The Primary Structure of Staphylococcal Enterotoxin B OF REDUCED AND AND THE COMPLETE AMINO ACID SEQUENCE* AMINOETHYLATED ENTEROTOXIN

Oxidized and reduced carboxymethylated enterotoxin B was digested with chymotrypsin. The peptides were fractionated on AGl-X2 and Dowex 5OW-X2, and further purified by paper chromatography and electrophoresis. A total of 67 pure peptides was isolated and their sequences were determined completely or partially. These peptides provide important overlaps of tryptic peptides previously isolated and sequenced. Since most of the overlaps involve only a single residue, the complete primary structure of enterotoxin B could not be elucidated.

la&one, as was expected from the number of methionine residues present in the enterotoxin B molecule. The cyanogen bromide peptides were fractionated on Sephadex G-50 and further purified by rechromatography on Sephadex G-50 or paper electrophoresis at pH 1.9. They contained a total of 239 amino acid residues.
Tryptic peptides of each of the cyanogen bromide peptides were purified and their amino acid composition was determined.
The order of these peptides in each cyanogen bromide peptide and the order of the cyanogen bromide peptides in enterotoxin B were established. Based on the information from tryptic, chymotryptic, and cyanogen bromide peptides, the complete amino acid sequence of enterotoxin B was established. Staphylococcal enterotoxin B is a single polypeptide chain containing 239 amino acid residues.
The amino acid sequence (partial or complete) of the tryptic and chymotryptic peptides of enterotoxin B has been elucidated (1,2).
These peptides account for 239 amino acid residues. Because of the large number of single residue overlaps, the complete amino acid sequence of enterotoxin B could not be determined. In order to link all of the tryptic peptides in one polypeptide chain, additional overlapping peptide information was essential. Chemical cleavage of the methioninyl bonds of the protein by cyanogen bromide (3) was chosen as a method to obtain the needed information.
Enterotoxin 13 contains 8 residues of methionine, but since in two instances 2 methionine residues appear together in the polypeptide chain (1) seven major peptides * This work was supported by Grant  of enterotoxin B peptides obtained after treatment with cyanogen bromide on reduced, aminoethylated enterotoxin B, and the complete amino acid sequence of enterotoxin B elucidated by utilizing information obtained from tryptic, chymotryptic, and cyanogen bromide digestion of enterotoxin B.

MATERIALS AND METHODS
Malerials-All materials used in this work have been described elsewhere (1, 2, 4).
Reduction and Aminoethylation of Enterotoxin B-The procedures used for reduction and aminoethylation of enterotoxin B were essentially the same as those reported previously (4). The NH2 and COOH termini and amino acid composition of reduced, aminoethylated enterotoxin B have also been described previously (4).
Cyanogen Bromide Cleavage of Enterotoxin B-The procedure used for the cleavage of methionyl bonds in reduced, aminoethylated enterotoxin B was essentially that of Steers et al. (5). Reduced, aminoethylated enterotoxin B (300 mg) was dissolved in 30 ml of 70% formic acid. Solid cyanogen bromide (600 mg) was added in an amount equal to twice the weight of the reduced, aminoethylated enterotoxin B, and the mixture was incubated for 24 hours at room temperature.
At the end of the reaction period, 300 ml of water were added to the reaction mixture and the latter was lyophilized.
The reaction proceeded to the extent of more than 95% as judged by amino acid analysis.

Separation of Cyanogen
Bromide Peptides of Enterotoxin B-The cyanogen bromide peptides were dissolved in 20 ml of 1 N acetic acid and applied to a column (1.9 x 150 cm) of Sephadex G-50 which had been equilibrated in 1 N acetic acid. The column was developed with 1 N acetic acid at a flow rate of 15 to 20 ml per hour at room temperature.
The effluent fractions (5.2 ml) were collected automatically.
The peptides in each fraction were detected by measurement of the absorption at 277 m/z and ninhydrin (6). Appropriate fractions were pooled and dried in a rotary evaporator under reduced pressure at 40". Further purification of the fraction was carried out by rechromatography of one-eighth of the fraction on the same column of Sephadex under the same conditions or by paper electrophoresis at pH 1.9 as described below.  c Qualitatively examined on paper by Ehrlich's reagent and, if positive, spectrophotometric determination in 0.1 N NaOH was made.
Tryptic Hydrolysis and Pur$ication of Tryptic Peptides-Each of the major cyanogen bromide peptides was digested with trypsin as described (1). Purification of the tryptic peptides was done by paper electrophoresis at pH 1.9 followed by paper chromatography (Solvent I) unless otherwise indicated.
NH&erminal Amino Acid Analysis-The NH2 terminus of each cyanogen bromide peptide was determined by a method described previously (1). Hydrolysis of DNPl-peptides was carried out in 6 N HCl at 105" for 16 hours and 12 N HCl at 105" for 6 hours. The latter condition was useful for DNP-proline or DNP-glycine.
Amino Acid Composition-The amino acid composition of the various peptides was determined on a Spinco model 120 B amino acid analyzer with the modification of Benson and Patterson (7). Corrections of 10 and 5% were made for losses of serine and threonine, respectively.
Tryptophan-The spectrophotometric method of Goodwin and Morton (8) was used to determine the content of tryptophan with the use of 0.1 N NaOH.
The cyanogen bromide and tryptic peptides which contained tryptophan were detected on paper chromatograms with Ehrlich's reagent (9). Nomenclature-The peptides obtained from cyanogen bromide digestion of reduced, aminoethylated enterotoxin B were given 1 The abbreviation used is: DNP-, 2,4-dinitrophenyl-. "is"

Puri$ication of Cyanogen Bromide Peptides
Seven major cyanogen bromide peptides plus 2 moles of homoserine or homoserine lactone per mole of protein were obtained from the treatment with cyanogen bromide. This is what was expected since enterotoxin B contains two pairs of methionine residues (1). Gel filtration on Sephadex G-50 was found to be the most satisfactory method for fractionating the mixture of cyanogen bromide peptides, the results of which are shown in Fig. 1. An aliquot of every fifth effluent sample was examined by paper electrophoresis at pH 5.6 and 1.9 and by end group analysis.
On the basis of these qualitative tests, fractions were pooled as shown in Fig. 1 A and dried in a rotary evaporator under reduced pressure.
These pooled fractions were reexamined by paper electrophoresis and by end group analysis.
End group analysis indicated the presence of two peptides in Fraction A, one with lysine and the other with tyrosine as the NH2 terminus.
These two peptides could not be separated by rechromatography on Sephadex G-50. Tryptic digestion of this fraction showed Fraction A to be the aggregated mixture of the two peptides purified from Fractions B and D described below. Purification of Fraction B, which contained both NHS-terminal lysine (+ + + +) and tyrosine (+ +), yielded BrCN III which contained only lysine as its NH2 terminus (Fig. 1B).
Peptide BrCN IX did not contain homoserine or homoserine lact,one and hydrazinolysis revealed lysine to be the COOH terminus; therefore, this peptide is the COOH-terminal part of enterotoxin B.
Purification of Fraction F, in which DNP-proline (+) was detected as the NH2 terminus (12 N HCl, g-hour hydrolysis), by paper electrophoresis at pH 1.9 yielded BrCN VI.
$ summary of the properties of the cyanogen bromide peptides and their designation is given in Table I. Peptides BrCN I and BrCN II contain glutamic acid as the NH2 terminus; however, it was determined from the tryptic and chymotryptic pept.ides (1, 2) that BrCN I is the NHz-terminal part of enterotoxin B. Only BrCN IV gave a positive test for tryptophan and, as discussed below, yielded a peptide upon tryptic digestion which corresponded to the single Ehrlich-positive peptide purified b\ paper electrophoresis at pH 1.9. The mole ratio of tyrosine to tryptophan was found by spectrophotometric analysis to be 9 for BrCN IV and 1 for the Ehrlich-positive tryptic peptide.

Digestion of Cyanogen Bromide Peptides zvith Trypsin
The major cyanogen bromide peptides, BrCN I, BrCN III, BrCN IV, BrCN VI, and BrCN IX, were further analyzed by digestion with trypsin.
The resultant tryptic peptides from BrCN I, BrCN IV, and BrCN VI were purified by paper electrophoresis at pH 1.9 followed by paper chromatography (Solvent I) (1). The peptides from BrCN III were chromatographed initially on a column of Dowex 5OW-X2 (7) (Fig. 2) followed by paper chromatography (Solvent I) except for Peptide BrCN III-T-15. This peptide was insoluble and was removed by centrifugation and purified by paper electrophoresis at pH 1.9. The peptides from BrCN IX were purified by paper chromatography only (Solvent I).
Peptide BrCN I-This peptide contains 21 amino acid residues and when digested with trypsin for 24 hours gave three peptides, BrCN I-T-l (Thr, 1.02; Gly, 0.97; Leu, 1.01; Phe, 0.87; home  and BrCN I-T-3 have the same amino acid compositions as OT-7 and OT-14, respectively (1). l'eptide BrCN I-T-2 was found to be the NH2 terminus of BrCN I and enterotoxin B. Peptide BrClj I-T-l which contains homoserine and homoserine lactone is the COOH terminus of BrCN I. It has the same amino acid residues as the first 5 residues of OT-18 (1). The sequence of this pept,ide is as shown in Fig. 3. Peptide BrCN II-As shown in Table I, this peptide contains only 3 residues with glutamic acid as its NH2 terminus.
The sequence of this peptide which is a part of OT-18 (1) is shown in Fig. 3.
Peptide T-7 is identical with the 3 NH*-terminal residues of OT-1 (1). Trypsin did not cleave the bond between S-/3-aminoethylcysteine and homoserine or homoserine lactone which is consistent with the failure of drypsin to cleave the peptide chain at the carboxyl side of lysine or arginine when these residues were adjacent to t#he COOH terminus of the peptide (     Only the order Roman numerals are placed on each methionine residue to express the COOH terminus of each cyanogen peptide. Peptide BrCN IX, the COOH-terminal peptide of enterotoxin B (from residue 223 to 239)) is not expressed.  (Table  I). The evidence for the sequence of BrCN III is given in Fig. 4, and the evidence that Peptides BrCN I, BrCN II, and BrCN III will appear in that order in the enterotoxin B chain is outlined in Figs. 3 and 4. Although the overlap of OT-3 and OT-22 by RC-39 is a single leucine residue, T-10 (OT-22) is the only tryptic peptide in BrCN III with leucine as the NH2-terminal residue. All of the residues in Peptides BrCN I, BrCN II, and BrCN III can be assigned to known tryptic peptides with only one possible arrangement for these peptides (Figs. 3 and 4).
Peptide BrCN IV-The amino acid composition and NH* terminus are out,lined in Table I The order in which these peptides appear in BrCN IV is given in Fig. 5. Peptide BrCN IV-T-l was the only peptide t.hat gave a positive reaction to Ehrlich's reagent. Spectrophotometric analysis established the more rat'io of tyrosine to tryptophan as 1, The location of tryptophan in this peptide is given in Fig. 5. This peptide is the COOH terminus of BrCN IV since it is the only tryptic peptide which contains homoserine and homoserine lactone.
The (1). Peptide BrCN IV-T-12, lysyl-lysine, is evidence that at least 3 consecutive lysine residues are present in BrCN IV which is confirmed by OC-21 (Fig. 5). Peptide BrCN IV-T-5 has the same amino acid composition as CT-1 except for the 3 NHz-terminal residues of OT-1.
This establishes T-5 as the NHz-terminal part of BrCN IV. Peptide OT-26 is placed on the carboxyl side of OT-1 since OT-26 (T-10) is the only tryptic peptide in BrCN IV with tyrosine as the NH2 terminus.
Peptides BrCN V and BrCN VII-Digestion with cyanogen bromide gave 2 moles of homoserine and homoserine lactone as was expected from the presence of two pairs of methionine residues in the enterotoxin B molecule (OT-28 (Fig. 5) and OT-23 ( Fig. 6)) (1, 2).
Peptide BrCN VI-T-3 has the same amino acid composition as OT-10, and T-2 the same composition as the COOH-terminal part of OT-28 (Fig. 6).
Peptide BrCN VIII-This peptide contains 6 amino acid residues including homoserine and homoserine lactone with tyrosine as the NH:! terminus (Table I). The sequence of this peptide is outlined in Fig. 7.
Peptide BrCN IX-The amino acid composition and NH2 terminus are outlined in Table I. This peptide is the COOHterminal part of enterotoxin B since it did not contain homoserine or homoserine lactone.
To complete the information on the tryptic peptides, T-5 has been labeled OT-15 as described previously (1).

Amino Acid Sequence
The complete amino acid sequence of enterotoxin B is outlined in Fig. 8. It is derived from Figs. 3 to 7 in which the order of placement of the cyanogen bromide peptides is outlined.
All of the residues in the cyanogen bromide peptides were assignable to known tryptic peptides and there was only one possible way to arrange each tryptic peptide into the cyanogen bromide peptides.

DISCUSSION
The results of the amino acid sequence studies show that enterotoxin B contains 239 amino acid residues with a molecular weight of 28,494. This is very near the molecular weight of 29,000 (242 residues) calculated from the amino acid composition by the minimum molecular weight method based on the presence of 2 residues of half-cystine.
Three different molecular weights have been published for enterotoxin 13, 24,000 f 3,000 (ll), 35,300 (299 residues) (12), and 30,000 f 1,000 (252 residues) (13). The first value was calculated from the sedimentation data with an assumed value for the diffusion constant which happened to be about 20% high. This value was more or less confirmed by minimum molecular weight calculations based on 1 residue of tryptophan.
Later values obtained for tryptophan were interpreted as indicat,ion of the presence of 2 tryptophan residues (12, 13). Actually, the tryptophan values proved to be high as only one tryptophan residue was revealed by the sequence studies. It should be expected that the tryptophan values would be inaccurate since the ratio of tyrosine to tryptophan in enterotoxin B is 21: 1. The value of 35,300, reported by Spero et al. (12), was obtained at least in part from the half-cystine value which was about 20% lower than that reported by Bergdoll et al. (13). The 30,000 value reported by the latter workers was a compromise between the value calcu-