1) Background: Exposure of biologically active materials to ionizing radiation at very low temperature leads to damage to macromolecules. Biochemical functions are lost at a rate directly dependent on the molecular mass of the active structures. This is the basis for radiation target analysis which is used to determine the size of the functional unit of enzymes, receptors, transporters and others that carry out important biological activities. These studies revealed new and unexpected aspects of biomedically important systems including basic differences between proteins and ribonucleic acid in their responses to ionizing radiation. Other biophysical techniques are used to learn other aspects of macromolecular function in living cells: ultracentrifugation of intact Euglena cells results in stratification of cellular components based on their respective densities. These cells are living and can recover, grow and divide. 2) Objective of present studies: a: Fundamental studies of the actions of ionizing radiation on macromolecules continue in order to define the exact nature of the damage in different species of molecules. Analyses of these effects by radiation target theory establishes a radiation-sensitive mass associated with measured biological activities. This reveals a fresh perspective in the structure-function relationship in these macromolecules. b: Application of the radiation technique to enzymes, binding sites, and transporters to determine the size of their active structures, which often is less than the mass of the entire complex. c: Analysis of the recovery processes in stratified Euglena cells to determine the mechanisms involved and the time course of restoration of normal cellular function. 3) Results during the past year: a: previous studies had shown dramatic differences between radiation effects on proteins compared to those on RNA. Although there is an enormous literature on radiation effects on DNA, none have considered the properties described in the above-mentioned RNA studies. Accordingly, a test system involving supercoiled DNA has begun. Supercoiled DNA is a double-stranded circular molecule which is further coiled. Cleavage of the backbone of one strand, as by radiation exposure, leads to an uncoiling of the molecule to an 'open circle' form. This is a double-stranded circular molecule with a broken bond in the backbone of one strand. A second break in the backbone of the same or opposite strand reputedly has no effect has no effect on the open circle form unless the new break is in the opposite strand close to the original break in the opposite strand. In that case, the circular structure is lost and the molecule assumes a linear conformation. Although the three forms (supercoiled, open circle and linear form) all have the same molecular weight, they can be separated by gel electrophoresis. Current studies of irradiated supercoiled DNA are revealing unexpected results. As expected, the supercoiled DNA form disappears as a simple exponential function of radiation dose, but the radiation target size appears to be only half of the expected mass - as if only one strand were damaged. Also as predicted, the quantity of open circle increases at low radiation exposures as the open circles are formed from damaged supercoiled molecules. After greater exposures, the amount of open circle molecules slowly decreases exponentially with radiation dose. This decrease yields a radiation target size ~300 bases. This is the distance along one strand from the break in the opposite strand which permits the open circle form to change, perhaps as by losing the circular structure. The linear form as well as biological function have also been determined in the same samples. b: Radiation target analysis of macromolecules requires very large exposures to ionizing radiation. The greatest radiation flux can be obtained from a LINAC - a linear accelerator which produces a beam of high energy electrons. This project has successfully used a 35 year-old machine which is now in need of considerable repair. An alternative machine has been located; it is much newer and has recently had a major overhaul. It can produce a pulsed beam of high energy electrons of the required energy (13 million electron volts; great enough to penetrate thick samples, but low enough not to induce artificial radioactivity). Preliminary experiments with this machine were promising. Although the beam intensity is considerably smaller than that previously used, the pulse rate is significantly greater. The required radiation doses can be obtained in only slightly longer time. The only remaining problem is to acheive beam uniformity over all the exposed samples. The electron beam is scattered to expose over 100 samples simultaneously to the same radiation dose. Determination of radiation dose is a complex measurement. For many years, this project has used thermoluminescent dosimeters which had a reputed error of +/- 6%. The new LINAC staff uses a newer method - the production of free radicals in alanine which are detected by electron spin resonance. Test experiments comparing the two methods have been completed; both agreed well in determination of radiation dose and the standard deviation among independent alanine dosimeters was much smaller that that among thermoluminescent dosimeters. Initial eperiments using this LINAC showed that three different enzymes were inactivated exactly as had been observed with the other LINAC. 4) Conclusions and significance. a) Radiation target analysis can be applied to DNA. In the radiation studies of supercoiled DNA, results suggest surprises about the nature of radiation energy transfer between and along individual DNA strands. The 10000 Dalton target size for the disappearance of the open circle DNA form indicates something about the distance between the original break in the opposing strand and the new break in the other strand. Either the two breaks do not have to be 'within a few bases' as previously believed, or else some of the radiation-deposited energy can be transferred along a stretch of 300 bases. This latter possibility is in contrast with previous observations with single-stranded RNA where the transfer distance was at most 3-5 bases.
