J. Biochem. 82, 1523-1531 (1977)
XI.
Complete Amino Acid Sequence of a Soybean Trypsin-Chymotrypsin-Elastase Inhibitor, C-II 1 Shoji ODANI and Tokuji IKENAKA Department of Biochemistry, Niigata University School of Medicine, Niigata, Niigata 951 Received for publication, May 16, 1977
Soybean inhibitor C-II, which inhibits trypsin, a-chymotrypsin, and elastase, was reduced and S-carboxymethylated, and digested with trypsin. The amino acid sequences of the resulting tryptic peptides were determined by conventional methods, establishing the complete 76-amino acid sequence of the inhibitor. Inhibitor C-II was found to be homologous with soybean (Glycine max) Bowman-Birk inhibitor and more closely related to an inhibitor from garden beans (Phaseolus vulgaris). The homology with these inhibitors and the limited proteolysis of C-II indicated the reactive sites of C-II for elastase and trypsin to be alanine-22 and arginine-49, respectively. Arginine-49 was also identified as a reactive site for a-chymotrypsin. It was found that only a few replacements of one or two amino acid residues around the reactive sites resulted in considerable alteration of the inhibitory specificity.
In the preceding paper (2) we have reported the isolation and partial characterization of four soybean double-headed proteinase inhibitors. One of them, inhibitor C-II, showed a unique inhibitory specificity. It bound one mole each of trypsin, a-chymotrypsin, and elastase. The reactive sites for trypsin and a-chymotrypsin appeared to be identical, since the trypsin-C-II complex no longer inhibited a-chymotrypsin. When a-chymotrypsin1
This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan. A preliminary account of this work has appeared (/). Abbreviations: PTH, phenylthiohydantoin; Cm, carboxymethyl; RCm, reduced and carboxymethylated; TPCK, l-tosylamidc-2-phenylethyl-chloromethyl ketone; TLCK, l-chIoro-3-tosylamido-7-amino-2-heptanone. Vol. 82, No. 6, 1977
C-ll complex was incubated with trypsin, chymotrypsin activity gradually appeared with a concomitant loss of trypsin activity. The amino acid sequences of several legume double-headed inhibitors with various inhibitory activities have been reported (3-6). It seems likely, therefore, that a comparative study of the primary structures of these inhibitors and soybean C-II may provide some explanation for the unique specificity and properties of C-II, as well as information about the structure-function relationships of the inhibitors. The present paper describes the complete amino acid sequence of inhibitor C-II, established by sequence analyses of the tryptic peptides and cyanogen bromide fragments of C-II.
1523
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Studies on Soybean Trypsin Inhibitors
1524
S. ODANI and T. IKENAKA
MATERIALS AND METHODS
Amino Acid Sequence Analysis—Stepwise degradation of the peptides was carried out by Edman's procedure (7). Sequence study was carried out according to the method of Edman and Begg (8) using a JEOL JS-47K sequence analyzer (0.5 M Quadrol buffer, single cleavage, Quadrol program). The semi-quantitative determination of PTHderivatives was done by measuring the absorbance at 269 nm before chromatography. PTH-derivatives were usually identified by thin-layer chromatography on Kieselgel F-254 sheets (9, 10). Gasliquid chromatography was carried out to confirm the identity of PTH-derivatives of alanine, glycine, proline, isoleucine (or leucine), methionine, and phenylalanine on a column (0.3x300 cm) packed with 10% SE-30 on chromosorb W (60-80 mesh) using a Shimadzu CG-4BM gas chromatograph according to the method of Pisano and Bronzert (//). For the identification of leucine and isoleucine, the PTH-derivatives were regenerated to amino acids by hydrolysis in 5.7 N HC1 containing 0.4% SnCl, (72). PTH-Cm-cysteine was resolved from PTH-aspartic acid on micropolyamide thin-layer sheets (75). Hydrazinolysis for determination of the carboxyl-terminal amino acid was carried out by the method of Fraenkel-Conrat and Tsung (14). Reduction and S-Carboxymethylation—This was done by a modification (75) of the procedure of Crestifield et al. (75). Tryptic Digestion of RCm-C-II—Thirty mg of RCm-C-II was digested with 0.6 mg of TPCK-
Cyanogen Bromide Cleavage and Separation of Peptides—The protein (5 mg, 0.62 ftmo\) was treated with a 200-fold molar excess of cyanogen bromide over methionine residues in 70% formic acid for 24 h at room temperature. After desalting by passage through a Sephadex G-15 column (1.5x20cm, 0.1 M formic acid), the protein was oxidized with performic acid by the method of Hirs (17) and then lyophilized. The oxidized material was placed on a DEAE-Sephadex A-25 column (0.9x55 cm) equilibrated with 0.02 M NH,HCO S and eluted with a linear gradient of the buffer to 1.0 M. Elution was monitored by measuring absorbance at 230 nm using a Hitachi double-beam effluent monitor. Limited Proteolysis of C-II by Trypsin—To identify the reactive site, the limited proteolysis technique of Ozawa and Laskowski (18) was employed. The inhibitor (3.4 mg) was incubated with 60 fig of TPCK-trypsin for 20 h at pH 3.5 and 20°C. The pH of the reaction mixture was brought to 7.8 with 2 N NaOH, and 27 /ig of carboxypeptidase B and 36 fig of carboxypeptidase A were added. After digestion for 30 h at 25°C, an aliquot was withdrawn and analyzed for liberated amino acids and inhibitory activities for trypsin, a-chymotrypsin, and elastase.
/ . Biochem.
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Materials—The inhibitors and most of the enzymes used in this study were the same preparations as in the previous paper (2). TPCKtreated trypsin and TLCK-treated chymotrypsin were products of Worthington Biochem. Corp. and Merck, respectively. Carboxypeptidases A and B treated with di-wo-propyl phosphorofluoridate were purchased from Worthington Biochem. Corp. Thin-layer precoated sheets, DCFertigplatten Kieselgel F-254, were a product of Merck. Reagents and solvents for Edman degradation were extensively purified from reagent grade preparations. Reagents for automated Edman degradation were purchased from Wako Pure Chemicals.
trypsin in 10 ml of 0.05 M Tris-HCl buffer, pH 8.0, for 4 h at 25°C and then lyophilized. The digest was dissolved in 2 ml of 0.05 M ammonium formate, pH9.1, and applied to a Bio-Gel P-4 column (1.5x200 cm) equilibrated with the same solution. Chymotryptic Digestion of Peptide T-l—A large tryptic peptide, fraction T-l (0.6 //mole as leucine residue), which contained a set of aminoterminal peptides, was further digested with 0.038 mg of TLCK-chymotrypsin (molar ratio, E : S = 1:400) in 2.2 ml of 0 . 0 1 M Tris-HCl buffer, pH 8.0, for 4 h at 40cC. The digestion was stopped by lowering the pH of the reaction mixture with acetic acid and the mixture was applied to a column of SP-Sephadex C-25 (0.6x60 cm) equilibrated with 0.02 M ammonium acetate, pH 4.0. The column was first developed with 90 ml of the same buffer and then with an exponential gradient produced by introducing a limiting buffer of 0.05 M ammonium acetate, pH 9.25, into a 40 ml mixing chamber filled with the equilibrating buffer. Peptides were detected by measuring the absorbance at 230 nm.
AMINO ACID SEQUENCE OF SOYBEAN INHIBITOR C-II
1525
E
RESULTS
T-1
75
175 125 Elution Volume (ml) Fig. 1. Separation of the tryptic peptides of RCm-C-II on Bio-Gel P-4. 30 mg of the digest was applied to a column (1.5x200 cm) equilibrated with 0.05 M ammonium formate, pH 9.1, and eluted with the same solution. Peptides were located by means of their absorbance at 230 nm.
TABLE I. Amino acid compositions of tryptic peptides from reduced and S-carboxymethylated C-II. Numbers in parentheses are those determined after sequence analysis. Amino acid Cm-Cysteine Aspartic acid Threoninc Serine Glutamic acid Proline Glycine Alanine Methionine Isoleucine Leucine
T-2A
4.00 (4) 1.66 (2) 1.05 (1)
T-2B 0.93 (1) 2.24 (2) 1.82 (2) 0.35
Vol. 82, No. 6, 1977
1.06 (1) 1.11 (1) 1.78 (2)
1.96 (2) 0.27 0.84 (1)
T-5
T-6
C-II (2)
2.06 (2)
1.02 (1)
14 13 4 12 4 5 1 4 3 1 3 1 1 3 3
1.69 (2) 0.12
0.98 (1)
1.08 (1)
0.65 1.00 1.07 1.01
(1) (1) (1) (1)
1.10 (1) 0.90 (1)
0.95 (1)
1.C0 (1)
0.93 (1) 0.92 (1) 2.00 (2) 0.96
Arginme
Yield (%)
T-3
1.81 (2) 1.91 (2) 1.20 (1) 2.00 (2)
1.18 (1) 0.94 (1) 0.94 (1)
Tyrosine Phenylalanine Lysine Histidine
Total residues
T-2C
m
0.96
m
1. 00 (1)
1. 00 (1)
1. 00 (1)
0.90 (1)
4 76
7
7
12
10
5
6
7
59
32
9
27
65
20
47
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Tryptic Peptides of RCm-C-H—Six peptide fractions, T-l to T-6, were obtained by gel-filtration of the tryptic digest of RCm-C-II on Bio-Gel P-4 (Fig. 1). Each fraction was evaporated to dryness and dissolved in 50 (i\ of pyridine-acetic acid buffer, pH 6.5 (19), then subjected to high-voltage paper electrophoresis for 1.5 h at pH 6.5 with a potential gradient of 40 volts per cm. T-2C was further purified on a SP-Sephadex C-25 column (0.9 x 60 cm) equilibrated with 0.02 M ammonium acetate, pH 4.0. An exponential gradient produced by introducing 0.05 M ammonium acetate, pH 8.6, into a mixing chamber (25 ml) filled with the equilibrating buffer was used to develop the column (data not shown). Seven peptides, T-2A, T-2B, T-2C, T-3, T-4, T-5, and T-6, were obtained in a pure state. Their amino acid compositions are shown in Table I. The overall yields of these peptides were between
c o m
1526
S. ODANI and T. IKENAKA
E
c o N (0
0.6
40 Fraotion Number Fig. 2
80
TABLE II. Amino acid compositions of T-1 and its chymotryptic peptides. Numbers in parentheses are those determined after sequence analysis. Chymotryptic peptides of T-1 Amino acid 4.47 4.85 1.00 5.96 1.68 1.98
T-1A
T-1B
T-1C
T-1D
T-1E
1.81 (2) 3.57 (4)
0.82 (1)
0.87 (1)
1.42 (1)
1.02 (1) 1.54 (1)
Cm-Cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Methionine Isoleucine Leucine
1.35 1.08 0.40 1.00
0.36
Tyrosine Phenylalanine Lysine Histidine
1.11 1.71
0.85 (1) 0.85 (1)
Arginine
0.39
Total Yield (%)
88
1.00 (1) 5.52 (6) 1.39 (1) 1.48 (1)
1.10 (1) 1.20 (1) 2.15 (2) 1.12 (1) 1.00 (1)
0.27 0.83 (1)
0.20 0.21 1.08 (1) 1.00 (1)
1.00 (1)
0.24 1.15 (1) 1.00 (1)
17
2
3
7
5
24
20
20
24
28
~t Molar ratio with respect to leucine taken as 1.00. J. Biochem.
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65 and 20% of the starting material, except for T-2C, the low yield (9%) of which may be attributed to the repeated chromatography and partial hydrolysis at the alanyl (22)-serine bond by trypsin (Fig. 4). A preliminary experiment showed that T-1 consisted of two or three peptides derived from the amino-terminal region of the protein, which could not be separated from each other. ThereFig. 2. Purification of chymotryptic peptides of T-1. SP-Scphadex C-25 equilibrated with 0.02 M ammonium acetate, pH 4.0, was developed first with 80 ml of the same buffer, then with an exponential gradient formed by introducing 0.05 M ammonium acetate, pH 9.25, into a 40 ml mixing chamber filled with the equilibrating buffer. Elution was monitored in terms of the absorbance at 230 nm. Fractions of 2.0 ml were collected. Drift of the base line at 230 nm is conpensated in the figure.
AMINO ACID SEQUENCE OF SOYBEAN INHIBITOR C-II
chymotryptic peptides are listed in Table II. The sum of the compostitions of T-1D and T-1E is identical with that of T-2C. Therefore, T-1D and T-1E appear to be derived from the T-2C region of the inhibitor molecule. The sum of the residues of the remaining three chymotryptic peptides of T-1 (T-1A, T-1B, and T-1Q and the tryptic peptides listed in Table I accounts for the total amino acid residues of the parent protein, Amino Acid Sequences of the Isolated Peptides—First, reduced and S-carboxymethylated inhibitor was subjected to manual Edman degradation and the first 21 residues were determined, There may be some heterogeneity at the aminoterminus, because a faint Pauli-positive reaction was observed in the aqueous phase of the first step of the Edman degradation.
TABLE III. Summary of sequence studies of C-II and its tryptic peptides.1 — ^ , Edman degradation; —*>, identification by amino acid analysis; -. _ ^ .
_>.
T-1A
Ser-Asp-His-Scr-Ser-Ser-Asp-Asp
T-1B
Cys-Met
T-1C
Cys.Thr.Ala
T-1D
Ser-Met,Pro,Pro,Glx,Cvs,His
T-1E
Cys, Ala, Asp, lie, Arg
T-2A
Ser-Ser-Asp-GIu-Asp-Asp-Asp
T-2B
Cys-Leu-Asp-Thr-Thr-Asp-Phe
T-2C
Ser-Met-Pro-Pro-Gln-Cvs-His-Cys-Ala-Asp-Ile-Arg
T-3
Leu-Asn-Ser-Cys-His-Ser-Ala-Cvs-Asp-Arg
T-4
Cvs-Ala-Cys-Thr-Arg
T-5
Cys-Tyr-Lys-Pro (Cys, Lys)
T-6
Ser-Met-P^-dty-Gin-Cys-Arg
» All the Cys residues were identified as Cys(Cm). Vol. 82, No. 6, 1977
.*_
Asp ^ _
Leu
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fore, the whole T-1 fraction was digested with chymotrypsin and the peptides were separated by SP-Scphadex C-25 chromatography (Fig. 2). The first peak was still heterogeneous and was applied to a column of Bio-Gel P-4 (1.5 x 200 cm, 0.05 M ammonium formate, pH9.1). Two peptide fractions were obtained (the elution pattern is not shown). One fraction, eluted at the breakthrough point, was a homogeneous peptide, T-1A, and the second still consisted of two peptides, T-1B and T-1C, which were separated by paper electrophoresis at pH 6.5. Peptides T-1D and T-1E were used for sequence analysis without further purification. T-l A', which was eluted just behind T-1 A fraction, had the same amino acid composition as T-1A. The amino acid compositions of T-1 and its
1527
S. ODANI and T. IKENAKA
1528
m.
be derived from the amino-terminal region of C-II, could not be fully purified, and structural investigation was not possible. The difference of amino acid compositions between the parent TABLE IV. Amino acid compositions of fragments obtained by cyanogen bromide cleavage of C-II and a chymotryptic peptide of CB-IV. One of the fragment, CB-I, could not be fully purified and is omitted from the table. Numbers in parentheses are those determined after sequence analysis. Amino acid
CB-II
CB-III
CB-IV
CB-IV-C3
Cysteic acid
1.17(1)
6.47 (6)
4.15(4)
1.37(1) 4.00(4)
Aspartic acid 0.42
3.40(3)
6.00(6)
Threonine
0.87(1)
1.12(1)
1.95(2)
Serinc
1.02(1)
3.24(3)
2.38(2)
1.73(2)
Glutamic acid
1.25(1)
2.29(2)
1.21(1)
Proline
1.97(2)
1.89(2)
1.09(1)
Glycine
Cyanogen Bromide Fragments—Figure 3 shows the elution pattern on a DEAE-Sephadex A-25 Alanine column of C-II treated with cyanogen bromide and Isoleucine then oxidized with performic acid. The first Leucine three peaks were not peptides, but were probably Tyrosine salts. Each fraction, except for CB-II, which was Phenylalanine essentially pure, was further purified on a Bio-Gel P-10 column (1.5x150 cm, 0 . 1 M formic acid). Lysine However, CB-I could not be freed from a con- Histidine taminant which seemed to be partially cleaved Arginine inhibitor. Amino acid compositions of the puri- Homoserine fied CNBr-fragments are listed in Table IV. Order of the Cyanogen Bromide Fragments— Total One of the CNBr fragments (CB-I), which should
CBIL-CBfll 40
80 Tu be Number
1.55(1) 1.00(1)
3.00(3) 0.97(1) 1.25(1)
1.24(1) 0.90(1) 0.91(1) 2.00(2)
2.11(2)
0.15
2.92(3)
1.07(1)
0.70(1)
1.01(1)
5
27
25
2.09(2)
11
"CBICB1\7 1 20
1 60
Fig. 3. Separation of CNBr-fragments on DEAE-Sephadex A-25. A column (0.9 X 55 cm) equilibrated with 0.02 M N H 4 H C 0 , was eluted with a linear gradient of NH 4 HCO, concentration to 1.0 M. Peptides were located by means of their absorbance at 230 run.
J. Biochem.
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The carboxyl-terminal residue of the protein was identified as aspartic acid by the hydrazinolysis method. The yield of aspartic acid was 20 mol % of the protein used. The sequence of the first eight amino acids of T-1A is identical with that of the original protein, and T-1B and T-1C seem to follow T-1A. The sequence of T-2C indicates that T-1D and T-1E were generated by chymotryptic cleavage of the His-Cys(Cm) bond in T-2C. T-5 was readily extracted into the organic phase during Edman degradation, so that the last two residues (Cm-cysteine and lysine) could not be identified. Since this peptide was produced by tryptic digestion, lysine should be placed at the carboxyl terminus. The amino acid sequences of the other tryptic peptides were unambiguously determined by the manual Edman degradation method. These results are summarized in Table
AMINO ACID SEQUENCE OF SOYBEAN INHIBITOR C-II
1529
1
10
20
Ser-Asp-His-Ser-Ser-Ser-Asp-Asp-Glu-Ser-Ser-Lys-Pro-Cya-Cys-Asp-Leu-Cys-Met-Cys•» y
T-1A
> 4-T-1B-* *— 1 i—
CB-I
io »o Thr-Ala-Ser-Met-Pro-Pro-Gln-Cys-His-Cys-Ala-Asp-Ile-Arg-Leu-Asn-Ser-Cys-His-Ser•T-1D • •T-3-T-1C-T-1ET-2C -CB-II-
-CB-IIISO
6 0
Ala-Cys-Asp-Arg-Cys-Ala-Cys-Thr-Arg-Ser-Met-Pro-Gly-Gln-Cys-Arg-Cys-Leu-Asp-Thr-
• T-4
T-6
-T-2B —
Hh
•CB-III-
-CB-IV-
Thr-Aap-Phe-CyB-Tyr-Lys-Pro-Cys-Lys-Ser-Ser-Asp-Glu-Asp-Asp-Asp. *• * T-5 *• •* T-2A > CB-IV•CB-IV-C-3-
Fig. 4. Alignment of the tryptic peptides of RCm-C-II by analysis of the CNBr-fragments. tion. One of the CNBr-fragments, CB-I, could not be isolated and is shown as a dashed line.
•, Edman degrada-
V.
Aap-Asp-Glu-Ser-Ser-Lya-Pro-Cya-Cys-Aap-fGln-jCysj
I
io
,
,
Ser-Aap-Hia-Ser-Ser-SerfAsp-Aap-Glu-Ser-Ser-Lya-Pro-Cya-CyB-ABp-jLeu-jCysl ~t I I 1 | io A8x,Hi8,A8x,Glx,HiB,Ser,Ser,Asx,Glx-Pro-Ser-}Glx-Ser-Ser T Pro-j-Pro-Cy8-CyB-ABX-Ile-{CyaAla-fCyB-Thr^-LYS-f 2
I °
L 12J
j-Met-rArg-Leu-Aan-Sor-Cya-His-
. Asn-jt
.
-
.
.
IleTArg-Leu-Asn-Ser-Cya-Hia-
I
MetyCyB-Thr-AIA-Serj-Met-jPro-Pro-Gln-CyBj-HiB-jCyi^Ala19
I
10
~~
BBI C-II GBI BBI C-II GBI
Val-j-Cya-Thr-ALA-Sarf Ile-jPro-Pro-Gln-Cya^ H e , Cya, Thr, Aax, Val) Arg-Leu-Asx-Ser-Cys-HisSer-Ala-Cys-Ly8-Sar-CyaTlle-'CyBjAla-LEU-!serYTyrJProj-Ala-Gln-CyaTPhe-7CysrVal|AspTlle-. to I n - l sol——i ' 1 ' ' ' ' lo-* Ser-Ala-CyafABp-ArgjCyBjAlaJCy8-Thr-ARG-Ser-Het-Pro-Gly-Gln-CyB-Arg-CyB-Leu-ABp-Thr-
C-II
Ser-Ala-CyB-LyB-Ser-CyajHet-Cys-Thr-ARG-Ser-Met-Pro-Gly-Lyaj-Cys-Arg-Cya-Leu-Aax-Thr-
GBI
Thr-Asp-Phe-Cys-TyrrGlu-Pro-CyB-LyB-Pro-SerjGlu-ABp-Asp-Lys-Glu-Asn.
BBI
Thr-Aap-Phe-Cys-Tyr-Lys-Pro-Cya-Lys-Ser-SeryAsp-Glu-Asp-Asp-Asp!
C-II
I
1
r^l
I
'
1
" |
Thr-AsxyTyr-Cys-Tyr-LyajSer-Cya-Lys-Ser-Aax-Ser-Gly-Glx-Aax-Asx.
BBI
GBI
Fig. 5. Comparison of the amino acid sequences of three legume inhibitors. BBI, soybean Bowman-Birk inhibitor (i); C-II, soybean inhibitor C-II (this work); GBI, garden bean inhibitor II (6). The reactive site residues are capitalized. Boxes show the homologous regions. Asx and Glx in GBI were assumed to be Asp and Glu, respectively. Vol. 82, No. 6, 1977
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protein and the sum of the three isolated CNBr of this fragment and also the order of the first fragments is close to the composition of the 19 two fragments as (CB-I>(CB-II). Since only amino-terminal amino acids, the sequence of which CB-IV contains no homoserine residue, this fragwas determined by Edman degradation of the ment should be placed in the carboxyl-terminal parent inhibitor (Table III). part of the molecule, and therefore, the order of Amino-terminal sequence analysis of the in- the four CNBr-fragments was assumed to be hibitor up to the 21st residue indicated the structure (CB-I)-(CB-II)-(CB-III)-(CB-IV).
1530
S. ODAN1 and T. IKENAKA
DISCUSSION There was some difficulty in sequencing the aminoterminal region of the protein because of partial
tryptic hydrolysis of the alanyl (22)-serine bond. As will be discussed later, this alanyl-serine bond seems to form the elastase reactive site of the inhibitor, and may be very susceptible to proteolytic digestion. A similar result was obtained in sequence analysis of a reduced and S-carboxymethylated Streptomyces subtilisin inhibitor, in which the subtilisin-reactive site methionyl (73)valine bond was extensively hydrolyzed by trypsin (21). Tan and Stevens (22) have also reported the chymotryptic cleavage of a lysyl-serine bond of lima bean inhibitor IV, which is the trypsin reactive site of this inhibitor. Therefore, the reactive site region of proteinase inhibitors appears to have some particular conformation extremely susceptible to proteinases even after reduction and S-carboxymethylation. Another anomalous tryptic hydrolysis occurred quantitatively at the phenylalanyl (63)Cm-cysteine bond. The corresponding bonds of Bowman-Birk inhibitor (IS) and D-II (23) were also hydrolyzed to some extent by TPCK-treated trypsin. As shown in Fig. 5, the sequence of C-II is highly homologous with those of Bowman-Birk and garden bean inhibitors. The elastase reactive site of garden bean inhibitor II, identified by Wilson and Laskowski, Sr., is alanine-25 and the corresponding position is also occupied by alanine (Ala-22) in soybean C-II. Therefore, it is likely that this alanine is the reactive site of C-II for elastase. The second reactive site residue of C-II is arginine (Arg-49), and it is directed to trypsin. It was shown that C-II also inhibits a-chymotrypsin at the reactive site for trypsin (2). This arginine residue is, therefore, suggested to be involved in the interaction of C-II with a-chymotrypsin. It is of considerable interest that garden bean inhibitor II, which has a reactive site sequence almost identical with that of C-II, showed no inhibitory action on bovine a-chymotrypsin. We have examined the chymotrypsin inhibitory activity of C-II in exactly the same way as that of garden bean inhibitor II (24), using a-N-benzoylL-tyrosine ethyl ester in 25% methanol (w/w, in the final assay medium), and found full inhibitory activity. Consequently the different behaviours of these inhibitors towards a-chymotrypsin are not due to a difference in the assay methods.
/. Biochem.
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Alignment of the Tryptic Pep tides and Complete Structure of the Inhibitor—This is shown in Fig. 4. Automatic amino-terminal sequence analysis of CB-III indicated the following sequence: Pro-Pro-Gln-X-His-X-Ala-Asp-Ile-Arg-Leu-(X denotes unidentified residues, probably cysteic acid residues). This result indicates the order (T-2Q(T-3), because there is only one tryptic peptide having amino-terminal leucine. T-4 is placed next to them and the two amino-terminal residues Ser-Met of T-6 are placed in the carboxyl terminus of the fragment. From the above results, CB-IV should begin with the third residue, proline, of T-6. T-2A is located at the carboxyl-terminal region of the fragment, since only this peptide has the same carboxyl-terminal residue (aspartic acid) as the parent molecule. To obtain additional information about the order of the remaining two peptides T-2B and T-5, CB-IV (0.04/*mol) was digested with 1 fig of chymotrypsin in 50 pt\ of 0.5 M NH4HCO, for 2 h at 40°C and the digest was separated by paper electrophoresis at pH 3.5 (50 volts/cm, 1.2 h). Fluorescent spots stained with 0.015% fluorescamine acetone solution (20) were cut out, eluted with dilute H O and hydrolyzed for amino acid analysis. The amino acid composition of one peptide, CB-IV-C3 (Table IV), indicates the order (T-5HT-2A). The complete amino acid sequence of C-II is shown in Fig. 4 and is compared with those of soybean Bowman-Birk inhibitor and the closely related garden bean inhibitor II in Fig. 5. Effects of Limited Proteolysis on the Inhibitory Activity—Carboxypeptidase digestion of the trypsin-treated inhibitor liberated 0.98 mol of arginine per mole of protein, and no other amino acid was released. As a result of this treatment, inhibitory activity for trypsin was completely lost and that for a-chymotrypsin decreased to 3% of that of the native inhibitor. However, 86% of the elastase inhibitory activity was retained. These results suggest that the trypsin and chymotrypsin reactive sites involve the same arginine residue, and that the elastase reactive site is different.
AMINO ACID SEQUENCE OF SOYBEAN INHIBITOR C-II
REFERENCES 1. Odani, S. & Ikenaka, T. (1976) / . Biochem. 80, 641-643 2. Odani, S. & Ikenaka, T. (1977) J. Biochem. 82, 1513-152' 3. Odani, S., Koide, T., & Ikenaka, T. (1971) Proc. Japan Acad. Al, 621-624 4. Odani, S. & Ikenaka, T. (1972) /. Biochem. 71, 839-848 5. Wilson, K.A. & Laskowski, M., Sr. (1974) in Proteinase Inhibitors (Fritz, H., Tschesche, H., Greene, L.J., & Truscheit, E., eds.) pp. 286-290, SpringerVerlag, Berlin 6. Wilson, K.A. & Laskowski, M., Sr. (1975) J. Biol. Chem. 250, 4261^267 7. Iwanaga, S., Wallen, P., Groendahl, N.J., Henschen, A., & Blombaeck, B. (1969) Eur. J. Biochem. 8, 189-199
Vol. 82, No. 6, 1977
8. Edman, P. & Begg, G. (1967) Eur. J. Biochem. 1, 80-91 9. Brenner, M., Niederwieser, A., & Pataki, G. (1969) in Thin-Layer Chromatography (Stahl, E., ed., Aschworth, M.R.F., translation) pp. 730-786, Springer-Verlag, Berlin 10. Jeppson, J.-O. & Sjoquist, J. (1967) Anal. Biochem. 18, 264-269 11. Pisano, J.M. & Bronzert, T.J. (1967) / . Biol. Chem. 244, 5597-5607 12. Mendez, E. & Li, C.Y. (1975) Anal. Biochem. 68, 47-53 13. Kulbe, K.D. (1974) Anal. Biochem. 59, 564-573 14. Fraenkel-Conrat, H. & Tsung, C M . (1967) in Methods in Enzymology (Hirs, C.H.W., ed.) Vol. 11, pp. 151-155, Academic Press, New York 15. Odani, S., Koide, T., & Ikenaka, T. (1972) /. Biochem. 71, 831-838 16. Crestfield, A.M., Moore, S., & Stein, W.H. (1963) /. Biol. Chem. 238, 622-627 17. Hirs, C.H.W. (1967) in Methods in Enzymology (Hirs, C.H.W., ed.) Vol. 11, pp. 197-199, Academic Press, New York 18. Ozawa, K. & Laskowski, M., Jr. (1966) /. Biol. Chem. 241, 3945-3961 19. Ryle, A.P., Sanger, F., Smith, L.F., & Kitai, R. (1955) Biochem. J. 60, 541-556 20. Vandekerckhove, J. & von Montagu, M. (1974) Eur. J. Biochem. 44, 279-288 21. Ikenaka, T., Odani, S., Sakai, M., Nabeshima, Y., Sato, S. & Murao, S. (1974) /. Biochem. 76, 11911209 22. Tan, C.G.L. & Stevens, F.C. (1971) Eur. J. Biochem. 18, 515-523 23. Odani, S. & Ikenaka, T. (1978) /. Biochem. in press 24. Wilson, K.A. & Laskowski, M., Sr. (1973) J. Biol. Chem. 248, 756-762 25. Odani, S. & Ikenaka, T. (1973) /. Biochem. 74, 697-715 26. Fritz, H., Schult, H., Meister, R., & Werle, E. (1969) Hoppe-Seyler's Z. Physiol. Chem. 350, 1531-1540
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Comparison of the nine-amino acid sequence between two half-cystine residues (which form a reactive site disulfide loop (25)) of the second domains of the two inhibitors indicates only one amino acid substitution between them, i.e., glutamine-lysine replacement in position 57 of garden bean inhibitor. Therefore, this replacement may result in a considerable difference in the affinity for bovine a-chymotrypsin, but not for trypsin, though this difference could also be attributed to the cumulative effects of amino acid substitutions throughout the molecules. The dissociation constant of C-II-bovine achymotrypsin complex is larger than that of the complex with trypsin (2). This may be in part due to the nature of the reactive site residue (arginine-49), which is primarily directed toward trypsin. A similar observation has been reported for bovine pancreatic trypsin inhibitor (Kunitz), which inhibits both trypsin and chymotrypsin at lysine-15, though the dissociation constant of the complex with a-chymotrypsin is far larger than that of the trypsin complex (26).
1531
XI.
Complete Amino Acid Sequence of a Soybean Trypsin-Chymotrypsin-Elastase Inhibitor, C-II 1 Shoji ODANI and Tokuji IKENAKA Department of Biochemistry, Niigata University School of Medicine, Niigata, Niigata 951 Received for publication, May 16, 1977
Soybean inhibitor C-II, which inhibits trypsin, a-chymotrypsin, and elastase, was reduced and S-carboxymethylated, and digested with trypsin. The amino acid sequences of the resulting tryptic peptides were determined by conventional methods, establishing the complete 76-amino acid sequence of the inhibitor. Inhibitor C-II was found to be homologous with soybean (Glycine max) Bowman-Birk inhibitor and more closely related to an inhibitor from garden beans (Phaseolus vulgaris). The homology with these inhibitors and the limited proteolysis of C-II indicated the reactive sites of C-II for elastase and trypsin to be alanine-22 and arginine-49, respectively. Arginine-49 was also identified as a reactive site for a-chymotrypsin. It was found that only a few replacements of one or two amino acid residues around the reactive sites resulted in considerable alteration of the inhibitory specificity.
In the preceding paper (2) we have reported the isolation and partial characterization of four soybean double-headed proteinase inhibitors. One of them, inhibitor C-II, showed a unique inhibitory specificity. It bound one mole each of trypsin, a-chymotrypsin, and elastase. The reactive sites for trypsin and a-chymotrypsin appeared to be identical, since the trypsin-C-II complex no longer inhibited a-chymotrypsin. When a-chymotrypsin1
This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan. A preliminary account of this work has appeared (/). Abbreviations: PTH, phenylthiohydantoin; Cm, carboxymethyl; RCm, reduced and carboxymethylated; TPCK, l-tosylamidc-2-phenylethyl-chloromethyl ketone; TLCK, l-chIoro-3-tosylamido-7-amino-2-heptanone. Vol. 82, No. 6, 1977
C-ll complex was incubated with trypsin, chymotrypsin activity gradually appeared with a concomitant loss of trypsin activity. The amino acid sequences of several legume double-headed inhibitors with various inhibitory activities have been reported (3-6). It seems likely, therefore, that a comparative study of the primary structures of these inhibitors and soybean C-II may provide some explanation for the unique specificity and properties of C-II, as well as information about the structure-function relationships of the inhibitors. The present paper describes the complete amino acid sequence of inhibitor C-II, established by sequence analyses of the tryptic peptides and cyanogen bromide fragments of C-II.
1523
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Studies on Soybean Trypsin Inhibitors
1524
S. ODANI and T. IKENAKA
MATERIALS AND METHODS
Amino Acid Sequence Analysis—Stepwise degradation of the peptides was carried out by Edman's procedure (7). Sequence study was carried out according to the method of Edman and Begg (8) using a JEOL JS-47K sequence analyzer (0.5 M Quadrol buffer, single cleavage, Quadrol program). The semi-quantitative determination of PTHderivatives was done by measuring the absorbance at 269 nm before chromatography. PTH-derivatives were usually identified by thin-layer chromatography on Kieselgel F-254 sheets (9, 10). Gasliquid chromatography was carried out to confirm the identity of PTH-derivatives of alanine, glycine, proline, isoleucine (or leucine), methionine, and phenylalanine on a column (0.3x300 cm) packed with 10% SE-30 on chromosorb W (60-80 mesh) using a Shimadzu CG-4BM gas chromatograph according to the method of Pisano and Bronzert (//). For the identification of leucine and isoleucine, the PTH-derivatives were regenerated to amino acids by hydrolysis in 5.7 N HC1 containing 0.4% SnCl, (72). PTH-Cm-cysteine was resolved from PTH-aspartic acid on micropolyamide thin-layer sheets (75). Hydrazinolysis for determination of the carboxyl-terminal amino acid was carried out by the method of Fraenkel-Conrat and Tsung (14). Reduction and S-Carboxymethylation—This was done by a modification (75) of the procedure of Crestifield et al. (75). Tryptic Digestion of RCm-C-II—Thirty mg of RCm-C-II was digested with 0.6 mg of TPCK-
Cyanogen Bromide Cleavage and Separation of Peptides—The protein (5 mg, 0.62 ftmo\) was treated with a 200-fold molar excess of cyanogen bromide over methionine residues in 70% formic acid for 24 h at room temperature. After desalting by passage through a Sephadex G-15 column (1.5x20cm, 0.1 M formic acid), the protein was oxidized with performic acid by the method of Hirs (17) and then lyophilized. The oxidized material was placed on a DEAE-Sephadex A-25 column (0.9x55 cm) equilibrated with 0.02 M NH,HCO S and eluted with a linear gradient of the buffer to 1.0 M. Elution was monitored by measuring absorbance at 230 nm using a Hitachi double-beam effluent monitor. Limited Proteolysis of C-II by Trypsin—To identify the reactive site, the limited proteolysis technique of Ozawa and Laskowski (18) was employed. The inhibitor (3.4 mg) was incubated with 60 fig of TPCK-trypsin for 20 h at pH 3.5 and 20°C. The pH of the reaction mixture was brought to 7.8 with 2 N NaOH, and 27 /ig of carboxypeptidase B and 36 fig of carboxypeptidase A were added. After digestion for 30 h at 25°C, an aliquot was withdrawn and analyzed for liberated amino acids and inhibitory activities for trypsin, a-chymotrypsin, and elastase.
/ . Biochem.
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Materials—The inhibitors and most of the enzymes used in this study were the same preparations as in the previous paper (2). TPCKtreated trypsin and TLCK-treated chymotrypsin were products of Worthington Biochem. Corp. and Merck, respectively. Carboxypeptidases A and B treated with di-wo-propyl phosphorofluoridate were purchased from Worthington Biochem. Corp. Thin-layer precoated sheets, DCFertigplatten Kieselgel F-254, were a product of Merck. Reagents and solvents for Edman degradation were extensively purified from reagent grade preparations. Reagents for automated Edman degradation were purchased from Wako Pure Chemicals.
trypsin in 10 ml of 0.05 M Tris-HCl buffer, pH 8.0, for 4 h at 25°C and then lyophilized. The digest was dissolved in 2 ml of 0.05 M ammonium formate, pH9.1, and applied to a Bio-Gel P-4 column (1.5x200 cm) equilibrated with the same solution. Chymotryptic Digestion of Peptide T-l—A large tryptic peptide, fraction T-l (0.6 //mole as leucine residue), which contained a set of aminoterminal peptides, was further digested with 0.038 mg of TLCK-chymotrypsin (molar ratio, E : S = 1:400) in 2.2 ml of 0 . 0 1 M Tris-HCl buffer, pH 8.0, for 4 h at 40cC. The digestion was stopped by lowering the pH of the reaction mixture with acetic acid and the mixture was applied to a column of SP-Sephadex C-25 (0.6x60 cm) equilibrated with 0.02 M ammonium acetate, pH 4.0. The column was first developed with 90 ml of the same buffer and then with an exponential gradient produced by introducing a limiting buffer of 0.05 M ammonium acetate, pH 9.25, into a 40 ml mixing chamber filled with the equilibrating buffer. Peptides were detected by measuring the absorbance at 230 nm.
AMINO ACID SEQUENCE OF SOYBEAN INHIBITOR C-II
1525
E
RESULTS
T-1
75
175 125 Elution Volume (ml) Fig. 1. Separation of the tryptic peptides of RCm-C-II on Bio-Gel P-4. 30 mg of the digest was applied to a column (1.5x200 cm) equilibrated with 0.05 M ammonium formate, pH 9.1, and eluted with the same solution. Peptides were located by means of their absorbance at 230 nm.
TABLE I. Amino acid compositions of tryptic peptides from reduced and S-carboxymethylated C-II. Numbers in parentheses are those determined after sequence analysis. Amino acid Cm-Cysteine Aspartic acid Threoninc Serine Glutamic acid Proline Glycine Alanine Methionine Isoleucine Leucine
T-2A
4.00 (4) 1.66 (2) 1.05 (1)
T-2B 0.93 (1) 2.24 (2) 1.82 (2) 0.35
Vol. 82, No. 6, 1977
1.06 (1) 1.11 (1) 1.78 (2)
1.96 (2) 0.27 0.84 (1)
T-5
T-6
C-II (2)
2.06 (2)
1.02 (1)
14 13 4 12 4 5 1 4 3 1 3 1 1 3 3
1.69 (2) 0.12
0.98 (1)
1.08 (1)
0.65 1.00 1.07 1.01
(1) (1) (1) (1)
1.10 (1) 0.90 (1)
0.95 (1)
1.C0 (1)
0.93 (1) 0.92 (1) 2.00 (2) 0.96
Arginme
Yield (%)
T-3
1.81 (2) 1.91 (2) 1.20 (1) 2.00 (2)
1.18 (1) 0.94 (1) 0.94 (1)
Tyrosine Phenylalanine Lysine Histidine
Total residues
T-2C
m
0.96
m
1. 00 (1)
1. 00 (1)
1. 00 (1)
0.90 (1)
4 76
7
7
12
10
5
6
7
59
32
9
27
65
20
47
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Tryptic Peptides of RCm-C-H—Six peptide fractions, T-l to T-6, were obtained by gel-filtration of the tryptic digest of RCm-C-II on Bio-Gel P-4 (Fig. 1). Each fraction was evaporated to dryness and dissolved in 50 (i\ of pyridine-acetic acid buffer, pH 6.5 (19), then subjected to high-voltage paper electrophoresis for 1.5 h at pH 6.5 with a potential gradient of 40 volts per cm. T-2C was further purified on a SP-Sephadex C-25 column (0.9 x 60 cm) equilibrated with 0.02 M ammonium acetate, pH 4.0. An exponential gradient produced by introducing 0.05 M ammonium acetate, pH 8.6, into a mixing chamber (25 ml) filled with the equilibrating buffer was used to develop the column (data not shown). Seven peptides, T-2A, T-2B, T-2C, T-3, T-4, T-5, and T-6, were obtained in a pure state. Their amino acid compositions are shown in Table I. The overall yields of these peptides were between
c o m
1526
S. ODANI and T. IKENAKA
E
c o N (0
0.6
40 Fraotion Number Fig. 2
80
TABLE II. Amino acid compositions of T-1 and its chymotryptic peptides. Numbers in parentheses are those determined after sequence analysis. Chymotryptic peptides of T-1 Amino acid 4.47 4.85 1.00 5.96 1.68 1.98
T-1A
T-1B
T-1C
T-1D
T-1E
1.81 (2) 3.57 (4)
0.82 (1)
0.87 (1)
1.42 (1)
1.02 (1) 1.54 (1)
Cm-Cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Methionine Isoleucine Leucine
1.35 1.08 0.40 1.00
0.36
Tyrosine Phenylalanine Lysine Histidine
1.11 1.71
0.85 (1) 0.85 (1)
Arginine
0.39
Total Yield (%)
88
1.00 (1) 5.52 (6) 1.39 (1) 1.48 (1)
1.10 (1) 1.20 (1) 2.15 (2) 1.12 (1) 1.00 (1)
0.27 0.83 (1)
0.20 0.21 1.08 (1) 1.00 (1)
1.00 (1)
0.24 1.15 (1) 1.00 (1)
17
2
3
7
5
24
20
20
24
28
~t Molar ratio with respect to leucine taken as 1.00. J. Biochem.
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65 and 20% of the starting material, except for T-2C, the low yield (9%) of which may be attributed to the repeated chromatography and partial hydrolysis at the alanyl (22)-serine bond by trypsin (Fig. 4). A preliminary experiment showed that T-1 consisted of two or three peptides derived from the amino-terminal region of the protein, which could not be separated from each other. ThereFig. 2. Purification of chymotryptic peptides of T-1. SP-Scphadex C-25 equilibrated with 0.02 M ammonium acetate, pH 4.0, was developed first with 80 ml of the same buffer, then with an exponential gradient formed by introducing 0.05 M ammonium acetate, pH 9.25, into a 40 ml mixing chamber filled with the equilibrating buffer. Elution was monitored in terms of the absorbance at 230 nm. Fractions of 2.0 ml were collected. Drift of the base line at 230 nm is conpensated in the figure.
AMINO ACID SEQUENCE OF SOYBEAN INHIBITOR C-II
chymotryptic peptides are listed in Table II. The sum of the compostitions of T-1D and T-1E is identical with that of T-2C. Therefore, T-1D and T-1E appear to be derived from the T-2C region of the inhibitor molecule. The sum of the residues of the remaining three chymotryptic peptides of T-1 (T-1A, T-1B, and T-1Q and the tryptic peptides listed in Table I accounts for the total amino acid residues of the parent protein, Amino Acid Sequences of the Isolated Peptides—First, reduced and S-carboxymethylated inhibitor was subjected to manual Edman degradation and the first 21 residues were determined, There may be some heterogeneity at the aminoterminus, because a faint Pauli-positive reaction was observed in the aqueous phase of the first step of the Edman degradation.
TABLE III. Summary of sequence studies of C-II and its tryptic peptides.1 — ^ , Edman degradation; —*>, identification by amino acid analysis; -. _ ^ .
_>.
T-1A
Ser-Asp-His-Scr-Ser-Ser-Asp-Asp
T-1B
Cys-Met
T-1C
Cys.Thr.Ala
T-1D
Ser-Met,Pro,Pro,Glx,Cvs,His
T-1E
Cys, Ala, Asp, lie, Arg
T-2A
Ser-Ser-Asp-GIu-Asp-Asp-Asp
T-2B
Cys-Leu-Asp-Thr-Thr-Asp-Phe
T-2C
Ser-Met-Pro-Pro-Gln-Cvs-His-Cys-Ala-Asp-Ile-Arg
T-3
Leu-Asn-Ser-Cys-His-Ser-Ala-Cvs-Asp-Arg
T-4
Cvs-Ala-Cys-Thr-Arg
T-5
Cys-Tyr-Lys-Pro (Cys, Lys)
T-6
Ser-Met-P^-dty-Gin-Cys-Arg
» All the Cys residues were identified as Cys(Cm). Vol. 82, No. 6, 1977
.*_
Asp ^ _
Leu
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fore, the whole T-1 fraction was digested with chymotrypsin and the peptides were separated by SP-Scphadex C-25 chromatography (Fig. 2). The first peak was still heterogeneous and was applied to a column of Bio-Gel P-4 (1.5 x 200 cm, 0.05 M ammonium formate, pH9.1). Two peptide fractions were obtained (the elution pattern is not shown). One fraction, eluted at the breakthrough point, was a homogeneous peptide, T-1A, and the second still consisted of two peptides, T-1B and T-1C, which were separated by paper electrophoresis at pH 6.5. Peptides T-1D and T-1E were used for sequence analysis without further purification. T-l A', which was eluted just behind T-1 A fraction, had the same amino acid composition as T-1A. The amino acid compositions of T-1 and its
1527
S. ODANI and T. IKENAKA
1528
m.
be derived from the amino-terminal region of C-II, could not be fully purified, and structural investigation was not possible. The difference of amino acid compositions between the parent TABLE IV. Amino acid compositions of fragments obtained by cyanogen bromide cleavage of C-II and a chymotryptic peptide of CB-IV. One of the fragment, CB-I, could not be fully purified and is omitted from the table. Numbers in parentheses are those determined after sequence analysis. Amino acid
CB-II
CB-III
CB-IV
CB-IV-C3
Cysteic acid
1.17(1)
6.47 (6)
4.15(4)
1.37(1) 4.00(4)
Aspartic acid 0.42
3.40(3)
6.00(6)
Threonine
0.87(1)
1.12(1)
1.95(2)
Serinc
1.02(1)
3.24(3)
2.38(2)
1.73(2)
Glutamic acid
1.25(1)
2.29(2)
1.21(1)
Proline
1.97(2)
1.89(2)
1.09(1)
Glycine
Cyanogen Bromide Fragments—Figure 3 shows the elution pattern on a DEAE-Sephadex A-25 Alanine column of C-II treated with cyanogen bromide and Isoleucine then oxidized with performic acid. The first Leucine three peaks were not peptides, but were probably Tyrosine salts. Each fraction, except for CB-II, which was Phenylalanine essentially pure, was further purified on a Bio-Gel P-10 column (1.5x150 cm, 0 . 1 M formic acid). Lysine However, CB-I could not be freed from a con- Histidine taminant which seemed to be partially cleaved Arginine inhibitor. Amino acid compositions of the puri- Homoserine fied CNBr-fragments are listed in Table IV. Order of the Cyanogen Bromide Fragments— Total One of the CNBr fragments (CB-I), which should
CBIL-CBfll 40
80 Tu be Number
1.55(1) 1.00(1)
3.00(3) 0.97(1) 1.25(1)
1.24(1) 0.90(1) 0.91(1) 2.00(2)
2.11(2)
0.15
2.92(3)
1.07(1)
0.70(1)
1.01(1)
5
27
25
2.09(2)
11
"CBICB1\7 1 20
1 60
Fig. 3. Separation of CNBr-fragments on DEAE-Sephadex A-25. A column (0.9 X 55 cm) equilibrated with 0.02 M N H 4 H C 0 , was eluted with a linear gradient of NH 4 HCO, concentration to 1.0 M. Peptides were located by means of their absorbance at 230 run.
J. Biochem.
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The carboxyl-terminal residue of the protein was identified as aspartic acid by the hydrazinolysis method. The yield of aspartic acid was 20 mol % of the protein used. The sequence of the first eight amino acids of T-1A is identical with that of the original protein, and T-1B and T-1C seem to follow T-1A. The sequence of T-2C indicates that T-1D and T-1E were generated by chymotryptic cleavage of the His-Cys(Cm) bond in T-2C. T-5 was readily extracted into the organic phase during Edman degradation, so that the last two residues (Cm-cysteine and lysine) could not be identified. Since this peptide was produced by tryptic digestion, lysine should be placed at the carboxyl terminus. The amino acid sequences of the other tryptic peptides were unambiguously determined by the manual Edman degradation method. These results are summarized in Table
AMINO ACID SEQUENCE OF SOYBEAN INHIBITOR C-II
1529
1
10
20
Ser-Asp-His-Ser-Ser-Ser-Asp-Asp-Glu-Ser-Ser-Lys-Pro-Cya-Cys-Asp-Leu-Cys-Met-Cys•» y
T-1A
> 4-T-1B-* *— 1 i—
CB-I
io »o Thr-Ala-Ser-Met-Pro-Pro-Gln-Cys-His-Cys-Ala-Asp-Ile-Arg-Leu-Asn-Ser-Cys-His-Ser•T-1D • •T-3-T-1C-T-1ET-2C -CB-II-
-CB-IIISO
6 0
Ala-Cys-Asp-Arg-Cys-Ala-Cys-Thr-Arg-Ser-Met-Pro-Gly-Gln-Cys-Arg-Cys-Leu-Asp-Thr-
• T-4
T-6
-T-2B —
Hh
•CB-III-
-CB-IV-
Thr-Aap-Phe-CyB-Tyr-Lys-Pro-Cys-Lys-Ser-Ser-Asp-Glu-Asp-Asp-Asp. *• * T-5 *• •* T-2A > CB-IV•CB-IV-C-3-
Fig. 4. Alignment of the tryptic peptides of RCm-C-II by analysis of the CNBr-fragments. tion. One of the CNBr-fragments, CB-I, could not be isolated and is shown as a dashed line.
•, Edman degrada-
V.
Aap-Asp-Glu-Ser-Ser-Lya-Pro-Cya-Cys-Aap-fGln-jCysj
I
io
,
,
Ser-Aap-Hia-Ser-Ser-SerfAsp-Aap-Glu-Ser-Ser-Lya-Pro-Cya-CyB-ABp-jLeu-jCysl ~t I I 1 | io A8x,Hi8,A8x,Glx,HiB,Ser,Ser,Asx,Glx-Pro-Ser-}Glx-Ser-Ser T Pro-j-Pro-Cy8-CyB-ABX-Ile-{CyaAla-fCyB-Thr^-LYS-f 2
I °
L 12J
j-Met-rArg-Leu-Aan-Sor-Cya-His-
. Asn-jt
.
-
.
.
IleTArg-Leu-Asn-Ser-Cya-Hia-
I
MetyCyB-Thr-AIA-Serj-Met-jPro-Pro-Gln-CyBj-HiB-jCyi^Ala19
I
10
~~
BBI C-II GBI BBI C-II GBI
Val-j-Cya-Thr-ALA-Sarf Ile-jPro-Pro-Gln-Cya^ H e , Cya, Thr, Aax, Val) Arg-Leu-Asx-Ser-Cys-HisSer-Ala-Cys-Ly8-Sar-CyaTlle-'CyBjAla-LEU-!serYTyrJProj-Ala-Gln-CyaTPhe-7CysrVal|AspTlle-. to I n - l sol——i ' 1 ' ' ' ' lo-* Ser-Ala-CyafABp-ArgjCyBjAlaJCy8-Thr-ARG-Ser-Het-Pro-Gly-Gln-CyB-Arg-CyB-Leu-ABp-Thr-
C-II
Ser-Ala-CyB-LyB-Ser-CyajHet-Cys-Thr-ARG-Ser-Met-Pro-Gly-Lyaj-Cys-Arg-Cya-Leu-Aax-Thr-
GBI
Thr-Asp-Phe-Cys-TyrrGlu-Pro-CyB-LyB-Pro-SerjGlu-ABp-Asp-Lys-Glu-Asn.
BBI
Thr-Aap-Phe-Cys-Tyr-Lys-Pro-Cya-Lys-Ser-SeryAsp-Glu-Asp-Asp-Asp!
C-II
I
1
r^l
I
'
1
" |
Thr-AsxyTyr-Cys-Tyr-LyajSer-Cya-Lys-Ser-Aax-Ser-Gly-Glx-Aax-Asx.
BBI
GBI
Fig. 5. Comparison of the amino acid sequences of three legume inhibitors. BBI, soybean Bowman-Birk inhibitor (i); C-II, soybean inhibitor C-II (this work); GBI, garden bean inhibitor II (6). The reactive site residues are capitalized. Boxes show the homologous regions. Asx and Glx in GBI were assumed to be Asp and Glu, respectively. Vol. 82, No. 6, 1977
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protein and the sum of the three isolated CNBr of this fragment and also the order of the first fragments is close to the composition of the 19 two fragments as (CB-I>(CB-II). Since only amino-terminal amino acids, the sequence of which CB-IV contains no homoserine residue, this fragwas determined by Edman degradation of the ment should be placed in the carboxyl-terminal parent inhibitor (Table III). part of the molecule, and therefore, the order of Amino-terminal sequence analysis of the in- the four CNBr-fragments was assumed to be hibitor up to the 21st residue indicated the structure (CB-I)-(CB-II)-(CB-III)-(CB-IV).
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S. ODAN1 and T. IKENAKA
DISCUSSION There was some difficulty in sequencing the aminoterminal region of the protein because of partial
tryptic hydrolysis of the alanyl (22)-serine bond. As will be discussed later, this alanyl-serine bond seems to form the elastase reactive site of the inhibitor, and may be very susceptible to proteolytic digestion. A similar result was obtained in sequence analysis of a reduced and S-carboxymethylated Streptomyces subtilisin inhibitor, in which the subtilisin-reactive site methionyl (73)valine bond was extensively hydrolyzed by trypsin (21). Tan and Stevens (22) have also reported the chymotryptic cleavage of a lysyl-serine bond of lima bean inhibitor IV, which is the trypsin reactive site of this inhibitor. Therefore, the reactive site region of proteinase inhibitors appears to have some particular conformation extremely susceptible to proteinases even after reduction and S-carboxymethylation. Another anomalous tryptic hydrolysis occurred quantitatively at the phenylalanyl (63)Cm-cysteine bond. The corresponding bonds of Bowman-Birk inhibitor (IS) and D-II (23) were also hydrolyzed to some extent by TPCK-treated trypsin. As shown in Fig. 5, the sequence of C-II is highly homologous with those of Bowman-Birk and garden bean inhibitors. The elastase reactive site of garden bean inhibitor II, identified by Wilson and Laskowski, Sr., is alanine-25 and the corresponding position is also occupied by alanine (Ala-22) in soybean C-II. Therefore, it is likely that this alanine is the reactive site of C-II for elastase. The second reactive site residue of C-II is arginine (Arg-49), and it is directed to trypsin. It was shown that C-II also inhibits a-chymotrypsin at the reactive site for trypsin (2). This arginine residue is, therefore, suggested to be involved in the interaction of C-II with a-chymotrypsin. It is of considerable interest that garden bean inhibitor II, which has a reactive site sequence almost identical with that of C-II, showed no inhibitory action on bovine a-chymotrypsin. We have examined the chymotrypsin inhibitory activity of C-II in exactly the same way as that of garden bean inhibitor II (24), using a-N-benzoylL-tyrosine ethyl ester in 25% methanol (w/w, in the final assay medium), and found full inhibitory activity. Consequently the different behaviours of these inhibitors towards a-chymotrypsin are not due to a difference in the assay methods.
/. Biochem.
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Alignment of the Tryptic Pep tides and Complete Structure of the Inhibitor—This is shown in Fig. 4. Automatic amino-terminal sequence analysis of CB-III indicated the following sequence: Pro-Pro-Gln-X-His-X-Ala-Asp-Ile-Arg-Leu-(X denotes unidentified residues, probably cysteic acid residues). This result indicates the order (T-2Q(T-3), because there is only one tryptic peptide having amino-terminal leucine. T-4 is placed next to them and the two amino-terminal residues Ser-Met of T-6 are placed in the carboxyl terminus of the fragment. From the above results, CB-IV should begin with the third residue, proline, of T-6. T-2A is located at the carboxyl-terminal region of the fragment, since only this peptide has the same carboxyl-terminal residue (aspartic acid) as the parent molecule. To obtain additional information about the order of the remaining two peptides T-2B and T-5, CB-IV (0.04/*mol) was digested with 1 fig of chymotrypsin in 50 pt\ of 0.5 M NH4HCO, for 2 h at 40°C and the digest was separated by paper electrophoresis at pH 3.5 (50 volts/cm, 1.2 h). Fluorescent spots stained with 0.015% fluorescamine acetone solution (20) were cut out, eluted with dilute H O and hydrolyzed for amino acid analysis. The amino acid composition of one peptide, CB-IV-C3 (Table IV), indicates the order (T-5HT-2A). The complete amino acid sequence of C-II is shown in Fig. 4 and is compared with those of soybean Bowman-Birk inhibitor and the closely related garden bean inhibitor II in Fig. 5. Effects of Limited Proteolysis on the Inhibitory Activity—Carboxypeptidase digestion of the trypsin-treated inhibitor liberated 0.98 mol of arginine per mole of protein, and no other amino acid was released. As a result of this treatment, inhibitory activity for trypsin was completely lost and that for a-chymotrypsin decreased to 3% of that of the native inhibitor. However, 86% of the elastase inhibitory activity was retained. These results suggest that the trypsin and chymotrypsin reactive sites involve the same arginine residue, and that the elastase reactive site is different.
AMINO ACID SEQUENCE OF SOYBEAN INHIBITOR C-II
REFERENCES 1. Odani, S. & Ikenaka, T. (1976) / . Biochem. 80, 641-643 2. Odani, S. & Ikenaka, T. (1977) J. Biochem. 82, 1513-152' 3. Odani, S., Koide, T., & Ikenaka, T. (1971) Proc. Japan Acad. Al, 621-624 4. Odani, S. & Ikenaka, T. (1972) /. Biochem. 71, 839-848 5. Wilson, K.A. & Laskowski, M., Sr. (1974) in Proteinase Inhibitors (Fritz, H., Tschesche, H., Greene, L.J., & Truscheit, E., eds.) pp. 286-290, SpringerVerlag, Berlin 6. Wilson, K.A. & Laskowski, M., Sr. (1975) J. Biol. Chem. 250, 4261^267 7. Iwanaga, S., Wallen, P., Groendahl, N.J., Henschen, A., & Blombaeck, B. (1969) Eur. J. Biochem. 8, 189-199
Vol. 82, No. 6, 1977
8. Edman, P. & Begg, G. (1967) Eur. J. Biochem. 1, 80-91 9. Brenner, M., Niederwieser, A., & Pataki, G. (1969) in Thin-Layer Chromatography (Stahl, E., ed., Aschworth, M.R.F., translation) pp. 730-786, Springer-Verlag, Berlin 10. Jeppson, J.-O. & Sjoquist, J. (1967) Anal. Biochem. 18, 264-269 11. Pisano, J.M. & Bronzert, T.J. (1967) / . Biol. Chem. 244, 5597-5607 12. Mendez, E. & Li, C.Y. (1975) Anal. Biochem. 68, 47-53 13. Kulbe, K.D. (1974) Anal. Biochem. 59, 564-573 14. Fraenkel-Conrat, H. & Tsung, C M . (1967) in Methods in Enzymology (Hirs, C.H.W., ed.) Vol. 11, pp. 151-155, Academic Press, New York 15. Odani, S., Koide, T., & Ikenaka, T. (1972) /. Biochem. 71, 831-838 16. Crestfield, A.M., Moore, S., & Stein, W.H. (1963) /. Biol. Chem. 238, 622-627 17. Hirs, C.H.W. (1967) in Methods in Enzymology (Hirs, C.H.W., ed.) Vol. 11, pp. 197-199, Academic Press, New York 18. Ozawa, K. & Laskowski, M., Jr. (1966) /. Biol. Chem. 241, 3945-3961 19. Ryle, A.P., Sanger, F., Smith, L.F., & Kitai, R. (1955) Biochem. J. 60, 541-556 20. Vandekerckhove, J. & von Montagu, M. (1974) Eur. J. Biochem. 44, 279-288 21. Ikenaka, T., Odani, S., Sakai, M., Nabeshima, Y., Sato, S. & Murao, S. (1974) /. Biochem. 76, 11911209 22. Tan, C.G.L. & Stevens, F.C. (1971) Eur. J. Biochem. 18, 515-523 23. Odani, S. & Ikenaka, T. (1978) /. Biochem. in press 24. Wilson, K.A. & Laskowski, M., Sr. (1973) J. Biol. Chem. 248, 756-762 25. Odani, S. & Ikenaka, T. (1973) /. Biochem. 74, 697-715 26. Fritz, H., Schult, H., Meister, R., & Werle, E. (1969) Hoppe-Seyler's Z. Physiol. Chem. 350, 1531-1540
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Comparison of the nine-amino acid sequence between two half-cystine residues (which form a reactive site disulfide loop (25)) of the second domains of the two inhibitors indicates only one amino acid substitution between them, i.e., glutamine-lysine replacement in position 57 of garden bean inhibitor. Therefore, this replacement may result in a considerable difference in the affinity for bovine a-chymotrypsin, but not for trypsin, though this difference could also be attributed to the cumulative effects of amino acid substitutions throughout the molecules. The dissociation constant of C-II-bovine achymotrypsin complex is larger than that of the complex with trypsin (2). This may be in part due to the nature of the reactive site residue (arginine-49), which is primarily directed toward trypsin. A similar observation has been reported for bovine pancreatic trypsin inhibitor (Kunitz), which inhibits both trypsin and chymotrypsin at lysine-15, though the dissociation constant of the complex with a-chymotrypsin is far larger than that of the trypsin complex (26).
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