Amino acid sequence of penicillopepsin. 111. Isolation and characterization of amino terminal, tryptic, and tryptophanyl peptides' Can. J. Biochem. Downloaded from www.nrcresearchpress.com by SAVANNAHRIVNATLABBF on 11/14/14 For personal use only.

G . IANHARRIS,= LETICIA ~ Q A BPAUL ,~ SHUTSA,ALEXANDER KUWOSKY,~ AND TWEO HOFMANN Drpartntent ofBiochemistry, tlni~vrsityof Toronto, Toronto, Qnt.. Cunuda M3S 1A8 Received April 15, 1976

Harris, C. I.. Rao, L., Shuesa, P., Kurosky, A. & H s h a n n , T. (1976) Amino acid sequence of penicillopepsin. 111. Isolation and characterization of amino terminal, tryptic, and tryptophanyl peptides. C'crn. J. BiocEtetaz. 54, 895-901 The amino acid sequences of peptides isolated from a tryptic digest of penicillopepsin (EC 3.4.23.71, a subtilisin (EC 3.4.21.14) digest of maleylated penicillopepsin, and a chymotryptic digest of penicillopepsin modified with dinitrophenylsulfenyl (DNPS) chloride have been determined. The first two digests identified four of the five lysyl residues of the enzyme as well as the N-terminal peptide. The third digest provided ove~lapsat three of the tryptophanyl residues. The DNPS-tryptophan peptides were isolated on an affinity column prepared by coupling dinitrophenyl antibody raised in sheep to cyanogen bromide-activated Sepharose. Harris, C. I., Wao, L., Shutsa, P., Kurosky, A . & Hofmann, T. (1976) Aminoacid sequence of penicillopepsin. 111. Isolation and characterization of amino terminal, tryptic. and tryptophanyl peptides. Can. 9. Biochem. 54,895-901 Nous avons determine la skqeaence des acides amines des peptides provenant d'un digestat trypsique de la pknicillopepsine (EC 3.4.23.7), d'un digestat B la subtilisine (EC 3.4.21.14jde la pknicillopepsine maleylee et d'un digestat chrymotnypsique de la penicillopepsine modifiee par le chlorure de dinitrophknylsulfenyle (DNPS). Les deux premiers digestats permettent #identifier quatre des cinq residus lysyl de I'enzyme ainsi que Ie peptide N-terminal. Le troisieme digestat fournit des chevauchements pour tsois des residus tryptophanyl. Les DNPS-tryptophane peptides sont is016s sur une colonne daffinite prkparee par coiaplage d'un anticosps de dinitrophewyl. produit chez le mouton, h la Sipharose activee pas le bromure de cyanogtne. [Traduit par le journal]

Pwaroducalon

In the first two papers of this series (1, 2) the isolation and sequences of ckymotryptic and thermolytic peptides are described. Although these peptides and fragments account for virtually all the amino acids of penicillopepsin (EC 3.4.23.3, a considerable number of overlaps were still required to complete the sequence. The present study was undertaken to provide as many of the overlaps as possible. Because penicillopepsin has only five lysine residues, a tryptic digest should yield six large fragments. Although many attempts, using different conditions and trypsin preparations, were made to

DNPS, 2,CdinitropAenylsulfenyl; ABBREVIATIONS: DNP, 2,Cdinitrophenyl; DCC, diphenyIcarbamylchloride: TPCK, (I-tosylamido-2-phenyl)ethyl chlorornethylketone. 'Supported by the Medical Research Council of Canada (grant No. MT- 1982). :Present address: Rowett Research Institute, Bucksbum, Aberdeenshire, Scotland. 3HHder of an MRCC Studentship. 4Holder of a Province of Ontario Graduate Fellowship. Present address: Department of Human Biologicail Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX. U.S.A. 77550.

confine the cleavage to the lvsine resid~mes.we always obtained other cleavages and complex mixtures of large fragments that were difficult to separate. Nevertheless, we were able to isolate four lysine-containing peptides which, in combination with the peptides obtained from the maleylated enzyme and from the them-molytic and chymotryptic digests, established the sequences arcsund the lysines. ILn addition. five lysine-free peptides which provided valuable overlaps (3) were isolated. Since all three enzymes cleaved a t tryptophanyl residues, it was also necessary to use a direct approach for obtaining the overlaps at thls amino acid. This was done by the method first used by Wilshek and Miron (4) in which the tryptophan residues are blocked by reaction with DNPS-chloride. The modified enzyme is then digested and the DNPS peptides are isolated on an affinity column prepared by coupling DNP antibody to Sepharose. The isolation and sequencing of those peptides which provided evidence for overlaps is described in thls paper. Materials and Methods

Enzymes DCC-treated trypsin BE& 3.4.21.4) and subtilisin (EC 3.4.21.14) were from Sigma Chemical Co.; TPCK-treated

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CAN. J. BIOGHEM. VOk. 54, 1976

tube number

PIG.1. Fractionation of fraction B (see text) on Sephadex G-75 medium (2.5 x 85 cm). Solvent, 0.05 N NH,; flow rate, 12 mllh: fraction volume, 3 mB. trypsin was from Worthington Biochemical C o p ; all other enzymes were obtained as reported (1). Bovine DNP-yglobulin and E-DNP-Sepharosewere gifts from Ms. Peggy Miniter. Maleic anhydride, dioxane sequanaJ grade, and DNPSchloride were from Pierce Chemical Co. A11 other chemicals were obtained as reported (1). Tryplic Digestinrts The tryptis digestions, from which the peptides listed in this paper were obtained, were carried out in a pH-Stat at pH 9.0, at penicillopepsin concentrations of 3 mg/ml, and protein-trypsin (DCC-treated) ratios of 50: 1 (by weight) for 3 h. The clear digest was applied to a G-25 (fine) Sephadex column (88 x 2.5 cm) in freshly prepared 0.85 N ammonia and eluted with the same solvent. Only two major fractions were obtained; the first (fraction A) consisted of a mixture of large fragments whereas the second (fraction B) was a mixture sf small peptides. Fraction A was applied to a 6-75 (medium) Sephadex column (2.5 x 85 cm) in 0.05 1V ammonia and eluted with the same solvent. The elution profile (absorbance at 280 nm) is shown in Fig. 1. Further purifications sf the peptides in peaks IV-Vl were carried out by high-voltage electrophoresis and paper chromatography. The peptides in fraction B were separated on a cation exchange column sf Technicon Chromobeads BQ126 x 0.6 cm) at 37 "C. The material was dissolved in 8.2 A4 acetate adjusted with pyridine to pH 3.1 and applied to the column in the same solvent. The peptides were eluted with a linear gradient of 258 mi 0.2 M pyridine-acetate, pH 3.1, and 250 rnl 2 M acetate adjusted with gylddine to pH 5.1. The peptides obtained were further purified by electrophoresis at pH 2.0. Carboxypeptidase digestions, pager chromatography, high-voltage electrophoresis, and peptide sequencing were carried out as described (1).

Reaction ofPesticillopepsin with Maleir Anhyeiride Penicillopepsin 630 mg) was dissolved in 15 ml of 0.1 M sodium p yrophosphate buffer, pH 9.0, and treated with 606) FB sf 1.0 M maleic anhydride in dioxane (BOO-fold excess over amino groups) at 4 "@. The maleic anhydride solution was added in eight portions and the pH of the mixture was maintained at 8.5-9.0 by the addition of 1 M NaBH. The solution of maleyl-penicillopepsin was then exhaustively dialyzed against 1% NH4PICO3. Dig esrion ~'I'th Stlbdilisin Subtilisin (0.3 mg in 0.1 ml water) was added to the maleyl-pemieilBopepsin in 0.1 M NH,H@O, and digestion was allowed to proceed for 6 h at 37 " C . The reaction was stopped by the addition of 100 pl diisopropyl phssphoroflusridate. Diagonal Electrophoresis Diagonal electrophoresis was carried out as described by Hartley (5). The volume of the digest was reduced to 1.5 ml by lyophilization and applied to Whatmam 3MM paper as a band (30 x 1 cm). The paper was subjected to high voltage electrophoresis at pH 6.5 in two dimensions (4Vlcm, 1.5 h). The rnaleyl groups were removed after the first dimension by heating the paper at 65 "C for 6 h in vapours of pyridine-acetate, pH 3.5 46). Preparation andPur@cution ofDNP Andibody Antisera were elicited in sheep by injection of 10 rng of bovine DNP-y-globulin in complete Freund's adjuvant at multiple intraderma1 sites. Booster injections of 18 mg antigen, each in Freund's adjuvant, were given three more times at three-weekly intervals. The antibody titer of the serum was determined by a precipitin reaction with a cross-reacting modified protein, human BNP- serum-

HARRIS ET AL.

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0-1 k4 BSrHCO,

H,O

-----------lo

% formic acid

FIG.2. Affinity chromatography of DNPS-tryptophan peptides. Column, DNP antibody - Sepharose,

2.5 x I4 cm. Flow rate. 60 ml/h; fraction volume, 5.5 ml.

albumin. The antibody ( I . 1 mgln.11) was purified by specific adsorption on an imrnuno-adsorbent column of c-NDNP-lysine-Sepharose. After elution with 0.1 1 3 4 dinitrophend, the latter was removed on a Dowex ,461-X8 column and the eluted antibody dialyzed against phosphate-saline biafer (0.01 NaKT,P04. 0.15 M NaCl, pH 7.3).

Prepariztion o f D h r PAntibody - Sephtrrose Collrnan The purified DNP antikody (about I .2 g) was coupled to CNBr-activated Sepharose 4B (about 60 g wet weight) by the method of Axen rr (11. (7).

Su!ferzylarion of T ~ 3 p ~ o p h irt a nPenEchllopep.si~r The method of Wilchek and Miron (4) was based with the following slight modifications. Freeze-dried peniciliopepsin (182 rng) was dissolved in 8.3 rnl of 90% formic acid. A 100-fold excess of DNPS-chloride (68 mg) was added gradually with stirring. After 5 h in the dark the excess DNPS-C1 was removed by centrifugation in the cold at 10 000 rpm for 15 min. Addition of cold 1 M HC1 in acetone precipitated the modified protein which was then washed three times with cold ether to ensure complete removal of the reagents and solvents. The precipitate was dissolved in water and lyophiiized. Digestion sj7DNP,Y-pe~zic.iflopc~psin The modified protein was digested in a pH Stat with a-chyrnotrypsin for 4+ h at room temperature and pH 8.3 using 21 substrate to enzyme ratio of 1:50. The reaction was stopped by lowering the pH to 4.5 with glacial acetic acid and freeze-drying the mixture. hcda~iorrofDMPS-tryptophan Pep tidcs

The Sepharose-conjugated DNP antibody was packed in a coIumn (2.5 x 15 cm) and washed with 0.1 A4 NaHCO,.

The digest was applied to the column and the ranadsorbed nontryptophan peptides were washed out with 0 . 1 ilig NaHCB, until the absorbance at 280 nm was less than 0.02 (Fig. 2). The adsorbed tryptophan peptides 6330 nm) were eluted first with water (peak Ch-II-a) and then with 18% formic acid (peak Ch-111-a). The peptides from fraction Ch-11-a were freeze-dried, dissolved in 70% formic acid, and separated on a Sephadex G-25 colirmn (2.5 x 10 cm) with the same solvent (Fig. 3). The peptides from fraction Ck-111-a were separated on a similar column, but the solvent was 45%) formic acid.

Results and Discussion Tryptic Peptides At the outset we had hoped to confine the tryptic cleavages to the Iysine residues of penicilBopepsin and obtain six fragments. Many attempts were made with various preparations of trypsin, including freshly prepared bovine P-trypsin (8), the single chain species which, according to KeiB-BBouhki er ( I ! . (9. LO), is more specific than the trypsins with internal nicks. However, in all cases evidence was found for partial cleavages at residues other than Iysine. The mixttires obtained consisted of large and small fragments, were often insoluble in most solvents, and could not be separated. DCC-trypsin gave a hil-ly extensive digest which allowed the isolation and purification of 22 peptides. Ten of these yielded unique sequences and (or) overlaps not found among the chyrnotryptic and thermolytis peptides. They are listed in Table 1. Peptides Tr-I, 73-2, Tr-8, Tr- B I , and Tr-22 were obtained from peak IV (Fig. I) and Tr-3, Tr-5, and Tr-6frorn peak V (Fig. I). Peak I

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CAN. 9. BIOCHEM. VOk. 54, 1976

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FIG.3. Rechromatography of fraction Ck-11-a (Fig. 2) on Sephadex G-25 (fine), 2.5 x 10 cm; solvent, 70% formic acid -0.02 MM-ethylmophsline; flow rate, 13.6 mllh; fraction volume, 2.5 ml. (Fig. 1) was not further analyzed. Peaks II and IIT (Fig. 1) consisted of complex mixtures of fairly large fragments. No paare fragments amenable to sequencing could be isolated. Peptides Tr-4 and Tr-7 were obtained from the ion-exchange column sf Raction A. The placing of the peptides in the partial sequence of penicillopepsin is given in the accompanying paper (3). Peptide Tr-8 is the C-terminal peptide obtained by cleavage at the Eys-Ser bond. Although the digestion with DCC-treated trypsin was relatively mild (3 h, 37 "C), several cleavages typical of chymotrypsin (tryptophan, tyrosine, phenylalanine, and leucine) had occurred. The reasons for this are not clear. The BCC-trypsin used showed no measurable activity towards acetyl tyrosine ethyl ester.

Beptidss J>om Maleylabed Penici!lc~pepsin The diagonal electropherograrn of the subtilisin digest of maleyl-penicillopepsin is given in Fig. 4. Although a considerable number of peptides were found off the diagonal, only five were finally obtained in sufficient yield for sequencing. The pep-

tides were further purified by high voltage electrophoresis at pH 2.0 and paper chromatography in pyridine - butanol- acetic acid - H,O. Table 2 gives the compositions, sequences, and mobilities before and after removal of the rnaleyl groups. Only three of the five lyslnes were obtained. These provided necessary overlaps (see accsmpanying paper, Ref. 3). The other two peptides do not contain lysine and therefore are candidates for the N-terminal of the intact enzyme. Since the manual dansyl-Edman procedure applied to the intact protein (11) gave the same sequence as that of peptide Mal-1, this was confirmed as the true N-terminal. originally, the reasons why Mal-3 appeared off the diagonal were not clear, but when an automatic sequenator became available and a number of preparations were examined, it became apparent that on prolonged storage as a freeze-dried powder penicillopepsin autolyzes, and the first auteslytic cleavage liberates tyrosine as a new N-terminal. Consequently, we assume that Mal-3 was also N-terminal in the preparation used for these experiments.

-0.55

nd

+0.27

Tr- 1

Tr-2

Tr-3

0

0

-0.31

0

0

Tr-6

Tr-7

Tr-8

Tr-1 1

Tr-22

-2

0

0

0

0

+1

+1

-2

Charge

"Relative to aspartic acid. 'Not corrected for losses.

+0.3

Tr-5

Tr-4

Mobility at pH 6.5"

Peptide

6

7

3.8

6

16.5

2.2

11.5

4.5

5.5

7.5

z b

Recovery,

7

I

- Gly

(Asp. Ile, Phe, 1 . 2 0.85 1.2

Leu) Lys 0.82

-Leu-Thr-Tyr 1+ 0 4 4 0&6

7

0.9

Phe O +

Ser 0.81 1+

0.99

- Lys 1.02 0.99

- Asp - Thr - Val

- Ser I

-Leu - Ala - Gln - Pro - Leu 0.81 1 4 5 2 9 1.09 (+) lZ

Ser - Glx - Tyr - Val -Val -Phe - Asx - Ser - Asx 0 2 9 1 e OA5 0 4 4 *1 0 3 l