Evidence That the Castor Bean Allergens Are the Albumin Storage Proteins in the Protein Bodies of Castor Bean

Abstract

The well characterized castor bean (Rhscm is L*) allergens were Identdfied as the low molecular weight albmin storae (12,14). A careful literature survey of the known properties of the castor bean allergens and of the albumin storage proteins revealed some similarities, such as the amino acid composition, the low mol wt, and the solubility in water. In this communication, we present evidence that the castor bean allergens are indeed the albumin storage proteins in the protein bodies of castor bean. MATERIALS AND METHODSPreparation of Protein Fractios. The matrix proteins, the lectins (4-6S albumins), the I IS globulins, the 2S4 albumins, and the total proteins of castor bean (Ricinus communis var. Hale) were purified as described (14, 15 at room temperature for 30 min, or at 100 C for 2 min in 0.01 M Tris-HCl (pH 6.8) and 1% SDS. Mol wt standards were used as described (14).Immun_okglcal Techniques. Each 2.27-kg New Zealand White rabbit was injected in the footpads and the back subcutaneously with 2 mg of CB-IA mixed in complete Freund's adjuvant. After I week, they were injected in the back subcutaneously with 2 mg of CB-IA mixed in incomplete Freund's adjuvant. They were bled 10 days after the second injection and the -y-globulins were purified by the procedure of Kendall (3). The y-globulins were dialyzed against 0.01 M Na-phosphate buffer (pH 7.5), 0.05'M NaCl, and after centrifugation at 10,000g for 30 min, the supernatant fraction was retained. y-Globulins were also prepared by an identical procedure from untreated rabbits as controls. Double diffusion tests were performed according to Ouchterlony (6). Equal amounts of protein samples to be tested were applied to each well except that twice the amount of the organelle matrix proteins and the total castor bean proteins were used.In the electrophoretic analysis of the antigenicity of the 2S albumins, 0.08 mg of the 2S albumins were mixed with 200 ,ul of 10 mm Na-phosphate buffer (pH 7.5) and 15 mm NaCl, and 200id of either anti-CB-lA y-globulins or unsensitized y-globulins.The solution was incubated at 23 C for 1 hr and then at 4 C for 16 hr. After centrifugation at 10,000g for 30 min, the supernatant fraction was lyophilized to dryness. The lyophilized powder was dissolved in 100 ,ul of 10 mm Na-phosphate buffer (pH 7.5) and 15 mm NaCl and 100 p1 of similar y-globulins. After similar incubation and centrifugation, the supernatant fractions were analyzed by SDS-polyacrylamide gel electrophoresis. RESULTSThe properties of the well characterized castor bean allergens, the CB-IA fraction, and our purified 2S albumin storage proteins were compared. SDS-Polyacrylamlde Gel Electropboresis and Mol Wt. After electrophoresis in SDS-polyacrylamide gels of high density (15%), the proteins of the isolated protein bodies of castor bean were resolved into two major groups (Fig. la) (Fig. la) and their estimated sedimentation value (15) are similar to those of the castor bean allergens reported earlier (9, 10). The isolated 2S albumins and the CB-IA had similar ...

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