studies on soybean trypsin inhibitors : 1. fragmentation of soybean trypsin inhibitor (kunitz) by...

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 KOIDE and Tokuji IKENAKA Department of Biochemistry, Niigata University School of Medicine

(Received July 5/September 21, 1972)

Soybean trypsin inhibitor (Kunitz) was divided into four peptide fragments by the combina- tion 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 struc- tures of the enzymes and their inhibitors is a pre- requisite to a complete understanding of the mecha- nism 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 amino-

Abbreviations. Cm, carboxymethyl ; R-Cm, reduced and

Enzym. Trypsin (EC 3.4.4.4). carboxymethylated.

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 (Laskows- ki 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

402

1

Fragmentation of Soybean %sin Inhibitor (Kunitz)

63 64 84 85

Eur. J. Biochem.

Trypsin 1 1 BrCN

BrCN A 0-C--D - L S x J & (Modified inhibitor) (Fragment A BC)

Reduct ion Car boxymethylat ion

Reduction Carboxymethylation 1

3-" + B-C-D-S . Cm

S .CmS .Cm S.Cm

Fig.l. The schematic representation of the fragnzentatiOn of soybean t y p i n inhibitor (Kunitz)

nor its lactone should represent one from the carboxyl-terminal of the protein.

These observations led us to make the scheme of the fragmentation of the inhibitor shown in Fig.1, and we refer to the fragment from the amino-terminal aspartic acid to the 63rd arginine residue as fragment A, the fragment from the 64th isoleucine residue to the first methionine as fragment B, the fragment following fragment B to the second methionine as fragment C, and the residual carboxyl-terminal fragment as fragment D.

The present paper describes the systematic fragmentation of soybean trypsin inhibitor (Kunitz) as the first step of the elucidation of complete amino acid sequence of the inhibitor.

MATERIALS AND METHODS Soybean trypsin inhibitor (Kunitz) was prepared

according to the procedure of Yamamoto and Ike- naka [32].

Limited Hydrolysis of Soybean Trypsin Inhibitor (Kunitz)

Modified inhibitor was prepared by the method of Ozawa and Laskowski [31]. Native inhibitor (500 mg) and trypsin (8 mg) were suspended in 45 ml of 0.05 M CaCI, and dissolved with drop-wise addition of 1 N hydrochloric acid, and the pH of the solution was adjusted to 3.75 with 0.1 N sodium hydroxide. The solution was allowed to stand for 24 h a t 25 "C. Since trypsin might interfere with later experiments, it was removed as trypsin * inhibitor complex by DEAE-cellulose chromatography. A chromatographic pattern is shown in Fig.2. The first small peak (the trypsin inhibitor complex) was discarded, and the second large peak (the modified inhibitor) was exhaustively dialyzed against distilled water and lyophilized.

1 .o

E, 0 m N

m - g 0 . 5 m L3

0 VI L

<

I I I 1 t

Modified inhibitor

, .rypsin inhibitor- complex

0 I i 500 1000 1500 2000 2500 0

Elution volume (ml)

Fig. 2. Chrmatographh elution pattern of modified soybean try'psin inhibitor f r m DEAE-cellulose. A column (3 x 45 em; was equilibrated with 0.1 M ammonium acetate pH 7.3 and eluted with an exponential gradient of ammonium acetate (0.1 M. pH7.3 t o 0.3M, pH6.4), using a mixing chamber which contained 1.1 1 of 0.1 M ammonium acetate buffer. The eluate was collected in 15-ml fractions and the

absorbance at 280 nm was measured

Reduction and Carboxymethylation of Modified Inhibitor

Reduction and carboxymethylation of modified inhibitor was carried out by a slight modification of the procedure described by Crestfield et al. [33]. Modified inhibitor was reduced with 0.5M 2-mer- captoethanol in 0.5 M Tris-HC1 buffer pH 8.6 containing 5O/, EDTA and 6 M guanidine hydrochlo- ride. After 4-h incubation a t 56 "C, a freshly prepared solution of 0.6 M iodoacetic acid dissolved in 1 N sodium hydroxide was added to the reaction mixture and the solution was maintained a t pH9.0 with drop-wise addition of 1 N sodium hydroxide for 15 min a t 25 "C.

Vo1.33, No.3, 1973 T. KOIDE and T. IKENAZA 403

Fractionation of R- Cm-Modified Inhibitor The concentrated preparations were chromato-

graphed on a column of Sephadex 6-75 (3.0 x 200 cm), equilibrated with deaerated 5001, acetic acid. Further purification of the fragment was achieved by rechromatography on the same column of Sephadex 6-75.

Cleavage of Methionyl Bonds in Modified Inhibitor

Modified inhibitor was dissolved in 70°/, formic acid to a concentration of 101,. A 100-fold molar excess of crystalline cyanogen bromide was added and the mixture was incubated for 48 h a t 38 "C. The reaction mixture was diluted with 9 volumes of water and lyophilized.

Fractionation of Cyanogen-Bromide-Treated Modified Inhibitor

The lyophilized preparations were dissolved in 0.2 N acetic acid and insoluble materials were removed by centrifugation. The soluble preparations were gel-atered on a Sephadex 6-50 column (2.5 x 200 cm). The absorbance a t 226nm [34] and 280nm was measured.

Cleavage of Methionyl Ron& in Virgin Inhibitor

Two methionyl bonds in virgin inhibitor were cleaved with cyanogen bromide by the similar procedure as described above.

Fractionation of Cyanogen-Bromide-Treated Inhibitor The lyophilized product was dissolved in a mini-

mum volume of formic acid-acetic acid-water (25:87:888, v/v/v), pH 1.9, and gel-filtered on a Sephadex G-100 column (1.8 x 200 cm), equilibrated with the same buffer. The absorbance at 280 nm was monitored (Fig. 5). Further purification of the fragments was achieved by rechromatography on the same column of Sephadex G-100.

Reduction and Carboxymethylation of Large Fragment ABC

The residual large fragment ABC after the remov- al of fragment D (see Fig.5) was reduced and carboxymethylated by a similar method as described above.

Fractionution of R-Cm-Fragment ABC The preparations were concentrated to a small

volume under reduced pressure, and gel-filtered on a 27.

Sephadex G-50 column (2.5 x 200 cm), equilibrated with formic acid-acetic acid-water (25 : 87 : 8Y8, v/v/v), pH 1.9 (Fig.6).

Amino-Acid Analyses Samples containing 0.05-0.1 pmol protein or

peptide were hydrolyzed with twice-distilled hydrochloric acid a t 105 "C in evacuated sealed tubes for 24 h, and for 48 or 72 h when necessary. The hydrolysate was concentrated to dryness a t 50 to 55 "C, and analyzed on a Hitachi Auto Amino Acid Analyzer KLA-3 by the method of Spackman et al. [35,36].

The cystine content of some peptides was determined as S-carboxymethyl-cysteine. The homoserine content was determined after opening the lactone ring of homoserine lactone by the procedure described by Ambler [37]. The tryptophan content was estimated by the method of Spies and Chambers [38,39], and by the method of Matsubara and Sasaki ~401.

Terminal Amino-Acid Analyses The amino-terminal amino acid and the ammo-

terminal sequence were determined by the Edman's method [41], and the carboxyl-terminal amino acid by carboxypeptidase procedure [42] and by hydrazinolysis [43].

RESULTS Fragmentations of soybean trypsin inhibitor

(Kunitz) were carried out as shown in Fig. 1.

Isolation of Fragment A Incubation of native inhibitor with catalytic

quantities of trypsin at pH 3.75 and 25 "C converted it into modified inhibitor by hydrolysis of the Arg'Js-Ile64 peptide bond [31]. Subsequent reduction and carboxymethylation of modified inhibitor, followed by gel filtration on a Sephadex G-75 column (3.0 x 200 cm) gave two peak fractions. The chromatographic elution pattern of R-Cm-modified inhibitor is shown in Fig.3. 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). The amino-terminal amino acid of the fragment was identified as aspartic acid and the carboxyl-terminal as aginine [44]. From these results the second peak was identified as fragment A. The yield of pure fragment A was 150 mg from 500 mg of the inhibitor. The first large peak with a shoulder contained modified inhibitor and fragment FL [31] (fragment BCD).

404 Fragmentation of Soybean Trypsin Inhibitor (Kunitz) Eur. J. Biochem.

1 .5

E = 1 .o 8 c-4 - 8 m r) 0 L

8 0.5

C

t I I f I

Modified inhibitor

Fragment BCD t

2 00 400 600 Elution volume (m l )

Fig. 3. Chromatographic elution pattern of R-Cm-modified inhibitor from Sephadex G-75. R-Cm-modified inhibitor was applied to a column (3 x 200 cm) of Sephadex G-75 previously equilibrated with 50% acetic acid and eluted with the same solvent. The column was operated at room temperature with a flow rate of 8 ml/h. The eluate was collected in 5-ml fractions, and the absorbance at 280 nm was monitored. Frac-

tions indicated by the bar were pooled and lyophilized

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 x 200 cm) separated one fragment from other fragments. Fig.4 shows the elution pattern of the cyanogen-bromide-treated modified inhibitor. 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 (1.3 x 145 cm). The purified second peak was identified as fragment B on the basis of amino acid analysis (presence of one mole of homoserine, Table 1) and the amino-terminal sequence as Ile-Arg-Phe-Ile-Ala- Glu-Gly- [44] which was identical [28] with that of the large fragment (FL) of [31]. The yield of pure fragment B was 24 mg from 280 mg of the inhibitor.

Isolation of Fragment D Cleavage of native inhibitor with a 100-fold

molar excess of cyanogen bromide in 70°/, formic acid was almost complete after 48 h of reaction. Amino acid analysis of a 24-h acid hydrolysate ofthe oxidized protein showed that less than 0.1 methionyl residue per molecule remained uncleaved. The cleavage products were fractionated on a Sephadex 6-100 column (1.8 x 200 cm) as shown in Fig. 5. Four peaks were obtained. The first small peak contained intact

I I

200 400 600 800 Elution volume (ml)

Fig.4. Chromatographic elution pattern of cyanogen-bromide- treated nutdified inhibitor from Sephadex G-50. Cyanogen- bromide-treated modified inhibitor was applied to a column (2.5 x 200 cm) of Sephadex G-50, previously equilibrated with 0.2 N acetic acid and eluted with the same buffer a t a flow rate of 4 ml/h; 3-ml fractions were collected and the absorbance at 226 nm and 280 nm was measured. Fractions indicat-

ed by the bar were pooled and lyophilized

g 1 .o w 0

.- m

D, % 0.5 0 4

0 200 300 400

Elution volume (ml)

Fig. 5. Chrornatographic elution pattern of cyanogen-bromide- treated inhibitor from Sephadex G-100. Cyanogen-bromide- treated inhibitor was applied to a column (1.8 x 200 cm) of Sephadex G-100, previously equilibrated with formic acid- acetic acid-water (25:87:888,v/v/v),pH 1.9.The column was eluted a t a flow rate of 6 ml/h. The eluate was collected in 3-ml fractions and the absorbance at 280 nm was monitored. Fractions indicated by the bars were pooled and lyophilized

Vo1.32, No.3, 1973 T. KOIDE and T. IHENAHA 405

Elution volume (ml)

Fig. 6. ChrowLatographic elution pattern of R- Crn-fragment ABC from Sephadex B-50. R-Cm-fragment ABC was applied to a column (2.5 x 200 cm) of Sephadex G-50, previously equilibrated with formic acid-acetic acid-water (25: 87: 888 v/vJv), pH 1.9. The column was eluted at a flow rate of 11 ml/h; 4-ml fractions were collected and the absorbance at 280 nm was measured. Fractions indicated by the bars were

pooled

inhibitor. The second and the third large peaks were rechromatographed and purified, respectively. 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. On the other hand, the purified third peak contained no homoserine or its lactone, 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 14451. 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). These results indicate that the third peak corresponds to the carboxyl- terminal fragment of the inhibitor, i.e. fragment D. The amino-terminal amino acid of fragment D was identified as aspartic acid and the carboxyl-terminal as leucine [45]. The yield of pure fragment D was 159 mg from 500 mg of the inhibitor.

Amino acid analysis showed that the fourth small peak had almost the same composition as that of the third peak, 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.

Isolation of Fragment C The purified fragment ABC, the second peak in

Fig. 5, was reduced and carboxymethylated as described in Methods. R-Cm- fragment ABC was gel- filtered on a Sephadex 6-50 column (2.5 x 200 cm), equilibraked with formic acid-acetic acid- water (25 : 87 : 888, v/v/v), pH 1.9. The elution pattern is

shown in Fig.6. The first small peak was parent fragment ABC not reduced or carboxymethylated. 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, Table l), the retention volume from the Sephadex G-50 column, and identification of the amino- terminal leucine [45]. 56 mg pure fragment C was obtained from 240 mg R-Cm-fragment ABC.

Isolation of Fragment AB

The second peak of Fig.6 was chromatographed on a column of Sephadex G-100 (2.5 x 200 cm), equilibrated with formic acid-acetic acid-water (25: 87: 888, v/v/v), pH 1.9, and purified by separation from a small amount of fragment ABC. The purified frac- tion was identified as fragment AB from the amino acid composition shown in Table 1.

DISCUSSION

The amino acid composition of soybean trypsin inhibitor (Kunitz) is listed in Table 1. Since the molecular weight of the inhibitor was presented as 21500 and the number of constituent amino acids of the inhibitor as 198 [25], most of the investigators have employed these values in their studies [31,32, 461. However, present study and the results of the sequence studies [44,45] showed that the molecular weight of the inhibitor should be 20100 and this inhibitor was composed of 181 amino acid residues.

Cyanogen bromide treatment of native inhibitor, without subsequent reduction and carboxymethylation made it possible to separate fragment D from fragment ABC, and fragment D contained two half- cystine residues, These observations indicate that one of the two disulfide bridges in the inhibitor is present in fragment D.

Fragments A and C were separated from modified inhibitor and fragment ABC, respectively, only after reduction and carboxymethylation of the reactants, and each fragment had one S-carboxymethyl cysteine. These observations confirm that the other disulfide bridge links fragment A with fragment C enclosing the reactive site in the S-S loop.

Fractionation of cyanogen-bromide-treated inhibitor gave the unexpected fourth peak a t a high retention volume behind fragment D (Fig. 5). The amino acid analysis of this peak showed the same composition as that of fragment D. It appears that some peptide bonds in fragment D (perhaps about middle of the peptide) have been cleaved during the cyanogen bromide treatment. Since the separation of the peptides in the fourth peak was unsuccessful, the Edman degradation was carried out using a mixture of peptides to identify the site of cleavage. One mai4

Tab

le 1

. Am

ino-

mid

com

posi

tions

of s

oybe

an t

ypsi

n in

hibi

tor

(Kun

itz)

and

of

frag

men

t.s A

, B, C

, D a

nd A

B

The

resu

lts a

re e

xpre

ssed

as m

olar

ratio

s with

resp

ect t

o am

ino

acid

den

oted

wit,

h ast

eris

k in

eac

h co

lum

n. T

he re

sulta

nt v

alue

s wer

e de

term

ined

aft

er s

eque

nce

anal

ysis

14

4,45

1

Frag

men

t A

B

Inhi

bito

r Fr

agm

ent

A

Frag

men

t B

Fr

agm

ent

C

Frag

men

t D

Am

ino

acid

fo

und

resu

ltant

fo

und

resu

ltan

t fo

und

resu

ltant

fo

und

resu

ltant

fo

und

resu

ltan

t 3

foun

d re

sulta

nt

~

Asp

artic

aci

d T

hreo

nine

a Se

rinen

G

luta

mic

aci

d Pr

olin

e G

lyci

ne

Ale

nine

H

alf-

cyst

ine

Val

ineb

M

ethi

onin

e ho

leuc

ine b

Le

ucin

e T

yros

ine

Phen

ylal

anin

e T

rypt

opha

n Ly

sine

H

istid

ine

Arg

inin

e H

omos

erin

e

25.9

26

9.

25

9 6.

9 7

4.80

5

10.4

11

4.

43

4 18

.8

18

5.46

5

10.2

10

3.

71

4 16

.3

16

8.00

* 8

8.2

8 3.

11

3 3.

1 4

0.54

c 1

13.8

14

3.

20

3 1.

9 2

0 0

13.6

14

5.

80

6 14

.7

15 (1

4)d

4.39

5

3.6

4 2.

39

3 9.

0*

9 1.

87

2 1.

7 2

0 0

9.7

10

0.97

1

1.7

2 0

0 8.

8 9

3.60

4

1.32

1

0 0

1.77

2

0.94

1

1.07

1

1.32

1

2.00

* 2

0 0

0.93

1

0 2.75

3

2.16

2

0 0

3.15

3

0 0

1.13

1

0.89

1

1.02

1

0.85

1

3.21

3

0.98

1

0.92

1

3.65

4

2.94

3

2.83

3

2.02

2

0.98

Q 1

4.03

4

0 2.07

2

2.00

* 2

0 0

0 0

0.81

1

2.00

2

0 0

0 0

0.80

1

13.0

9 1.

02

3.57

7.

47

1.73

3.

82

1.23

2.

03

5.68

0 3.

28

5.15

0.

96

4.00

* 0.

57

6.00

0.

91

4.10

13

9.82

10

"g

6 $

1

5.08

5

z 4

6.38

8

6.31

6

2 5.

47

5 4

9.39

9

4 1

6.47

5

5 2

0.85

l

C

B 6

3.83

4

0 0

0 3

8.93

9

7.00

* 7

2.82

3

8.

6 (5

)d

1

3 m 2f

4 4.

83

5 1

0 0

6 2.

10

2 1

0.87

1

4 5.

01

5 0.

90

1

c, tj"

x h

p?

Tot

al re

sidu

es

181

1180

bd

63

21

30

67 (

661d

84

g

Val

ues

extr

apor

ated

to z

ero

hour

. b

Val

ues

from

the

72-h

hyd

roly

sat,e

. 0

Val

ues

dete

rmin

ed a

s C

m-c

yste

ine.

d

Val

ues

in p

aren

thes

es s

how

that

som

e of

the

inhi

bito

r lack c

arbo

xyl-

term

inal

leuc

ine.

Vo1.32, No.3.1973 T. KOIDE and T. IKENAKA 407

and some minor spots were detectable on a thin- layer chromatogram of phenylthiohydantoin derivatives of amino acid a t each step of the Edman degradation. The analyses of main derivatives at each step made it possible to establish one sequence as Asp-Gly-Trp-Phe, which is identical with the amino-terminal sequence of fragment D [45]. It is suggested from these results that the fourth peak in Fig.5 was composed of the large amino-terminal peptide of fragment D and some other peptides.

In the present study, an isolation technique for fragment AB has been established. Once the inhibitor has been inactivated, it is impossible to isolate fragments A and B, because limited proteolytic hydrolysis of the reactive site, an arginyl-isoleucine bond, by trypsin occurs only with active inhibitor. Therefore, 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.

We wish to express our gratitude to Prof. Y. Mateushima (Osaka University) for his kind guidance throughout this investigation. This study was supported in part by a grant for Scientific Research from the Ministry of Education of Japan.

1.

2. 3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

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T. Koide and T. Ikenaka Department of Biochemistry Niigata University School of Medicine Asahimachi-Dori, 1-Bancho, Niigata, Japan

studies on soybean trypsin inhibitors : 1. fragmentation of soybean trypsin inhibitor (kunitz) by...

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