[62] Proteinase inhibitors from cereal grains

[62]

PROTEINASE INHIBITORS FROM CEREAL GRAINS

723

[52] P r o t e i n a s e I n h i b i t o r s f r o m C e r e a l G r a i n s

By YEHUDITH BIRK Trypsin inhibitors are widely distributed among the cereal grains including 1 wheat, corn, rye, oats, buckwheat, barley, rice, and sorghum. 2 As stated earlier, 3 they have been isolated from whole wheat flour, corn seed, rye and wheat germ, barley, and oats and recently also from rice 4 and rye seeds2 The amino acid sequence of the corn seed inhibito# and the partial sequences of the rye and wheat germ inhibitors 7 have been reported. The preparation and characterization of the trypsin inhibitor from barley, as reported by Mikola and Suolinna,s was described in detail in this series2 Mikola and Kirsi 9 later showed that trypsin inhibitors are present both in the embryos and endosperms of grains of barley, wheat, and rye, the inhibitors in the two respective organs being dissimilar to each other. The presence, in barley, of inhibitors of Aspergillus oryzae proteinases has been known since 1955.l° They were further studied by Mikola and Suolinna, 11 who have purified and characterized a group of isoinhibitors from barley that inhibit the alkaline proteinases of Aspergillus oryzae, Bacillus subtilis, Streptomyces griseus, and Alternaria tenuissima and chymotrypsin. These inhibitors have been selected for detailed description herein. Studies have also been carried out on formation of trypsin inhibitors and of Aspergillus-proteinase inhibitors in developing barley grain 12 and in germinating barley embryos, la Recently, the wheat inhibitor of 1 I. E. Liener and M. L. Kakade, in "Toxic Constituents of Plant Foodstuffs" (I. E. Liener, ed.), p. 8. Academic Press, New York, 1969. J. Xavier-Filho, J. Food Sci. 39, 422 (1974). 3 B. Kassell, this series Vol. 19 [66a]. 4 T. Horiguchi and K. Kitagishi, Plant Cell Physiol. 12, 907 (1971). A. Polanowski, Acta Soc. Bot. PoI. 43, 27 (1974). 6 K. Hochstrasser, K. Illchmann, and E. Werle, Hoppe-SeyIer's Z. Physiol. Chem. 351, 721 (1970). 7 K. Hochstrasser, M. Muss, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem. 350, 249 (1969). '~J. Mikola and E.-M. Suolinna, Eur. J. Biochem. 9, 555 (1969). 0j. Mikola and M. Kirsi, Acta Chem. Scand. 26, 787 (1972). 10K. Matsushima, J. Agric. Chem. Soc. Jpn. 29, 405 (1955). 11j. Mikola and E.-M. Suolinna, Arch. Biochem. Biophys. 144, 566 (1971). 15M. Kirsi, Physiol. Plant. 119, 141 (1973). 1~ M. Kirsi, Physiol. Plant. 32, 89 (1974).

724

[62]

NATURALLY OCCURRING PROTEASE INHIBITORS

Tribolium

castaneum

larval

proteinases 14 has

been

purified

and

characterized. ~5

Aspergillus Proteinase Inhibitor from Barley A s s a y M e t h o d 11

Principle. The inhibition of hydrolysis of casein by the alkaline proteinase of Aspergillus oryzae is measured by the change in absorbance at 280 nm of a trichloroacetic acid supernatant of the reaction mixture. Reagents Buffer 1 : sodium glycinate, 50 mM, pH 10.3 Substrate: casein, 1%, in buffer 1 Buffer 2: buffer 1 containing 20 mM EDTA. Enzyme solution: Aspergillus oryzae proteinase (crude, type II Sigma Chemical Corp., St. Louis, Missouri) 600 t~g per milliliter of buffer 2 Inhibitor solution: Dilute with buffer 2 to a concentration to give about 50% inhibition of the enzyme. Trichloroacetic acid: 5% in water (w/v)

Procedure. Pipette 1 ml of substrate into a series of test tubes in a 35 ° bath. In a second series of small test tubes, place 2 ml of enzyme solution plus 2 ml of diluted inhibitor for unknowns. In the same way pipette 2 ml of enzyme solution and 2 ml of buffer for control containing no inhibitor. Allow to stand at room temperature for 10 min. Timing with a stopwatch, add 1 ml of the enzyme and inhibitor mixture (or enzyme and buffer mixture) to the substrate and mix well. Incubate at 35 °. Exactly 30 rain later, stop the reaction by the addition of 3 ml of 5% trichloroacetic acid. Allow the tubes to stand at room temperature for 30 min for precipitation. Then clarify by centrifugation. Measure the absorbance at 280 nm against a blank without enzyme or inhibitor. Subtract the absorbance of the unknowns from the absorbance of the enzyme standard. Definition o] Unit. One unit of enzyme is the amount that causes an increase of 1 unit of absorbance at 280 nm per minute of digestion of casein under the standard conditions. Enzyme-inhibiting activity is expressed as units of enzyme inhibited. This is based on the casein digestion method of Kunitz, TM originally devised for the determination of 14S. W. Applebaum and A. M. Konijn, J. Insect Physiol. 12, 665 (1966). 12R. Lippmann, Y. Birk, and S. W. Applebaum, personal communication. le M. Kunitz, J. Gen. Physiol. 30, 291 (1947).

[62]

725

P R O T E I N A S E I N t I I B I T O R S FROM CER EA L GRAINS

TABLE I PURIFICATION OF Aspergillus PROTEINASE INHIBITOR FROM PIRKKA BARLEYa

Fraction Extract Ammonium sulfate precipitate, 20-40 % Same after dialysis DEAE-cellulose peak Diethylether precipitate Sephadex G-75 peak, freeze-dried

Volume (ml) 2160 232 280 342 60 190 mg

Total activity (units) 268,000 196,000 180,000 123,000 108,000 98,000

Specific activityb Re(units/ eovery E2s0) (%) 19c 32 102 370 580

100 92 64 55 50

j. Mikola and E.-M. Suolinna, Arch. Biochem. Biophys. 144, 566 (1971). b Ratio of activity (units/ml) to the E280 of the solution (1.0-cm cuvette). c Determined separately after removal of smMl-molecular compounds by gel filtration.

a

trypsin and later applied to the determination of trypsin inhibitors and chymotrypsin. The original method is described in detail in a previous volume of this series. 17 Purification Procedure n The procedure is summarized in Table I. All operations are effeeted at -I-5 °, unless otherwise stated. Between the different stages of purification the inhibitor solutions should be frozen rapidly and then stored at - - 1 8 ° . Step I. Extraction. The starting material is P i r k k a barley (a Finnish six-row variety). Mix thoroughly 1600 g of finely ground barley with 3200 ml of water. Incubate the thick slurry (pH 5.9) at 5 ° for 2 hr without agitation. Then separate the extract by centrifugation and allow to stand overnight a 5 °. Step 2. Ammonium Sulfate Precipitation. Add solid a m m o n i u m sulfate to 20% saturation, stir the suspension for half an hour, and remove the inactive precipitate by centrifugation. To precipitate the active m a t e rial, add to the supernatant fraction ammonium sulfate to 40% saturation and stir for 1 hr. Collect the active precipitate by centrifugation and dissolve in one-tenth the original volume of water. Remove the small amount of insoluble material by ccntrifugation. Dialyze the active solution extensively against 35 m M Tris chloride buffer, p H 7.5. 1~M. Laskowski, St., this series Vol. 2 [3].

726

NATURALLY OCCURRING PROTEASE INHIBITORS

[62]

Step 8. Chromatography on DEAE-Cellulose. Prepare a 5 X 14 cm column of DEAE-eellulose, equilibrated with 35 mM Tris chloride buffer, pH 7.5--the starting buffer. Apply half of the solution of step 2 (about 140 ml) to the column. Elute inert proteins first with 100 ml of the starting buffer and then elute the active material with 125 mM Tris chloride buffer, pH 7.5. Treat the other half of the solution similarly, and combine the active eluates. Step 4. Precipitation by Ethanol and Diethyl Ether. All the operations should be effected at --15 °, and the organic solvents should be chilled to --18 ° before use. Mix 1 volume of the active fraction from step 3 with 3 volumes of 94% ethanol. After 30 min, remove the large, inactive precipitate by centrifugation. To the supernatant fraction add 2 volumes of diethyl ether. After 30 min collect the active precipitate by centrifugation. Dissolve in one-fifth the original volume of 25 mM acetic acid and lyophilize. Step 5. Gel Filtration on Sephadex G-75. Prepare a 2.5 X 95 cm column of Sephadex G-75, equilibrated with sodium acetate buffer pH 4.9, ionic strength 0.1. Dissolve the active lyophilization residue from step 4 in a small volume of the same buffer and apply to the column. Elute with the same buffer, collecting 6.7-ml fractions at the rate of 30 ml/hr at a temperature of 25 °. The active peak appears approximately in tubes 200-300. Pool the active peak and dialyze against 9 volumes of water for 18 hr. Adjust the dialyzed solution to pH 7.5 with 1 N NaOH and precipitate the inhibitors with ethanol and ether exactly as in step 4. Dissolve the precipitate in 25 mM acetic acid and lyophilize. Properties m

Stability. The inhibitor is heat labile. Incubation at 100 ° for 15 min at pH 5.4 and ionic strength of 0.05 results in complete inactivation. Purity. As found from polyacrylamide gel electrophoresis and isoelectric focusing, the purified inhibitor consists of 4-5 isoinhibitors, similar in molecular size but with different isoelectric points, which were already present at the beginning of the purification procedure. The inhibitor preparation is relatively free from contaminating proteins. Physical Properties. The molecular weight is 25,000 as figured from the elution volume of the active peak from the Sephadex G-75 column. A solution of 1 mg of inhibitor per milliliter has an absorbance of 0.882 at 280 nm. Specificity. The inhibitor (preparation from step 5) inhibits the alkaline proteinases of Aspergillus oryzae, Streptomyces griseus (casein substrate, pH 10.3), Alternaria tenuissima (casein substrate pH 9.5), and

[62]

PROTEINASE INHIBITORS FROM CEREAL GRAINS

727

TABLE II AMINO ACID COMPOSITION OF BARLEY Aspergillus PROTEINASE I N H I B I T O R a Residues/MW 25,000 in Amino acid Aspartic acid Glutamic acid Glycine Alanine Valine Leucine Isoleucine Serine Threonine Half-cystine Methionine Proline Phenylalanine Tyrosine Histidine Lysine Arginine Amide-NH2 Tryptophan

20-hr hy- 72-hr hydrolyz~te drolyzate 15.1 21.3 12.7 18.8 19.5 8.1 7.6 14.0 8.9 0.4 6.2 20.3 4.2 2.6 1.6 11.8 8.7 12.0 ND ~

14.1 21.2 13.3 19.8 25.1 9.0 11.3 12.5 8.4 0.4 6.1 21.3 4.9 2.4 1.7 12.0 8.9 -ND

Mean 14.6 21.3 13.0 19.3 25.1 b 8.6 11.3 b 14.4 c 9.1 ¢ 0.4 6.2 20.9 4.6 2.5 1.7 11.9 8.8 11.1 a ND

a j. Mikola and E.-M. Suolinna, Arch. Biochem. Biophys. 144, 566 (1971). b 72-hr value. c Extrapolated to zero time. Corrected for destruction of serine and threonine. Not determined.

Bacillus subtilis (casein s u b s t r a t e , p H 10.3) a n d also c h y m o t r y p s i n (casein s u b s t r a t e , p H 7.6, and g l u t a r y l - L - p h e n y l a l a n i n e p - n i t r o a n i l i d e s u b s t r a t e , p H 7.6). I t does n o t i n h i b i t t r y p s i n ( B A P A s u b s t r a t e , p H 8.2), t h e e n d o p e p t i d a s e s of g e r m i n a t i n g b a r l e y ( g e l a t i n s u b s t r a t e , p H 5.4), p e p s i n ( h e m o g l o b i n s u b s t r a t e , p H 2.0), a n d p a p a i n (casein s u b strafe, p H 7.6). I t also has no effect on t h e n e u t r a l p r o t e i n a s e s of Aspergillus oryzae, Streptomyces griseus, a n d Bacillus subtilis (casein s u b s t r a t e s , p H 6.5). Kinetic Properties. T h e i n h i b i t i o n of Aspergillus p r o t e i n a s e is l i n e a r up to 65% a n d r e a c h e s its m a x i m u m a t a b o u t 95% i n h i b i t i o n . T h a t of Bacillus subtilis is l i n e a r up to 3 0 % , w i t h a m a x i m u m of a b o u t 75% inhibition. I n h i b i t i o n of Streptomyces griseus a n d Alternaria tenuissima p r o t e i n a s e s has no l i n e a r section, a n d it reaches m a x i m u m i n h i b i t i o n a t

728

NATURALLY OCCURRING PROTEASE INHIBITORS

[63]

75% and 80%, respectively. Inhibition of chymotryptic digestion of casein is linear up to 20% with a m a x i m u m of 75%, and t h a t of glutarylL-phenylalanine p-nitroanilide up to 70% with a m a x i m u m of 95%. A m i n o Acid Composition. The amino acid composition expressed as residues per molecular weight of 25,000 is shown in T a b l e II. The inhibitor contains no carbohydrates.

[63] P r o t e i n a s e

Inhibitors

from Potatoes

B y YEHUDITH BIRK

A large n u m b e r of proteinase inhibitors have been separated and isolated from potatoes. I n spite of a certain confusion in the nomenclature used by different groups of investigators, the potato proteinase inhibitors seem to belong to four m a j o r groups. The chymotrypsin inhibitor I, described in an earlier volume in this series, 1 has been found to be similar to potato proteinase inhibitor I. ~,3 C h y m o t r y p s i n Inhibitor I is composed of four subunit components, 4,5 which will be described in detail in the present section. The second group comprises proteinase inhibitors I I a and I I b , studied extensively by I w a s a k i and associates2 -1° Both of them inhibit stoichiometrically chymotrypsin and the bacterial proteinase nagarse, but inhibitor I I a inhibits also trypsin. Inhibitor I I a is p r o b a b l y identical with inhibitor A7b described by Belitz and associates as one of thirteen inhibitors of trypsin and chymotrypsin isolated by isoelectric focusing21-~3 To the 1C. A. Ryan and B. Kassell, this series Vol. 19 [66f]. 2 T. Kiyohara, T. Iwasaki, and M. Yoshikawa, J. Biochem. (Tokyo) 73, 89 (1973). T. Kiyohara, M. Fujii, T. Iwasaki, and M. Yoshikawa, J. Biochem. (Tokyo) 74, 675 (1973). 4j. C. Melville and C. A. Ryan, Arch. Biochem. Biophys. 138, 700 (1970). 5j. C. Melville and C. A. Ryan, J. Biol. Chem. 247, 3445 (1972). T. Iwasaki, T. Kiyohara, and M. Yoshikawa, J. Biochem. (Tokyo) 70, 817 (1971). ' T . Iwasaki, T. Kiyohara, and M. Yoshikawa, J. Biochem. (Tokyo) 72, 1029 (1972). ST. Iwasaki, T. Kiyohara, and M. Yoshikawa, J. Biochem. (Tokyo) 73, 1039 ( 1973). T. Iwasaki, T. Kiyohara, and M. Yoshikawa, J. Biochem. (Tokyo) 74, 335 (1973). I°T. Iwasaki, T. Kiyohara, and M. Yoshikawa, J.Biochem. (Tokyo) 75, 843 (1974). ~1H. D. Belitz, K. P. Kaiser, and I/:. Santarius, Biochem. Biophys. Res. Commun. 42, 420 (1971). ~2K. Santarius and H. D. Belitz, J. Biochem. (Tokyo) 154, 206 (1974).

1, K. P. Kaiser, L. C. Bruhn, and H. D. Belitz, Z. Lebensm.-Unters. Forsch. 154, 339 (1974).