The Primary Structure of the Phase-l Flagellar

Protein of Salmonella typhimurium

I. THE TRYPTIC l’lWTIDES*

(Received for publication, April 14, 1972)

TERENCE _\I. Jous ASD VIKTORIJA RANKIS

Fwxn The Departme~rt of Microbiology, U~~iwrsily of Oyego?l Medical School, Pwtland, Oregon 97201

SUMMARY

Salmonella PQrQtyphi B SL 877 was shown to be a suitable source of flagellar protein whose amino acid sequence was determined by the H-l structural gene of Salmonella fy- phimurium. Isolated flagellin gave one band at a variety of pH levels in acrylamide gel electrophoresis in the presence of disaggregating agents and contained a small amount of carbohydrate. The amino acid content of this flagellin was determined and the NHp-terminal amino acid was identified as alanine. Analysis of the flagellin by gel filtration on aga- rose in 6 M guanidine HCl-0.1 M Z-mercaptoethanol and acrylamide gel electrophoresis in 0.1% sodium dodecyl sul- fate both indicated a molecular weight of 49,000 for the protein, a value not in agreement with previous suggestions. Digestion of the flagellin with trypsin was performed in the presence of 2 M urea, and 29 peptides were isolated from the resulting digest by combinations of column and paper chro- matography. The combined amino acid content of the puri- fied peptides corresponded to 46% of the total amino acids present in the molecule (based on a mol wt of 49,000). The amino acid sequence was determined for the peptides isolated and four peptides were found to contain adjacent glycine residues.

Bacterial flagella have been used as models for study of the immunological response (I), the nature of antigen specificity (2, 3), the aggregation of protein polymers (4), the mechanism of function of contractile proteins (4), and the mechanism of epistatic control (5). Despite such wide interest, little is known of the structure of these organelles except that they may be disaggregated by a variety of agents to a protein monomer, termed flagellin, which comprises 95c0 or more of the material present in intact flagella. Studies of flagellins isolated from a variety of bacterial species have been mainly limited to deter-

* This work was supported by (irant aI 08253 from the National Institutes of Health. Part of this work was briefly reviewed at the 71st annual meeting of the American Society for Microbiology at Minneapolis in 1971.

1 Recipient of Research Career Development Award AI 38337 from the National Institutes of Health.

minations of molecular weight and total amino acid content (see References 6 and 7 for review). This series of papers describes investigations which attempt to define a flagellar protein in terms of its primary structure.

The phase-l flagella of Salmonella typhimurium were selected as a source of protein as these have been studied genetically (8) and serologically (2) and the flagellin has been isolated (9). S. typhimurium possesses two distinct structure genes (H-Z and B-d) for flagellar protein and regulates flagella production so that at a given time only one of these genes is expressed and flagella are polymerized from a single flagellin type (for review see 6 and 7). Alternation in such gene expression gives rise to phase variation (10). Because of the relatively high rate of change between antigenic phases (II), it is impossible to obtain large numbers of cells all possessing the sarne flagellar antigen. In addition, cells of this species possess an act’ive nml gene (12), the product of which causes N-methylation of some of the lysine residues present in the flagellin, resulting in an increased re- sistance of the molecule to digestion with trypsin (13). In order to obtain only one flagellar protein free of e-N-meth>lly- sine, Salmonella paratyphi l3 SL 877 was used as a source of flagella. This strain is an nml - , monophasic transductant of S. paratyphi Js SW 543 which produces only phase-l flagella whose protein is determined by a structural gene (H-li) derived from S. typhimurium. Flagellin has been isolated from SL 877 and its amino acid content determined (9).

The first paper in this series describes some properties of the flagellin molecule isolated from SL 877 and an analysis of the products of its tryptic digestion.

MATERIALS .4ND METHODS

Bacterial Strains and Flagellin Isolation-S. paratyphi 13 SI,

877 and S. paratyphi R SL 167 were obtained from Dr. B. A. 1). Stocker (Stanford University Medical School, Stanford, Calif. 94305). Salmonella adelaide was obtained from Dr. W. II. Ewing (Communicable Diseases Center, Atlanta, Ga. 30333). Flagellin was isolated by the method of McDonough (9), with omission of precipitation by (NHd)?SOd.

Acrylamide Gel Electrophoresis-Electrophoresis was carried out in acrylamide gels using the preliminary equilibration tech- nique (14) in the presence or absence of 4 M urea at the following constant pH: pH 2, pH 4.3, pI1 7.0, and pH 9.3.

dlolecular Weight Determinalions---Scrylamide gel electro

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1)lroresis in the presence of 0.1 ‘,i SIX+ was carried as described by Weber and &born (15). I;sing the same reagent recipes, electrophoresis was also carried out with the samples introduced into a slab (14 x 15 x 0.3 cm) of acrylamide (E. C. Apl)aratus). Gel filtration in 6 I\I guanidine HCl-0.1 M 2-mercaptoethanol, 1)H 6.5, was carried out on liio-Gel A-h (Uio-Rad Labs., Lot 9034) as recommended by Fish et al. (16). Samples, including flagellin, were reduced in 0.1 M 2-mercaptoethanol at 20 mg per ml before use (16). Column fractions were assayed by the turbidimetric technique (16) and blue destran 2000 (Pharmacia) was used to determine the column void volume, B,,.

Awino Acid A?zalvses-Samples of flagellin and peptides were hydrolyzed in 6 4 HCl at 110” in evacuated tubes for various times (9). Ak commercial analyzer was not available and quan- titative amino acid analyses were carried out using a Moore- Stein two-column system in combination with a modified pep- tide analyzer (Technicon model Pl). Basic amino acids were analyzed on a column (5 cm x 0.9 cm diameter) of DC-2 resin (l)urrum Corp.) at pH 5.35. Acidic and neutral amino acids were analyzed on a column (57 cm x 0.9 cm diameter) of DC-la resin (Durrum Corp.) using a two-buffer system, pH 3.35 and pH 4.40. Buffer flow through both columns was maintained at 95 ml per hour. Only the section of the analyzer for unhydro- lyzed analysis (17) was used and the tube sizes were modified to permit analysis of the column effluent at 80 ml per hour. The recorder supplied was incapable of rapid print-out and the reac- tioit of ninhydrin with the column effluent was monitored at 570 IIII~ ill a continuous record. As a result, the conventional dot- counting procedure could not be used to calculate the areas of the peaks produced and it was necessary to measure the peak widths directly using vernier calipers. The system required 150 min for completion of each analysis and was calibrated using a standard mixture of 18 amino acids each at 0.25 pmole per ml (Technicon). For hydrolysates of fiagellin, an additional com- plete analysis was carried out using a calorimeter at 450 nm and the proline content only was determined.

In order to estimate glutamine and asparagine quantitat’ively, aminopeptidase M digests were analyzed using the lithium buffer system of Benson et al. (I 8) with a single column (58 cm X 0.9 diameter) of DC-la resin (Durrum). A third buffer (0.1 M

lithium citrate, 1.5 &f lithium chloride, pII 6.5) was added to the system after the elution of phenylalanine in order to elute the basic amino acids. The analysis was standardized using a mis- ture of 20 amino acids (the usual 18 amino acids plus asparagine at~d glutamine) each at 0.25 pmole per ml 0.1 M lithium citrate.

~\7H2-terminal Amino Acid T)etermination-The NIlz-terminal amino acid of flagellin was determined by the dansylation method of Gros and Labouesse (19). Preparation and identifi- cation of standard dansyl amino acids is described below.

Tryptic DigestioTx-Lyophilized flagellin (600 mg) was dis- solved in 120 ml of water, and the solution was heated to 95” for 5 min, rapidly chilled, and lyophilized. The resulting dena- tured flagellin was dissolved in 60 ml of 6 M urea-O.3 M NaHCOa, allowed to stand at room temperature for 30 min, diluted to 2 M urea by the addition of water, and the $-I adjusted to 8.5 by the addition of 0.1 s HCl. Trypsin L-l-tosylamide-2-phenylethyl chloromethyl ketone (15 mg) (Worthington), freshly prepared as :t 2.5 mg per ml solution in water, was added and digestion allowed to proceed at room temperature for 3 hours with manual adjustment of the pH to 8.5 at 15.min intervals. A second

1 The abbreviations Ilsed are: SDS, sodilnn dodecyl sulfate;

ideiltical addition of trypsin was then made and digestion WII-

tinued for 2 hours more. The final digest was lyophilized. Thermolysin Digestion-Peptide (0.3 pmole) was dried and

resuspended in I ml of water. Thermolysin solution (0.5 ml) (1 mg of thermolysin, 120 rng of tris(hydroxymethyl)amino- methane, 30 mg of CaCl?.2HsO, 10 ml of water) was added, the solution adjusted to pH 8.2 by addition of 0.1 N HCl, and irl- cubated at 37” for 4 hours. Following digestion, 6 N HCI was added to the digest to lower the pI-I to 2.1. The final solution was analyzed on a column (23 cm x 0.9 cm diameter) of Amines A-5 (Bio-Rad) at 50” using a linear gradient between 0.2 M pyridine acetate, pH 3.1, and 2 $1 pyridine acetate, pH 5.0, pumped through the colurm at 30 ml per hour. The effluent was analyzed in the same manner a:: the Sephades G-25 effluent (described below).

Aminopeptidase X Digestion-Peptide (0.2 pmole) was dried and resuspended in 0.8 ml of 0.1 K tris(hydroxymethyl)amino- methane-I-IQ pH 8.0. Aminopeptidase XI (equivalent to al)- prosimately 1000 milliunits of enzyme) was added and digestioll allowed to proceed at 37” overnight. The digest was adjusted to pH 2.2 with 1 1\~ HCl and applied directly to 1X-la resin in the lithium buffer system.

Hydrolysis with Acetic Acid---Peptide (0.3 prnole) was dried in a 5-ml ampoule and 2 ml of 0.5 &I acetic acid were added. The ampoule was briefly evacuated, sealed, placed at 110” over- night, opened, and the contents applied directly to a column of Aminex A-5 resin. Analysis of the digest using this resin was carried out in the same manner as the analysis of the thermolysiir digest (see above).

Peptide Isolation-The lyophilized tryptic digest was dis- solved in I5 ml of 0.1’): collidine (2,4,6-trimethylpyridine) acetate, ~1-1 8.0, and 3-ml aliquots were fractionated on Sepha- dex G-25 Fine (Pharmacia) contained in two columns (200 cm x 0.9 cm diameter) arranged in series (20). The Sephadex G-25 was previously equilibrated with 0.17; collidine acetate, pH 8.0, and the digest was eluted with the same solvent pumped through the column at 15 ml per hour at roorn temperature. The column effluent was analyzed using a peptide analyzer (Technicon model P-l) which monitored the reaction of the effluent, with ninhydrin both before and after hydrolysis with SaOH (17). The ana- lyzer was modified to utilize 0.9 ml per hour for unhydrolyzed analysis and 1.8 ml per hour for hydrolyzed analysis; the excess effluent stream was collected in tubes changed at 20.min inter- vals. Appropriate fractions corresponding to the peaks ob- tained were pooled and lyophilized.

Fractioils obtained from Sephadex G-25 were further analyzed using a column (23 rm x 0.9 cm diameter) of Aminex A-5 resin (ISio-Rad) at 50”. Buffers were made at various pH levels bJ mixing either 0.05 RI pyridine acetate, pH 2.5, with 0.2 M l)yri- dine acetate, pH 3.1, or 0.2 M pyridine acetate, pH 3.1, with 2 RI pyridine acetate, pH 5.0. Prior to sample application, columns were equilibrated with the lowest pH buffer to be used. Buffer gradients were obtained using a gradient device (21) and pre- liminary experiments were performed to select gradients giving adequate separation. Columbus were washed with 2 M pyridine acetate, pH 6.5, after completion of the gradient. Isuffers were pumped through the column at 30 ml per hour, part of the ef- fluent stream was analyzed as for the Sephadex G-25 effluent, and the remainder of the effluent was collected in tubes changed at 7+-rni11 intervals. Appropriate fractions corresponding to the Amines h-5 peaks were pooled, lyophilized, and dissolved in a small volume of 0.1 C; acetic acid. Samples of Amines A-5 fractions were examined by paper chroin:ttogi,ilpli~ on Whatman

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5182

No. 3MM paper using l-butanol-acetic acid-water (200:30:75, v/v) (Solvent A) and l-butanol-pyridine-acetic acid-water (60: 40 : 12 : 18, v/v) (Solvent B) as solvent systems (22) and by high voltage paper electrophoresis at pH 6.5 (23). Papers were sprayed with 0.25% ninhydrin in acetone and heated to 70” for 30 min for detection of peptide material. Those fractions which showed a single ninhydrin-reactive spot in both solvents and in electrophoresis were judged to be pure, a judgment later tested by amino acid analysis. Those fractions which produced two spots were separated into their component peptides on paper using the solvent that produced the best separation (24). For this purpose, each impure fraction was applied to a separate sheet of Whatman No. 3MM paper (182 x 223 inches) as a band 1 inch long on a line 23 inches from the narrow edge and also as a spot 1 inch along from the edge of the band. Loading was between 0.2 and 2.0 pmoles of peptide on the band and 0.01 and 0.1 pmole on the spot. The paper was then developed for maximal separation of the components present and dried. A strip of paper containing the material originally in the spot was cut out and stained with ninhydrin to act as a guide strip. Areas in the unstained paper corresponding to ninhydrin-positive regions in the guide strip were cut out and eluted by descending chromatography with 10% acetic acid to yield pure peptides.

Samples of purified peptides were hydrolyzed in 6 N HCl for 18 hours at 110” and analyzed for quantitative amino acid composition. No correction was made for the partial destruc tion of threonine and serine under these conditions or for the incomplete release of isoleucine, leucine, and valine. Peptides containing aspartic acid residues, glutamic acid residues, or both were digested with aminopeptidase M and the digests analyzed in the lithium buffer system for quantitative estimation of amide residues present.

Amino Acid Sequence Determination-The amino acid sequence of all of the peptides isolated were first determined using the sequential Edman degradation plus dansylation method of Gray (25), hereafter called the DNS-Edman method. The procedure was modified in that 25 ~1 of N-ethylmorpholine were added during the coupling of the phenylisothiocyanate to the peptide to maintain the optimal pH. Standard dansyl amino acids were prepared by the method of Boulton and Bush (26). Unknown dansyl amino acids were examined by thin layer chro- matography on acrylamide plates (Brinkmann) using benzene- acetic acid (9:1, v/v) as solvent (27) and detected by their yellow fluorescence under short wave ultraviolet irradiation (Mineralight UVS-11, Ultraviolet Products). Tentative identi- fication was based upon the RF values determined. Confirma- tion was obtained by direct examination of the unknown, to- gether with the suggested identical standard dansyl amino acid and dansyl amino acids with similar RF properties, on acrylamide plates using three solvent systems (27) : benzene-acetic acid (9 : 1, v/v) ; water-907c formic acid (200 :3, v/v) ; and n-heptane-l- butanol-acetic acid (3 : 3 : 1, v/v).

Confirmation of the sequences, and identification of the amide residues present, was obtained using a rapid Edman degradation procedure (28) with direct identification of the phenylthiocar- bamyl derivatives released after their conversion to the PTH form by thin layer chromatography; hereafter termed the “direct-Edman method.” Peptide (0.2 to 0.3 pmole) was dis- solved in 100 ~1 of coupling buffer (0.4 M dimethylallylamine in 60% aqueous pyridine adjusted to pH 9.5 with 10% trifluoro- acetic acid) and 5 ~1 phenylisothiocyanate added. Coupling was allowed to proceed in an atmosphere of nitrogen at 50” for 20 min. The solution was then extracted once wit,h 0.3 ml of

benzene and dried at 60” in a vacuum desiccator (25). Trifluoro- acetic acid (100 ~1) was added, and cleavage was induced in an atmosphere of nitrogen at 50” for 7 min. Trifluoroacetic acid was removed in a gentle stream of nitrogen, and the dried prod- ucts were extracted once with 0.3 ml of ethylene chloride. The phenylthiocarbamyl amino acid in the ethylene chloride extract was converted to the PTH form using the 1 x HCI method of Edman (29), and the remaining peptide, insoluble in ethylene chloride, was dried and recycled. This procedure required I+ hours for each cycle of Edman degradation. Unknown PTII- amino acids were identified by thin layer chromatography on plates of silica gel containing fluorescent indicator (Silica GF, Analtech) using ethylene chloride-acetic acid (30:7, v/v) (29) for polar PTH-amino acids and heptane-propionic acid-ethylene chloride (58:17 :25, v/v) (30) for nonpolar PTH-amino acids. Each unknown PTH-amino acid was examined directly in a posi- tion adjacent to the suspected standard PTH-amino acid, based upon the DNS-Edman results, and PTH-amino acids with similar migration characteristics. Standard PTH-amino acids were obtained commercially. PTH-amino acids were detected by quenching of the fluorescence of the plate indicator under short wave ultraviolet irradiation and confirmed further by color reaction with the ninhydrin-collidine reagent (31). PTH histidine and PTH-arginine were identified by specific chemical reactions (29). Identification of asparagine and glutamine residues was compared with amino acid compositions of amino- peptidase M digests.

Peptides with sequences which indicated adjacent identical amino acid residues were examined by a subtractive Edman degradation procedure. Each indicated peptide was passed through the necessary steps of Edman degradation, using the rapid method described above, to remove those amino acids prior to the suspected pair. ,4 sample was removed for acid hydrolysis and quantitative amino acid analysis and the re- mainder was further cycled through the Edman procedure with samples for similar analyses taken at each cycle. Amino acids removed at each stage are indicated in boldface print.

Chemical Assays-Carbohydrate was determined by the an throne test (32) and by the thymol-sulfuric acid test (33). Pro- line was detected qualitatively by the isatin reagent (34).

Flagella Staining-Flagella were stained by Liefson’s method

(35). Antiserum Production-Specific anti-i serum was prepared as

previously described (2) in rabbits maintained in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care.

Proteins and Reagents-Bovine serum albumin was obtained from Calbiochem; phosphorylase a (rabbit muscle), catalase, and ovalbumin from Worthington; transferrin (bovine), y-globu- lin (human), apoferritin (horse), and lysozyme (egg white) from Schwartz-Mann; glutamate dehydrogenase (bovine liver), fumarase (pig heart), and aldolase (rabbit muscle) from Boeh- ringer-Mannheim; and chymotrypsinogen _4 (bovine pancreas) from Sigma. Trypsin L-l-tosylamido-2-phenylethyl chloro- methyl ketone was obtained from Worthington, thermolysin (B grade) from Calbiochem, and aminopeptidase M from Henley.

Chromatographically pure amino acids, purified urea, purified guanidine hydrochloride, and standard PTH-amino acids were obtained from Schwartz-Mann, N-Dimethylallylamine was ob- tained from Pierce. Acetic acid and pyridine w-ere distilled from ninhydrin. Collidine, phenyl isothiocyanate, AT-ethyl- morpholine, and butyl acetate were distilled. Reagents for the

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rapid-Eclman procedure were purified as recommended by Ed- man (29). All remaining chemicals were reagent grade.

RESULTS

After passage through semi-solid medium, the strain of SL 877 was found to be heavily flagellated in stained preparations. One hundred single colonies tested were all agglutinated by specific anti-i serum, and a heavy inoculum of the strain failed to produce spreading growth through semi-solid medium in the presence of anti-i serum (2). These results confirmed the monophasic nature of SL 877 and its production of flagella with the i flagellar antigen of S. typhimurium.

The flagellin preparation was found to contain 2 to 47;, carbo- hydrate, expressed as milligram equivalents of gluco*e per g dry weight, a similar value to that reported for a variety of flagellins derived from different Salmonella flagellar antigens (9, 13).

Flagellin yielded a single stained band in acrylamide gel elec- trophoresis at the pH values tested provided that a high con- centration of urea was present, indicating the probable purity of the protein isolated. In the absence of urea, multiple lines were formed together with material unable to enter the gel, which showed that the flagellin monomers readily aggregated under the conditions used. Replacement of urea as a disaggregating agent by 0.1 y0 SDS also resulted in the production of a single band in this technique.

Fig. 1 shows the relationship between the molecular weights and electrophoretic mobilities in SDS-acrylamide gel electro- phoresis of 12 standard proteins using individual gels (65 mm X 6 mm diameter) (15). Each result represents the average of four to ten experiments. Similar results were obtained using a slab of acrylamide gel. The mobility obtained for SL 877 flagel-

Glutamic Dehydrogenose -

Fumarose 8 8-Globulin (Hchoin)

\ Ovalbumin

Aldolasek

\ Chymotrypsinogen A

&-Globulin (L chain) t

.

\ Apoferritin ‘0

\

\ Lysozyme .

FIG. 1. The relationships between electrophoretic mobility and log molecular weight of 12 standard proteins in acrylamide gel in the presence of 0.1% SDS. Each resldt shown is the aver- age of four to ten experiments. The Q~TOZU indicates the mobility of the flagellins of SL 877, SL 167 and Salmonella adelaide and cor- responds to a molecular weight of 49,000. BSA, bovine serum albumin.

lill corresponded to a molecular weight of 49,000. In order to confirm the result, the SL 877 flagellin was examined by this technique in mixture with ovalbumin, glutamate dehydrogenase, y-globulin, and fumarase. Under the standard conditions of Weber and Osborn (15), and after 3 cm migration through the acrylamide gel in the slab method, the flagellin separated from both ovalbumin and glutamate dehydrogenase but never sepa- rated from either fumarase or the H-chain of y-globulin, which were themselves inseparable. Identical results were obtained using the flagellins of SL 167, which produced the i flagellar antigen with some of the lysine residues n-methylated (9) and of X. adelaide (H antigen f, g).

Fig. 2 shows the relationships between the molecular weight and V, : V. for seven standard proteins in gel filtration chroma- tography on agarose in 6 M guanidine HCl-0.1 M 2-mercapto- ethanol. Each result represents the average of three experi- ments and the relationship found confirms the report of Fish et

al. (16), although the actual V,: Bo values determined here were not identical with those reported because a different batch of agarose @o-Gel Lot 9034) was used. The V,:Vo values ob- tained for the flagellins of SL 877, SL 167, and S. adelaide are shown in Fig. 2 and indicate a molecular weight for flagellin of 49,000 in agreement with the results obtained in SDS-acrylamide gel electrophoresis (above).

The results obtained in quantitative amino acid analysis of the SL 877 flagellin, following various times of hydrolysis, were calculated on the basis of a molecular weight of 49,000 and are shown in Table I. For comparison, the results of McDonough (9) were calculated for this molecular weight and, as can be seen, they show good agreement with the results obtained here. NHZ- terminal amino acid analysis by the dansyl technique (19) yielded a single amino acid which was readily identified as DNS-alanine, in confirmation of the previously unpublished report (rited in

2- \ Hemoglobin l

FIG. 2. The relationship between the V,: V0 and log molecular weight of seven standard proteins by column chromatography on agarose (Bio-Rad A-5m) in the presence of 6 M guanidine HCI-0.1 ~~ 2-mercaptoethanol. Each result shown was the average of three exoeriments. The arr’ou, indicates the V,:V, value of the _ .,_ “-__ flagellins of SL 877, SL 167 and Salmonella. actelaide and corre- sponds to a moleclllar weight of 49,000. RSA, bovine serum albumin.

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Amino acid composition of SL 877 jfagellin

l+:xpressed as micromoles of each amino acid detected. The number of residues was calculated for a molecular weight of 49,000. The threonine and serine values were calculated bv extrapolation to zero time, and the values for valine, leucine,

and isolencine were calculated using the 72.hour hydrolysis re- sult,s. The values obtained by McDonough (9) are shown re-

calclllated for molecular weight of 49,000. Tryptophan and cyst,ine were absent (9).

Amino acid

Hydrolysis time

24 1 48 / 72

L,vsine. 2.16 Histidine 0.08 Arginiile.. 0.99 Aspartic acid. 5.86 Threonine 4.01 Serille. 2.50 (ilutarnic acid 3.53

(:Iycine.. 3.33 Alanine.. 4.20 Valine. 2.0.5

Methionine 0.17 Tsolellcine 1.4.5 Leucine 2.85

Tyrosine 0.77 Pherlylalanine. 0.50 I’roline 0.43

hrs

2.18

0.08 1.01 3.89

3.96 2.43

3.62

3.20

4.42

2.38

0.17

1 .so

2.93

0.80

0.54

0.4j

2.19 2.18 28.7

0.08 0 .08 1.05 1.00 1.00 13.2 5.88 3.88 77.3

3.90 4.07 33.5

2.37 2..i7 33.8

3.59 3..i8 47.1

3.27 3.26 42.9.

4..i2 4.38 .i7.6

2.39 2.39 31.4

0.17 0.17 2.2

1 ..52 1.52 20.0

2.92 2.92 38.4

0.77 0.78 10.3

0.30 0.51 6.7

0.44 0.44 t, '8

Average Residues present

Mc- Donough:

residues present

28.8

1.8 12.j 73.3

.53.9

34.3

46.2

38.6

5.5.1 31.0

2.7

22.8

38.6

10.7

6.6 5.8

Keference 9) that alanine was the KHz-terminal amino acid of the flagellin isolated from flagella with antigen i.

The fiagellin was denatured by heat prior to digestion in order to render it sensitive to trypsin. AddiCon of trypsin to the denatured fiagellin in the absence of urea, at pH 8.5 and tern- peratures in the range of 2Op38”, resulted in the rapid formation of a heavy flocculant precipitate which could not be dissolved in 8 RI urea, 80’2, acetic acid, or 1 M NaOH. However, digestion with trypsin in the presence of 2 M urea resulted in only the oc- casional occurrence of a fine precipitate which was removed by rentrifugation from digests in which it formed. Gel filtration on Sephadex G-25 of the flagellin, digested with trypsin in the presence of urea, yielded an effluent divisible into six fractions (Fig. 3). The first two fractions (I and II), comprising t.he larger l)el)tides, have Ijroven resistant to further analysis. All at- tempts to conc*entrate these fractions, viz. lyophilization, rotary evaporation, and membrane ultrafiltration, yielded an insoluble aggregate which resembled the product of urea-free tryptic digestion in al)l)earance and insolubilit,y in 8 RI urea, SOO/O acetic acid, and 1 M NaOH. The remaining Sephadex G-25 fractions (III, IV, V, and VI) were soluble and were further fractionated using ;2minex A-5. The results of such fractionations are shown itI Figs. 4 to 6, in which only the record of analysis after hy- drolysis is shown for reasons of clarity. The record obtained for Fraction VI is not shown as this fraction consisted only of

urea and TP 27. Where necessary, further purification was per- formed on paper (see “Materials and Methods”). Peptides isolated were assigned arbitrary numbers as they were identified with the prefix TP to indicate tryptic peptide. The resulting 29 different purified peptides were quantitatively analyzed for amino acid content after 18 hours of acid hydrolysis, and the number of residues identified was baaed uljon int,egral values of

ilme (hrrl

E’IG. 3. Fractionation of a tryptic digest of 120 mg of SL 87i flagellin, performed in the presence of urea, using a column (400 cm X 0.9 cm diamet,er) of Sephadex (;-25 at room temperature. Collidine acetate (O.lyO,), pH 8.0, was pumped through the column at 1.5 ml per hour and a portion of the effluent stream was COII- tinuously monitored for reaction with ninhydrin by a Technicon model P-l peptide analyzer. The record shows results with both unhydrolyzed (---) and hydrolyzed (- - -) effluent. The bars under the peaks indicate the material pooled for each fraction. Additional details may be found in the text.

FIG. 4. Purification of the Sephadex Fraction III on Arninex Ad. The pH profile of the eltlates is indicated in the figure. De- tails of the method are described in the text. Peaks of ninhydrin reactivity corresponding to material later separated into two peptides are indicated.

9 II 13 15 17 Time (MS)

FIG. 5. Purification of the Hephadex Fraction IV on Aminex A-j. The pH profile of the eluates is shown in the figure. De- tails of the method are explained in the text.

lysine and arginine, the known specificity of trypsin (24), and later results of sequence analysis. The results of these analyses are shown in Table II, which also shows the identification of proline residues based upon chemical tests, the elution pH of each peptide from the Amines A-5 column, and the migrations

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I -- -- I I I 1 I

I3 15 Time (hrs)

FIG. 6. Purification of the Sephadex Fract,ion V on Aminex A-5. The pH profile of the eluates is shown in the figllre. Details of the method are explained in the text.

of each peptide in paper chromatography. Peptides possessing aspartic acid or glutamic acid residues were digested with amino- peptidase M and analyzed in the lithium buffer system for quan- titation of amide residues (Table II). Each peptide was sub- jected to sequence analysis (summary of results in Table III) and the results are described individually below.

Peptides in Sephadez G-25 Fraction V

Because of their elution properties in relat.ion to urea on the Sephadex G-25, these peptides were suspected of being dipep tides and were tentatively sequenced on the basis of their amino acid composition and the known specificity of trypsin.

Peptide TP 29:Tyr-Q/s--Both the DNS-Edman and the direct-Edman techniques indicated the sequence shown, in COII- firmation of the tentative result.

Peptide TP %?:Val-Arg-Both the IINS-Edman and the direct-Edman techniques indicated the sequence shown in COW firmation of the tentative sequence.

Peptide TP 27: Tyr-Tyr-Ala-Lys-This peptide was found in the Sephadex G-25 Fraction V but 80% of it was present with the urea in Fraction VI. The late elution of TP 27 from the Sephades G-25 was thought to be due to its high tyrosine con- tent (36). 130th the DNS-Edman and the direct-Edman meth- ods indicated the sequence shown, which was confirmed by the subtractive Edman analysis:

Step 1: Lys, 1.00 (1); Ala, 1.04 (1); Tyr, 1.06 (1) Step 2: Lys, 1.00 (1); Ala, 1.05 (1); Tqr, 0.21 (0)

Peptide TP 26:Ser-Arg-The tentative sequence was corn firmed by both the DNS-Edman and direct-Edman techniques.

Peptides in Sephadez G-25 Fraction IV

Peptide TP 25:Ala-Ala-Gly-His-Asp-Phe-Lys-The DNS- Edman method indicated the sequence shown. The direct- Edman technique confirmed this sequence and showed that asparagine was not present, in agreement with the analysis of the aminopeptidase M digest (Table II). The existence of adjacent alanine residues was proven by the subtractive Edman procedure.

Step 1: Lys, 1.00 (1); His, 0.93 (1); Gly, 1.12 (1); Ala, 1.24 (1); Phe, 0.85 (1).

Step 2: Lys, 1.00 (1); His, 0.87 (1); Gly, 1.06 (1); Ala, 0.35 (0); Phe, 0.96 (1).

Peptide TP 24: Leu-Ser-Ser-Gly-Leu-Arg-The sequence shown was obtained by both the DNS-Edman and the direct-Edman techniques. The subtractive Edman method confirmed the presence of the adjacent serine residues.

Step 1: Arg, 1.00 (1); Ser, 1.61 (2); Gly, 1.04 (1); Leu, 0.98 (1). Step 2: Arg, 1.00 (1); Ser, 1.01 (1); Gly, 1.05 (1); Leu, 0.88 (1). Step 3 : Arg, 1 .OO (1) ; Ser, 0.34 (0) ; Gly, 0.94 (1); Leu, 0.89 (1).

Peptide TP 23: Thr-T,yr-Asn-Ala-Ser-Lys-The sequence shown was indicated by both the DNS-Edman and direct-Edman techniques. The latter identified the asparagine as its PTH derivative, in agreement with the analysis of the aminopeptidase M digest (Table II).

Peptide TP 22: Zle-Asn-Ser-illa-Lgs-The sequence shown was obtained using both the DNS-Edman and the direct-Edman techniques. The asparagine residue found in aminopeptidase M digests of TP 22 (Table II) was identified and positioned in the direct-Edman tests.

Peptide TP 21: Thr-Ala-Leu-Asn-Lys -Both the DNS-Edman and direct-Edman methods gave the sequence shown and the asparagine residue, detected in aminopeptidase M digests (Table II), was positioned directly.

Peptide TP ZO:Phe-Thr-$la-Asn-Zle-Lys-Both the DNS- Edman and direct-Edman techniques yielded t,he sequence shown and the asparagine residue, detected in aminopeptidase M digests of TP 20 (Table II), was positioned directly.

Peptide TP i9:Gl~-Leu-Thr-Gln-.4la-Ser-iirg--J~ot,h the DiSd- Edman and the direct Edman methods yielded the sequence shown with the glutamine residue, detected in aminopeptidase M digests of TP 19 (Table II), positioned directly.

Peptides in Sephadez G-25 Fraction III

Peptide TP lS:Leu-Asn-Glu-Zle-Asp-Arg--Arlalysis of the aminopeptidase M digest’ of TP 18 (Table II), indicated the presence of 1 glutamic acid, 1 aspartic acid, and I asparagine residue per residue of lysine. The sequence shown, which shows the positions of the aspartic acid and asparagine residues, was obtained using the direct-Edman procedure and was confirmed, without identification of amides, by the DNS-Edman met’hod.

Peptide TP 17:Zle-Asp-Gly-Asp-Leu-Ly-This peptide rluted at pH 3.40 from -4mines-A5 together with 1‘1’ 16, from which it was separated using paper chromatography in Solvent A\. Analysis of the aminopeptidase 11 digest of TP 17 (Table II) indicated the absence of amide residues; the sequence indicated was obtained by both the DNS-Edman and direct-Edman methods.

Peptide TP 16:Leu-Gly-Gly-Ala-Asp-Gly-Lys-This pept’ide was eluted with TP 17 from Amiues-95 and purified from it by paper chromatography in Solvent A. The sequence sholvn was obtained using the DNS-Edman procedure. The direct-Edman method confirmed this sequence and indicated the absence of amide residues in agreement with analysis of the products of aminopeptidase M digestion (Table II). The existence of adja- cent glycine residues was confirmed by the subtractive Edman method.

Step 1: Lys, 1.00 (1); Asp, 1.10 (1); Gly, 3.10 (3); Ala, 1.05 (1); Leu, Lx-ace (tr) (0).

St,ep 2: Lys, 1.00 (1); Asp, 1.12 (1); Gly,2.11 (2); Ala, 0.98 (1). Step 3: Lys, 1.00 (1); 4sp, 1.16 (1); (;I), 1.32 (I); Ala, 1.13 (1).

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TABLE II

Amino acid content of tryptic peptides

The molar rat,ios of the amino acids are shown and the values in parentheses are the assumed number of residues, except for proline which was determined by a qualitative chemical test. Both the results from hydrolysis in 6 N HCl for 18 hours at 110“ (isoleucine, leu- tine, serine, t,hreonine, and valine were not corrected for the time of hydrolysis) and digestion with aminopeptidase M are shown. Also show11 are the RF values of the peptides in paper chromatography using Solvents A and B and their elution pH from Aminex A-5 resin (see text for details).

TP 1 I

TP 2 TP 3 TP 4 TP 5 TP 6

I

1 -

Acid :ulirlopep- Acid Aminopep- Acid kinopep- Acid Arninopcp- Acid ,k2r.linopep- Acid Aminopep-

Amino hydro- tidase M hydra- tidase M hydra- tidase M hydro- tidase M hydro- tidase M hydro- tidase ?I

acid l.ysi.5 digestion lysis digestion lysis digestion lysis digestion lysis digestion lysis digestion

LYS 1.00(l) 1.00(l) 1.00(l) 1.00(l) 1.00(l) i.OO(l) 1.00(l) 1.00(l) 1.00(l) 1.00(l)

'jcld 25% 147: 10% 59 n 5% 14% ---.__-. ___

X(SolvenL A) 0.16 0.23 0.19 0.31 0.53 0.30

tT(SolVcnL I:) 0. jl 0.56 0.30 0.64 0.89 0.69 __- -___

mi~lcx A-5

2.80 2.805 2.81 2.83 2.86 2.86

,lu:ioil ;,I! -

1

Peptide TP 15: Phe-Asp-Asp-Thr-Thr-Gly-Lys--The DNS-

Edman procedure yielded the sequence shown and this was con-

firmed by the direct-Edman method. The absence of amide residues was shown by the latter procedure and in the analysis of the amino-peptidase M digest of TP 15 (Table II). The presence of adjacent aspartic acid and threonine residues was confirmed by the subtractive Edman method.

Step 1: Lys, 1.00 (1); Asp, 2.12 (2); Thr, 1.87 (2); Gly, 1.12 (1);

Peptide TP 14:Net-Ser-Tyr-T1ar-Asp-Asn-Asn-Gly-Lys-Anal-

ysis of the aminopeptidase M digest of this peptide (Table II) indicated the presence of both aspartic acid (1 residue) and as- paragine (2 residues). The sequence shown was obtained from the direct-Edman procedure and was confirmed, without identi- fication of amides, b!- the DNS-Edman method. The presence of adjacent -1s~ residues was confirmed by the subtractive Edman procedure.

Phe, 0.10 (0). Step 2: Lys, 1.00 (1); Asp, I .13 (1); Thr, 2.03 (2); Gly, 1.34 (1). Step 4: Lys, 1.00 (1); Asp, 2.86 (3); Thr, 0.21 (0); SW, 0.12

St,ep 3: Lys, 1.00 (l);Asp,0.40 (0); Thr, 1.82 (2); Gly, 1.02 (1). (0) ; Gly, 1.34 (1) ; Met, (tr) (0) ; Tyr, tr (0).

Step 4: Lys, 1.00 (l);Asp,0.20 (O);Thr, 1.06 (1); Gly, 1.32 (1). Step 5: Lys, 1.00 (1); Asp, 1.94 (2); Thr, tr (0); Ser, tr (0); Step 5: Lys, 1.00 (I); -4sp, 0.20 (0); Tlrr, 0.33 (0); Gly, 1.12 (1). my, 1.04 (1).

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TABLE II-Continued

-

-

I

-.

-.

-

-_

_1

TP 9 TP 10 TP 11 TP 12 i

TP 8 TP 7

----I-

-- Acid Aminopep-

hydro- tidasc M

Acid Aminopep

hydra- tidase ?l Amino

acid lysis digestia

1.00(l) 1.00(l)

1.98(Z) 0.95(l) 1.08(l) 0.87(l) 1.91(Z) 0.84(l)

His

2.56(3) 0.87(l) 0.38(l) 1.89(Z) 1.92(2)

0.80(l) 3.94(l) 0.91(l) 0.98(l)

0.87(l) 3.93(l)

Glu 0.92(l)

1.17(l)

3.18(j) 1.00(l) 0.98(l) 1.92(2) 1.73(2) 1.06(l) 1.12(l)

0.94(l), 1.06(l) 1.16(l) 0.79(l) 0.96(l) 1.05(l)

Gln

2.03(2) 1.76(2)

Gly 1.09(l)/ 0.91(l)

Ala 1.01(l) 1 1.11(l)

Val 3.96(l)/ 0.87(l) 1.77(2: 0.87(l) 1.07(l) 0.99(l) 0.76(l)

-i-- Met

0.81(1) 0.92(l) --I-- - 0.92(l) 1.04(l)

+ 1.84(2) 1.86(2)

12 I 3 16% 13:: 18% 23% 20% 10%

- 0.26 to.29 0.33 0.09

0.73 1.55 0.96 0.44

2.92 2.98 2.93 3.15

0.26

0.58

2.92

St,ep 6: Lys, 1.00 (1); Asp, O.Y2 (1); Gly, 1.18 (1). St.ep 7: Lys, 1.00 (1); Asp, 0.36 (0); Gly, 1.14 (1).

Peptide TP 1.2: Tyr-Thr-Ala-Asp-Asn-Gly-Thr-Ser-Lys--This

pept’ide was eluted with TP 13 from Aminex A5 and was purified using paper chromatography in Solvent B. The sequence shown was obtained by the direct-Edman technique, wkh identifica- tion of PTH-aspartic acid and PTH-asparagine, and was con- firmed (without distinction of amide residues) by the DNS- Edman method. The existence of both aspartic acid and aspara- gine residues was evident from the analysis of an aminopeptidase M digest of TP 12 (Table II). The existence of adjacent Ass residues was confirmed by the subtractive Edman procedure.

Step 3: Lys, 1.00 (1); Asp, 2.04 (2); Thr, 0.96 (1); Ser, 0.94 (1); Gly, 1.03 (1); Ala,0.29 (0); Tyr, tr (0).

Step 4: Lys, 1.00 (1); Asp, 1.18 (1); Thr, 1.03 (1); Ser, 0.90 (1); Gly, 1.05 (1); Ala, 0.24 (0).

Step 5: Lys, 1.00 (1); Asp, 0.34 (0); Thr, 0.94 (1); Ser, 0.90 (1); Gly, 0.98 [I); Ma, 0.17 (0,.

Peptide TP 11: Thr-Ile-Asp-Gly-Gly-Leu-Ala-Val. Lys-This

Peptide TP lS:Asp-Gly-Tyr-Tyr-Glu-Val-Ser-Val-Asp-Lys -

This peptide was eluted from Amines A-5 at pH 3.15 together with TP 12, from which it was separated using paper chroma- tography in Solvent B. The sequence shown was defined using the DNS-Edman technique and was confirmed by the direct- Edmail method. Both the latter and digestion with amino- peptidase M (Table II) indicated the absence of amide residues. The presence of adjacent tyrosine residues was confirmed by the subtractive Edman method.

Step 2: Lys, 1.00 (1); Asp, 1.26 (1); Ser, 0.97 (1); Glu, 0.90 (1); Gly, 0.10 (0); Val, 2.13 (2); Tyr, 1.86.

Step 3: Lys, 1.00 (1); Asp, 1.22 (1); Ser, 1.21 (1); Glu, 1.00 (1); Gly, 0.14 (0); Val, 1.93 (2); Tyr, 0.99 (1).

Step 4: Lys, 1.00 (1); Asp, 1.24 (1); Ser, 0.85 (1); Glu, 0.99 (I); Gly, 0.09 (0); Val, 1.89 (2); Tyr, 0.21 (0).

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TABLE II-Continued _-

TP 15 TP 16 TP 17 TP 18 I

Acid Aminopep-

hydra- tidase M

lysis digestion -

1.00(l) 1.00(l)

Acid Aminopep- Acid Aminopep- Acid Aminopep-

hydro- tidase M hydro- tidase >I hydro- tidase M

Sysis digestion lysis digestion lysis digestion

1.00(l) 1.00(l) 1.00(l) 1.00(l)

1.00(l) 1.00(l)

1.96(2) 1.71(2)

i.86(2) 1.85(2)

1.06(l) 1.07(l) 1.85(2) 2.21(2) 1.97(2) 1.12(l)

0.89(l); 0.99(l) 0.92(l) 0.95(l) '0.98(1! 1.03(l)

7

317 I I

19% 19% 46% ---

3.26

-- Tyr 1.89(2) 1.82(2) 0.87(l) 1.15(i)

Phf

Pro

Total 10

Yield 6%

Kf(SotvenL A) 0.23

Rf(Solvent B) 0.8;

Aminex A-5 3.15

elurion pE

9

8%

0.15

0.53

3.17

peptide was eluted at pH 2.98 from Aminex A-5 with TP 10 from which it was purified using paper chromatography in Solvent A. The DES-Edman procedure indicated the sequence shown. The direct-Edman method confirmed the sequence and indicated the absence of amide residues, in agreement with the analysis of the aminopeptidase M digest (Table II). The adjacent glycine residues were confirmed using the subtractive Edman technique.

St,ep 3: Lys, 1.00 (1); Asp, 0.21 (0); Thr, 0.12 (0); My, 2.23 (2); Ala, 1.12 (1); Val, 0.92 (1); Ile, 0.12 (0); Leu, 1.21 (1).

Step 1: Lys, 1.00 (1); Asp, 0.20 (0); Thr, 0.11 (0); Gly, 1.24 (I); Ala, 1.05 (1) ; Val, 1.03 (1) ; Ile, 0.09 (0) ; Lell, 0.93 (1).

St,ep 5: Lys, 1.00 (l);Asp,0.13 (O);Thr,O.lO (O);Gly,O.Sh (0); Ala, 1.08 (1); Val, 0.95 (1); Ile, 0.08 (0); Leu, 0.80 (1).

Peptide TP IO:Ser-8sp-Leu-Gly-Ala-Val-Gin-Asn-Arg-Tllis

peptide was eluted from Amines -1-5 with TP 11 and was purified using paper chromatography in Solvent A. The sequence was obtained using the DNS-Edman procedure and was confirmed,

and the amide residues identified, by the direct-Edman method. The presence of 1 aspartic acid, 1 asparagine, and 1 glutamine residue was confirmed by analysis of the aminopeptidase JI digest (Table II).

Peptide TP 9: Val-Thr-Val-Thr-Gly-Gly-Thr-Gly-Lys- ‘I’llis peptide was eluted at pH 2.92 frorn dminex A-5 together with TI’ 8 and further purified using paper chromatography in SolvelIt 11. The sequence was determined by the DXS-Edrnan technique and confirmed by the direct-Edman method. Subtractive Edman degradation was used to confirm the presence of adja- cellt glycine residues.

Step 4: Lys, 1.00 (1); ‘1’111, 1.26 (1); my, 3.OG (3); 1.4, 0.17

(0). St,ep 5: Lys, 1.00 (1) ; Thr, 1.06 (1) ; Gly, 2.27 (2); Val, 0.12 (0). St,ep G: Lys,l.OO (l);Thr,0.98 (l);Gly,1.4,3 (l);Val,O.ll (0).

Peptide TP 8: Val-Gly-dsp-Asp-Tyr-Tyr-Ser-Ala-Thr-Gin-Asp-

Lus---This nelltide was eluted with TP 9 from Amines h-5 and

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5159

TABLE II-Continued

TP 19 TP 20 TP 21 TP 22 TP 23 'IX' 24

Acid Aminopep- Acid Aminopep- Acid Aminopep- Acid Aminopep- Acid Aminopep- Acid Aminopep-

Amino hydro- tidase M hydro- tidase M hydra- tidase PI hydro- tidase M hydro- tidase F! hydro- tidase M

acid lysis digestion lysis digestion lysis digestion lysis digestion lysis digestion lysis digestion

LYS 1.00(l) 1.00(l) 1.00(l) 1.00(l) 1 00(l) 1.00(l) 1.00(l) 1.00(l)

His

A% 1.00(l) 1.00(l) 1.00(l)

ASP 1.07(l) 1.01(l) 0.94(l) 0.96(l)

Thr 0.95(l) 0.87(l) 0.92(l) 0.97(l) 0.90(l) 0.95(l) 3.91(l) 0.87(l)

Ser 0.91(l) 0.93(l) 0.88(l) 0.97(l) 0.87(l) 0.79(l) 1.85(2)

ASll 0.89(l) 0.93(l) 0.94(l) 1.14(l)

Glu 0.97(l)

Gln 1.13(l)

GUY 0.93(l) 0.87(l) 0.95(l)

Ala 0.98(l) 1.03(l) 0.93(l) 0.97(l) 0.97(l) 1.03(l) 0.95(l) 1.04(l) 0.97(l) 1.12(l)

Val

bfet

Ile 0.83(l) 0.89(l) 0.84(l) 0.79(L)

LIZ11 0.90(l) 0.87(l) 0.90(l) 0.94(l) 1.80(2)

Tyr 0.95(l) 0.85(l)

Phe 1.02(l) 1.03(l)

Pro -

Total 7 6 5 5 6 6

Yield 47% 25% 46% 36% 33% 36%

itf(So~vc11t A) 0.29 0.56 0.30 0.22 0.14 0.60

Rf(Solrent C) 0.84 0.97 0.79 0.61 0.60 0.98

Aminfx A-5 3.23 3.32 3.36 3.39 3.49 3.56

elution pl:

was further purified using paper chromatography in Solvent B. The sequence was obtained using the DNS-Edman procedure and was confirmed by the directEdman method, which identi- fied 1 glutamine residue, in agreement with the analysis of the aminopeptidase M digest (Table II). The subtractive Edman procedure gave the following results in confirmation of the deter- mined sequence.

Step 2: Lys, 1.00 (1); Asp, 3.12 (3); Thr, 0.98 (1); Ser, 0.96 (1); Glu, 0.97 (I); Gly, 0.46 (0); Ala, 1.04 (1); Val, 0.13 (0); Tyr, 1.89 (2).

Step 3: Lys, 1.00 (l);Asp,2.16 (2);Thr,0.94 (1); Ser, 1.02 (1); Glu, 1.04 (1); My, 0.26 (0); Ala, 1.02 (1); Val, tr (0); Tyr, 1.77 (2).

Step 4: Lys, 1.00 (1); Asp, 1.28 (1); Thr, 0.91 (1); Ser, 0.92 (I); Mu, 0.98 (1); Gly, 0.21 (0); Ala, 1.04 (1); Tyr, 1.75

(2). Step 5: Lys, 1.00 (1); Asp, 1.13 (1); Thr, 0.90 (1); Ser, 0.94 (1);

Glu, 1.03 (1); Gly, 0.15 (0); Ala, 0.99 (1); Tyr, 1.02

(1).

Step 6: Lys, 1.00 (1); Asp, 1.12 (1); Thr, 0.93 (1); Ser, 0.89 (1); Mu, 0.96 (1); Gly, 0.13 (0); Ala, 1.05 (1); Tyr, 0.35 (0).

Peptide TP 7:Xer-Gin-Ser-Ala-Leu-Gly-Thr-Ala-Ile-Glu-Arg- The sequence was determined by the DNS-Edman procedure. The presence of 1 glutamic acid and 1 glutamine residue 1~s evident from the analysis of t’he aminopeptidase M digest (Ta- ble II) and these residues were identified by the direct-Edman method, which also confirmed the sequence.

Peptide TP 6: Thr-Thr-Glu-Asn-Pro-Leu-Gin-Lys-This pep- tide was eluted from hmines A-5 at pH 2.86 with TP 5 and was further purified by paper chromatography using Solvent B. The sequence was obtained by the DNS-Edman method and confirmed by the direct-Edman method, which identified the amide residues and confirmed the results of analysis of the aminopeptidase M digest (Table II). The subtractive Edman procedure confirmed the presence of the adjacent threonine residues.

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T.IRLE II-Continuerl

- GlU 14

Gln

GUY 1.04(l) 1.13(l)

Ala 1.89(Z) 2.21(2)

Val -. Met

lie

LeU -_-

Tyr

PIIt? 0.93(l) 1.10(l)

--F--̂ Pro - ___-_ Total 7 2

Yield 442 34% -.-

I:f(Solvc!l( A) 0.14 0.22

i-3 (Solvi,nt C) 0. 56 0.55 -. -.____.- .-

iminex A-5 5.0 3.55

elutim pl:

27 - -

1.00(l) 21

0.93(l) 12

1

10

12

1.87(2) 0.96(l) 10

4

- - - 1

4 2 2 217

33% 23% 32%

0.60 0.66 0.46

0.99 0.94 0.91

3.70 4.10

Estimated

Residues

in

Flagellin

2

20 I 38

10

-+--I

] 470

St,ep 1: Lys, 1.00 (1); Asp, 0.92 (1); Thr, 0.92 (1); Glu, 1.97 (2); Leu, 0.85 (1); Pro, f.

Step 2: Lys, 1.00 (1); Asp, 1.01 (I); Thr, 0.31 (0); Glu, 2.02 (2); Leu, 0.89 (1); Pro, +.

Peptide TP 5: Thr-Glu-Val-Val-Thr-Ile-Asn-Gly-Lys--Thiu peptide eluted from Aminex A-5 with TP 6 and was purified using paper chromatography in Solvent B. The sequence was determined by the DNS-Edman procedure and confirmed by the directEdman method, which identified the asparagine resi- due detected in the aminopeptidase M digest (Table II). The adjacent valine residues were confirmed by the subtractive Edman method.

Step 2: Lys, 1.00 (1); Asp, 0.91 (1); Thy, 0.98 (1); Glu, 0.24 (0); Gly, 1.09 (1); Val, 1.82 (2); Ile, 0.82 (1).

Step 3: Lys, 1.00 (1); Asp, 0.90 (1); Thr, 0.90 (1); Glu, 0.21 (0); Gly, 1.13 (1); Val, 0.92 (1); Ile, 0.87 (1).

Step 4: Lys, 1.00 (1); Asp, 1.21 (1); Thr, 0.95 (1); Glu, 0.19 (0); Gly, 0.98 (1); Val, 0.41 (0); Ile, 0.87 (1).

Peptide TP 4: Val-Asn-Gly-Gin-Tlzr-Gin-Phe-Ser-Gly-Val-Lys

-The sequence was determined by the l)NS-Edman method. The amide residues were identified, in agreement with the re- sults of analysis of the aminopeptidase M digest (Table II), by the directEdman procedure which confirmed the sequence.

Peptide TP S:Asp-Asp-Ala-Ala-Gly-Gin-Ala-Ile-Alu-Asn-drg -The sequence was determined by the 1)X+-Edman method.

Aminopeptidase NI digestion revealed (Table II) the presence of 1 asparagine and 1 glutamine residue. The sequence n-as confirmed and the amide residues were positioned by the di- reckEdman procedure. The results of the subtractive Edman procedure confirmed the adjacent aspartic acid and alanine residues.

Step 1: Arg,l.OO (l);Asp,2.04(2);Glu,1.00 (l);Gly,l.O9 (1); Ala, 4.01 (4); Ile, 0.82 (1).

Step 2: Arg, 1.00 (1); Asp, 0.97 (1); Glu, 0.89 (1); Gly, 1.09 (1); Ala, 4.30 (4); Ile, 0.78 (1).

Step 3: Arg, 1.00 (1); Asp, 1.02 (1); Glu, 0.96 (1); Gly, 1.12 (1); Ala, 2.90 (3); Ile, 0.72 (1).

Step 4: Arg, 1.00 (1); Asp, 1.18 (1); Glu, 1.00 (1); Gly, 1.08 (1); Ala, 2.24, (2); Ile, 0.80 (1).

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TP

1

2

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

TABLE III

Amino acid sequences of tryptic peptides

sequence

Ala-Ser-Ala-Thr-Gly-Leu-Gly-Gly-Thr-Asp-Glu-Lys

Asp-Gly-Ser-Ile-Ser-Ile-Asp-Thr-Thr-Lys

Asp-Asp-Ala-Ala-Gly-Gin-Ala-Ile-Ala-Asn-Arg

Val-Asn-Gly-Gln-Thr-Gln-Phe-Ser-Gly-Val-Lys

Thr-Clu-Val-Val-Thr-Ile-Am-Gly-Lys

Thr-Thr-Mu-Am-Pro-Leu-Gln-Lys

Ser-Gln-Ser-Ala-Leu-Gly-Thr-Ala-Ile-Glu-Arg

Val-Gly-As?-Asp-Tyr-Tyr-Ser-Ala-Thr-Gin-Asp-Lys

Val-Thr-Val-Thr-Gly-Gly-Thr-Gly-Lys

Ser-Asp-La,-Gly-Ala-Val-Gin-Asn-Arg

Thr-Ile-Asp-Gly-Gly-Leu-Ala-Val-Lys

Tyr-Thr-Ala-Asp-Asn-Gly-Thr-Ser-Lys

Asp-Gly-Tyr-Tyr-Glu-Val-Ser-Val-Asp-Lys

Met-Ser-Tyr-Thr-Asp-Asn'Asn-r-ly-Lys

Phe-Asp-Asp-Thr-Thr-Gly-Lys

Leu-Gly-Gly-A%-Asp-Gly-Lys

Ile-Asp-Gly-Asp-Leu-Lys

Leu-Am-Glu-Ile-Asp-Arg

Gly-Leu-Thr-Gln-Ala-Ser-Arg

Phe-Thr-Ala-Am-Ile-Lys

Thr-Ala-Lru-Am-Lys

Ile-Am-Ser-Ala-Lys

Thr-Tyr-Am-Ala-Ser-Lys

Leu-Ser-Ser-Gly-Leu-Arg

Ala-Ala-Gly-His-Asp-Phe-Lys

Ser-Arg

Tyr-Tyr-Ala-Lys

Val-Arg

Tyr-Lys

Peptide TP ~:Asp-Gly-Ser-Ile-Ser-Ile-Asp-Thr-Thr-Lys--This

peptide eluted from Aminex A-5 at pH 2.805 and was contam- inated with some TP 1 which was removed by paper chroma- tography in Solvent A. The sequence was determined by the DNS-Edman method and confirmed by the directEdman pro- cedure, which demonstrated the absence of amide residues in agreement with the analysis of the aminopeptidase M digest (Table II). TP 2 was subjected to hydrolysis with 0.5 M acetic acid, and the hydrolysate was analyzed on the Aminex A-5 column (see “Materials and Methods”). Three peaks of nin hydrin-positive material were obtained and material correspond- ing to each was dried, hydrolyzed with 6 K HCl, and subjected to amino acid analysis. The materials were labeled AC-~, AC-~, etc. to indicate the use of acetic acid.

TP $? AC-~ :Asp-This material eluted from Aminex A-5 at pH 3.2 and was deduced to be the aspartic acid released in the acetic acid hydrolysis.

TP 2 Ac-2:Ser, 1.76 (3); Gly, 1.00 (1); Tie, 1.68 @--This material eluted from the Aminex A-5 at pH 2.92 and was de- duced to correspond to residues two to six of TP 2.

TP 2 AC-S:Lys, 1 .OO (1); Thr, 1.6s (!+This material eluted from t.he Aminex A-5 at pH 3.76 and was deduced to corre- spond to residues eight to ten of TP 2. On the basis of the known specificity of trypsin, which places lysine at the COOH terminal end of the peptide, the isolation of TP 2 AC-S was taken as evidence for the existence of adjacent threonine resi- dues, in agreement with the over-all sequence determined for TP 2. In confirmation, the DNS-Edman procedure yielded the sequence Thr-Thr-Lys for TP 2 AC-S.

Peptide TP 1: Ala-Ser-Ala-Thr-Gly-Leu-Gly-Gly-Thr-Asp-Glu-

Lys-This peptide eluted from Aminex A-5 at p1-I 2.80 was purified by paper chromatography in Solvent A to remove traces of TP 2. The sequence was determined by the DNS- Edman method. Analysis of the aminopeptidase M digest of this peptide (Table II) indicated the absence of amide residues, which was confirmed by the direct-Edman method during the confirmation of the sequence. The peptide was digested with thermolysin, and the digest was analyzed on a column of Xmi- nex A-5 (see “Materials and Methods”). Two peaks of ninhy- drin-positive activity were obtained, and the corresponding material was analyzed for total amino acid content. The ma- terial was labeled Th-1 and Th-2 to indicate the use of ther- molysin.

Peptitie TP 1 Th-i:Thr, 1.00 (1); Ser, 0.98 (1); Gly, 0.96 (1);

Ala, 1.85 (@-This peptide eluted from Aminex A-5 at pH 3.47 and was deduced to correspond to the first 5 residues of TP 1.

Peptide TP 1 Th-2:Lys, 1.00 (1); Asp, 1.02 (1); Thr, 0.9s (1);

Glu, 0.9s (1); Gly, 1.86 (6); Leu, 0.82 (I)-This peptide eluted from Aminex A-5 at pH 3.87 and was subjected to subtractive Edman degradation.

Step 1: Lys, 1.00 (1); Asp, 1.05 (1); Thr, 1.03 (1); Glu, 1.06: (1); Gly, 1.93 (2); Leu, 0.34 (0).

Step 2: Lys, 1.00 (1); Asp, 0.98 (1); Thr, 0.89 (1); Glu, 1.16 (1); Gly, 1.05 (1); Leu, 0.19 (0).

Step 3: Lys, 1.00 (1); Asp, 1.03 (1); Thr, 0.87 (1); GIli, 1.03 (1); Gly, 0.36 (0); Leu, 0.11 (0).

These results confirm the existence of adjacent Gly residues.

The reported derivation of SL 877 (9), its inability to give spreading growth through semi-solid medium containing anti-i serum, and its rapid agglutination with specific anti-i serum make it valid to conclude that the flagella isolated were poly- mers of a flagellin whose primary structure was determined by the phase-l structural gene (H-li) of X. typhimurium. The mode of isolation of the flagellin, the results of analysis of its amino acid content, and the identification of alanine as its NH?-terminal amino acid, demonstrated that the flagellin iso- lated was identical with previously investigated flagellin from SL 877 (9). The observation of only one band in acrylamide gel electrophoresis in the presence of disaggregating agents, and the obtaining of only one amino acid in NHz-terminal amino acid analysis, suggested that a single protein was present in the flagellin preparation.

The molecular weight of the phase-2 flagellin (H-antigen 1, 2) of 8. typhimurium has been established as 40,000 by the technique of analytical ultracentrifugation in three independent investigations (9, 37, 38), and it has been assumed that this

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5192

figure is uniform for all Sahnonella flagellins (9), a concept sup- ported by analysis using the same technique of the flagellin of S. adelaide (39). Centrifugation of flagellin in the presence of either 6 M guanidine HCl and O.lScxj SI)S has been reported (9) but no attempt was made to use the data obtained to cal- culate molecular weight values. Attempts to estimate the molecular weight of a sample of SL 877 flagellin (phase-l anti- gen i) by sedimentation velocity and sedimentation equilibriurn (undertaken by Worthington Analytical Services) were un- successful due to the formation of aggregates, even after transi- tory treatment with urea to disaggregate polymers (a technique used successfully by McDonough (9)). The two methods de- scribed here investigated the behavior of SL 877 flagellin in the presence of d&aggregating agents and both techniques yielded a value of 49,000 for the molecular weight of this pro- tein. The discrepancy between these estimates and the pre- viously reported value of 40,000 appeared to be due to the different techniques used as, in SDS-acrylamide gel electro- phoresis, the phase-l and phase-2 flagellins of S. typhimurium could not be distinguished2 and both SL 167 flagellin and S. adelaide flagellin were inseparable from fumarase and the H- chain of y-globulin. The value of 49,000 for the molecular weight of SL 877 flagellin was preferred because of the extensive experimental data on which it was based and because the num- ber of tyrosine residues already confirmed in the peptide anal- yses (total = 10) was greater than the number predicted from total amino acid analyses (total = 9) based on a molecular weight of 40,000 (9).

The 29 tryptic peptides isolated had a total amino acid COII- tent of 2L7 residues (Table II) and accounted for 46.2yc of the total residues present. The remaining 14 anticipated tryptic peptides contained a total of 253 ammo acid residues and were present in the Sephadex G-25 Fractions I and II, which have proved refractile to further differentiation.

Despite the fact that some of the peptides were obtained in low yields (TP 4, TP 5, TP 13, and TP 14) it appeared that they were bona fide regioiis of the SL X77 fiagellin. Acrylamide gel electrophoresis in the presence of disaggregating agents, and SHz-terminal amino acid analysis indicated that only one pro- t,ein was present so that these peptides were unlikely to be tryptic products of a contaminating protein. In the sequence analyses, the COOHterminal lysine and arginine residues were identified in all cases and occurred in the predicted positions caalculated on the basis of total amino acid composition. Also, after sufficient steps of Edman degradation to remove the ly- sine or arginine residues, no amino acids could be detected. These results demonstrated that the peptides obtained in low yields did not represent nontryptic cleavage products. To date, 10 different preparations of SL 877 flagellin, each re- sulting from a different starting clone of cells, have been sub- jected to tryptic digestion and the peptides described here have been recovered in pure form from all digests.

Examination of the amino acid sequences of the purified peptides revealed the presence of adjacent glycine residues in f%r peptides (TP 1, TP 9, TP II, aild 1’1’ 16). Attention has been drawn to the role of glycine in permitting flexibility of a polypeptide chain (40-42). Wu and Kabat (43) have recently suggested that regions of urn~sual flexibility, termed llivot position, exist in the light and heavy chains of human and mouse immunoglobulins and consist of adiacent glycine residues or 2 glycine residues separated by a glut’amine, :I pro-

2 T. &I. Joys, unpublished data.

line, or a11 arginine residue. The known cylindrical helix structure of bacterial flagella cannot be explained by the cur rent model of flagella structure, based up011 electron microscope studies, iu which the individual flagellin molecules are consid- ered to be identical, rigid spheres (see References 6 and 7 for review) and it has been proposed that flagellin molecules must exist in me&stable states (37). It is tempting to hypothesize that the four -Gly-Gly- regions identified in the flagellin are regions of flexibility within the molecule that allow conforma- tional changes to occur resulting in the known structure of flagella and possibly allowing waves of movement to be trans- ferred down the flagellum filarnent with consequent locomotion of the bacterial cell.

Acknowledgments-The authors wish to express appreciation to Drs. Hernadine Rrirnhall, John A. Hlack, and Richard T. Jones for helpful discussion in all phases of the work and to Dr. I-T. I). Niall for details of the rapid Edman degradation technique. The excellent technical assistance of James F. Martin is gratefully acknowledged.

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Terence M. Joys and Viktorija Rankis: I. THE TRYPTIC PEPTIDES

Salmonella typhimuriumThe Primary Structure of the Phase-1 Flagellar Protein of

1972, 247:5180-5193.J. Biol. Chem. 

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