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The ionizing radiation sensitivity of ricin, a disulfide-linked heterodimeric protein, was studied …


Biology Articles » Biophysics » Medical Biophysics » Radiation inactivation of ricin occurs with transfer of destructive energy across a disulfide bridge » Results

Results
- Radiation inactivation of ricin occurs with transfer of destructive energy across a disulfide bridge

Frozen solutions containing intact ricin, or ricin in which the disulfide bond between the A and B chains had been reduced, or isolated ricin A chain were exposed to increasing doses of high-energy electrons (8). The radiation-dependent inactivation of enzymatic activity in each preparation then was assayed by determining the ability of the irradiated sample to inhibit protein synthesis in a rabbit reticulocyte in vitro translation system. Since A chain activity is maximal after reduction of the interchain disulfide bond (11), samples containing intact ricin were reduced with mercaptoethanol after irradiation but prior to the assay.

Irradiation of both reduced ricin and isolated ricin A chain resulted in monoexponential inactivation of enzymatic activity; typical experiments are shown in Fig. 2. Target analysis revealed average target sizes of 28 ± 3 kDa and 31 + 1 kDa, respectively (Table 1); both of these are in good agreement with the known molecular size of the A chain (Table 1). The experiment using isolated A chain can be considered an internal control, and irradition of the reduced ricin dimer provides insights into the role of noncovalent bonds in energy transfer. Reduction of intact ricin under these conditions did not result in dissociation of the two subunits as assayed by chromatography on a Bio-Rad Bio-Gel P-100 column (12) even though the interchain disulfide bond was reduced; complete reduction of the bond was confirmed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (12). Thus, some of the noncovalent interactions between the two chains remain under the conditions in which reduced ricin was irradiated. Since the target size of reduced ricin matches the size of the monomeric A chain, these noncovalent interactions between A and B chains of ricin were not adequate to transfer destructive energy from the B to the A chain.

Irradiation of intact ricin caused a monoexponential inactivation of protein synthesis inhibitory activity (Fig. 2). The inactivation curves from several experiments corresponded to an average target size of 63 ± 4 kDa, in good agreement with the molecular size of dimeric ricin (Table 1). This is so even though the measured biochemical activity resides only in the A chain and the postirradiation assay was performed in the presence of mercaptoethanol, which disrupts the disulfide bond connecting the two chains.


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