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Isolation of the pI 4.5 soybean trypsin inhibitor by isoelectric focusing
76
l~IOCm.~m:A ET BIOPHYSI(A ACTA
I~BA35321 ISOLATION OF T H E pI 4.5 SOYBEAN TRYPSIN I N H I B I T O R BY ISOELECTRIC FOCUSING
N. CATSIMPOOLAS, C. E K E N S T A M AND E. W. M E Y E R
Protein Research Laboratory, Central Soya-Chemurgy Division, Chicago, Ill. 60639 ( U.S.A .) (Received A u g u s t 22nd, I968)
SUMMARY
Soybean trypsin inhibitor was isolated from commercial crude soybean trypsin inhibitor and soybean whey protein by isoelectric focusing in the region between pH 3 and pH IO. The inhibitor was isoelectrically focused at pH 4.5. Disc electrophoresis in I i °/0 polyacrylamide gels and immunoelectrophoresis in agar gel, using anti-crude soybean trypsin inhibitor and anti-soybean whey protein sera, indicated that the isolated inhibitor was not contaminated by other proteins.
INTRODUCTION
Isoelectric focusing in stable pH gradients 1,2 is a relatively new technique for fractionation of macromolecular ampholytes. The method has been successfully applied to studies on the heterogeneity of a number of proteins (see review by HAGLUND 3) .
The present communication describes a procedure based on the electrofocusing technique for the isolation of the pI 4.5 soybean trypsin inhibitor (SBTIA2) from crude commercial soybean trypsin inhibitor and soybean whey proteins. The isolated inhibitor was found to be homogeneous by disc electrophoresis and immunoelectrophoresis in agar gel. MATERIALS AND METHODS
Materials
The crude soybean trypsin inhibitor was a commercial preparation (Mann Lot T-2178) obtained according to the procedure of RACKIS et al. 4 and corresponds to their chromatographic Component VI. Soybean whey proteins were prepared from defatted flakes of Harosoy 63 variety soybeans as described by RACKIS et al. 4. Carrier Abbreviation: SBTIA2, m a j o r c o m p o n e n t in the commercial p r e p a r a t i o n of s o y b e a n trypsin inhibitor obtained according to the procedure of RACKIS et al. 4.
l~iochirn. Biopp, yS. Acta, 175 (1969) 76 8l
SOYBEAN TRYPSIN INHIBITOR
77
ampholytes were obtained from L K B Instruments, Inc., Rockville, Md. All the other chemicals were of reagent grade. A o.25% solution of crude soybean trypsin inhibitor, and a I } o solution of soybean whey proteins in p H 7 buffered saline, mixed and homogenized with an equal w~lume of Freund's complete adjuvant (Difco), were used for the intraperitoneal immunization of New Zealand white rabbits. The immunizing dosage of each protein solution was i ml the first week, 2 ml the second week, and 5 ml the third week. After a 3o-days rest period, the rabbits were given a 5-ml booster injection. Test bleedings were used to determine if the precipitating antibody titer was adequate before the final bleeding was performed. Some of the animals produced satisfactory antibody response before the booster injection and were bled by cardiac puncture. The antisera were stored at 4 ° after filtration sterilization and addition of I :IO ooo merthiolate. Immunoelectrophoresis in agar gel was carried out by the general procedure described by GRABAR AND WILLIAMS5 as modified by SCHEIDEGGER6. The gel medium consisted of 1% Ionagar No. 2 (Oxoid) in Tris-barbital-sodium barbital buffer (pH 8.8 0.05 I) (Gelman). Electrophoresis was carried out for 2.5 h with a current of 5 mA per microscope slide. Staining of tile precipitin arcs was done with Ponceau S as described by URIEL v.
Disc electr@horesis Polyacrylamide gel electrophoresis in Tris~lycine buffer was carried out as described b y Davis 8. The gels were i I % with respect to the acrylamide and were polymerized with riboflavin and light 9, since the persulfate catalyst causes artifact bands when soybean trypsin intfibitor is analyzed by disc electrophoresis ~°. Densitometric tracings and integration of areas under the trace were performed with a Canalco Model F nficrodensitometer.
Isoelectric focusing An L K B 81o2 electrofocusing column of 44 ° ml capacity (LKB Instruments, Inc.) was used for these experiments. Stabilization against convection was achieved by using a density gradient prepared stepwise from one dense and one less dense solution". The carrier ampholyte was selected to give pH gradient between pH 3 and p H IO. Preparation of the solutions and of the density gradient was performed as described in the preliminary instruction sheet and its addendum supplied by L K B instruments. The anode solution was placed at the bottom of the colunm and the cathode solution at the top. The sample was prepared by dissolving IOO mg of the lyophilized soybean whey protein or 5o mg of the crude soybean trypsin inhibitor in 13.8 ml of the less dense solution and centrifuging at io ooo × g for 15 rain to remove insoluble material. The protein sample then replaced the less dense solution in the preparation of Fractions No. 23, 24, and 25 of tile density gradient. Electrofocusing was performed for i6 tl with a potential of 5o0 V (at IO°). Fractions (5 lnl) were collected and A280 mu of each fraction was determined using a I-Cm cell, with a Beckman DU spectrophotometer equipped with thermospacers and photomultiplier attachment. The pH of each fraction was also measured at 25 ° with a Beckman expandomatic p H meter equipped with a Beckman expandomatic range selector. Selected fractions were pooled and dialyzed against several changes of water Biochim. Biophvs. Acla, I75 (I969) 76 81
7~
N. (%-VI'SIMI)OOLAS, ('. I{KENSTAM, E. \V. MEVEI,'
at 5 ° for 8 days to remove sucrose and ampholytes. The dialyzed material was then freeze-dried to provide samples for analysis. Trypsin inhibitory activity was measured according to the casein digestion method of KUNITZ11.
1.8 q
1"6I 1.4
1.2 S 1.0
pH 6
0.8
< 0.6
0.4
4
ri
I I
0.2 '
1~
L 20
310 I
40 50 60 FRACTION NUMBER
70
80
90
100
Fig. I. isoelectric focusing of commercial crude s o y b e a n t r y p s i n inhibitor in the region between p H 3 and p H io. 0 - - - 0 , A2s0 m~ using I - c m cell; O C~, p H gradient measured at 25 °. The dashed-line brackets indicate the region of electrofocusing, and the arrow the p I 4.5 s o y b e a n t r y p s i n inhibitor peak.
RESULTS AND DISCUSSION
Isolation of the pI 4.5 inhibitor from crude soybean trypsin inhibitor The commercial crude soybean trypsin inhibitor when subjected to isoelectric focusing in the p H range between p H 3 and pH IO exhibits the pattern shown in Fig. i. The dashed-line brackets in the diagram indicate the area of electrofocusing. Areas outside the brackets represent the electrolytes of the anode and cathode. Although two peaks were found in the acidic electrolyte, only traces of protein were recovered indicating an artifact of unknown cause. At least four peaks were demonstrated in the electrofocusing range between pH 3 and pH IO. The major peak (indicated by an arrow in Fig. I) was eluted between p H 4.3o and pH 4.80. The pH at the m a x i m u m A2s0 ms value of the peak was 4.5 o. In order to avoid possible contamination from adjacent fractions, the p I 4.5 inhibitor was collected between p H 4.40 and pH 4.60. Isoelectric focusing did not affect the trypsin inhibitory activity of the inhibitor. The pH at m a x i m u m of the other contaminating components was found to be 3.93, 5.97, 6.38, and 7.11. The p I 3.93 protein was the major contaminating component Biochim. Biophys. dcta, 175 (1969) 76--81
79
SOYBEAN TRYPSIN INHIBITOR
and migrated ahead of the pI 4.5 inhibitor in the disc electrophoresis gels. No further studies were performed on the characterization of these components at present. KUNITZn reported the isoelectric point of crystalline soybean trypsin inhibitor to be at pH 4.5- This is in agreement with the results obtained by isoelectric focusing. RACKIS et al. 12 suggested that the SBTIA 2 inhibitor has similar physical properties to the KUNITZ' inhibitor, but a comparison ofisoelectric points was not made. This report shows that the SBTIA 2 inhibitor and KUNITZ' inhibitor exhibit identical isoelectric points. 1.8
[I I 1.4
¢ g
1.2
1.0 pH 6
0.8
~6
oE 0.2
I
I I
L
I I
[
I FRACTION
I
50
I
I
60 NUMBER
I
I
70
]
I
80
I
I
90
I
2
100
Fig. 2. Isoelectric focusing of s o y b e a n w h e y p r o t e i n in t h e region b e t w e e n p H 3 a n d p H ~o. O - - Q , A.~so ,n. u s i n g t - c m cell; O - - O , p H g r a d i e n t m e a s u r e d a t 25 °. T h e dashed-line b r a c k e t s indicate t h e region of electrofocusing, a n d t h e arrow t h e p I 4.5 s o y b e a n t r y p s i n inhibitor peak.
Isolation of the p I 4.5 inhibitor from soybean whey protein fraction The pI 4.5 soybean trypsin inhibitor can be isolated from soybean whey protein by a one-step electrofocusing procedure. The elution diagram of soybean whey protein after 16 h electrofocusing in the pH 3 IO region is shown in Fig. 2. The pI 4-5 inhibitor was eluted between pH 4.28 and pH 4.80 with peak at maximmn at pH 4.5 ° (see arrow in Fig. 2). Sixteen h of electrofocusing time was adequate for the isolation of the inhibitor. However, isolation of other soybean whey protein components was achieved after 48 h of isoelectric focusing 13. Disc electrophoresis analysis of the contaminating whey protein components has been described in detail 13. Data on the trypsin inhibitory and hemagglutinating activity of certain fractions have also been given. Disc electrophoresis and immunoelectrophoresis Fig. 3 shows disc electrophoresis patterns of the purified pI 4.5 inhibitor (A), unfractionated soybean whey protein (B), and commercial crude soybean trypsin Biochim. Bioph3,s. Ac/a, I75 (I969) 76 8I
~O
N. (AI'SIMtR)OI.A>, t'. EKI,INSTAM, E. kV. MI';YEI,~
inhibitor (C). The purified inhibitor (either from whey protein or commercial crude preparation) was found to be 99~)i, tmre by microdensitometry of the stained gels. Only a trace of a slow-migrating b a n d was detected as a contaminant. The commercial crude preparation has been demonstrated to contain at least 22')i, of impurities by densitometry 1°. The crude soybean inhibitor preparation exhibited at least four contaminating components. The whey protein fraction contained nmltiple contaminating components, the isoelectric points and relative electrophoretic mobilities of which have been described as. Disc electrophoresis was performed in i I °/o polyacrylanfide gels polymerized with riboflavin and light because the inhibitor forms artifact bands with persulfatO °, and lower concentrations of acrylamide result in incomplete resolution of the contanfinating components ~°.
! A
o
I m _ _ ~
R o
1
1 !
Fig. 3- Disc electrophoresis in t i o/,,o polyacrylamide gels of (A) purified pl 4,5 soybean, trypsin inhibitor; (B) soybean whey protein; and (C) commercial crude soybean trypsin inhibitor. Fig. 4- Schematic diagram of immunoelectrophoresis in agar gel patterns of (A) purified pl 4.5 soybean trypsin inhibitor against anti-crude soybean trypsin inhibitor serum i68 or anti-soybean whey protein serum; (B) crude soyhean trypsin inhibitor against antiserum 168. We have previously demonstrated 1° t h a t tile soybean trypsin inhibitor is antigenic and can be detected by several immunochemical methods such as double gel diffusion, immutmelectrophoresis, single diffusion, and complement fixation. These studies were performed with antiserum IO6 which was found to be specific for the p I 4.5 inhibitor when reacted with the total soybean whey protein fraction. This antiserum forms one specific immunoprecipitin arc with the purified inhibitor. Antiserum 168 described in this report was prepared by immunizing rabbits with the crude soybean trypsin inhibitor. This antiserum can be used to detect immunoehemically not only the p I 4,5 inhibitor but also the contaminating components, Immunoelectrophoresis patterns with antiserum I68 are shown in Fig. 4- The purified p I 4.5 inhibitor exhibited only one immunoprecipitin arc whereas at least three additional contaminating precipitin bands were detected in the crude trypsin inhibitor preparation. Reaction of the purified p I 4.5 inhibitor with anti-total soybean Biochim. Biophys. Acts, i75 (1969) 76 8i
SOYBEAN TRYPSIN INHIBITOR
81
whey protein serum showed only one immunoprecipitin arc. Thus, the pI 4.5 inhibitor purified by isoelectric focusing appears to be pure by both the disc electrophoresis and immunoelectrophoresis techniques. The isoelectric focusing method appears to be very promising for the isolation of other trypsin inhibitors and helnagglutinins present in the soybean whey protein fraction la. We have demonstrated that the pI 4.5 soybean trypsin inhibitor can be isolated either from total soybean whey protein or from crude commercial soybean trypsin inhibitor by one-step fractionation procedure based on electrofocusing. REFEF, ENCES i H. SVI~XSSON, Arch. Biochem. Biophys. ,Suppl. 1 (1962) 132. 2 (). VESTERBERG AND H . SVENSSON, Acta Chem. Scan&, 2o (1960) $2o. 3 H. HAGLUND, Sci. Tools, 14 (1967) I8. 4 J- J. P~ACKIS, H. A. SASAME, 1{. L. ANDERSON AND A. ](. SMITH, J. ~1~1. Chem. Soc., 81 (19.59)
()2() 5. 5 P. GRABAR AND C. A. \VILLIAMS, Biochim. Biopkvs. Acta, lO (1953) 193. 6 J. J. SCHEIDEGGER, Intern. Arch. Allergy AppI. Immunol., 7 (1955) lO3. 7 J. URIEL, in P. GRABAR AND P. BURTIN, Immuno-electrophoretic Analysis, Elsevier, N e w York, 1964, p. 3 o. 3; D. J. DAVIS, Ann. N . Y . Acad. Sci., 121 (1964) 256. 9 M. M. BREWER, Science, i56 (1967) 256. 10 N. CATSIMPOOLAS, D. A. ROGERS AND E . W . MEYER, Cereal Chem., in the press. i i M. KU.NITZ, J. Gen. Physiol., 3 ° (1947) 29I. 12 J. J. RACKIS, H . A. SASAME, R . I(. MANN, R . L. ANDERSON AND A. I~. SMITH, AI'ck. Biochem. Biophys., 98 (1962) 47 I. 13 N. CATSIMPOOLAS, C. EKENSTAM AND g . W. MEYER, Cereal Chem., in the press.
Biochim. Biophys. Acta, i75 (I90Q) 76 ~I
l~IOCm.~m:A ET BIOPHYSI(A ACTA
I~BA35321 ISOLATION OF T H E pI 4.5 SOYBEAN TRYPSIN I N H I B I T O R BY ISOELECTRIC FOCUSING
N. CATSIMPOOLAS, C. E K E N S T A M AND E. W. M E Y E R
Protein Research Laboratory, Central Soya-Chemurgy Division, Chicago, Ill. 60639 ( U.S.A .) (Received A u g u s t 22nd, I968)
SUMMARY
Soybean trypsin inhibitor was isolated from commercial crude soybean trypsin inhibitor and soybean whey protein by isoelectric focusing in the region between pH 3 and pH IO. The inhibitor was isoelectrically focused at pH 4.5. Disc electrophoresis in I i °/0 polyacrylamide gels and immunoelectrophoresis in agar gel, using anti-crude soybean trypsin inhibitor and anti-soybean whey protein sera, indicated that the isolated inhibitor was not contaminated by other proteins.
INTRODUCTION
Isoelectric focusing in stable pH gradients 1,2 is a relatively new technique for fractionation of macromolecular ampholytes. The method has been successfully applied to studies on the heterogeneity of a number of proteins (see review by HAGLUND 3) .
The present communication describes a procedure based on the electrofocusing technique for the isolation of the pI 4.5 soybean trypsin inhibitor (SBTIA2) from crude commercial soybean trypsin inhibitor and soybean whey proteins. The isolated inhibitor was found to be homogeneous by disc electrophoresis and immunoelectrophoresis in agar gel. MATERIALS AND METHODS
Materials
The crude soybean trypsin inhibitor was a commercial preparation (Mann Lot T-2178) obtained according to the procedure of RACKIS et al. 4 and corresponds to their chromatographic Component VI. Soybean whey proteins were prepared from defatted flakes of Harosoy 63 variety soybeans as described by RACKIS et al. 4. Carrier Abbreviation: SBTIA2, m a j o r c o m p o n e n t in the commercial p r e p a r a t i o n of s o y b e a n trypsin inhibitor obtained according to the procedure of RACKIS et al. 4.
l~iochirn. Biopp, yS. Acta, 175 (1969) 76 8l
SOYBEAN TRYPSIN INHIBITOR
77
ampholytes were obtained from L K B Instruments, Inc., Rockville, Md. All the other chemicals were of reagent grade. A o.25% solution of crude soybean trypsin inhibitor, and a I } o solution of soybean whey proteins in p H 7 buffered saline, mixed and homogenized with an equal w~lume of Freund's complete adjuvant (Difco), were used for the intraperitoneal immunization of New Zealand white rabbits. The immunizing dosage of each protein solution was i ml the first week, 2 ml the second week, and 5 ml the third week. After a 3o-days rest period, the rabbits were given a 5-ml booster injection. Test bleedings were used to determine if the precipitating antibody titer was adequate before the final bleeding was performed. Some of the animals produced satisfactory antibody response before the booster injection and were bled by cardiac puncture. The antisera were stored at 4 ° after filtration sterilization and addition of I :IO ooo merthiolate. Immunoelectrophoresis in agar gel was carried out by the general procedure described by GRABAR AND WILLIAMS5 as modified by SCHEIDEGGER6. The gel medium consisted of 1% Ionagar No. 2 (Oxoid) in Tris-barbital-sodium barbital buffer (pH 8.8 0.05 I) (Gelman). Electrophoresis was carried out for 2.5 h with a current of 5 mA per microscope slide. Staining of tile precipitin arcs was done with Ponceau S as described by URIEL v.
Disc electr@horesis Polyacrylamide gel electrophoresis in Tris~lycine buffer was carried out as described b y Davis 8. The gels were i I % with respect to the acrylamide and were polymerized with riboflavin and light 9, since the persulfate catalyst causes artifact bands when soybean trypsin intfibitor is analyzed by disc electrophoresis ~°. Densitometric tracings and integration of areas under the trace were performed with a Canalco Model F nficrodensitometer.
Isoelectric focusing An L K B 81o2 electrofocusing column of 44 ° ml capacity (LKB Instruments, Inc.) was used for these experiments. Stabilization against convection was achieved by using a density gradient prepared stepwise from one dense and one less dense solution". The carrier ampholyte was selected to give pH gradient between pH 3 and p H IO. Preparation of the solutions and of the density gradient was performed as described in the preliminary instruction sheet and its addendum supplied by L K B instruments. The anode solution was placed at the bottom of the colunm and the cathode solution at the top. The sample was prepared by dissolving IOO mg of the lyophilized soybean whey protein or 5o mg of the crude soybean trypsin inhibitor in 13.8 ml of the less dense solution and centrifuging at io ooo × g for 15 rain to remove insoluble material. The protein sample then replaced the less dense solution in the preparation of Fractions No. 23, 24, and 25 of tile density gradient. Electrofocusing was performed for i6 tl with a potential of 5o0 V (at IO°). Fractions (5 lnl) were collected and A280 mu of each fraction was determined using a I-Cm cell, with a Beckman DU spectrophotometer equipped with thermospacers and photomultiplier attachment. The pH of each fraction was also measured at 25 ° with a Beckman expandomatic p H meter equipped with a Beckman expandomatic range selector. Selected fractions were pooled and dialyzed against several changes of water Biochim. Biophvs. Acla, I75 (I969) 76 81
7~
N. (%-VI'SIMI)OOLAS, ('. I{KENSTAM, E. \V. MEVEI,'
at 5 ° for 8 days to remove sucrose and ampholytes. The dialyzed material was then freeze-dried to provide samples for analysis. Trypsin inhibitory activity was measured according to the casein digestion method of KUNITZ11.
1.8 q
1"6I 1.4
1.2 S 1.0
pH 6
0.8
< 0.6
0.4
4
ri
I I
0.2 '
1~
L 20
310 I
40 50 60 FRACTION NUMBER
70
80
90
100
Fig. I. isoelectric focusing of commercial crude s o y b e a n t r y p s i n inhibitor in the region between p H 3 and p H io. 0 - - - 0 , A2s0 m~ using I - c m cell; O C~, p H gradient measured at 25 °. The dashed-line brackets indicate the region of electrofocusing, and the arrow the p I 4.5 s o y b e a n t r y p s i n inhibitor peak.
RESULTS AND DISCUSSION
Isolation of the pI 4.5 inhibitor from crude soybean trypsin inhibitor The commercial crude soybean trypsin inhibitor when subjected to isoelectric focusing in the p H range between p H 3 and pH IO exhibits the pattern shown in Fig. i. The dashed-line brackets in the diagram indicate the area of electrofocusing. Areas outside the brackets represent the electrolytes of the anode and cathode. Although two peaks were found in the acidic electrolyte, only traces of protein were recovered indicating an artifact of unknown cause. At least four peaks were demonstrated in the electrofocusing range between pH 3 and pH IO. The major peak (indicated by an arrow in Fig. I) was eluted between p H 4.3o and pH 4.80. The pH at the m a x i m u m A2s0 ms value of the peak was 4.5 o. In order to avoid possible contamination from adjacent fractions, the p I 4.5 inhibitor was collected between p H 4.40 and pH 4.60. Isoelectric focusing did not affect the trypsin inhibitory activity of the inhibitor. The pH at m a x i m u m of the other contaminating components was found to be 3.93, 5.97, 6.38, and 7.11. The p I 3.93 protein was the major contaminating component Biochim. Biophys. dcta, 175 (1969) 76--81
79
SOYBEAN TRYPSIN INHIBITOR
and migrated ahead of the pI 4.5 inhibitor in the disc electrophoresis gels. No further studies were performed on the characterization of these components at present. KUNITZn reported the isoelectric point of crystalline soybean trypsin inhibitor to be at pH 4.5- This is in agreement with the results obtained by isoelectric focusing. RACKIS et al. 12 suggested that the SBTIA 2 inhibitor has similar physical properties to the KUNITZ' inhibitor, but a comparison ofisoelectric points was not made. This report shows that the SBTIA 2 inhibitor and KUNITZ' inhibitor exhibit identical isoelectric points. 1.8
[I I 1.4
¢ g
1.2
1.0 pH 6
0.8
~6
oE 0.2
I
I I
L
I I
[
I FRACTION
I
50
I
I
60 NUMBER
I
I
70
]
I
80
I
I
90
I
2
100
Fig. 2. Isoelectric focusing of s o y b e a n w h e y p r o t e i n in t h e region b e t w e e n p H 3 a n d p H ~o. O - - Q , A.~so ,n. u s i n g t - c m cell; O - - O , p H g r a d i e n t m e a s u r e d a t 25 °. T h e dashed-line b r a c k e t s indicate t h e region of electrofocusing, a n d t h e arrow t h e p I 4.5 s o y b e a n t r y p s i n inhibitor peak.
Isolation of the p I 4.5 inhibitor from soybean whey protein fraction The pI 4.5 soybean trypsin inhibitor can be isolated from soybean whey protein by a one-step electrofocusing procedure. The elution diagram of soybean whey protein after 16 h electrofocusing in the pH 3 IO region is shown in Fig. 2. The pI 4-5 inhibitor was eluted between pH 4.28 and pH 4.80 with peak at maximmn at pH 4.5 ° (see arrow in Fig. 2). Sixteen h of electrofocusing time was adequate for the isolation of the inhibitor. However, isolation of other soybean whey protein components was achieved after 48 h of isoelectric focusing 13. Disc electrophoresis analysis of the contaminating whey protein components has been described in detail 13. Data on the trypsin inhibitory and hemagglutinating activity of certain fractions have also been given. Disc electrophoresis and immunoelectrophoresis Fig. 3 shows disc electrophoresis patterns of the purified pI 4.5 inhibitor (A), unfractionated soybean whey protein (B), and commercial crude soybean trypsin Biochim. Bioph3,s. Ac/a, I75 (I969) 76 8I
~O
N. (AI'SIMtR)OI.A>, t'. EKI,INSTAM, E. kV. MI';YEI,~
inhibitor (C). The purified inhibitor (either from whey protein or commercial crude preparation) was found to be 99~)i, tmre by microdensitometry of the stained gels. Only a trace of a slow-migrating b a n d was detected as a contaminant. The commercial crude preparation has been demonstrated to contain at least 22')i, of impurities by densitometry 1°. The crude soybean inhibitor preparation exhibited at least four contaminating components. The whey protein fraction contained nmltiple contaminating components, the isoelectric points and relative electrophoretic mobilities of which have been described as. Disc electrophoresis was performed in i I °/o polyacrylanfide gels polymerized with riboflavin and light because the inhibitor forms artifact bands with persulfatO °, and lower concentrations of acrylamide result in incomplete resolution of the contanfinating components ~°.
! A
o
I m _ _ ~
R o
1
1 !
Fig. 3- Disc electrophoresis in t i o/,,o polyacrylamide gels of (A) purified pl 4,5 soybean, trypsin inhibitor; (B) soybean whey protein; and (C) commercial crude soybean trypsin inhibitor. Fig. 4- Schematic diagram of immunoelectrophoresis in agar gel patterns of (A) purified pl 4.5 soybean trypsin inhibitor against anti-crude soybean trypsin inhibitor serum i68 or anti-soybean whey protein serum; (B) crude soyhean trypsin inhibitor against antiserum 168. We have previously demonstrated 1° t h a t tile soybean trypsin inhibitor is antigenic and can be detected by several immunochemical methods such as double gel diffusion, immutmelectrophoresis, single diffusion, and complement fixation. These studies were performed with antiserum IO6 which was found to be specific for the p I 4.5 inhibitor when reacted with the total soybean whey protein fraction. This antiserum forms one specific immunoprecipitin arc with the purified inhibitor. Antiserum 168 described in this report was prepared by immunizing rabbits with the crude soybean trypsin inhibitor. This antiserum can be used to detect immunoehemically not only the p I 4,5 inhibitor but also the contaminating components, Immunoelectrophoresis patterns with antiserum I68 are shown in Fig. 4- The purified p I 4.5 inhibitor exhibited only one immunoprecipitin arc whereas at least three additional contaminating precipitin bands were detected in the crude trypsin inhibitor preparation. Reaction of the purified p I 4.5 inhibitor with anti-total soybean Biochim. Biophys. Acts, i75 (1969) 76 8i
SOYBEAN TRYPSIN INHIBITOR
81
whey protein serum showed only one immunoprecipitin arc. Thus, the pI 4.5 inhibitor purified by isoelectric focusing appears to be pure by both the disc electrophoresis and immunoelectrophoresis techniques. The isoelectric focusing method appears to be very promising for the isolation of other trypsin inhibitors and helnagglutinins present in the soybean whey protein fraction la. We have demonstrated that the pI 4.5 soybean trypsin inhibitor can be isolated either from total soybean whey protein or from crude commercial soybean trypsin inhibitor by one-step fractionation procedure based on electrofocusing. REFEF, ENCES i H. SVI~XSSON, Arch. Biochem. Biophys. ,Suppl. 1 (1962) 132. 2 (). VESTERBERG AND H . SVENSSON, Acta Chem. Scan&, 2o (1960) $2o. 3 H. HAGLUND, Sci. Tools, 14 (1967) I8. 4 J- J. P~ACKIS, H. A. SASAME, 1{. L. ANDERSON AND A. ](. SMITH, J. ~1~1. Chem. Soc., 81 (19.59)
()2() 5. 5 P. GRABAR AND C. A. \VILLIAMS, Biochim. Biopkvs. Acta, lO (1953) 193. 6 J. J. SCHEIDEGGER, Intern. Arch. Allergy AppI. Immunol., 7 (1955) lO3. 7 J. URIEL, in P. GRABAR AND P. BURTIN, Immuno-electrophoretic Analysis, Elsevier, N e w York, 1964, p. 3 o. 3; D. J. DAVIS, Ann. N . Y . Acad. Sci., 121 (1964) 256. 9 M. M. BREWER, Science, i56 (1967) 256. 10 N. CATSIMPOOLAS, D. A. ROGERS AND E . W . MEYER, Cereal Chem., in the press. i i M. KU.NITZ, J. Gen. Physiol., 3 ° (1947) 29I. 12 J. J. RACKIS, H . A. SASAME, R . I(. MANN, R . L. ANDERSON AND A. I~. SMITH, AI'ck. Biochem. Biophys., 98 (1962) 47 I. 13 N. CATSIMPOOLAS, C. EKENSTAM AND g . W. MEYER, Cereal Chem., in the press.
Biochim. Biophys. Acta, i75 (I90Q) 76 ~I