Immunochemical methods for detection and quantitation of Kunitz soybean trypsin inhibitor

ANALYTICAL

BIOCHEMISTRY

lmmunochemical of

31, 437447

Methods for Kunitz Soybean

N. CATSIMPOOLAS Protein

(1969)

Detection Trypsin AND

and Quantitation Inhibitor

E. LEUTHNER

Research Labor.atory, Central Soya-Chemurgy Chicago, Illinois 60639

Division,

Received June 9, 1969

The development of immunochemical methods for the detection and quantitative estimation of the multiple proteinase inhibitors present in soybean seeds (l-7) may contribute to the solution of problems involving identification, structural interrelationship, and correlation of antigenic and inhibitory activities. Preliminary results obtained with Kunitz trypsin inhibitor using water extract antiserum (8) did not show the full sensitivity that can be achieved by the preparation of specific antisera. In the present study, data that were obtained by using specific Kunitz soybean trypsin inhibitor antisera indicate that as little as 0.3 ,ug of the protein can be measured by radial immunodiffusion. The techniques of single diffusion and quantitative precipitation were found to be less sensitive and a relatively large amount of antiserum was required per assay. Immunochemical detection of the inhibitor was performed by the techniques of double gel diffusion, immunoelectrophoresis, and immunoelectrofocusing. A method for isolation of soybean trypsin inhibitor antibodies by formation of water-insoluble polymers of the protein is also described. MATERIALS

AND

METHODS

Soybean Trypsin Inhibitor. Commercial soybean trypsin inhibitor (lot T-4901) was purchased from Mann Research Laboratories. This commercial preparation was obtained according to the procedure of Rackis et al. (7) and corresponds to their chromatographic component VI. The majo,, component (SBTIA,) of this preparation is identical to the classic inhibitor described by Kunitz (1). Carrier ampholytes were obtained from LKB Instruments, Inc., Rockville, Maryland. All other chemicals were of reagent grade. The principle and application of the isoelectric focusing method 437

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LETJTHNER

in sucrose gradient have been described (9, 10). A LKB 8102 electrofocusing column of 440 ml capacity (LKB Instruments, Inc.) was used for these experiments. The carrier ampholyte (Ampholine, LKB) was selected to give a pH gradient between pH 3 and 6. Preparation of the solutions and of the density gradient was performed as described in the preliminary instruction sheet and its addendum supplied by LKB Instruments (11). The sample was prepared by dissolving a total of 50 mg of the commercial trypsin inhibitor (Mann Research Laboratories) in fractions No. 23, 24, and 25 of the density gradient and centrifuging at 10,OOOg for 15 min to remove insoluble material. The anode electrolyte solution was placed at the bottom of the column and the cathode solution at the top. Electrofocusing was performed for 18 hr with a final potential of 500 V (at 10’). Draining of the contents of the column was carried out slowly through the bottom tubing. Fractions (3.0 ml) were collected and the absorbance of each fraction at 280 rnp was determined using a 1 cm cell, with a Gilford 2000 spectrophotometer. The pH of each fraction was measured at 25O with a Beckman Expandomatic pH meter equipped with a Beckman Expandomatic range selector. Selected fractions were pooled and dialyzed against several changes ef water to remove sucrose and ampholytes. The dialyzed material was then lyophilized to provide a sample for analysis. The purified inhibitor exhibited stoichiometric inhibitory activity toward trypsin. Preparation of Antisera. The immunization procedure was similar to that described for other soybean proteins (12, 13). A 0.25% solution of Kunitz soybean trypsin inhibitor in pH 7.0 buffered saline (8.71 gm Na2HP04*7H,0, 2.31 gm NaH,PO,*H:O, 8.77 gm NaCl, made to 1 liter with H,O) was mixed and homogenized with an equal volume of Freund’s complete adjuvant (Difco) and then used for the intraperitoneal immunization of New Zealand white rabbits. The immunizing dosage of each protein sample was 1 ml the first week, 2 ml the second week, and 5 ml the third week. After a 30 day rest period, the rabbits were given a 5 ml booster injection. The animals were bled two weeks after the booster injection. The antisera were stored at 4” after filtration and addition of 1: 10,000 Merthiolate. Some of the animals produced satisfactory antibody response before the booster injection and were bled by cardiac puncture. Thirty per cent of the rabbits died during the immunization procedure. These animals refused food and their blood appeared to be hemolyzed to a significant extent.

SOYBEAN

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439

Double Gel Immunodiffusion. This test was carried out in 50 X 12 mm plastic Petri dishes according to the general procedure described by Ouchterlony (14). The gel medium consisted of 1% Ionagar No. 2 (Oxoid) in pH 8.8 Tris-barbital-sodium barbital 0.05 ionic strength buffer (Gelman). An immunodiffusion punch set (Shandon) was used for punching 7 mm and 9 mm diameter holes for the sample wells. fmmunoelectrophoresis in Agur Gel. The general procedure described by Grabar and Williams (15) as modified by Scheidegger (16) was employed for these analyses. The gel medium was prepared from 1% Ionagar No. 2 solution pH 8.8 Tris-barbital-sodium barbital 0.05 ionic strength buffer. Electrophoresis using a 0.05 to 0.1 mg soybean trypsin inhibitor sample was carried out for 2.5 hr with a current of 5 mA per microscope slide. Analytical Disc Electrofocusing and Disc Immunoelectrofocusing. Separation of proteins by isoelectric focusing on polyacry-

lamide gel columns similar to those used for disc electrophoresis has been ,described in detail (17). The experimental procedure for disc immunoelectrofocusing and immunodiffusion against specific antisera has also been reported (18). Radial Immunodiffusion. A 1% solution of Ionagar No. 2 (Oxoid) in pH 8.8 Tris-barbital-sodium barbital 0.05 ionic strength buffer (Gelman) was prepared by dissolving the agar in the buffer over a steam bath. The 1% agar solution was cooled to 60”, mixed with the appropriate volume of antiserum, and placed in 50 x 12 mm plastic Petri dishes. The agar-antiserum solution (5 ml per plate) was then allowed to cool and solidify on a horizontally leveled surface. An immunodiffusion punch set (LKB Instruments) equipped with a die bearing standard 3 mm well cutters was used for punching holes in the gel in a staggered row fashion. The distance between the centers of two adjacent holes is 16.5 mm. The protein sample (10 ~1) containing different amounts of soybean trypsin inhibitor was then placed in the wells with a microliter syringe No. 701 (Hamilton). The use of this type of syringe is necessary to avoid bubble formation in the wells. The Petri dishes were then covered and placed in a humid chamber at 20’ for 24 hr. Radial diffusion was determined by measuring the diameter of the precipitin zones with a measuring magnifier (Bausch & Lomb) precise to 0.1 mm. Measurements were performed under dark field illumination. Several dilutions of antiserum in agar solution have to be examined for optimum results with given amounts of trypsin inhibitor.

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In this case, a mixture of 1 part undiluted antiserum to 10 parts 1% agar gave very good precipitin zones with concentrations of trypsin inhibitor ranging from 0.3 to 10 pg per 10 ~1sample. Single Immunocliffusion. Glass tubes of 2 mm i.d. and approximately 100 mm long were coated with 0.1% Ionagar (Oxoid) and dried at ‘70”. For the single diffusion procedure, these tubes were half filled with 0.6% Ionagar in pH 8.8 T.ris-barbital-sodium barbital 0.5 ionic strength buffer and antisoybean trypsin inhibitor serum at a final dilution of 1 :lO. Soybean trypsin inhibior dispersed in the above-described gel diffusion buffer (0.5 ml containing from 10 pg to 2 mg of inhibitor) was layered above the gel and the tubes were capped with Vaspar (50: 50 white petroleum :paraffin) . Migration of the antigen-antibody precipitate was measured with a measuring magnifier after incubation of the tubes at 20” for 20 hr. Quantitative Precipitin Test. Serial dilutions of an 1 s solution of soybean trypsin inhibitor in pH 7.0 phosphate buffered saline were prepared, and 0.5 ml of each dilution was added to 0.4 ml of antisoybean trypsin inhibitor serum. The test tubes containing the reaction mixtures were incubated at 3’7” for 30 min and then stored for 72 hr at 4”. The resultant immunoprecipitates were centrifuged, washed twice with pH 7 buffered saline, and dissolved in 3 ml of 0.1 N NaOH. The absorbance of the solutions was measured at 280 rnp in a Beckman DU spectrophotometer against 0.1 N NaOH solution. Purification of the Soybean Trypsin Inhibitor Antibody. The purification was carried out according to the method of Avrameas and Ternynck (19) by using ethyl chloroformate to convert the antigen into an immunoadsorbent. The purified soybean trypsin inhibitor (50 mg) was dissolved in 5 ml of pH 7.0 phosphate-buffered saline and, while the solution was stirred, 0.2 ml of ethyl chloroformate was added dropwise. After 45 min of stirring, a precipitate appeared, and the mixture was adjusted to pH 7.0 with 1 N NaOH. The mixture was then left without stirring for 1 hr at room temperature and was subsequently centrifuged at 10,OOOgfor 10 min. The precipitate was suspended and washed several times with a large volume of phosphate-buffered saline (pH 7.0) until the optical density of the eluates was 0 at 280 rnp. The washing procedure was repeated with 0.2 M glycine-HCl (pH 2.2) buffer. The precipitate was then washed with phosphate-buffered saline (pH 7.0) until the pH of th eluates was neutral. The suspension was then centrifuged at 10,OOOgfor 10 min. The polymerized trypsin inhibitor was suspended in 35 ml of phosphate-buffered saline, and 5 ml of pooled

SOYBEAN

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trypsin inhibitor antiserum was added. The mixture was stirred gently at 37O for 90 min and then centrifuged at 10,OOOgfor 20 min. The precipitate was washed several times with phdsphatebuffered saline until the optical density of the eluates was 0 at 280 mp. To elute the adsorbed antisoybean trypsin inhibitor antibody, the precipitate was stirred for 15 min with 5 ml portions of 0.2 M glycine-HCl (pH 2.2) buffer and centrifuged at 10,OOOgfor 15 min after each elution. Most of the antibody was eluted with the first 5 ml portion of the pH 2.2 buffer as determined by the 280 rnp absorbance of the eluates. The combined eluates were neutralized carefully with NaOH solutions (0.1 N NaOH was used at the beginning and 0.02 N NaOH to the end of the titration) and dialyzed against phosphate-buffered saline at 4”. The purity of the antibody preparation was determined by immunoelectrophoresis against goat antirabbit serum (Hyland) and quantitative precipitation (90% precipitable by soybean trypsin inhibitor). Approximately 5 mg of purified antibody was obtained by using 50 mg of trypsin inhibitor and 5 ml of antiserum. RESULTS

The commercial soybean trypsin inhibitor subjected to isoelectric focusing in the pH region between pH 3 and 6 exhibited the pattern shown in Figure 1. The inhibitor was focused between pH 4.30 and 4.80 with a peak at pH 4.50 but only the material collected in the center of the peak (shaded area, Fig. 1) was free of impurities. The major contaminating component was focused approximately between pH 3.90 and 4.10. Other minor contaminants were focused at pH values lower than 4.3 and higher than 4.5 The isolated inhibitor showed only one band by disc electrofocusing (pH3-10) on polyaerylamide gel Isolation

(16).

of Soybean

Trypsin

Inhibitor.

Imrnunodiffusion Methods and Quantitative Precipitation. Double gel immunodiffusion, immunoelectrophoresis, and immunoelectrofocusing analyses of the purified trypsin inhibitor are shown in Figure 2. The patterns were developed by using specific soybean trypsin inhibitor antisera. The immunoelectrofocusing experiments were performed in two pH regions, namely, between pH 3 and 10, and between pH 3 and 6. The standard curve obtained from single diffusion experiments with purified soybean trypsin inhibitor in agar gel containing soybean trypsin inhibitor antiserum is shown in Figure 3. The log of inhibitor concentration approximated a straight line when plotted

442

CATSIMPOOLAS

30

50

AND

LELJTHNER

IO

90

m Fraction

130

150

Number

FIG. 1. Isoelectric focusing of commerical soybean trypsin inhibitor (Mann) (50 mg) in region between pH 3 and 6. The solid line represents absorbance at 280 rn& (1 cm cell). The dotted line represents pH gradient. The shaded area shows fractions that contained the inhibitor free of impurities.

against the distances of migration of the precipitin bands in a 20 hr diffusion period at 20”. Concentrations of the inhibitor in the range of 0.2 to 2 mg/0.5 ml sample can thus be estimated by this method. However, a very significant increase in sensitivity is obtained by the radial immunodiffusion technique (Fig. 4). Amounts of the inhibitor in the 0.3 to 10 pg range can be quantitated by using as little as 10 ~1 of sample. The precipitin curve shown in Figure 5 demonstrates results obtained by quantitative precipitation of the purified soybean trypsin inhibitor with specific antiserum. The precipitin curve shows a typical antigen-antibody reaction. Soybean Trypsin Inhibitor Antibody. Ethyl chloroformate polymerized soybean trypsin inhibitor was found to be an efficient and specific immunoadsorbent for the purification of antisoybean trypsin inhibitor antibody. Polymerization of the protein occurred readily, and the water-insoluble polymer retained its immunological properties. After immunoadsorption, the purified antibody formed an immunoprecipitin arc with the trypsin inhibitor (Fig. 2) and it was found to be 90% precipitable. When the electrophoresed trypsin

SOYBEAN

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+ FIG. 2. Double gel diffusion (a), immunoelectrophoresis (b) , pH 3-10 immunoelectrofocusing (c) , and pH 3-6 immunoelectrofocusing (d) patterns of Kunitz soybean trypsin inhibitor developed with its specific antiserum or specific antibody.

inhibitor antiserum and purified antibody anti-rabbit serum, the antibody exhibited the yG-globulin region (Fig. 6) .

were developed with goat a strong precipitin line in

DISCUSSION

A method for the isolation of the soybean trypsin inhibitor by isoelectric focusing in the pH region between pH 3 and 10 has been described (20). Since, most of the impurities present in the commercial preparation are focused in the pH region between 3 and 6 (21) it appeared that electrofocusing in this pH region may be of advantage over the previously described pH range. Indeed, the results obtained indicated that improved resolution in the separation of the inhibitor from contaminating components was achieved by electrofocusing between pH 3 and 6.

444

CATSIMPOOLAS

100 0

1

2

3 Migralion(mm)

FIG. 3. Single diffusion of isolated bean trypsin inhibitor serum.

TRYPSIN

FIG. 4. Radial immunodiffusion antisoybean

trypsin

inhibitor

AND LEUTHNER

4

5

soybean trypsin

6

7

inhibitor

against

antisoy-

soybean trypsin

inhibitor

against

INHIBITOR(,a~)

of isolated serum.

SOYBEAN

TRYPSIN

01I

100

IO Trypsin

445

INHIBITOR

Inhibitor(pgl0.2ml

1000 serum)

FIG. 5. Quantitative precipitin curve. Increasing amounts trypsin inhibitor added to standard volume of antiserum. ~JJ indicates amount of antigen-antibody complex.

of isolated soybean Absorbance at 280

The production of specific soybean trypsin inhibitor antibodies in rabbits by immunization with Kunitz trypsin inhibitor confirms the earlier observations (8, 20) that the inhibitor is antigenic. Since several trypsin inhibitors have been reported to be present in the soybean (l-7)) it would be of interest to examine the antigenicity

FIG. 6. Immunoelectrophoresis and purified antisoybean trypsin antirabbit serum.

of antisoybean trypsin inhibitor antibody (b)

inhibitor serum (a) developed with goat

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CATSIMPOOLAS

AND LEUTHNER

of all the isolated inhibitors. This may lead to a new nomenclature based on immunochemical specificity and identification of the various inhibitors. The isoelectric point-immunodiffusion profile technique described previously (23) may be helpful in this respect. Qualitative immunochemical detection of the inhibitor can be performed by double gel diffusion, immunoelectrophoresis, and immunoelectrofocusing. The latter method offers excellent resolution (21) and may be advantageous in the immunochemical characterization of other inhibitors. Quantitatively, the inhibitor can be determined by both single and radial immunodiffusion. However, the latter technique is considerably more sensitive since amounts of the inhibitor in the 0.3 to 10 ILg range per assay can be quantitated. Thus, in studies in which comparative data on the immunochemical reaction of intact or modified inhibitor are desirable, the order of preference in respect to sensitivity of detection is (1) radial immunodiffusion, (2) quantitative precipitation, and (3) single diffusion. The sensitivity of radial immunodiffusion method may be utilized in determining the degree of cross-precipitation of trypsin inhibitor derivatives, in inhibition studies of trypsin-trypsin inhibitor complexes, and in detecting local conformational changes of antigenic determinants. This method has been successfully applied to the quantitation of Kunitz trypsin inhibitor in extracts of germinating soybean seeds (unplublished results) . Isolation of the trypsin inhibitor antibodies by formation of biologically active water-insoluble polymers of the inhibitor may be useful in producing strong precipitating antibody solutions from weak antisera. This method may have applications other than immunological when a bioactive water-insoluble polymer of the inhibitor is desirable. The availability of specific antisera produced by immunization of animals with various trypsin inhibitors would offer simple and rapid methods for their detection, quantitative estimation, and study of their interrelationships. SUMMARY

Immunization of rabbits with Kunitz soybean trypsin inhibitor results in the formation of antisera that can be utilized for the detection and quantitative determination of this protein. The trypsin inhibitor was isolated from a commercial preparation by isoelectric focusing in the pH region between 3 and 6. The inhibitor was studied by several immunochemical methods including double gel diffusion, immunoelectrophoresis, immunoelectrofocusing, quantitative

SOYBEAN

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precipitation, single diffusion, and radial immunodiffusion. The quantitative aspects of some of these methods are discussed. A water-insoluble polymer of the trypsin inhibitor obtained by the reaction of the protein with ethyl chloroformate was used as an immunoadsorbent for the purification of the antitrypsin inhibitor antibody. REFERENCES KUNITZ, M., J. Gen. Physiol., 30, 291 (1947). BIRK, Y., GERTLER, A., AND KHALEF, S., Biochem. J., 87,281 (1963). RACKIS, J. J., AND ANDERSON, A. L., Biochem. Biophys. Res. Commun., 15, 230 (1964). 4. YAMAMOTO, M., AND IKENAKA, T., J. Biochem. (Tokyo), 62,141 (1967). 5. FRATTALI, V., AND STEINER, R. F., 7, 521 (1968). 6. FRATTALI, V., J. Biol. Chem., 244, 274 (1969). 7. RACKIS, J. J., SASAME, H. A., ANDERSON, R. L., AND SMITH, A. K., J. Am. Chem. SOL, 81, 6265 (1959). 8. CATSIMPOOLAS, N., ROGERS, D. A., AND MEYER, E. W., Cereal Chem., 46, 136 (1969). 9. SVENSSON, H., Arch. Biochem. Biophys., Suppl. 1, 132 (1962). 10. VESTERBERG, O., AND SVENSSON, H., Acta Chem. Sand., 20, 820 (1966). 11. LKB Instruments, Inc., “LKB 8102 Electrofocusing Column-Preliminary Instruction Sheet and Addendum,” Rockville, Maryland, 1967. 12. CATSIMPOOLAS, N., AND MEYER, E. W., J. Agr. Food Chem., 16, 128 (1968). 13. CATSIMPOOLAS, N., AND MEYER, E. W., Arch. Biochem. Biophys. 125, 742 (1968). 14. OUCHTERLONY, O., Acta Path. Microbial. Stand., 26, 507 (1949). 15. GRABAR, P., AND WILLIA~~IS, C. A., Biochim. Biophys. Acta, lo, 193 (1953). 16. SCHEIDEGGER, J. J., Znt. Arch. Allergy Appl. Zmmun. 7, 103 (1955). 17. CATSIMPOOLAS, N., Anal. Biochem., 26, 480 (1968). 18. CATSIMPOOLAS, N., Immunochemistry, 6, 501 (1969). 19. AVRAMEAS, S., AND TERNYNCK, T., J. Biol. Chem. 242, 1651 (1967). 20. CATSIMPOOLAS, N., EKENSTAM, C., AND MEYER, E. W., Biochim. Biophys. Acta, 175, ‘76 (1969). 21. CATSIMPOOLAS, N., Sqi. Tools. 16, 1 (1969). 22. CATSIMPOOLAS, N., Biochim. Biophys Acta, 175, 214 (1969). 1. 2. 3.

Biochemistry,