Purified ricin was a generous gift from Daniel Cawley (Department Biological Chemistry, University of California, Los Angeles), and isolated ricin A chain was obtained from Worthington. Reduced ricin was prepared by incubating ricin (0.1 mg/ml) in Tris-HCl (20 mM, pH 8.0) containing 2- mercaptoethanol (120 mM) for 45 min at room temperature. Irradiations were carried out at - 1350C with a 13-MeV electron beam produced by a linear accelerator as described elsewhere (8) on the following preparations sealed in 2-ml glass ampules: 1.0 jig of intact ricin in 0.3 ml of Tris'HCl (20 mM, pH 7.4) containing transferrin at 1.0 mg/ml as a carrier protein; 1.0 Ag of reduced ricin in 0.3 ml of Tris HCl (20 mM,. pH 7.4) containing transferrin at 1.0 mg/ml and 120 mM mercaptoethanol; and 0.1 ml of isolated ricin A chain (0.15 mg/ml) prepared by mixing three parts of the commercial preparation with seven parts .of 0.1 M NaCi containing 20% (vol/vol) glycerol as a cryoprotectant.
The enzymatic activity associated with ricin was determined by measuring its ability to inhibit a rabbit reticulocyte in vitro translation system that was a modification of the method of Pelham and Jackson (9). Ricin solutions were diluted to the desired concentration in Tris HCl (30 mM, pH 7.4) containing transferrin (1.0 mg/ml) and mercaptoethanol (100 mM) and preincubated for 30 min at 30'C. The solution containing ricin (10 1.l) was added to a reaction mixture (30 1.l) containing 40% reticulocyte lysate, amino acids (0.2 mM each of 19 amino acids but no phenylalanine), phosphocreatine (20 mM), creatine kinase (38 tkg/ml), hemin (10 AM), potassium acetate (240 mM), Tris HCl (4 mM, pH 8.0), and dithiothreitol (10.6 mM). The ricin/lysate mixture was incubated for 20 min at 30°C, then translation was started by adding a solution (20 ,u1) containing L-[ring-2,6-3H]phenylalanine (20 AM, 1.6 ,Ci, New England Nuclear; (1 Ci = 37 GBq) and of magnesium acetate (7.2 mM). After a 35-min incubation at 30°C, aliquots (15 ,ul) of the reaction mixture were spotted onto filter paper and placed in 10% trichloroacetic acid. The filter papers were boiled for 10 min, rinsed with 10% trichloroacetic acid, ethanol, and diethyl ether, then digested with 90% Protosol (New England Nuclear) before liquid scintillation counting. Approximately 10,000 cpm was incorporated in the absence of ricin and approximately 1500 cpm was incorporated when translation was maximally inhibited with ricin (1.5 ng per reaction). Radiation dose-dependent inactivation of ricin activity was measured by comparing the translation-inhibiting activity in the irradiated ricin samples with a standard curve constructed by plotting translation activity versus concentration of unirradiated reduced ricin or ricin A chain. In a typical assay, translation was reduced to 37% of the control by approximately 50 pg of reduced ricin per reaction. The logarithm of the fractional surviving activity was a linear function of radiation dose [in megarads (1 rad = 0.01 gray)] and the slope of this function, K, was determined by least-squares regression analysis constrained to the normalized control value of 1.0 at zero radiation dose. The molecular weight of the functional unit was calculated from the relationship M, = 6.4 x 105 SK, in which S, is a temperature factor with a value of 2.8 for -1350C (10).