Eur. J. Biochem.
32, 401-407 (1973)
Studies on Soybean Trypsin Inhibitors
1. Fragmentation of Soybean Trypsin Inhibitor (Kunitz) by Limited Proteolysis and by Chemical Cleavage
Takehiko KOIDEand Tokuji IKENAKA
Department of Biochemistry, Niigata University School of Medicine (ReceivedJuly 5/September 21, 1972)
Soybean trypsin inhibitor (Kunitz) was divided into four peptide fragments by the combination of limited hydrolysis with trypsin a t acidic pH, chemical cleavage of methionyl bonds with cyanogen bromide, and reduction and carboxymethylation of &sulfides in protein or peptide. Each fragment was separated and purified by gel filtration on Sephadex 6-50, G-75 or G-100, and referred to as fragments A, B, C, and D from the amino- to the carboxyl-terminal region of the inhibitor. Soybean trypsin inhibitor (Kunitz) consisted of 181 amino acid residues and its molecular weight was proved to be 20100. Fragments A, B, C, and D consisted of 63,21,30, and 67 amino acid residues, respectively. One of the two disulfide bridges in the inhibitor was involved in fragment D, and the other linked fragment A with fragment C.
Naturally occurring protein proteinase inhibitors are widely distributed in plants and animals; many of them have been isolated and studied for chemical and physicochemical properties [l,2,3]. Knowledge of the amino acid sequence and three-dimensional structures of the enzymes and their inhibitors is a prerequisite to a complete understanding of the mechanism of interaction between them. The amino acid sequences of several proteinase inhibitors have been reported, e.g. bovine pancreatic basic [4,5,6], bovine pancreatic Kazal’s , bovine colostrum [8,9], porcine pancreatic secretory I and I1 [lo, 111, ovine pancreatic basic , ovine pancreatic secretory , ascaris , corn , peanuts , and lima bean I V  inhibitors. Two well-known soybean proteinase inhibitors have been reported; one is so-called soybean trypsin inhibitor (Kunitz) originally isolated and crystallized by Kunitz [is], and the other is called Bowman-Birk inhibitor  whose complete amino acid sequence has recently been reported by us [20,21,22]. Soybean trypsin inhibitor (Kunitz) is the most extensively studied of proteinase inhibitors and a lot of research of its chemical and physicochemical properties has been carried out [3,23,24]. This protein consists of a single polypeptide chain, of which the molecular weight has been reported as 21500 . The aminoAbbreviations. Cm, carboxymethyl ; R-Cm, reduced and carboxymethylated. Enzym. Trypsin (EC 22.214.171.124).
27 Eur. J. Biochem., V01.32
terminal amino acid is aspartic acid  and the carboxyl-terminal amino acid is leucine . Amino acid sequences of the amino-terminal region [ZS] and cystine-containing peptides  have been reported. Finkenstadt and Laskowski, Jr.  reported that the interaction of trypsin with soybean trypsin inhibitor (Kunitz)resulted in the proteolytic splitting of a peptide bond in the inhibitor. Later, Ozawa and Laskowski, Jr.  presented evidence that it was an arginyl-isoleucine bond between residues 64 and 65 (in our results, between residues 63 and 64). This modified inhibitor (Laskowski et al. referred to the inhibitor whose Arg63-Iles4 bond was specifically hydrolyzed as “modified” inhibitor ) gave two fragments after reduction and carboxymethylation, and one which represented the amino-terminal 63 residues contained no methionine. Soybean trypsin inhibitor (Kunitz) contains two methionine residues. Native inhibitor, however, when treated with cyanogen bromide and chromatographed on Sephadex gel without reduction and carboxymethylation, gave two fragments : one contained one mole of cystine and neither homoserine nor its lactone, and the other two moles of homoserine or its lactone. The results show that one of two methionine residues in the inhibitor is involved in a S-S loop and the other is located out of the loop. The fragment which contains neither homoserine
After 4-h incubation a t 56 "C. using a mixing chamber which contained 1.402
Fragmentation of Soybean %sin
Eur.3 t o 0.g 0 . and the second large peak (the modi. Limited Hydrolysis
of Soybean Trypsin Inhibitor (Kunitz)
try'psin inhibitor f r m DEAE-cellulose.6 experiments. Since trypsin might interfere with later captoethanol in 0. A column (3 x 45 em.1 M. E. . Native inhibitor of Modified Inhibitor (500 mg) and trypsin (8 mg) were suspended in 45 ml of 0.CmS .and the solution was maintained a t pH9.1 1 of 0.complex fragment as fragment D. Chrmatographh elution pattern of modified soybean
MATERIALS AND METHODS
Soybean trypsin inhibitor (Kunitz) was prepared according to the procedure of Yamamoto and Ikenaka . The solution of 0.5 M Tris-HC1 buffer p H 8. The eluate was collected in 15-ml fractions and the absorbance at 280 nm was measured
Modified inhibitor was prepared by the method Reduction and Carboxymethylation of Ozawa and Laskowski . J . pH7. pH6. oxide. Fig. Biochem. it was removed as trypsin * inhibitor containing 5O/.1 M ammonium acetate pH 7.3M. The solution was allowed to stand for 24 h Modified inhibitor was reduced with 0.rypsin inhibitor.m ment A.2.inhibitor was carried out by a slight modification of tion was adjusted t o 3. the fragment from the 64th isoleucine resi. Modified inhibitor These observations led us to make the scheme of the fragmentation of the inhibitor shown in Fig. EDTA and 6 M guanidine hydrochlocomplex by DEAE-cellulose chromatography.o oxyl-terminal of the protein.the procedure described by Crestfield et al.6 M iodoacetic acid dissolved in 1 N first small peak (the trypsin inhibitor complex) sodium hydroxide was added to the reaction mixture was discarded. distilled water and lyophilized..3 and eluted with an exponential gradient of ammonium acetate (0. and the residual carboxyl-terminal < .05 M CaCI. The schematic representation of the fragnzentatiOn of soybean t y p i n inhibitor (Kunitz)
I I I 1 t nor its lactone should represent one from the carb1 .1. A ride. 5 due to the first methionine as fragment B. and the p H of the solu.Cm
S.4).l.m ment following fragment B to the second methionine as fragment C.Cm
Fig. The present paper describes the systematic frag0 I i mentation of soybean trypsin inhibitor (Kunitz) as 0 500 1000 1500 2000 2500 Elution volume (ml) the first step of the elucidation of complete amino acid sequence of the inhibitor. N m and we refer to the fragment from the amino-terminal 0 aspartic acid to the 63rd arginine residue as frag. the frag. a freshly prepared chromatographic pattern is shown in Fig. was equilibrated with 0.0 with fied inhibitor) was exhaustively dialyzed against drop-wise addition of 1 N sodium hydroxide for 15 min a t 25 "C.
S .1 M ammonium acetate buffer. and dissolved with drop-wise addiReduction and carboxymethylation of modified tion of 1 N hydrochloric acid.1 N sodium hydr.75 with 0.5M 2-mera t 25 "C. 2.
63 6 4
A 0-C--D LSxJ
(Modified inhibitor) Reduct ion Car boxymethylat ion
(Fragment A BC) Reduction Carboxymethylation
8 x 200 cm). p H 1. Cleavage o f Methionyl Ron& in Virgin Inhibitor Two methionyl bonds in virgin inhibitor were cleaved with cyanogen bromide by the similar procedure as described above. The amino acid analysis of the second peak agreed reasonably well with that of the small fragment (Fs) reported by Ozawa and Laskowski  (Table 1).
T.KOIDE and T. Fractionution of R-Cm-Fragment ABC The preparations were concentrated t o a small volume under reduced pressure. and for 48 or 72 h when necessary. Subsequent reduction and carboxymethylation of modified inhibitor.0 x 200 cm) gave two peak fractions. Further purification of the fragment was achieved by rechromatography on the same column of Sephadex 6-75. Reduction and Carboxymethylation o f Large Fragment ABC The residual large fragment ABC after the removal of fragment D (see Fig. The yield of pure fragment A was 150 mg from 500 mg of the inhibitor. Fractionation o f Cyanogen-Bromide-Treated Inhibitor The lyophilized product was dissolved in a minimum volume of formic acid-acetic acid-water (25:87:888. The reaction mixture was diluted with 9 volumes of water and lyophilized. The tryptophan content was estimated by the method of Spies and Chambers [38. Terminal Amino-Acid Analyses The amino-terminal amino acid and the ammoterminal sequence were determined by the Edman's method . The amino-terminal amino acid of the fragment was identified as aspartic acid and the carboxyl-terminal as aginine . and gel-filtered on a Sephadex G-100 column (1. Further purification of the fragments was achieved by rechromatography on the same column of Sephadex G-100.6).
Amino-Acid Analyses Samples containing 0.
.Vo1. v/v/v).9 (Fig. The absorbance a t 226nm  and 280nm was measured. acetic acid. Cleavage of Methionyl Bonds in Modified Inhibitor Modified inhibitor was dissolved in 70°/.9. The homoserine content was determined after opening the lactone ring of homoserine lactone by the procedure described by Ambler . The cystine content of some peptides was determined as S-carboxymethyl-cysteine. Fractionation o f Cyanogen-Bromide-Treated Modified Inhibitor The lyophilized preparations were dissolved in 0. and gel-filtered on a
27.75 and 25 "C converted it into modified inhibitor by hydrolysis of the Arg'Js-Ile64peptide bond .
Isolation o f Fragment A Incubation of native inhibitor with catalytic quantities of trypsin at pH 3. formic acid t o a concentration of 101. 1.
Sephadex G-50 column (2. 5). followed by gel filtration on a Sephadex G-75 column (3. [35.3.5 x 200 cm).39]. IKENAZA
Fractionation o f R-Cm-Modified Inhibitor The concentrated preparations were chromatographed on a column of Sephadex 6-75 (3. equilibrated with deaerated 5001. The chromatographic elution pattern of R-Cm-modified inhibitor is shown in Fig. The absorbance at 280 nm was monitored (Fig.0 x 200 cm).2 N acetic acid and insoluble materials were removed by centrifugation. The hydrolysate was concentrated to dryness a t 50 to 55 "C.36].1 pmol protein or peptide were hydrolyzed with twice-distilled hydrochloric acid a t 105 "C in evacuated sealed tubes for 24 h. and analyzed on a Hitachi Auto Amino Acid Analyzer KLA-3 by the method of Spackman et al.5) was reduced and carboxymethylated by a similar method as described above. The first large peak with a shoulder contained modified inhibitor and fragment FL  (fragment BCD). pH 1. v/v/v).3. From these results the second peak was identified as fragment A. and by the method of Matsubara and Sasaki ~401.
Fragmentations of soybean trypsin inhibitor (Kunitz) were carried out as shown in Fig. equilibrated with formic acid-acetic acid-water (25:87 :8Y8. and the carboxyl-terminal amino acid by carboxypeptidase procedure  and by hydrazinolysis .33. equilibrated with the same buffer. No. The soluble preparations were gel-atered on a Sephadex 6-50 column (2. A 100-fold molar excess of crystalline cyanogen bromide was added and the mixture was incubated for 48 h a t 38 "C..5 x 200 cm).05-0.
The eluate was collected in 5-ml fractions. The first small peak contained intact
Elution volume (ml)
Isolation of Fragment D Cleavage of native inhibitor with a 100-fold molar excess of cyanogen bromide in 70°/.8 x 200 cm) of Sephadex G-100. Cyanogenbromide-treated modified inhibitor was applied t o a column (2. which was identical  with that of the large fragment (FL)of . Table 1) and the amino-terminal sequence as Ile-Arg-Phe-Ile-AlaGlu-Gly.1 methionyl residue per molecule remained uncleaved. Biochem. The column was operated at room temperature with a flow rate of 8 ml/h. Fig.
% 0. 5
= 1 .
Fig. The first large peak was not studied. Fractions of the second peak indicated by the bar were collected and rechromatographed on a Sephadex G-25 column (1.5
Elution volume ( m l )
Fig.2 N acetic acid and eluted with the same buffer a t a flow rate of 4 ml/h. 5.pH 1. Chromatographic elution pattern of R-Cm-modified inhibitor from Sephadex G-75. Fractions indicated by the bar were pooled and lyophilized
g 1 .5 x 200 cm) of Sephadex G-50.
1.The column was eluted a t a flow rate of 6 ml/h.5 x 200 cm) separated one fragment from other fragments. Amino acid analysis of a 24-h acid hydrolysate ofthe oxidized protein showed that less than 0. 3-ml fractions were collected and the absorbance at 226 nm and 280 nm was measured. and the absorbance at 280 nm was monitored. Fractions indicated by the bar were pooled and lyophilized
400 600 Elution volume (ml)
Isolation of Fragment B
Chemical cleavage of two methionyl bonds of modified inhibitor with cyanogen bromide and following gel filtration on a Sephadex 6-50 column (2.m
D. Cyanogen-bromidetreated inhibitor was applied t o a column (1.404
Fragmentation of Soybean Trypsin Inhibitor (Kunitz)
Eur. J.8x 200 cm) as shown in Fig. The purified second peak was identified as fragment B on the basis of amino acid analysis (presence of one mole of homoserine.v/v/v). Chrornatographic elution pattern of cyanogen-bromidetreated inhibitor from Sephadex G-100. The cleavage products were fractionated on a Sephadex 6-100 column (1. 5.4.o
8 0. Chromatographic elution pattern of cyanogen-bromidetreated nutdified inhibitor from Sephadex G-50. The eluate was collected in 3-ml fractions and the absorbance at 280 nm was monitored.formic acid was almost complete after 48 h of reaction. Four peaks were obtained.4 shows the elution pattern of the cyanogen-bromide-treated modified inhibitor.o
w . previously equilibrated with formic acidacetic acid-water (25:87:888. 3. previously equilibrated with 0. The yield of pure fragment B was 24 mg from 280 mg of the inhibitor.3x 145 cm). Fractions indicated by the bars were pooled and lyophilized
. R-Cm-modified inhibitor was applied to a column (3x 200 cm) of Sephadex G-75 previously equilibrated with 50% acetic acid and eluted with the same solvent.9.
32. KOIDE and T.45] showed that the molecular weight of the inhibitor should be 20100 and this inhibitor was composed of 181 amino acid residues.e. the retention volume from the Sephadex G-50 column. the purified third peak contained no homoserine or its lactone. the second peak in Fig.A total amino acid composition of the second and the third peaks accounts closely for the amino acid composition of the intact inhibitor (Table 1). On the other hand. and fragment D contained two halfcystine residues. R-Cm-fragmentABC was applied to a column (2. fragment D. the Edman degradation was carried out using a mixture of peptides to identify the site of cleavage. 5). The elution pattern is
The amino acid composition of soybean trypsin inhibitor (Kunitz) is listed in Table 1.Vo1. most of the investigators have employed these values in their studies [31. only after reduction and carboxymethylation of the reactants.9. i. and no homoserine or its lactone.
inhibitor. pH 1. without subsequent reduction and carboxymethylation made it possible to separate fragment D from fragment ABC. These observations confirm that the other disulfide bridge links fragment A with fragment C enclosing the reactive site in the S-S loop. The third peak was identified as fragment C on the basis of amino acid analysis (presence of one mole each of homoserine and carboxymethyl cysteine. 6. IHENAHA
shown in Fig. and identification of the aminoterminal leucine . The purified fraction was identified as fragment AB from the amino acid composition shown in Table 1. No. The column was eluted at a flow rate of 11 ml/h. v/v/v).5 x 200 cm). Fractionation of cyanogen-bromide-treated inhibitor gave the unexpected fourth peak a t a high retention volume behind fragment D (Fig.3. The second and the third large peaks were rechromatographed and purified. v/v/v). previously equilibratedwith formic acid-acetic acid-water (25:87: 888 v/vJv). and each fragment had one S-carboxymethyl cysteine. 1973
T. was reduced and carboxymethylated as described in Methods. equilibraked with formic acid-acetic acid. The first small peak was parent fragment ABC not reduced or carboxymethylated. Table l). Fragments A and C were separated from modified inhibitor and fragment ABC. Fractions indicated by the bars were pooled
The second peak of Fig. The amino acid analysis of this peak showed the same composition as that of fragment D.9. The purified second peak was identified as the large fragment ABC on the basis of amino acid analysis (presence of two moles of homoserine or its lactone) and the retention volume from a Sephadex 6-100 column. and purified by separation from a small amount of fragment ABC. It appears that some peptide bonds in fragment D (perhaps about middle of the peptide) have been cleaved during the cyanogen bromide treatment. One mai4
.water (25 :87 :888. Since the separation of the peptides in the fourth peak was unsuccessful. p H 1. Amino acid analysis showed that the fourth small peak had almost the same composition as that of the third peak. 5. p H 1. Cyanogen bromide treatment of native inhibitor. respectively.6. However.9. ChrowLatographic elution pattern of R-Crn-fragment ABC from Sephadex B-50. The amino-terminal amino acid of fragment D was identified as aspartic acid and the carboxyl-terminal as leucine . The yield of pure fragment D was 159 mg from 500 mg of the inhibitor. 56 mg pure fragment C was obtained from 240 mg R-Cm-fragment ABC.32. These observations indicate that one of the two disulfide bridges in the inhibitor is present in fragment D. Since the molecular weight of the inhibitor was presented as 21500 and the number of constituent amino acids of the inhibitor as 198 . 461. equilibrated with formic acid-acetic acid-water (25:87: 888. R-Cm-fragment ABC was gelfiltered on a Sephadex 6-50 column (2. The electrophoretic pattern of this peak gave two overlapping ninhydrin-positive spots but the separation of a mixture by column chromatography on Whatman DE-32 was unsuccessful.
Isolation of Fragment AB
Elution volume (ml)
Fig. respectively.6 was chromatographed on a column of Sephadex G-100 (2. and the hydrazinolysis and the carboxypeptidase digestion of the fragment gave the same carboxyl-terminal amino acids (serine and leucine) as those of the native inhibitor 1 4 4 5 1 . present study and the results of the sequence studies [44. These results indicate that the third peak corresponds to the carboxylterminal fragment of the inhibitor.5x 200 cm). 4-ml fractions were collected and the absorbance at 280 nm was measured.5 x 200 cm) of Sephadex G-50.
Isolation of Fragment C The purified fragment ABC.
87 0 0.e.47 9.23 2.07 1.09 1.83 0 2.32 2.98 0.60 1.6 14.57 6.7 3.83 2.39 6.9 6.00 0.71 8.00* 0.20 0 5.80 4.91 4.54c 3.25 4.Table 1.87 5. Values from the 72-h hydrolysat.10 3.47 1.3 8. 0 Values determined as Cm-cysteine.9 13.7 1.0* 1.10 0.57 7.00*
9 5 4 5 4 8 3 1 3 0 6 5 3 2
2 f 8.31 5.68 0 3.07 2.94 1.h asterisk in each column.451
Inhibitor found resultant found resultant resultant found resultant Fragment A Fragment B Fragment C found Fragment D resultant Fragment A B found resultant
13 1 4 8 2 4
10 5 6 6 5 9 5 4 3 3 2 1 4
1 0 2 1 1 1 2 0 1
9.4 18.7 8.00* 0 0.94 2.28 5.sA .02 0.03 0 2.00* 3.93 0 2.73 3. The resultant values were determined after sequence analysis 144.00* 0 0 0.8 26 7 11 18 10 16 8 4 14 2 14 15 (14)d 4 9 2 10 2 9 9.82 1.39 2.43 5.80 2 2 0 0 1 2 0 0 1 30
4 67 (661d
2.32 0 1.8 10.82 5.96 4.13 0.15 0.
1 0 4
2 6 0 3 6 (5)d 1 4 1 6
3 2 0 3 0 1 1
Aspartic acid Threoninea Serinen Glutamic acid Proline Glycine Alenine Half-cystine Valineb Methionine holeucine b Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine Homoserine
25.15 0 1.
. Amino-mid compositions of soybean typsin inhibitor (Kunitz) and of fragment.6 9.01 0. d Values in parentheses show that some of the inhibitor lack carboxyl-terminal leucine.39 1.98 4.2 16.00 0 0 0.08 6.93
7. C.80 4.
c . B.77 0.1 13.7 9.65 2.11 0.8 1.90
4 0 9 7 3 5 0 2 1 5 1 84
181 1180bd 63
Values extraporated to zero hour.97 0 3.85
13.46 3.75 2.92 3.2 3.47 0.82 4.21 0.02 3.81 2.83 0 8.89 1.85 3. D and A B The results are expressed as molar ratios with respect to amino acid denoted wit.03 5.02 0.9 10.38 6.16 0 3.
H. 155. R.
We wish to express our gratitude to Prof. 11. (1966) J. 241. T. (Tokyo) 69. & Stein. 350. & Koide. & Werle. Laskowski. 30.T. R. Biochem. (1970) Hoppe-Seyler's Z . 25. R. Hochstrasser. KOIDE and T. H. (1968) Collection Czech. 20. & fiorm. (1969) Biochem. Biophys. M. P. T. 28. 244. A..
. D. Chern. 621. 13. & Neurath. J . PhysioZ. Once the inhibitor has been inactivated. Chem. (1946) J. & Moore. 893. 29. J. Bwl. &chov&. 17. € Schwartz. Res.3. & Ikenaka. Japan
.. 14.Vo1. S. 35.. 5. K. K. (1962) Biochemistry. H. (1967) Methods Enzymol. & Acher. & Laskowski. Bwl. 507.V. & Ikenaka. 38. 151. 24. V. 155. D. 32 P. Biochem. 1185. 2646. S.. M. Cechovh. & Ikenaka. I. 30. 18. Ambler. (1971) Proc. 246. REFERENCES
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T. Koide. J . 32.. In the present study. Hochstrasser. 1-Bancho. W. Odani. E. Iwanaga. which is identical with the amino-terminal sequence of fragment D . (Tokyo) 54. J. (1971) The Enzym (Boyer. Greif. 2824.. & Wachter. Chem. Jr. 141. 2218. (196G) Biochem. Ikenaka. 46.
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and some minor spots were detectable on a thinlayer chromatogram of phenylthiohydantoin derivatives of amino acid a t each step of the Edman degradation. J. W. F.. Y. Biochem. C. W.. & Chamber. & sorm. 175. Koide. H.. T. This study was supported in part by a grant for Scientific Research from the Ministry of Education of Japan. Biol. C. 33. Spies. Biochem. (1969) FEBS Lett. 23. 547. 16. 10. Soc. & Liener. T. K. 139. J . E. Kunitz. T. 244. Bwphys. Hochstraeaer. It is suggested from these results that the fourth peak in Fig. P. (1970) Bethods Enzymol. 36. 240. C. Biophys. (1969) J. T. Commun. & Greene..D. R. D. (1948) A d . Physiol. & Chamber. W. Werle. L. Academic Press. PospigilovB. (1965) Bwchem. Med.. ed.5 was composed of the large amino-terminal peptide of fragment D and some other peptides. & Laskowski. & Tsum. H. M. Jackson. & Him. 187. H. the isolation technique of fragment AB is useful in the study of chemical modification of some amino acid residues in the inhibitor and its inhibitory activity. G. Henschen. 21. 408-416. (Tokyo) 63. Chem. (1963) J . Chem.
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New York. Bb l. V. (1966) B h h i m . Finkenstadt. 12. Acta. Ozawa. (1969) Eur. 7.. L. Spackman. Chem. 463. an arginyl-isoleucine bond. 1363. Bbl. E. (1971) J . Fraefel. . (1969) Hoppe-Seyler's 2. H. W. I . Commun. R. & Stevens. Koide. Berlin. K.698. F. 351. Odani. (1970) Hoppe-Seyler's 2. Ikenaka Department of Biochemistry Niigata University School of Medicine Asahimachi-Dori. R. Z. Chim. Biochem. S. Y. Fritz. Spackman. Hoessl. Odani. T.. (1965) J. N. Yamamoto. W. Biochem. (1968) J. 32. Jr. Biol. Chem. . K... Chent. P.. Tsunasawa S. Biochem.. W. 3955. Ikeda. J. E. F.. Mateushima (Osaka University) for his kind guidance throughout this investigation. 624.. Meloun. Koide. H. Walter de Gruyter. N. (1971) Eur. F.
15. 27. Ambler. 561. & Sealock. Jap. 41. Chem. & Bohak. B. (Tokyo) 62. S. & Tschmche. Svestkod. Biol. Crestfield. (1970) Eur. 615. H. Hochstrasser. (1971) J. 4. 21. 45. & Sorm. A d . L. P h y k l . (1967) J. M. Hamaguchi. 3. Commun. No.. D. 20. Moore. (1970) Hoppe-Seyler's Physid. Moore. WallBn. K.. Vogel. M. Y. J. v. Chem.. & Laskowski. Biol. D. Koide and T. F. Res. R. Greene. 8. 3. 1190. (1967) Hethods 43. E. Odani. 1503. & Ikenaka. E. K. B. A. D. 11973) Eur. & Scheraga. & Ikenaka. 6. R. T. Tsunasawa. K. Chem. The analyses of main derivatives at each step made it possible to establish one sequence as Asp-Gly-Trp-Phe.
2. Shimada. 351. 149. 185. T. R. 375. K.