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 [7], bovine colostrum [8,9], porcine pancreatic secretory I and I1 [lo, 111, ovine pancreatic basic [12], ovine pancreatic secretory [13], ascaris [14], corn [15], peanuts [16], and lima bean I V [17] 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 [19] 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 [25]. The aminoAbbreviations. Cm, carboxymethyl ; R-Cm, reduced and carboxymethylated. Enzym. Trypsin (EC
27 Eur. J. Biochem., V01.32

terminal amino acid is aspartic acid [26] and the carboxyl-terminal amino acid is leucine [27]. Amino acid sequences of the amino-terminal region [ZS] and cystine-containing peptides [29] have been reported. Finkenstadt and Laskowski, Jr. [30] 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. [31] 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 [30]) 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

Chrmatographh elution pattern of modified soybean L L3 0 VI MATERIALS AND METHODS Soybean trypsin inhibitor (Kunitz) was prepared according to the procedure of Yamamoto and Ikenaka [32]. 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 .402 Fragmentation of Soybean %sin Inhibitor (Kunitz) Eur.2.3 and eluted with an exponential gradient of ammonium acetate (0. A ride.1 M. a freshly prepared chromatographic pattern is shown in Fig.1 N sodium hydr.Cm S. and the residual carboxyl-terminal < .1 1 of 0. using a mixing chamber which contained 1.75 with 0. it was removed as trypsin * inhibitor containing 5O/. and the second large peak (the modi.inhibitor was carried out by a slight modification of tion was adjusted t o 3. oxide. Limited Hydrolysis of Soybean Trypsin Inhibitor (Kunitz) try'psin inhibitor f r m DEAE-cellulose. Native inhibitor of Modified Inhibitor (500 mg) and trypsin (8 mg) were suspended in 45 ml of 0. EDTA and 6 M guanidine hydrochlocomplex by DEAE-cellulose chromatography. was equilibrated with 0. [33].o oxyl-terminal of the protein..complex fragment as fragment D. Modified inhibitor These observations led us to make the scheme of the fragmentation of the inhibitor shown in Fig.the procedure described by Crestfield et al. the frag. distilled water and lyophilized.1. After 4-h incubation a t 56 "C.4). 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 [31].1 M ammonium acetate buffer. The solution was allowed to stand for 24 h Modified inhibitor was reduced with 0.5 M Tris-HC1 buffer p H 8. J .m ment A. and dissolved with drop-wise addiReduction and carboxymethylation of modified tion of 1 N hydrochloric acid. pH7. 1 63 6 4 84 85 1 A 0-C--D LSxJ Trypsin & BrCN 1 1 3-" BrCN (Modified inhibitor) Reduct ion Car boxymethylat ion (Fragment A BC) Reduction Carboxymethylation B-C-D-S . 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. Since trypsin might interfere with later captoethanol in 0.0 with fied inhibitor) was exhaustively dialyzed against drop-wise addition of 1 N sodium hydroxide for 15 min a t 25 "C.CmS .1 M ammonium acetate pH 7.m ment following fragment B to the second methionine as fragment C.Cm S . .Cm + Fig.3 t o 0. Fig.5M 2-mera t 25 "C. The solution of 0.g 0 .rypsin inhibitor.6 experiments.and the solution was maintained a t pH9. pH6. the fragment from the 64th isoleucine resi. and the p H of the solu. 5 due to the first methionine as fragment B.05 M CaCI. 2. E.l. Biochem. N m and we refer to the fragment from the amino-terminal 0 aspartic acid to the 63rd arginine residue as frag.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.3M. A column (3 x 45 em.

p H 1. formic acid t o a concentration of 101.1 pmol protein or peptide were hydrolyzed with twice-distilled hydrochloric acid a t 105 "C in evacuated sealed tubes for 24 h. The chromatographic elution pattern of R-Cm-modified inhibitor is shown in Fig. followed by gel filtration on a Sephadex G-75 column (3. The reaction mixture was diluted with 9 volumes of water and lyophilized.39]. 1973 T. RESULTS Fragmentations of soybean trypsin inhibitor (Kunitz) were carried out as shown in Fig. and for 48 or 72 h when necessary. 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.9 (Fig.75 and 25 "C converted it into modified inhibitor by hydrolysis of the Arg'Js-Ile64peptide bond [31].36].2 N acetic acid and insoluble materials were removed by centrifugation. The soluble preparations were gel-atered on a Sephadex 6-50 column (2. [35. A 100-fold molar excess of crystalline cyanogen bromide was added and the mixture was incubated for 48 h a t 38 "C. and analyzed on a Hitachi Auto Amino Acid Analyzer KLA-3 by the method of Spackman et al.5 x 200 cm). Further purification of the fragment was achieved by rechromatography on the same column of Sephadex 6-75. 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. Further purification of the fragments was achieved by rechromatography on the same column of Sephadex G-100. The amino-terminal amino acid of the fragment was identified as aspartic acid and the carboxyl-terminal as aginine [44]. v/v/v). and gel-filtered on a Sephadex G-100 column (1. v/v/v).33. The absorbance a t 226nm [34] and 280nm was measured. equilibrated with formic acid-acetic acid-water (25:87 :8Y8. equilibrated with deaerated 5001.5) was reduced and carboxymethylated by a similar method as described above. Reduction and Carboxymethylation o f Large Fragment ABC The residual large fragment ABC after the removal of fragment D (see Fig. Terminal Amino-Acid Analyses The amino-terminal amino acid and the ammoterminal sequence were determined by the Edman's method [41]. The absorbance at 280 nm was monitored (Fig. 5). . The first large peak with a shoulder contained modified inhibitor and fragment FL [31] (fragment BCD).3.KOIDE and T.Vo1. Subsequent reduction and carboxymethylation of modified inhibitor.05-0. The tryptophan content was estimated by the method of Spies and Chambers [38. 1. From these results the second peak was identified as fragment A. The amino acid analysis of the second peak agreed reasonably well with that of the small fragment (Fs) reported by Ozawa and Laskowski [31] (Table 1). equilibrated with the same buffer.0 x 200 cm). The homoserine content was determined after opening the lactone ring of homoserine lactone by the procedure described by Ambler [37]. Fractionation o f Cyanogen-Bromide-Treated Modified Inhibitor The lyophilized preparations were dissolved in 0. The yield of pure fragment A was 150 mg from 500 mg of the inhibitor. pH 1. No.3. IKENAZA 403 Fractionation o f R-Cm-Modified Inhibitor The concentrated preparations were chromatographed on a column of Sephadex 6-75 (3. and by the method of Matsubara and Sasaki ~401. and the carboxyl-terminal amino acid by carboxypeptidase procedure [42] and by hydrazinolysis [43].5 x 200 cm). and gel-filtered on a 27.. Fractionution of R-Cm-Fragment ABC The preparations were concentrated t o a small volume under reduced pressure. Cleavage of Methionyl Bonds in Modified Inhibitor Modified inhibitor was dissolved in 70°/. Isolation o f Fragment A Incubation of native inhibitor with catalytic quantities of trypsin at pH 3.0 x 200 cm) gave two peak fractions. The hydrolysate was concentrated to dryness a t 50 to 55 "C. The cystine content of some peptides was determined as S-carboxymethyl-cysteine. Amino-Acid Analyses Samples containing 0.9.8 x 200 cm). Sephadex G-50 column (2.6). acetic acid.

404 Fragmentation of Soybean Trypsin Inhibitor (Kunitz) Eur. Cyanogen-bromidetreated inhibitor was applied t o a column (1. Chromatographic elution pattern of R-Cm-modified inhibitor from Sephadex G-75. The first large peak was not studied.5 x 200 cm) separated one fragment from other fragments.5 C 2 00 400 Elution volume ( m l ) 600 Fig.pH 1. The first small peak contained intact 0 200 300 400 Elution volume (ml) Fig. Fractions indicated by the bar were pooled and lyophilized g 1 . Cyanogenbromide-treated modified inhibitor was applied t o a column (2. Fractions of the second peak indicated by the bar were collected and rechromatographed on a Sephadex G-25 column (1.formic acid was almost complete after 48 h of reaction. 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.5 x 200 cm) of Sephadex G-50.8x 200 cm) as shown in Fig.9. Fig. Table 1) and the amino-terminal sequence as Ile-Arg-Phe-Ile-AlaGlu-Gly. Chrornatographic elution pattern of cyanogen-bromidetreated inhibitor from Sephadex G-100. Fig. and the absorbance at 280 nm was monitored.m D. The eluate was collected in 5-ml fractions. Fractions indicated by the bar were pooled and lyophilized I I 200 400 600 Elution volume (ml) 800 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. 5. Amino acid analysis of a 24-h acid hydrolysate ofthe oxidized protein showed that less than 0. The cleavage products were fractionated on a Sephadex 6-100 column (1.1 methionyl residue per molecule remained uncleaved. The eluate was collected in 3-ml fractions and the absorbance at 280 nm was monitored. 1. Four peaks were obtained. Fractions indicated by the bars were pooled and lyophilized . J.o 0 w . The purified second peak was identified as fragment B on the basis of amino acid analysis (presence of one mole of homoserine.3x 145 cm). The column was operated at room temperature with a flow rate of 8 ml/h.The column was eluted a t a flow rate of 6 ml/h.4.5 4 Isolation of Fragment D Cleavage of native inhibitor with a 100-fold molar excess of cyanogen bromide in 70°/. 3. previously equilibrated with 0. 5.2 N acetic acid and eluted with the same buffer a t a flow rate of 4 ml/h. The yield of pure fragment B was 24 mg from 280 mg of the inhibitor. Biochem.v/v/v).8 x 200 cm) of Sephadex G-100.o E r ) L 0 8 0. Chromatographic elution pattern of cyanogen-bromidetreated nutdified inhibitor from Sephadex G-50. previously equilibrated with formic acidacetic acid-water (25:87:888.[44] which was identical [28] with that of the large fragment (FL)of [31]. 3-ml fractions were collected and the absorbance at 226 nm and 280 nm was measured.4 shows the elution pattern of the cyanogen-bromide-treated modified inhibitor. 5 t I I f I Modified inhibitor t Fragment BCD 8 c-4 8 m = 1 . 0 % 0.

the second peak in Fig.9. Fractions indicated by the bars were pooled The second peak of Fig. IHENAHA 405 shown in Fig. 5.water (25 :87 :888. v/v/v). On the other hand. Isolation of Fragment AB Elution volume (ml) Fig. 6. R-Cm-fragmentABC was applied to a column (2.e. only after reduction and carboxymethylation of the reactants. p H 1. The first small peak was parent fragment ABC not reduced or carboxymethylated. p H 1. without subsequent reduction and carboxymethylation made it possible to separate fragment D from fragment ABC. and each fragment had one S-carboxymethyl cysteine.5x 200 cm). present study and the results of the sequence studies [44.32. Since the molecular weight of the inhibitor was presented as 21500 and the number of constituent amino acids of the inhibitor as 198 [25]. These observations confirm that the other disulfide bridge links fragment A with fragment C enclosing the reactive site in the S-S loop. KOIDE and T. 56 mg pure fragment C was obtained from 240 mg R-Cm-fragment ABC. Cyanogen bromide treatment of native inhibitor. It appears that some peptide bonds in fragment D (perhaps about middle of the peptide) have been cleaved during the cyanogen bromide treatment. 4-ml fractions were collected and the absorbance at 280 nm was measured. most of the investigators have employed these values in their studies [31.9. 5). Fractionation of cyanogen-bromide-treated inhibitor gave the unexpected fourth peak a t a high retention volume behind fragment D (Fig. Isolation of Fragment C The purified fragment ABC. and no homoserine or its lactone. 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. pH 1.5 x 200 cm). was reduced and carboxymethylated as described in Methods. The amino-terminal amino acid of fragment D was identified as aspartic acid and the carboxyl-terminal as leucine [45]. and identification of the aminoterminal leucine [45].9.3.45] showed that the molecular weight of the inhibitor should be 20100 and this inhibitor was composed of 181 amino acid residues. Fragments A and C were separated from modified inhibitor and fragment ABC. One mai4 . 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. However.6 was chromatographed on a column of Sephadex G-100 (2.32. fragment D. i. DISCUSSION inhibitor. 1973 T. R-Cm-fragment ABC was gelfiltered on a Sephadex 6-50 column (2. These observations indicate that one of the two disulfide bridges in the inhibitor is present in fragment D. The purified fraction was identified as fragment AB from the amino acid composition shown in Table 1.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). and fragment D contained two halfcystine residues. equilibraked with formic acid-acetic acid. The second and the third large peaks were rechromatographed and purified. The elution pattern is The amino acid composition of soybean trypsin inhibitor (Kunitz) is listed in Table 1. The yield of pure fragment D was 159 mg from 500 mg of the inhibitor. and purified by separation from a small amount of fragment ABC. respectively. equilibrated with formic acid-acetic acid-water (25:87: 888. No. Table l). The amino acid analysis of this peak showed the same composition as that of fragment D. the retention volume from the Sephadex G-50 column. Amino acid analysis showed that the fourth small peak had almost the same composition as that of the third peak. The column was eluted at a flow rate of 11 ml/h. the purified third peak contained no homoserine or its lactone. 461. v/v/v).5 x 200 cm) of Sephadex G-50. These results indicate that the third peak corresponds to the carboxylterminal fragment of the inhibitor. respectively.6.Vo1. previously equilibratedwith formic acid-acetic acid-water (25:87: 888 v/vJv). the Edman degradation was carried out using a mixture of peptides to identify the site of cleavage. Since the separation of the peptides in the fourth peak was unsuccessful. 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 . 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. ChrowLatographic elution pattern of R-Crn-fragment ABC from Sephadex B-50.

28 5.47 0.39 6.80 4.93 0 2.85 3.9 13.87 5.03 5.38 6.7 9.10 0. C. 0 1 0 4 2 6 0 3 6 (5)d 1 4 1 6 1 tj" 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 25.07 1.11 0.08 6.89 1.32 2.03 0 2.73 3.00* 3.80 4.20 0 5.6 9.16 0 3.6 14.75 2.02 3.47 9. Values from the 72-h hydrolysat. c .Table 1.9 6.97 0 3.8 1.7 1.85 1 1 21 13.39 1.68 0 3.77 0.h asterisk in each column.81 2.31 5.07 2.7 3.00* 4 5 B 9 5 4 5 4 8 3 1 3 0 6 5 3 2 2 f 8.65 2.e.15 0 1.46 3.93 7.21 0.98 0.10 3.92 3.9 10.47 1.00* 0.7 8. The resultant values were determined after sequence analysis 144.23 2.0* 1.82 1.8 26 7 11 18 10 16 8 4 14 2 14 15 (14)d 4 9 2 10 2 9 9.09 1.94 2.01 0.00 0 0 0.82 4.96 4.00* 0 0.13 0.54c 3.39 2. Amino-mid compositions of soybean typsin inhibitor (Kunitz) and of fragment.25 4.451 Inhibitor found resultant found resultant resultant found resultant Fragment A Fragment B Fragment C found Fragment D resultant Fragment A B found resultant Amino acid found 3 ~ 3 1 1 "g z 13 1 4 8 2 4 1 $ 3 10 5 6 6 5 9 5 4 3 3 2 1 4 l C m 1 0 2 1 1 1 2 0 1 Q 9. d Values in parentheses show that some of the inhibitor lack carboxyl-terminal leucine.83 0 8.1 13.00* 0 0 0.94 1.57 7.60 1.00 0.02 0.98 4.8 10.2 16.87 0 0.4 18. .90 4 0 9 7 3 5 0 2 1 5 1 84 p? Total residues 181 1180bd 63 g b Values extraporated to zero hour.02 0.57 6.32 0 1.3 8.71 8. B.15 0.83 0 2.43 5.2 3.80 2 2 0 0 1 2 0 0 1 30 x 4 67 (661d h 2. 0 Values determined as Cm-cysteine.83 2.91 4.sA .82 5. D and A B The results are expressed as molar ratios with respect to amino acid denoted wit.

an isolation technique for fragment AB has been established. (Tokyo) 69.. 3. . 39. (1967) Hethods 43. BioZ. 32. 2. M. Ambler. Chem. 18.(1965) Bwchem. V. 25. 37. & Bohak.. & Matsushima. Biochem. S. 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. * Enzymol. Hamaguchi. 96. 11973) Eur. Chem. (1971) J . Hochstrasser. J. J. Exp. & Blombiick. N. & Sealock. C. Matsubara. J..5 was composed of the large amino-terminal peptide of fragment D and some other peptides. 30. L.. Kunitz. W. 26. Fritz. Hoessl. Walter de Gruyter. 2646. Moore. 185. Jr. H. 33. (1955) J. B. 15. 925. Trautachold. 31. K. 14. P. Chern. & Laskowski.. K. Meloun. 16. 7. 244. D. it is impossible to isolate fragments A and B. & Stein. (1965) Bwchem. S. (1958) Anal.. & Ikenaka. 30. 20. R. Chim. &chov&. H. R. Biochek. Chem. Y. 6. 30. 35. & Ikenaka. J. T.. D. M. (1968) Natural Proteinme Inhibitors. G. S.) Vol. M. Koide. M. W. 5. Iwanaga. In the present study. 1503. L. K. Res. 8. & Ikenaka. & Schwartz. (1969) J . 193. W. J. P. 18. Chem. T. Illchmann. K. T. C. (1972) J. & Laskowski. Biol. Tsunasawa S. K. (1968) Collection Czech. H. D.. Odani. 4. 11. Commun. 24. 20. 22. T. Biochem. Physiol.. Fraenkel-Conrat. Chern. S.T. & Bartelt. H. Res.. Kassell. & Stein. (1962) Biochemistry. 20. Stein. Tan. Koide. Biophys. 3. & Stevens. Chem. 244. Biochem. p. T. W. Mateushima (Osaka University) for his kind guidance throughout this investigation. 36. 23. . T. I. Tsunasawa. H. J. (1972) J . 985. Werle. Tschesche. Biochem. 151. PC 962. Physiol. Med. Dlouh&. Hochstrasser. 35. Bartelt. 175. R. 3955. H. Jr. Biol. Academic Press. Jr. (1971) Eur. F. by trypsin occurs only with active inhibitor. V. W. Kassell. & Tsum. Yamamoto. 155. Bb l. 251. 17. 29. (Tokyo) 63. Res. 350. (1971) Proc. Chauvet. (1963) J . (1971) J. T. 662. D. 16. & Acher. E. & Laskowski. Biochem. Biochem. Bowman.. Keil. Henschen. K. Chem. (Tokyo) 70. (1969) J. J . € Schwartz. & Scheraga. E.. E. T. 21. 32. T. Biochem. 2218. The analyses of main derivatives at each step made it possible to establish one sequence as Asp-Gly-Trp-Phe. D. Koide. B.. E. E. (1971) in Proceed9. (1949) Anal. & Werle. Fritz.698. Greif..V. J. 515. Biochem. & Koide. (1969) Eur. ed.. & Him. because limited proteolytic hydrolysis of the reactive site. P. Greene. (1970) Bethods Enzymol. & Wachter. 212. We wish to express our gratitude to Prof. Chem. 1185. C. 463. Schramm. J. Biophys. R. (1971) J. L. W. Hochstraeaer.. F. 839. J . S. It is suggested from these results that the fourth peak in Fig. Biol.. I. Gen. Berlin. 41. Lerman. (Tokyo) 71.. 10. H. Ikenaka. H. an arginyl-isoleucine bond. Bwphys. (1969) Hoppe-Seyler's 2. Y. 245. (1946) Proc. which is identical with the amino-terminal sequence of fragment D [45]. Academic Press. P. H. R. & Werle. I .. (196G) Biochem. Bwchem. . Sr. (1965) J.. 1249. K. & fiorm. Wu. Z.. S. 40. 615. Davie. N. V. S... E. Shimada. S. D. & Ikenaka. C. & Neurath. 238.T. 839. 32 P. P h y k l . 375. 831. R. K. Bwl. E. (1970) Hoppe-Seyler's 2. 408-416. 141. DiBella.. (1963) J . (1966) J. & Chamber. Grondahl.D. Bwl. 246. H. 4. K. Crestfield. 624. (1970) Hoppe-Seyler's Physid. 49. 11. Chent. (1970) Eur. Laskowski. (1967) Bull. (1946) J. Ikenaka. Soc. & Chamber. 11. . H. Chem. Biochem. (1958) A d . Biochem. KOIDE and T. T. Hochstrasser. R. Jap. Odani. B. J . B. D. PhysioZ. 1. (1966) B h h i m . & Sorm. 23. 28. R. & Liener. T.1973 T. C. A.Vo1. (1967) J. Moore. 507. 241. F. M. & Sasaki. Odani. 893. Spies. M.-M. 547. 139. Cechovh. 187. 38. E. 30. R. (1970) J . & Werle. Brown. & Ikenaka. 33. & Tschmche. Spies. W. S. & Ikenaka. & Acher. S. L. 46. Koide. Biophys. 155. J. (1968) J. 29. WallBn. 621. 47.. 105.. 561. W. F. F. D. Japan . P. E. B. Commun. W. Acta. Koide and T. Therefore. PospigilovB. Vogel. 1363. Bbl. REFERENCES 1. Chem. B. T. 721. .. (Tokyo) 54. Jonrikov&. Sm. Y.. Koide. IKENAKA 407 and some minor spots were detectable on a thinlayer chromatogram of phenylthiohydantoin derivatives of amino acid a t each step of the Edman degradation. D. 8. 19. 149. 351. Schramm.32. H. & sorm. Chem. Ozawa. Niigata. 1190. J . 45. 32. M. 63. H. 521. (Tokyo) 62. (1971) The Enzym (Boyer. Ikeda. Illchmann. 19. A. Spackman..3. Odani. 351. 21. Chem. 240. Finkenstadt. H. T.. C. 27. Biol. M . inga of the International Research Conference on Proteinase Inhibitors (Fritz. a. eds) p. 1-Bancho. Commun. Chem. & Ikenaka. A. 13. Chem.. W. Svestkod. Ambler.. & Greene. Ikenaka Department of Biochemistry Niigata University School of Medicine Asahimachi-Dori. R. R. (Tokyo) 71. & Ikenaka. 417-431. New York. No. 2824. This study was supported in part by a grant for Scientific Research from the Ministry of Education of Japan. (1970) Hoppe-Seyler's Z . & Moore. (1969) Biochem. 351. A d . R. 154. 34. T. (1967) Methods Enzymol. Biol. Yamamoto.. v. Once the inhibitor has been inactivated. (1973) Eur. M. Spackman. 4 4 . 42. & Werle. (1948) A d . (1969) FEBS Lett. Commun. 12.. Chem. D. C. New York. Jackson. C. K. Fraefel. T. S. R.

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