The action ofγ-rays on sodium deoxyribo-nucleate in solution

Abstract

The effect of irradiation byγ-rays from a cobalt-60 source, without exclusion of oxygen, on the molecular structure of herring sperm sodium deoxyribonucleate (DNA) has been studied with the following aims: (i) to obtain evidence on the existence of the double-helicalDNAstructure in aqueous solution by testing the application of recent theoretical treatments for the random fracture of single-chain macromolecules (Charlesby 1954); and (ii) to study the effects of dosages ofγ-rays lower than those which have been shown to disrupt the constituent nucleotides. Changes in viscosity, electrometric titration behaviour, ultra-violet absorption, precipitability and other chemical properties have been investigated. The viscosity of solutions ofDNAin 0·1 M-sodium chloride was decreased on irradiation by an amount which was independent of the dose rate and was determined by the ratio (R, in eV/atom ofDNAphosphorus) of dosage in eV entering each ml. of solution to concentration ofDNAphosphorus. The reduced specific viscosity of irradiatedDNAsolutions was a linear function of concentration and obeyed the Huggins equation (Huggins 1942). The intrinsic viscosity [ƞ] decreased with increasingRand, on a logarithmic plot, the relation was linear with a negative slope of 1.9 at the higherRvalues. Comparison of this value with that expected from the theory of random degradation leads to the proposal that a decrease in molecular weight, and thus in [ƞ], of theDNAcan occur only when two independent breaks occur at approximately opposite positions in each of two intertwined polynucleotide chains; this indicates that the double-helical structure adduced for the moist solid state persists in aqueous solution. This is confirmed by the observation that aDNApreparation which had undergone mild acid treatment before irradiation, in order to disrupt the complementary cross-linking hydrogen bonds (Watson & Crick 1953a) and there by the double-helical structure, exhibited a less steep decrease of [ƞ] with increasingR(i.e. a slope of — 1·2 on the logarithmic plot). The earlier theory for the viscosity of a single-chain macromolecule undergoing random breakdown is extended to the case of degradation of a cross-linked molecule consisting of two intertwined chains. The difference between the forward-and backward-titration curves characteristic of the hydrogen-bondedDNAstructure decreased onγ-irradiation, which indicates a progressive disruption of the complementary hydrogen bonds and of the double-helical structure that they hold together; this was confirmed by observed parallel increases in ultra-violet absorption. The asymmetrical character of the displacement suggests, though not quite conclusively, that the hydrogen bonds linking adenine and thymine were ruptured more readily than those joining guanine and cytosine. The combined rupture of all hydrogen bonds had initially a very highGvalue, of the order of sixty base pairs severed per 100 eV, but this decreased with increasing dosage. This efficient rupture of complementary hydrogen bonds would presumably impair the duplication of theDNAhelicesin vivoand may underlie some of the biological effects of low dosages of ionizing radiation. The titration studies demonstrated the release at highRof approximately equal quantities of acidic groups withpK'aof about 4·5 to 5·5 and of 5·5 to 8·5; this observation is discussed briefly in connexion with the detailed chemical mechanism of the rupture of phospho-ester linkages. The indirect nature of the action ofγ-rays under the present conditions was con-confirmed by the determinative character of the ratioRand by the inhibitory effects of certain com pounds known to combine with the free radicals produced in the water.