A complete amino acid analysis is presented for staphylococcal enterotoxin B. The protein is composed solely of amino acids and is particularly rich in aspartic acid and lysine. It has no free sulfhydryl groups and only one disulfide bridge. On the basis of the amino acid composition the molecular weight is 35,380, the isoionic point is 8.70, and the partial specific volume is Q WJtaphylococcal enterotoxin B is the emetic material elaborated by the S-6 strain of Staphylococcus aureus. A purification procedure permitting the isolation of large amounts of an immunologically and physically homogeneous material is described in an accompanying paper (Schantz, et al., 1965). The toxin is devoid of any lipid, carbohydrate, or nucleic acid. This report presents a complete amino acid analysis of the protein, using the automatic amino acid analyzer, and a comparison with an earlier, largely microbiological analysis (Hibnick and Bergdoll, 1959). It also describes the identification of the amino-(N-) and carboxyl-(C-) terminal amino acids of the toxin, and quantitation of these residues. The implications these analyses have on the structure and conformation of the protein are discussed.
Materials and MethodsMaterials. The enterotoxin was prepared according to the method of Schantz et al. (1965). It was stored as the lyophilized powder in a deep freeze. All the solvents used in chromatography were reagent grade. Anhydrous hydrazine was prepared by distillation under reduced pressure in a nitrogen atmosphere from sodium hydroxide pellets and was stored in a desiccator in foilcovered tubes. Benzaldehyde was freshly distilled before use. Dioxane was distilled over sodium, frozen, and stored in a deep freeze. Phenyl isothiocyanate was purified by distillation in vacuo.
Amino Acid AnalysisPreparation of Samples. Samples containing approximately 2 mg/ml protein were pipetted into 40-ml Pyrex freeze-drying ampoules. An equal volume of concen-* From the
Uredospores of Puccinia graminis var. tritici (race 56) were analyzed quantitatively for total free amino acids and ninhydrin-positive substances by ion-exchange chromatography. Extracts of these substances were obtained by leaching the spores and by re-extracting leached spores with boiling water. Thirty-five ninhydrin-positive compounds were found and identified. The leach extract differed quantitatively from the extract obtained by boiling although both contained the same 35 substances. It is proposed that there are easily extractable ninhydrin-positive substances coating the spore wall and ninhydrin-positive substances in the protoplasm that can be extracted only with difficulty.
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