the amino acid sequence of viscotoxin b from the european mistletoe (viscum album l, loranthaceae)

Eur. J. Biochem. 21 (1971) 86-89

The Amino Acid Sequence of Viscotoxin B from the European Mistletoe (Viscum aZbum L, Loranthaceae)

Gunnar SAMTJELSSON and Barbro M. PETTERSSON Farmakognostiska Institutionen, Farmaceutiska Fakulteten, Stockholm

(Received February 17/April28,1971)

Viscotoxin B from Viscum album has been oxidized with performic acid and digested with trypsin and chymotrypsin. The resulting peptides have been separated by ion exchange chromatography and subjected to amino acid sequence determination by Edman degradation. Free lysine, six tryptic and three chymotryptic fragments are primary digestion products and permit deduction of the following amino acid sequence for the 46 amino acids of viscataxin B : Lys-Ser-Cys-Cys-Pro-Asn-Thr-Thr-Gly-Arg-Asn-Ile-Tyr-Asn-Thr-Cys-Arg-Leu-Gly-Gly-Gly- Ser- Arg-Glu-Arg-Cys-Ala-Ser-Leu-Ser-Gly-Cys-Lys-Ile-Ile-Ser-Ala-Ser-Thr-Cys-Pro-Ser-Tyr- Pro- Asp-Lys.

plant.

I 1 I

I I I

This sequence is very similar to the amino acid sequence of viscotoxin A3 from the same

Viscotoxin is a mixture of pharmacologically acti- ve, small, basic proteins isolated from the European mistletoe, Viscum album L [1,2]. Three main compo- nents-viscotoxin A2, viscotoxin A3 and viscotoxin B-have been isolated by chromatography on cellu- lose phosphate [3] and SE-Sephadex [a]. These three substances are all composed of 46 amino acid residues and have molecular weights around 5000 [3,4]. The amino acid sequence of viscotoxin A3 has been determined [5]. This paper describes the determina- t'ion of the amino acid sequence of viscotoxin B.

EXPERIMENTAL PROCEDURE

Materials Viscotoxin B was prepared by previously described

methods [3,4]. Carboxypeptidase A was obtained from Worthington Biochemical Corp. (Freehold, New Jersey, U.S.A.). The enzyme (2 x crystallized, batch COA 726, 52 U/mg protein) was treated with DFP as previously described [5]. DFP-treated carboxypeptidase B was also purchased from Worthing- ton (Batch COBDFPSlA, 6 mg/ml, 109 U/mg). Chymotrypsin was a 3-times crystallized preparation from Worthington (Batch CDl 6114-5). Leucine aminopeptidase was a preparation type I11 from hog kidney obtained from Sigma Chemical Company (St. Louis, MO, U.S.A., Lot 108B-0540, 5 mg protein/ml). Trypsin was a Worthington 2-times crystallized salt free preparation, batch 6118. The enzyme was treated

Enzymes. Carboxypeptidase A (EC 3.4.2.1) ; carboxy- peptidsse B (EC 3.4.2.2); chymotrypsin (EC 3.4.4.5); leucine aminopeptidase (EC 3.4.1.1); trypsin (EC 3.4.4.4).

with ~-(l-tosylamido-2-phenyl) ethyl-chloromethyl ketone to reduce chymotryptic activity [6]. Cation exchanger Dowex 5OW-XZ was purified and subjected to hydraulic separation of particle sizes as described by Moore et al. [7] with the exception of treatment with NaOH which was performed a t room temperature instead of on the steam bath. The fraction collected a t a water flow of 517-650 ml/min was used for packing the column. Sephadex G-10 and G-15 were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden).

Performic Acid Oxidation 500 mg of viscotoxin B was oxidized with performic

acid a t 0 "C, according to Hirs [8].

Digestion with Trypsin and Chymotrypsin 150 mg oxidized viscotoxin B was digested with

trypsin for 20 h a t pH 8.5 and a temperature of 37 "C. The digestion was performed in a pH-stat as previously described [5]. The enzyme- protein ratio was 1 : 50 (w/w).

The same technique was used for digestion of 300 mg oxidized viscotoxin B with chymotrypsin for 15 h a t 37 "C, pH 8.5 and an enzyme-protein ratio of 1 :30 (w/w).

Chromatography of Peptides on Dowex 50W-X2 and Dowex 1-X2

The peptides from the tryptic and chymotryptic digestions were separated by chromatography on Dowex 50W-X2 or Dowex 1-X2 as described by

Vol.31, xo.1, 1971 G. SAMUELSSOX and B. &I. PETTERSSON 87

Schroeder et al. [9] with some minor modifications [5] . After lyophilization, the peptides were further purified by gel filtration on Sephadex G-10 or G-15, using 100/,, acetic acid as eluant.

Peptide Maps The purity of the isolated peptides was checked

by peptide mapping using the thin layer techniques of Ritschard [ 101 with previously described modifications [5].

Quantitative Amino Acid Analysis Peptides were hydrolyzed with constant boiling

hydrochloric acid in sealed evacuated tubes a t 110 "C for 24 h. The acid was removed in vmuo in a rotary evaporator and the amino acids determined with an automatic amino acid analyzer according to Spack- man et al. [ill as modified by Samuelsson [12]. Norleucine and guanidino amino propionic acid were added to the hydrolysates as internal standards.

Amino Acid Xequence Analysis Subtractive Edman-degradation was used as

previously described [5]. Whenever possible the results were confirmed by thin layer chromatographic identification of the liberated phenylthiohydantoin derivatives of amino acids using the system of Jepp- son and Sjoquist [13].

Digestion with Carboxypeptidase and Leucine Aminopeptidase

Digestions were performed as previously described [5] on 0.2-0.3 pmoles of the peptides.

RESULTS AND DISCUSSION TRYPTIC PEPTIDES

Chromatography of a tryptic hydrolysate of oxidized viscotoxin B on Dowex 50W-X2 (Annex, Fig.1) yielded seven main peaks, numbered 1-7. Peptide mapping and amino acid analyses showed peaks 2, 3 , 4 , 6 and 7 to contain pure peptides. They were designated as peptides OT3, OT4, OT5, OT6 and OT7 respectively. Peak 1 represented a mixture of peptides and was subjected to chromatography on Dowex 1-X2 yielding two main peptides designated OT1 and OT2. Peak 5 was shown to be free lysine in a yield of 6.7 pmoles. The amino acid compositions and the yields of the isolated peptides are presented in Annex Table I. The yields, which were based on the amino acid analyses and were not corrected for losses during purification, ranged from 4-17 pmoles from approximately 24 pmoles of viscotoxin B.

The determination of amino acid sequences of the peptides was founded on the results of Edman degradation of the peptides, followed by amino acid analysis of an aliquot of the rema.ining peptide frag-

ment after each degradation. I n most cases these results could be confirmed by thin layer chromatographic identification of the phenylthiohydantoin derivatives of the liberated amino terminal amino acids.

Peptide OT1 Ser-Cys(S0,H)-Cys(S0,H)-Pro- Asn-Thr-Thr-

Gly-Arg The results of Edman degradation of this peptide

are presented in Annex, Table 2. The degradation was carried through 8 steps (7 presented in Annex, Table 2). After step 8 the presence of free arginine was demonstrated. Carboxypeptidase A + B a t 37 "C for 24 h liberated gIycine, threonine and asparagine in the ratio 1 ~ 2 ~ 0 . 3 (arginine not determined). These results prove the amino acid sequence of peptide OTI.

Peptide OT2 Cys(S0,H)-Ala-Ser-Leu-Ser-Gly-Cys(S0,H)-Lys Assuming lysine to be in the normal C-terminal

position the sequence of this peptide was proven by six steps of Edman degradation (Annex, Table 3).

Peptide OT3 Ile-Ile-Ser-Ala-Ser-Thr-Cys(S0,H)-Pro-Ser-

(Tyr Pro)-Asp-Lys The peptide was taken through nine steps of Ed-

man degradation (Annex, Table 4). From these results the amino acid sequence of the first 9 amino acids could be deduced. 0.2 pmoles of the peptide was hydrolyzed with 0.01 M HCl at 110 "C for 24 h. Amino acid analysis of the hydrolysate yielded 0.050 pmoles lysine and 0.057 pmoles aspartic acid. No other free amino acids were detected. Since aspartic acid is known to be the amino acid which is most easily split off by acid hydrolysis this indicates the C-terminal sequence -Asp-Lys. Digestion with carboxypeptidase A + B confirmed this result and proved that the dicarboxyl amino acid is aspartic acid and not asparagine. This leaves the sequence of the two amino acids tyrosine and proline undetermined. They must be placed between the third serine and aspartic acid but the evidence so far discussed, does not show their mutual order.

Peptide OT4 Lys-Ser-Cys(S0,H)-Cys(S0,H)-Pro-Asn-Thr-

Thr-Gly- Arg Except for an additional residue of lysine the

amino acid composition of this peptide is the same as that of peptide OTl. One step of Edman degradation of intact viscotoxin B yielded the derivative of lysine indicating this amino acid to be amino terminal. A substantial amount of free lysine was found in the

88 The Amino Acid Sequence of Viscotoxin B from Viscum album Eur. J. Biochem.

tryptic hydrolysate of oxidized viscotoxin B. This indicates peptide OT4 to be the amino terminal peptide of viscotoxin B from which partial tryptic digestion liberates lysine yielding peptide OTI. This was confirmed by two steps of Edman degradation of peptide OT4 which clearly showed the sequence of the first two amino acids to be Lys-Ser-.

Peptide OT5 Asn-Ile-Tyr-Asn-Thr-Cys(S0,H)-Arg

The results of Edman degradation as presented in Annex, Table 5 and the presence of free arginine in the aqueous phase after the 6th degradation step, established the sequence of this peptide. Digestion with leucine amino peptidase liberated asparagine, isoleucine, tyrosine and threonine in the ratio 2: I : 1 :0: 7.

Peptide OT6 Glu-Arg

Free arginine was detected in the aqueous phase after one step of Edman degradation. Digestion with leucine amino peptidase yielded glutamic acid and arginine.

Peptide OT7 Leu-Gly-Gly-Gly-Ser- Arg

Results of Edman degradation are presented in Annex, Table 6. Free arginine was detected after the 5th step.

CHYMOTRYPTIC PEPTIDES

The products of a chymotryptic hydrolysate of oxidized viscotoxin B were separated by chromatography on Dowex 50W-X2 (Annex, Fig.2). Four main peaks were obtained, numbered 1-4. The material from peak I was chromatographed on Dowex I-X2 yielding one main peptide designated as OCI. Chro- matography of the material from peak 2 on Dowex 1- X2 yielded one main peak, which however did not represent a pure peptide. Further purification was achieved by preparative thin layer chromatography on I mm thick layers of Kieselgel H (Merck) in the system n-butanol-acetic acid-water (4: I : 5, v/v/v), using the upper phase for development. This yielded another pure peptide called OC2. Peaks 3 and 4 were denoted peptides OC3 and OC4 respectively.

Peptide OCl Ser- Gly-Cys(S0,H) -Lys-Ile-Ile- Ser- Ala-

(2 Ser, Thr, Cys(SO,H), 2 Pro, Tyr)-Asx-Lys The results of 8 steps of Edman degradation of

this peptide are presented in Annex, Table 7. The Ile-Ile bond is very resistant to acid hydrolysis. Hydrolysis for 24 h liberated only one residue of isoleucine. I n steps 3-6 it was therefore necessary

also to analyse samples hydrolyzed for 72 h to de- monstrate the Ile-Ile sequence of the peptide. After coupling with phenylisothiocyanate, lysine gives low values in the amino acid analysis due to incom- plete hydrolysis of the phenylthiocarbamate of the €-amino group. This causes difficulties in demonstra- tion of the presence of a lysine residue in the amino acid sequence when using the subtractive Edman degradation method. The position oflysine as the fourth amino acid of the peptide was confirmed by dansy- lation of an aliquot of the residual peptide after the third Edman degradation step and by identification of the derivative of lysine after four degradation steps. Partial acid hydrolysis liberated equal amounts of aspartic acid and lysine indicating these two amino acids to be C-terminal (see discussion of peptide OT3). The partial amino acid sequence and the amino acid composition shows that peptide OCI forms an overlap between the tryptic peptides OT2 and OT3.

Peptide OC2 Lys-Ser-Cys(S0,H)-Cys(S03H)-Pro-(2Asx,

2Thr, Gly, Arg, 1le)-Tyr 5 steps of Edman degradation were performed

(Annex, Table 8). Assuming normal chymotryptic activity tyrosine must be in the C-terminal position. The partial amino acid sequence and the amino acid composition shows that peptide OC2 forms an overlap between the tryptic peptides OT4 and OT5.

Peptide OC3 Asx-Thr-Cys(SO3H)-Arg-Leu-G1y(2Gly, 2Ser, 2Arg, Glx, Cys(SO,H), A1a)-Leu

Six steps of Edman degradation yielded this partial amino acid sequence (Annex, Table 9). The spe- cificity of chymotrypsin suggests the second leucine residue to be in the C-terminal position. The amino acid composition of peptide OC3 indicated that the peptide was not completely pure. The high figures for arginine and glycine indicated admixture of peptide OC4 which is eluted close to peptide OC3 from the Dowex 5OX-2 column. There was also a progres- sive decrease in the content of glycine in the first 3 degradation steps, which is in agreement with the amino acid sequence of peptide OC4 (see below). The partial amino acid sequence and the amino acid composition of peptide OC3 permit the arrangement of the tryptic peptides OT2, OT5, OT6 and OT7 in the correct sequence.

Peptide OC4 Gly-Gly-Gly-Ser-Arg-Glu-Arg-Cys(S0,H)-

Ala- Ser - Leu The results of 9 steps of Edman degradation are

presented in Annex, Table 10. Free leucine was demonstrated in the aqueous phase after the 10th de-

Vol.21, No. 1, 1971 G. SAMUELSSON and B. M. PETTERSSON 89

-- - - - - - -0c3 -F------- - OT5 + I -0T7- -1le -Tyr l A s n -Thr - C i s - Arg $Leu [ G ly -GIy -GIy -

of-4 ---- - J 12 13 ’ 1 4 15 16 17 18 19 20 21

I -C:s - LysJ-lle - Ile - Ser -Ala- S e r - Thr -Cys - 32 33 34 35 36 37 38 39 L O

-4 _-_.-----

- OT3 -4 - Pro - Ser - Tyr - Pro - Asp - Lys I

41 42 4 3 LL 45 46

Pig. 1. Amino acid sequence of viscotoxin B

gradation step. Peptide OC4 forms an overlap between the tryptic peptides OT2, OT6 and OT7, and confirms the sequence deduced from the analysis of peptide OC3. The presence of both peptide OC3 and OC4 in the chymotryptic hydrolysate indicates that the Leu-Qly bond in peptide OC3 is relatively resistant to hydrolysis by chymotrypsin.

THE COMPLETE AMINO ACID SEQUENCE O F VISCOTOXIN B

Overlapping sequences in the tryptic and chymotryptic series of peptides from oxidized viscotoxin B permit the establishment of the complete amino acid sequence of viscotoxin B as presented in Fig. 1.

The only sequence which is not definitely proven by the results of Edman degradation and the action of leucine aminopeptidase and carboxypeptidase is the Tyr-Pro bond (residues 43 -44). However, the failure of chymotrypsin to attack this bond is a fairly good indication that this sequence is correct.

It is interesting to note that this sequence is very similar to the sequence of viscotoxin A3 [5] . The sequences of amino acids 1 - 18 are identical with the exception of amino acid no. 15 which is alanine in viscotoxin A3. Amino acid 23 is arginine in both proteins. The sequences of residues 26-46 are also almost the same in both proteins, the exceptions being residues no. 28 and 37 which are lysine and glycine respectively in viscotoxin A3.

This work was supported by grants from the Swedish Medical Science Research Council (Projects No B69-13X- 2084-038, B70- 13X-2084-04B and B7 1 -13X-2084-05C).

ANNEXES

The following documments have been deposited a t the Archives Originales du Centre de Documentation du C.N.R.S. (15 Quai Anatole-France, F-75 Paris 7 , France) where they may be ordered as microfiche or photocopies. Reference No. :

Annex, Table 1. Amino acid composition of tryptic

Annex, Table 2. Edman degradation of peptide OT1. Annex, Table 3. Edman degradation of peptide OT2. Snnex, Table 4. Edman degradation of peptide OT3. Annex, Table 5. Edman degradation of peptide OT5. Annex, Table 6. Edman degradation of peptide OT7. Annex, Table 7. Edman degradation of peptide OC1. Annex, Table 8. Edman degradation of peptide OC2. Annex, Table 9. Edman degradation of peptide OC3. Annex, Table 10. Edman degradation of peptide OC4. Annex, Fig. 1. Chromatography of a tryptic hydrolysate

of 150 mg oxidized viscotoxin B on Dowex 50W-X2. Gradient elution with pyridine-acetic acid buffers of increasing ionic strength and pH. The total volume of the gradient was 10500 ml. The figure shows the chromatogram only for the first 4000ml of eluate. No more peaks were obtained a t higher buffer concentrations or with 2 N NaOH.

Annex, Fig. 2. Chromatography of a chymotryptic hydrolysate of 300 mg oxidized viscotoxin B on Dowex 50W-X2. Conditions as in Annex, Fig. 1. The chromatogram is shown only for the first 3500 ml of eluate. No more peaks were obtained at higher buffer concentrations.

A.O. - 496.

peptides from oxidized viscotoxin B.

1.

2. 3. 4.

5.

6.

7.

8. 9.

10. 11.

12. 13.

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Hirs, C. H. W., J . Bid . Chem. 219 (1956) 611. Schroeder, W. A., Jones, R. T., Cormick, J., and McCal-

Ritschard, W. J., J . Chromatog. 16 (1964) 327. Spackman, I). H., Stein, W. H., and Moore, S., Anal.

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la, K., Anal. Chem. 34 (1962) 1570.

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(1967) 264.

G. SamueIsson and B. Pettersson Institutionen for Farmakognosi, Farmaceutiska Fakulteten Lindhagensgatan 128, S-112 51 Stockholm, Sweden

the amino acid sequence of viscotoxin b from the european mistletoe (viscum album l, loranthaceae)

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