Radical Transfer in Irradiated Nucleohistone

When nucleohistone was irradiated in a aqueous solution, the radicals produced in DNA seemed to be transferred to histone in the absence of Ca ion. By this mechanism DNA may be protected by histone associat ing with the former. The transfer of radicals from DNA to histone was prevented in the presence of Ca ion. INTRODUCTION A variety of radiobiological evidences in vitro and in vivo suggests that ionizing radiation weakens the protein-nucleic acid linkage1'2>. In a previous paper we demon strated that complex between Ca and two neighbouring phosphates of DNA after dissociation of deoxyribonucleohistone (DNH) into DNA and histone with radiation was formed in the presence of Ca ion 3). The purpose of this article is to show the different nature of intermolecular radical transfer in irradiated DNH solution with or without bivalent ions. ESR measurements have provided convincing evidence for free radical transfer from DNA to histone in DNH solution in the absence of Ca ion. MATERIALS AND METHODS DNA was prepared from calf thymus by the methods of Kay, Simmons and Dounce4>. The DNA was dissolved in 0.15M NaCI solution. Calf thymus histone was purchased from Sigma Chemical Co. and dissolved in 0.15M NaCl. The small amount of insoluble histone was removed by centrifugation. DNH was prepared from calf thymus by the method of Zubay and Doty5). The viscous solution of DNH was dialysed against four changes of 0.7 mM phosphate buffer at pH 7.8. 0.024 M NaEDTA was added to the first and second buffer solutions to remove Ca ions. When assayed with a flame photometer the Ca content in each solution was found to be negligible. The concentration of DNA, histone and DNH used for this experiment was 1 mg/ml. When ESR spectra were measured in the presence of Ca ions, CaC12 (10,tg/ml) was added to the DNH solution. 3H20 (500 mCi/ml) 3 ray was chosen as an internal source of irradiation, since it allows one to avoid the background of irradiated glass in ESR spectrum'). 0.2 ml 3H20 (500 mCi/ml) was added to 0.2 ml of sample solution. The estimated dose rate was approximately 3 x 1015 ev/ml. An X-band spectrometer with a reflection cavity and a modulation frequency of 100 kc/sec has been used. All spectra recorded, after phase sensitive detection, yield the first derivative of absorption cur-v e. The number of radicals in irradiated sample was determined by the comparison with a standard sample of DPPH. 0.4 ml of sample solution (DNA, histone, DNH or DNH plus Ca) was put into a quartz tube of 3 mm inner diameter. The sample tube was degassed by freezing-pumping thawing method and sealed up at a pressure of 10-3 to 10-4 mmHg. The frozen samples were put rapidly into liquid nitrogen. ESR measurements were carried out at 77°K after warming the sample at 150°K for 3 minutes. RESULTS AND DISCUSSION Before ESR measurements of the sample solution, the stability of the radiation-induced radicals produced in water was examined by subjecting the water irradiated (1019 ev) at 77°K to heat-treatment. It was indicated that the radical produced in water was completely disappeared by subsequent heat treatment ranging 120°K to 140°K. This result is in agreement with the data obtained by Sanner'). Fig. 1. ESR spectra of DNA, histone, DNH or DNH plus Ca solution. Sample solutions were irradiated with 1019 ev/ ml at 77°K and recorded after heat treatment at 150°K for 3 minutes. Radi cal yield is expressed as G-value. The ESR spectra of sample solu tions exposed to a dose of 1019 ev/ml and subsequently heated to 150°K are shown in the Fig. 1. Spectra to suc cesive heat treatment (130°K to 190°K) of the irradiated samples have been reported elsewhere 8). The radicals re maining in the sample solutions after heat-treatment to 150°K must have been produced in solute molecules, since the radical in water was shown to disappear by reacting with solute molecule during to heat treatment') The spectrum of the DNA solution is quite different from that of the histone solution, while the spectrum of DNH solution closely resembles to that of the his tone solution. The most plausible explanation of the close resemblance between the spectrum of the irradiated histone and the irradiated DNH seems to be that radicals formed in DNA molecules have been transferred to histone molecules. This view is supported by the fact that G-value for DNH is twice as large as either for DNA or histone (see Fig. 1). If it is assumed that radicals produced in water attack exclusively histone molecules in DNH, G-values for DNH and histone must be equal. On the other hand, Lloyd and Peacocke9> reported that the histone which has been dissociated by irradiation does not re-associated with DNA. Hence we must explain the reason why radical transfer occurs after dissociation of DNH into DNA and histone. It seems to be that histone and DNA, initially associated by electro static force, are dissociated by irradiation and again they come to be re-associated by weak forces, such as interaction of water molecule and close range Van der