation of the phosphate group and of the N-7 guanine by Tb3+ simultaneously alters the geometry of the sugar-phosphate backbone and the stacking interaction between the bases in double-stranded DNA.

An electro-optical study of the mechanisms of DNA condensation induced by spermine

Condensation of DNA by spermine has been studied by electric dichroism, electric birefringence and rotational relaxation times at 1 mM ionic strength. Using Mannings theory, we found that condensation occurs for a fraction of neutralized phosphate charges (r) equal to 0.90, in good agreement with previous studies using spermidine, synthetic polyamines and trivalent cations (e.g. Co(NH3)36 +, Tb3 +). Our results are compatible with the presence in solution of torus-shaped condensed structures in a narrow range of spermine concentration; further addition of the polyamine produced precipitation due to the self-aggregation of several toroids. For spermine concentrations lower than that required for collapse, important changes of the orientation mechanism in the electric field and of DNA stiffness were observed. Whereas free DNA was mainly oriented by a fast-induced polarizability mechanism, DNA-spermine complexes displayed an important permanent dipole component, in the spermine concentration range where extension of the DNA molecules was present. The birefringence relaxation times suggested that, in the first step, the stiffness of the DNA molecules increased, and then, at higher spermine concentration, bending of the DNA molecules occurred so that condensation into toroidal particles became possible.

Comparative binding study of the interaction of quinacrine and ethidium bromide with DNA and nucleohistone.

Abstract The degree of binding of quinacrine dihydrochloride and ethidium bromide to DNA and nucleohistone has been determined by direct and indirect methods. The results obtained by the equilibrium dialysis experiments have been analyzed in terms of various theoretical models and have led us to propose for the interaction of the dyes with DNA at low ionic strength a structural scheme where the external binding sites are next to the intercalative ones. The equilibrium dialysis results were used to check those obtained by the indirect methods, i.e. absorption and fluorescence titrations, and to identify the origin of the discrepancies. The comparative study of the binding of these dyes to DNA and nucleohistone has shown that the accessible part of DNA in the nucleoprotein is to some extent different from free DNA itself.

Electro-optical properties of nucleic acids and nucleoproteins I. Study of the gel-forming deoxyribonucleohistone

Abstract An apparatus allowing the measurement of birefringence and dichroism and displayed by macromolecular solutions in high electric fields (up to 14 kV/cm) is described. Some new developments concerning the determination of dichroism and the calculation of relaxation times are detailed. Birefringence, dichroism and relaxation times have been used to characterize the changes in size and shape of the particles. Solutions of deoxyribonucleohistone (gel fraction) have been studied under various experimental conditions. The electro-optical parameters were influenced by the different factors investigated in the following manner: 1. 1. The curves of the measured quantities versus field strength indicated the approach to saturation. The relaxation times increased sharply with decreasing field strength. 2. 2. At a given field strength, the birefringence ( Δn ) was approximately proportional to the deoxyribonucleohistone concentration while the dichroic ratio ( D ) was independent of it. A slight increase of relaxation times with increasing concentration was observed. 3. 3. The three electro-optical parameters were sharply diminished by increase in the ionic strength and by decrease in the pH from 7 to 5·5. 4. 4. The value of Δn decreased slightly from 350 to 550 mμ; D increased towards the maximum of the ultraviolet absorption band at 260 mμ. 5. 5. By slow enzymic degradation, concomitant with a loss of histone, the gel properties of the deoxyribonucleohistone disappeared while Δn and D increased. The relaxation times were only slightly lowered by this degradation and were much less influenced by the field strength and by the ionic strength. These results are consistent with the presence of aggregates in the gel-like solutions which could be disordered by increasing the ionic strength and decreasing the pH. When histone was removed by enzymic degradation, the optical anisotropy increased up to values comparable with those obtained for DNA alone. The spectra of the ultraviolet dichroism indicated the presence of an n → π ∗ transition at about 280–290 mμ. Other unknown transitions appeared below 260 mμ, the moments of which were not perpendicular to the helical axis. The results are considered in connexion with other physico-chemical properties.

Alteration in the nucleosome and chromatin structures upon interaction with platinum coordination complexes.

The interaction of various platinum coordination complexes with nucleosomes and chromatin has been investigated by ultraviolet absorption spectrophotometry, circular and electric linear dichroism, and thermal denaturation, at low binding ratios (r less than 0.1-0.2). The general trend of the changes in these physicochemical properties is similar to that observed for the DNA-platinum complexes, which indicates that the same binding sites are involved in the platinum interaction with DNA and with its nucleoprotein complex. The cis-bidentate ligands, cis-dichlorodiammine, diaminocyclohexane and ethylenediamine platinum(II), showed a distinct behavior, with a more important destabilization of the DNA structure in the nucleoprotein than the trans-bidentate ligand, trans-dichlorodiammine-Pt(II), and monodentate ligand, diethylenetriamine-Pt(II). The drastic decrease of the negative electric dichroism in the 260 nm absorption band of the bases, observed with the five ligands, indicates a profound alteration of the DNA arrangement in chromatin and nucleosomes, attributed to a condensation of its superhelical structure. Some differences with previous observations on DNA complexes with the same platinum compounds indicate the possible formation of protein-DNA crosslinks in chromatin and nucleosomes. These could have some importance for the biological effects.

Gel-forming properties of thymus nucleoproteins☆

Abstract Deoxyribonucleoproteins from calf thymus have been separated in two components by centrifugation at very low ionic strength or by salt fractionation. The major component forms gel-like suspensions; the other one is well dispersed and is probably the product of enzymic hydrolysis of the former. Both components do not contain polysaccharides, lipidsoor RNA. The sizes of their particles are very much alike, as indicated by viscosity measurements. They have different protein contents, the ratios protein/DNA being respectively 1.3 and 1.1 for the gel fraction and for the dispersed one. The rigidity properties of the gel fraction have been investigated in dependence of time, pH and concentration. It is concluded that the gel fraction is closer to the native deoxyribonucleoproteins in nuclei particularlyas regards its protein content. The higher protein content is responsible for the formation of the gel network.

Electric birefringence of DNA and chromatin: Influence of divalent cations

The effects of divalent cations on the DNA and chromatin conformation have been investigated by electric birefringence and birefringence relaxation measurements at low and constant ionic strength (0.001). An important decrease of the intrinsic optical anisotropy of DNA has been found in the presence of Mn2+ and Cu2+, but not with Mg2+. A complex variation of the mean relaxation time with the ratio I/P of ion to DNA-phosphate molar concentration has been evidenced in the presence of Mn2+ and Cu2+, while the mean relaxation time monotonously decreased in the presence of Mg2+. These observations are interpreted in terms of a specific organization of DNA in a compact, rigid structure, in the presence of Mn2+ and Cu2+, and a non-specific coiling in the presence of Mg2+. Drastic conformational changes encountered by chromatin in the presence of Mg2+ and Mn2+ cations have also been evidenced through electric birefringence measurements. They are interpreted by the formation of a superhelical compact arrangement of nucleosome strings which yielded a reversal of the birefringence sign with respect to the negative anisotropy observed in the presence of Na+ ions. The removal of the histone H1 prevented the appearance of this quaternary structure. More extended fragments of the chromatin chain obtained by ECTHAM chromatography of sonicated chromatin could not afford such compact arrangements.

Enzymic degradation of deoxyribonucleic acid II. Enzymic properties of thymus acid deoxyribonuclease

Abstract The degradation of deoxyribonucleic acid by thymus acid deoxyribonuclease was studied by viscosity, formation of acid-soluble products, spectrophometry, and pH measurements. The influence of enzyme and substrate concentration is examined. Divalent cations, Mg, Mn, Ca and Zn are activators at low concentrations and inhibitors at higher ones. There is a stoichiometric relationship between the concentrations of substrate and cations at the point of maximum activation. Spectral changes and pH variation are discussed in relation with structural modifications of the degradation products. From the viscometric experiments, the mechanisms of action of the two DNases (neutral and acid) appear to be quite different.

Enzymic degradation of deoxyribonucleic acid I. Purification of acid deoxyribonuclease

Abstract A procedure for the purification of calf thymus and spleen acid deoxyribonuclease by means of ammonium sulphate precipitation and chromatography on calcium phosphate is described. Enzyme having a high specific activity is produced with a good yield.