and the Complete Amino-Acid Sequence of Soybean Trypsin Inhibitor (Kunitz) ... trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the ...
Eur. J. Biochem. 32, 417-431 (1973)
Studies on Soybean Trypsin Inhibitors 3. Amino-Acid Sequence of the Carboxyl-Terminal Region and the Complete Amino-Acid Sequence of Soybean Trypsin Inhibitor (Kunitz) Takehiko KOIDEand Tokuji IKENAKA Department of Biochemistry, Niigata University School of Xedioine (Received July 5/September 21, 1972)
For the elucidation of the amino-acid sequence of the carboxyl-terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50x2 or by gel filtration on BioGel P-4. Further fractionation and purification of the peptides were performed by ion-exchange chromatography on Dowex 1x2, by gel filtration on Bio-Gel P-2 or by high-voltage paper electrophoresis a t pH 1.9 and 3.6. Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino-acid sequences were determined by the direct Edman method and by carboxypeptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D. Nine main peptides and nine minor peptides were obtained. The amino acid composition and the partial amino-acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino-acid sequence of the carboxyl-terminal region of the inhibitor. The complete amino-acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman-Birk inhibitor, another well-known soybean protejnase inhibitor.
In the preceding papers [1,2] the fragmentation of soybean trypsin inhibitor (Kunitz) into four peptide fragments (A, B. C and D) and the amino-acid sequence of fragments A and B, the amino-terminal half region of the inhibitor, have been reported. I n this paper we shall describe the isolation and sequence determination of the tryptic, the chymotryptic and the thermolysin peptides of fragments C and D, and the complete amino-acid sequence of soybean trypsin inhibitor (Kunitz) reconstructed from this information. Circular dichroism and optical rotatory dispersion studies of the inhibitor [3,4]have predicted that this protein should have not helical structure, which is one of the most interesting structural features of the inhibitor. The discussion will be concerned with helix in the protein as re%ected by the amino-acid -
Abbreviations. Cm, carboxymethyl ; Hse, homoserine. Enzymes. Carboxypeptidase A (EC 3.4.2.1): carboxypeptidase B (EC 3.4.2.2); trypsin (EC 3.4.4.4); chymotrypsin (EC 3.4.4.6); thermolysin (EC 3.4.4.-). A preliminary account of this work has appeared in J. Biochem. (Tokyo) 71, 165 (1972). 28 Eur.J. Bioehem., Vo1.32
sequence and also with the occurrence of repetitive sequences. MATERIALS AND METHODS
Only those materials and methods not described in the preceding papers [1,2] are documented in the present communication. Fragments C and D were prepared as described in the preceding paper [l]. Trypsin treated with ~-(l-tosylamino-2-phenyl)ethyl chloromethyl ketone was used for the enzymatic digestion of fragments C and D. Thermolysin was a kind gift of Drs. M. Ebata and K. Morihara (Shionogi Pharm. Co., Ltd, Osaka).
Digestion with Trpsin Fragment C was digested with treated trypsin for 6 h at an enzyme/substrate ratio of 1 O l 0 (wlw). Fragment D was digested with treated trypsin for
6 h a t a n enzyme/substrate ratio of 2 O / , (wlw).
418
The Complete Amino-Acid Sequence of Soybean Trypsin Inhibitor (Kunitz)
Digestion with Chymotrypsin C-Ti peptide was digested for 30min a t a n enzyme/substrate ratio of 1,/ (wlw). Fragment D was digested for 2 h a t a n enzyme/ substrate ratio of l o l o (wlw).
Digestion with Thermolysin Sample peptide D-Cl2 was dissolved in water with a concentration of 1pmol/ml and the pH of the solution was adjusted to 8.0 with 1 M ammonium acetate buffer pH 8.7. Thermolysin was dissolved to a O.Oio/, solution in 0.8 M ammonium acetate buffer pH 8.0. The peptide was digested for 3 h a t an enzyme/substrate ratio of O.lo/, (wlw) a t 40 “C. The digestion was terminated by the drop-wise addition of glacial acetic acid, and the digest was lyophilized.
High-voltage paper electrophoresis was also performed in formic acid-acetic acid- water buffer, (25:87:888, v/v/v) p H 1.9.
H ydrazinol y sis The procedure described by Fraenkel-Conrat and Tsung [9] was used.
Nomenclature and Symbob The letters TH and PA refer to peptides obtained by the digestion with thermolysin and by partial acid hydrolysis, respectively. The head letters C and D, respectively, refer to peptides derived from fragments C and D. Others are the same as those described previously [2]. RESULTS
Partial Acid Hydrolysis Partial acid hydrolysis was performed as described by Light [ 5 ] .The peptide (2-3 pmol) was hydrolyzed with 3 ml of 0.03 N hydrochloric acid at 105 “C for 16 h in a n evacuated sealed tube.
Fractionation of the Peptides The conditions for the column chromatographic separation of the tryptic peptides of fragment D on Dowex 50x2 were identical t o those described in the preceding paper [2] for the separation of the tryptic peptides of fragment A. The separation of the peptides of peak V in Fig. 3 was accomplished by chromatography on a column (0.6 x 60 cm) of Dowex 1 x 2 operated as described by Schroeder [6]. The peptides were monitored by ninhydrin analysis subsequent to alkaline hydrolysis c71. Gel filtrations of the tryptic peptides of fragment C, the chymotryptic peptides of fragment D and the thermolysin peptides of D-C12 peptide were carried out a t a flow rate of 3 m l / h using a Bio-Gel P-4 column (1.5 x 200 om) equilibrated with 0.2 N acetic acid. The eluate was collected in 2-ml fractions. The tryptic peptides of fragment C were detected by measurement of the absorbance a t 280 nm and by the ninhydrin reaction after alkaline hydrolysis. The chymotryptic peptides of fragment D were detected by measurement of the absorbance a t 226 nm [S] and 280nm. The thermolysin peptides of D-C12 peptide were detected by measurement of the absorbance a t 226 nm. Separation of the peptides obtained by further chymotryptic hydrolysis of C-Ti peptide was performed by chromatography on a Bio-Gel P-2 column (1.5x 140 cm). The operations were identical to those described for gel filtration on Bio-Gel P-4.
Eur. J. Biochem.
Tryptic Peptides of Fragment C Fig.1 shows the elution pattern of the tryptic peptides of fragment C fractionated on Bio-Gel P-4. Four peak fractions were pooled as indicated by bars. The purity of each peak was examined by highvoltage paper electrophoresis a t p H 1.9. Peaks 11, I11 and IV, respectively, consisted of almost pure peptides C-Ti, C-T2 and C-T3. P e a k 1 was the parent peptide (fragment C) not digested. The amino acid compositions of the three tryptic peptides are listed in Table 1.
1.4
C-TI
1.2
1
E 1.0 C
0
% m
0.6
E
C-T3
.0 r
0.8
0.4 m
ar
ar
5 0.6
c
e
-P
:: 0.4
0.2
:: 9
0.2
0 100
200
E l u t i o n volume ( m l )
300
0
Fig. 1. Chromatographic elution pattern of the tryptic peptides of fragment C from Bio-Gel P-4. The column ( 1 . 5 ~ 2 0 cm) 0 was equilibrated with 0.2 N acetic acid and operated at a flow rate of 3 ml/h a t room temperature. Effluent was collected in 2-ml fractions. Peptides were detected by measurement of the absorbance of the fractions at 280nm (0-0) and also by ninhydrin reaction after alkaline absorbance at 570 nm) hydrolysis (0-0,
T. KOIDEand T. WENAKA
Vol.32, No.3,1973
Chymotryptic Peptides of Peptide G-TI Fig.2 shows the elution pattern of the chymotryptic digest of peptide C-T1 fractionated on Bio-Gel P-2. Two peak fractions indicated by bars were pooled. PeakI, monitored by only the ninhydrin reaction, contained peptide C-Tl-C2 and peak 11, monitored by both the ninhydrin reaction and measurement of the absorbance a t 280 nm, consisted of peptide C-Ti-Ci. Both peptides were obtained in
Peptides C-T2 and C-T3 were used in sequence determination without further purification. Peptide C-Ti was further digested by chymotrypsin preceding sequence studies.
Table 1. Amino-acid compositions of the tryptic peptides from fragment C The results are expressed as molar ratios with respect to the amino acid denoted with an asterisk in each column and were obtained by analyses of 24-h hydrolysates a t 105 "C without corrections. Values in parentheses are the numbers of residues obtained by sequence analyses Amino acid
C-Ti
C-T2
C-T3
1.07 (1)
1.13 (1)
Cm-cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tryptophan Lysine Homoserine
0.81 (1) 1.07 (1) 0.86 (1) 1.06 (1) 3.00* (3) 2.94 (3) 2.03 (2) 1.08 (1) 3.04 (4). 0.00 0.91 (1) 1.84 (2) 0.51 (1) 0.87 (1)
Total residues
22 (85- 106)
(107- 111)
(112-114)
47
74
37
419
C-T1 -C1 0
r ln '
& 0.4
LOO* (1) 0.96 (1)
LOO* (1) 0.00 0.93 (1)
0 96 (1)
0.00
0.91 (1) -60
Yield (Ole)
5
80
100
120
140
160
Elution volume ( m i )
3
Fig.2. Chromatogvaphy of the peptides obtained by further chymotryptic hydrolysis of peptide C-TI (cf. Fig. I ) . The Bio-Gel P-2 column (1.5 x 140 cm) was operated in the manAbsorbance a t 280 nm; ner indicated under Fig.1. (0-0) (-0) absorbance at 570 nm
Sequence analyses reveal that the pept,ide contains Val-Val, giving a low recovery by incomplete hydrolysis of the bond [21.
Table 2. Amino-acid compositions of the chyrnotryptic peptides from C-TIpeptide and the peptides obtained by partial acid hydrolysis of C - T I 4 2 peptide The results are expressed as molar ratios with respect to the amino acid denoted with an asterisk in each column and were obtained by analyses of 24-h hydrolysates at 105 OC without corrections. Values in parentheses are the numbers of residues obtained by sequence analyses Amino acid
Cm-cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glvcine Ahne Valine Isoleucine Leucine Tryptophan Lysine Total residues Yield ('J/J 8
b
28.
C-Ti-Ci
C-Ti42
C-TI-C2-PAi
C-TI-CZ-PA2
C-Tl-CZ-PA3
0.63 (1) 0.92 (1) 0.94 (1) 1.05 (1) 1.04 (1) 1.14 (1)
.,
0.94 (1) 1.02 (ij l.OO'(1) -I- (1). 9 (85-93) 38
0.98 1.92 1.87 1.09 1.01 2.09
(1) (2) (2)
0.76 (1) LOO* (1)
(1)
(ij (3)b ..
0.94 (2)b ,.
1.20 1.93 1.01 0.98 1.30
(1) (2) 11)
(ij (1) ._
1.07 0.92 LOO* 0.95
(1) (1)
(ij (1)
.,
1.00* (1)
1.00* (1)
0.72 (1)
0.87 (1)
0.94 (1)
8 (99-106)
5 (102-106)
13 (94- 106)
4 (94-97)
72
Ehrlich reaction was positive and absorption curve around 280 nm was typical of tryptophan. Sequence analyses reveal that the peptide contains Val-Val, giving a low recovery by incomplete hydrolysis of the bond [2].
The Complete Amino-Acid Sequence of Soybean T r y p i n Inhibitor (Kunitz)
420
pure form and used for sequence studies without further purification. The amino acid compositions of the two chymotryptic peptides are shown in Table 2. Peptide C-Tl-C2 was partially hydrolyzed with dilute hydrochloric acid as mentioned in Methods. Two main peptides C-Ti-C2-PAi and C-Tl-C2-PA2 and one minor peptide C-Tl-C2-PA3 were isolated by paper electrophoresis at pH 3.6. The amino acid compositions of these peptides are listed in Table 2.
Tryptic Peptides of Fragment D Fig.3 shows the elution pattern of the tryptic peptides of fragment D fractionated on Dowex 50x2. Eleven peak fractions were pooled as indicated by bars. The purity of each peak was examined by paper electrophoresis at pH 3.6. Peaks I, 11, VI, VII, VIII and IX, respectively, contained almost pure peptides D-T10, D-T4, D-T6, D-T5, D-T2 and D-T7. Peak I11 was produced by the overlap of peaks I1 and IV, consisting of peptides D-T4 and D-T8. PeakIV was purified by paper electrophoresis a t pH 3.6 isolating peptide D-T8. Peak V contained peptides D-T3 and D-T9, which were separated by a column chromatography on Dowex 1 x 2 as shown in Fig.4. The first peak contained peptide D-T9 and the second peak peptide D-T3. It was observed that peptide D-Ti was eluted as two peaks, X and XI. Insoluble material (D-T insoluble) was found to be pure peptide D-T4 by amino acid analysis. Table 3 lists the amino acid compositions of the ten tryptic peptides isolated in pure form.
r
0
I
I
200
I
I
I
I
I
I
400 Elution volume ( m l )
I
I
I
I
600
I
I
800
Fig. 3. Chromatographic elution pattern of the tryptic peptides of fragment D from Dowex 50x2. The digest was applied to a column (1.2 x 120 em) equilibrated with 0.2 M pyridineacetic acid buffer pW 3.0. The elution was initiated with this buffer a t 38 "C, followed by a gradient set up with 333 ml of 0.2 M buffer p H 3.0 placed in the mixer and 666 ml of 5 M buffer pH5.0 in the reservoir. The flow rate was 16 ml/h and 2-ml fractions were collected. The fractionation was monitored by application of the ninhydrin reaction t o 0.2 ml of samples from each fraction previously subjected to alkaiine hydrolysis. (0-0) Absorbance at 570 nm; (0-0) pH of the eluate
Eur. J. Biochem.
T. KOIDEand T. I~ENAEA
Vol. 82, No.3,1973
421
10
2N acetic acid
g 0.8
-9
D-T3
-/
D-C12-THl
0.3
8
-7 6
s -
5 % 4 3
(D
z0.2 m
2 I
80
-
a, 0
c m
1 I
I
I
n
10
I
100 120 140 160 Elution volume ( m l )
L
*
0
180
n
