Radiation inactivation of ricin occurs with transfer of destructive energy across a disulfide bridge
HARRY T. HAIGLER*, DIXON J. WOODBURY*, AND ELLIS S. KEMPNER**
*Department of Physiology and Biophysics, College of Medicine, University of California, Irvine, CA 92717; and **National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20205 Communicated by W. A. Hagins, April 19, 1985
The ionizing radiation sensitivity of ricin, a disulfide-linked heterodimeric protein, was studied as a model to determine the ability of disulfilde bonds to transmit destructive energy. The radiation-dependent loss ofA chain enzymatic activity after irradiation of either intact ricin or ricin in which the interchain disulfide bond was disrupted gave target sizes corresponding to the molecular size of dimeric ricin or monomeric A chain, respectively. These results clearly show that a disulfide bond can transmit destructive energy between protein subunits.
Proc. Natl. Acad. Sci. USA. Vol. 82, pp. 5357-5359, August 1985.
Ionizing radiation damages molecular structure. Chemical bonds in macromolecules are broken and new ones form, leading to changes in physical, chemical, and biological properties. In a solid, changes in irradiated matter are due to the direct action of radiation on the molecules in question. The initial interaction is a primary ionization of an orbital electron in which large amounts of energy [about 1500 kcal/mol (1 kcal = 4.18 kJ)J are transferred to macromolecules. The subsequent events are only partially understood, but they clearly involve the transfer ofenergy to other regions of the molecule. In addition to the theoretical interest in these processes, this subject has attracted considerable research because high-energy radiation inactivation can be used- to measure the functional size of biologically active macromol'- ecules (see refs. 1 and 2 for reviews). The method is conceptually simple: if a polypeptide chain is "hit" it is completely inactivated; if a "hit" does not occur on a polypeptide it is not affected. By exposing biologically active molecules to increasing doses of radiation and measuring the surviving activity, the functional size of the structure(s) responsible for the activity can be determined by target analysis.
The effects of ionizing radiation depend on the structural requirements for transfer of the energy deposited by a primary ionization. Evidence has been presented that radiation damage in synthetic polymers occurs at considerable distance from the primary ionization (3, 4) and throughout a polypeptide chain no matter where the original hit occurs (5). These data provide no insight into the radiation sensitivity of disulfide bonds, which often play a critical role in the structure and function of proteins. In this report the disulfidelinked heterodimeric protein ricin was studied as a model to determine the ability of disulfide bonds to transmit destructive energy.
Ricin is a toxic plant lectin that consists of two polypeptide chains, A and B, ofknown amino acid sequence (6, 7) that are joined by a single disulfide bond (Fig. 1). The B chain is a lectin that binds the toxin to cells and facilitates the entry of the A chain into the cytosol. The A chain is an enzyme capable of inactivating the protein-synthesizing capacity of eukaryotic ribosomes. The enzymatic assay for A chain activity was used to determine if destructive energy could be transferred to the A chain after a primary ionization on the B chain. If transfer occurs, irradiation ofintact ricin should give a target size corresponding to the molecular size of dimeric ricin, but if the A and B chains are energetically isolated with regard to ionizing radiation, the expected target size should correspond to the size of the monomeric A chain. In control experiments the radiation sensitivity of A chain enzymatic activity was me. sured after irradiation of either isolated A chain or ricin thaL was reduced with mercaptoethanol to form A and B chains that were not covalently associated.