For many years this laboratory has been developing and using radiation target analysis, a method for determining the mass of biologically active molecules. The laboratory has developed a variety of experimental techniques to properly utilize the inactivation of biochemically active material by ionizing radiation, specifically that from high-energy electrons. Radiation target theory has been successfully extended to complex biological systems. These techniques and theories have been utilized in studies of enzymes, receptors, transporters, and other structures of wide interest in the biomedical field. Often these radiation studies resulted in discovery of unexpected features or properties of these biologically active materials. The objectives of present studies are to determine the precise nature of damage in macromolecules caused directly by ionizing radiation and to utilize this knowledge in the application and extension of radiation target analysis. Several projects have been brought to fruition during the past year. A fundamental question of radiation target theory was addressed and solved. The concept of a radiation-sensitive volume for biologically active molecules was always accepted until it was questioned on theoretical grounds. Some proteins are known to change their hydrodynamic volume at low pH. Now the radiation sensitivity of several proteins irradiated at both neutral and acid pH has been examined. Some of these proteins do not change their sedimentation properties at low pH, while others that were tested did show such change. The radiation sensitivity of each protein was found to be invariant with pH. Thus neither the hydrodynamic volume nor the molecular shape has any effect on the target size. In agreement with the theoretical physics argument, the radiation sensitivity of proteins is dependent only on the molecular mass. A separate project, now completed, concerns the hepatic sinusoidal reduced glutathione transport system. Radiation inactivation analysis of both the high- and low-affinity units. The high-affinity structure was found to be 70 kDa, indistinguishable from a similar unit found in the canalicular transporter. However, the low-affinity unit was hardly inactivated by even massive doses of radiation, implying a very small structure is required, much smaller than either the high-affinity transporter. It is also much smaller than the analogous transporter in the canalicular membranes and is free of the masking effect seen in those membranes. The method of radiation target analysis continues to reveal unique molecular properties of biologically active structures. Often these properties were unanticipated from studies using other techniques. This new knowledge has given a greater understanding of the mechanisms of these biological functions. In some cases these properties offer the possibility of external control or modification of these active structures. - Enzymes, structure-function, radiation, target analysis
