Structure and Orientation of Free Radicals Formed by Ionizing Radiations in Certain Native Proteins.

Earlier studies1' 2 of electron spin resonance of free radicals produced by ionizing irradiations in proteins revealed two characteristic patterns which occur for many different kinds of proteins at room temperatures, usually separately but sometimes in combination. One of these patterns is a doublet like that observed for irradiated acetylglycine, glycylglycine, and certain other peptides.1' 3 This doublet arises from the interaction of the electron spin density with the nuclear moment of a single proton. Originally it was suggested1 that the proton giving rise to this interaction was possibly a proton involved in one of the hydrogen bridges. Later work in this laboratory showed that this could not be true. First, the orientation-dependence of the doublet spacing found for silk strands2 did not correspond to that expected from the known directions of the hydrogen bridges in silk. Second, substitution of deuterium4 for the bridging NH hydrogens in the proteins and simple peptides did not alter the characteristic doublet patterns. Furthermore, studies on a number of dipeptides in the powdered form' and the measurement and detailed analysis of the resonances in single crystals of alanine5 and the dipeptides acetylglycine6 and glycylglycine7 have all indicated that the doublet must arise from a proton of the CH group within the free radical. The theoretical prediction of negative electron spin densities by McConnell and Chesnut8 and the finding by Miyagawa and Gordy5 that the isotropic Fermi contact coupling A, and the dipole-dipole interaction A4 are of opposite sign in the CH group of the free radical of irradiated alanine have made possible the present interpretation of the orientation-dependence of the proton doublet in silk on the basis of its arising from a CH proton. Analysis of the Electron Spin Resonance of Irradiated Silk.-The measurements2 of the electron spin resonance of silk strands as a function of the orientation in the static magnetic field revealed a doublet of 26 gauss separation for the silk strands parallel to the field and an incompletely resolved doublet of 13 gauss separation for the strands perpendicular to the field. The resonance was found to be axially symmetric about the silk strands, as would be expected since the various groups of the twisted strands have random orientations about the axis of the strand. However, the components of the doublets are noticeably broader for the perpendicular than for the parallel orientation. This suggests that the axial symmetry of the resonance arises from the random orientation of the free radicals in the perpendicular plane rather than from symmetry axes of the individual free radicals. The measurements were made at three widely differing frequencies: 2.9 kMc/sec, 9 kMc/sec, and 24 kMc/sec. The doublet spacing was found to be independent of frequency. The component widths were measurably broader at the higher frequency, and this broadening was more noticeable for the perpendicular than for the parallel orientation. We have found it possible to account for all these observed features on the assumption of the free radical