crystalline trypsin inhibitor from colostrum

CRYSTALLINE TRYPSIN INHIBITOR FROM COLOSTRUM*

BY M. LASKOWSKI, JR.,t AND M. LASKOWSKI

(From the Department of Biochemistry, Marquette University School of Medicine, Milwaukee, Wisconsin)

(Received for publication, December 18, 1950)

A chance observation was made in this laboratory that raw cream pos- sessed trypsin-inhibiting activity. The inhibiting activity of various samples varied greatly. The explanation for this variation became apparent when it was found that bovine colostrum inhibited trypsin to a much greater extent than did cream. While in the case of cream the detection of the inhibitor was possible only after a considerable concentration of the inhibitory fraction, in colostrum the inhibitor could be detected directly after 1: 10 dilution.

The purpose of this paper is to present methods for the concentration of the trypsin inhibitor from bovine colostrum, the preparation of the crystalline trypsin-trypsin inhibitor compound, and, finally, the crystallization of the free inhibitor. Some properties of both crystalline substances are described.

EXPERIMENTAL

The inhibitor was determined by the spectrophotometric method of Ku- nitz (2). Recrystallized trypsin from Armour and Company has been used as a standard. It gave an activity curve considerably lower than that described by Kunitz. For routine work, a solution of trypsin containing 500 y per ml. was prepared and kept frozen in 0.2 ml. aliquots in small stoppered test-tubes. Just before use it was thawed and diluted to contain 10 y per ml. In testing for activity it was found convenient to use a minimum of two sets of four test-tubes each. The tubes of the first set contained no inhibitor and 0, 2, 4, and 8 y of trypsin respectively. The second set contained the same gradation of trypsin plus 0.1 ml. of the solution of inhibitor in all four of the test-tubes. When the amount of the inhibitor could not be predicted, the third and fourth sets were added, containing the same gradation of trypsin and 0.1 ml. of solution of inhibi-

*Aided by a research grant from the National Institutes of Health, United States Public Health Service, and by a grant from Clara Woltring Memorial admin- istered by the Marquette University School of Medicine. A preliminary note de- scribing the discovery of the inhibitor in colostrum and its partial purification has been published (1).

t Present address, Department of Chemistry, Cornell University, Ithaca, New York.

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564 TRYPSIN INHIBITOR FROM COLOSTRUM

tor diluted 1: 10 and 1: 100 respectively. The amount of the inhibitor was expressed in actual micrograms of trypsin inhibited. Only in the experiments with crystalline inhibitor was the activity of trypsin read from the standard curve of Kunitz (2) and expressed in his units. The degree of the purification was followed not by the total N, but by the spectrophotometric reading at 280 mp. The potency of the inhibitor solution was expressed by means of the ratio, micrograms of trypsin inhibited to Ezso.

It soon became evident that the inhibitor from colostrum and trypsin form a stoichiometric compound. Even with a crude solution of the inhibitor, the direct proportionality was apparent (Fig. 1).

Before we attempted the purification of the inhibitor, its distribution in bovine and human colostrum was investigated. Only one experiment was

ml.OF INHIBITOR

FIG. 1. Stoichiometric relationship between the inhibitor from colostrum and trypsin. Solution of inhibitor after step (2) of purification procedure.

carried out on the colostrum of the same cow. The following figures were obtained: 1 ml. of the 1st days colostrum inhibited 600 y of trypsin, while 1 ml. of the 2nd days colostrum inhibited 200 y of trypsin. Colostrum of the 1st day from different cows inhibited from 120 to 600 y of trypsin per ml.

The colostrum of ten human subjects has been investigated. The human colostrum contained less inhibitor; the highest figure obtained was 60 y of trypsin inhibited per ml. The maximum individual content oc- curred between the 1st and 3rd days after delivery. Fig. 2 represents the results of three selected cases to indicate this variation. On the 5th day no inhibitor could be detected in human milk by direct measurement.

Early during this work an assumption was made that the inhibitor from colostrum has at least some properties of solubility similar to those of the

1 We are indebted to Dr. W. A. D. Anderson and Dr. H. B. Benjamin for supply- ing us with these samples.


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M. LASKOWSKI, JR., AND M. LASKOWSKI 565

trypsin inhibitor from pancreas. This assumption was found justified. The general trend of the purification procedure resembled, therefore, that of Kunitz and Northrop (3). The method finally adopted differed from the procedure previously described (1) in that the first three steps were omitted, although, as a result, yield was sacrificed in favor of speed of operation. By omitting prolonged filtrations of large volumes and by in- troducing a direct precipitation of the bulk of proteins with 2.5 per cent trichloroacetic acid, the method was simplified,, but the loss in the first

FIQ. 2. Variation in the content of trypsin inhibitor in human milk during the first 5 days after delivery.

step was approximately 75 per cent of the total inhibitor. The authors still believe that in laboratories where the handling of large volumes of liquid does not offer a major difficulty the original method would be both cheaper and more efficient. The final procedure is described in detail below.

1. To each liter of colostrum, 1 liter of water and 1 liter of 7.5 per cent trichloroacetic acid are added (final concentration 2.5 per cent in respect to trichloroacetic acid). The mixture is heated to 80 with constant stirring and allowed to stand at that temperature for 5 minutes. It is then cooled and filtered by suction with Whatman filter paper No. 1 or No. 4. The heavy cheese-like precipitate is discarded. The filtrate is brought to 80


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per cent saturation by addition of solid ammonium sulfate (603 gm. per liter) and allowed to stand overnight at room temperature. The slight precipitate which floats on the surface is removed by filtration with suction through Whatman filter paper No. 4. The filtrate is discarded.

2. The precipitate (plus crude fractions from previous preparations) is dissolved in 7 volumes of water with the aid of a Waring blendor, and enough trichloroacetic acid is added to attain a final concentration of 2.5 per cent. The mixture is heated to 80 for 5 minutes, cooled, and filtered with suction through Whatman filter paper No. 4. The precipitate is washed with 2.5 per cent trichloroacetic acid and discarded. The com- bined filtrate and washings are brought to 80 per cent saturation of ammonium sulfate and the mixture is filtered with suction through Whatman filter paper No. 4. The filtrate is discarded.

3. After the precipitate has been dissolved in 5 volumes of water (War- ing blendor), the solution is adjusted to pH 6.5 with 1 N NaOH (glass electrode) and brought to 30 per cent saturation of ammonium sulfate (22.6 gm. per 100 ml). After addition of 5 gm. of Celite 545 (Johns-Man- ville) per 100 ml., the mixture is filtered with suction through Whatman filter paper No. 4. The filtration is slow. The dark precipitate is discarded. The filtrate is brought to 70 per cent saturation of ammonium sulfate (26.7 gm. per 100 ml.), which results in a formation of rubber-like precipitate. The latter is filtered with suction through Whatman filter paper No. 4, and kept as Precipitate 3. Some inhibitor can be saved by adjusting the filtrate to pH 2 and 80 per cent saturation, filtering, and adding it to the next preparation (step (2)).

4. Precipitate 3 is dissolved in 5 volumes of water, trichloroacetic acid is added to attain a concentration of 2.5 per cent, and the solution is shaken thoroughly in a separatory funnel with an equal volume of ether. It is then allowed to stand for 2 hours. The water layer is separated and kept. The ether layer is washed with one-half of the previous amount of water, the washings are added to the next preparation, and the rest is discarded. The liquid, which is still saturated with ether, is brought to 30 per cent saturation of ammonium sulfate (22.6 gm. per 100 ml.). The rubber-like precipitate which forms is filtered through Whatman filter paper No. 4 and the filtrate is discarded.

5. The precipitate is dissolved by stirring in a minimum amount of water and the solution is dialyzed against distilled water overnight.2 The dialyzed liquid is adjusted to pH 5.5 with 1 N NaOH and is treated with an equal volume of methanol. The solution is allowed to stand in a deep

* Sizable amounts of inhibitor are lost during dialysis, since the inhibitor slowly passes across the cellophane membrane. The dialysis was, however, necessary for the succeeding precipitation with methanol.


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freeze (-18) for an hour and is centrifuged in a conical head of a refrigerated centrifuge at -10 for 15 minutes at 3500 r.p.m. The precipitate may be used in the next preparation (step (2)). The slightly cloudy supernatant is treated with 4 times the previous volume of methanol (a total of 5 volumes). It is set in the deep freeze for an hour and is centrifuged at - 10 for 20 minutes at 3500 r.p.m. The supernatant is discarded.

Crystallization of Trypsin-Trypsin Inhibitor Compound-Precipitate 5 is dissolved in a minimum amount of water and the solution is adjusted to pH 3. The amount of trypsin required to neutralize the inhibitor com- pletely is accurately determined on an aliquot. The calculated amount of recrystallized trypsin (Armour and Company) is dissolved in a small vol-

FIG. 3 FIG. 4

FIG. 3. Crystalline trypsin-trypsin inhibitor compound (usual form), X 240. FIG. 4. A different form of crystalline trypsin-trypsin inhibitor compound, X 300.

ume of 0.0025 N HCl and added to the solution of inhibitor. The solution is brought to pH 8, with the use of Kunitz borate buffer of pH 9, and is allowed to stand in the refrigerator for an hour, after which it is readjusted to pH 5.5 with 1 N HCl. If a precipitate appears at that time, it should be centrifuged, washed with a little acetate buffer at pH 5.5, and discarded. The solution should be free of the excess of either trypsin or inhibitor when tested at 1: 100 dilution. If it is not, the whole step should be repeated. To the solution an equal volume of saturated ammonium sulfate is added and then a few drops more, until the sign of first turbidity. After seeding with the crystals of trypsin-trypsin inhibitor compound, the solution is allowed to stand for 3 to 4 days at room temperature. The mixture first becomes gelatinous; then needles of the crystalline compound slowly form (Fig. 3). If the crystallization does not occur at that time, the compound should be precipitated with ammonium sulfate at 70 per cent saturation,


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redissolved in a minimum amount of water, adjusted with the saturated solution of ammonium sulfate to the first sign of turbidity, and seeded again. Crystals are separated by centrifugation with the high speed attachment of the refrigerated centrifuge. Recrystallization is carried out by dissolving the crystals in a minimum amount of water and adjusting with a saturated solution of ammonium sulfate until the faint turbidity. Only once crystals were observed in a different shape (Fig. 4). On recrystallization the usual needle form was obtained.

Crystallization of Free Trypsin Inhibitor from Colostrum-The twice recrystallized trypsin-trypsin inhibitor compound is dissolved in a small amount of water. An equal volume of 5 per cent trichloroacetic acid is

FIG, 5. Crystalline trypsin inhibitor from colostrum, X 240

then added and the solution is allowed to stand for an hour. The precipitated trypsin is centrifuged and may be saved for the next preparation. The solution of inhibitor is heated to 80 for 5 minutes, cooled to 25, and filtered through a small fluted filter (Whatman No. 4) to remove a slight precipitate, which is discarded. The filtrate is brought to 80 per cent saturation with ammonium sulfate and centrifuged in the high speed attachment of the refrigerated centrifuge at about 25,000 r.p.m. The precipitate is dissolved in a minimum amount of 0.05 M acetate buffer at pH 5.5, an equal amount of saturated ammonium sulfate is added, and then very carefully an excess of a few drops until the minute turbidity appears. Crystals form almost immediately (Fig. 5). The solution is allowed to stand overnight and is centrifuged in a high speed attachment. Recrystallization is carried out in the same manner as the first crystallization.


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The balance sheet of one of the experiments on purification up to the stage of formation of trypsin-trypsin inhibitor compound is shown in Ta- ble I. The purification was also followed by an occasional electropho- retie pattern.3 Fig. 6 shows the results of electrophoresis of purified inhibitor after step (5). Fig. 7 shows the pattern of twice recrystallized trypsin-trypsin inhibitor compound, indicating the presence of at least one major impurity. Fig. 8 shows the pattern obtained on the inhibitor SO~U- tion after splitting the complex, but prior to crystallization of the free inhibitor. The pattern is much more uniform and indicates the presence of only small amounts of impurities. The isoelectric point of the inhibitor is somewhat lower than pH 5.47. As yet it has not been possible to secure enough of the recrystallized inhibitor to obtain its pattern.

The recrystallized inhibitor was dialyzed against distilled water for 24 hours and lyophilized. Several samples were weighed and the extinction

TABLE I Balance Sheet of Experiment with 4 Gallons of Colostrum

Purification step No. Total amount of trypsin inhibited potency trypain inhibited (7) lzlso

Sm.

1 1.7 43 2 1.1 130 3 1.0 220 4 0.6 550 5 0.4 730

coefficient at 280 rnp was determined. The inhibitor was again dialyzed and lyophilized, and the extinction was determined once more. No significant changes in coefficient were found after the second dialysis. The average value for the factor was found to be 2.00, which is very high when compared with 0.500 for chymotrypsin CZ, 0.585 for trypsin, 0.600 for chymotrypsin B, and 1.10 for soy bean inhibitor. The high value of the factor suggests that the total amount of tyrosine plus tryptophan in the inhibitor molecule is low.

Fig. 9 shows the ultraviolet absorption spectra of the solution of 0.9 mg. per ml. of recrystallized inhibitor in 0.1 N NaOH and 0.1 N HCl. The curve in 0.1 M acetate buffer, pH 5.5, was almost identical with the acid curve and is not reproduced. It is interesting to note the striking differ- ence between acid and alkaline spectra, which is characteristic for predom- inance of tyrosine. The maximum in acid (277 mp) and the minimum in alkali (276 mp) almost correspond to the same point. The content of

3 A more detailed electrophoretic study of the trypsin-trypsin inhibitor compound will be published separately.


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tyrosine and tryptophan was calculated according to Goodwin and Mor- ton (4) from the 0.1 N NaOH curve and the following figures were obtained: tyrosine 5.3 per cent, tryptophan 0.15 per cent. Since the method gives accurate results only when the ratio of the two components is within

AmA- ASCENDING - - DESCENDING

FIG. 6. Electrophoretic pattern of the inhibitor preparation after step (5) in 0.1 p acetate buffer at pH 5.5, after 180 minutes.

ASCENDING - -DESCENDING

FIG. 7. Electrophoretic pattern of twice recrystallized trypsin-trypsin inhibitor compound in 0.1 p acetate buffer at pH 5.2, after 100 minutes.

DESCENDING- - ASCENDING FIG. 8. Electrophoretic pattern of free inhibitor obtained after splitting the

trypsin-trypsin inhibitor compound, but prior the crystallization of the pure inhibitor. Acetate buffer 0.1 /J, pH 5.47,80 minutes.

the limits of 1: 20 or 20 : 1, the presence of even a small amount of tryptophan may be doubted.

The potency of the new trypsin inhibitor could have been expressed either in actual micrograms of trypsin inhibited or in relative potency compared to the soy bean trypsin inhibitor. The preparation of trypsin used throughout this work gave a lower activity curve than the standard curve reported by Kunitz (2). Hence, the values obtained in a neutrali- zation experiment would have been significant only in respect to the prepa-


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ration used. The second alternative was therefore chosen, and the new inhibitor was compared to soy bean inhibitor.4 The experiment was prepared as follows: Each tube contained 25 y of trypsin. To one series of tubes crystalline soy bean inhibitor was added in steps of 2.5 r; to a second, crystalline inhibitor from colostrum was added in steps of 1.25 y. The activity of all tubes was read from the standard curve of Kunitz and

1400 -

1200 - > !I g? 1000 -

1700 1700

IGOO IGOO

1400

1200 > !I g? 1000

;: 800 u k 0 GO0

400

200

240 ZGO 280 3~0 320 340 WAVELENGTH irIp

FIG. 9. Ultraviolet absorption spectra of recrystallized inhibitor from colostrum. Solution of inhibitor 0.9 mg. per ml., 1 cm. silica cell, Beckman spectrophotometer.

the degree of inhibition was calculated in the units of Kunitz. The results are shown in Fig. 10. It is obvious that, per unit of weight, inhibitor from colostrum is 2.3 times as potent as soy bean inhibitor. Since both inhibitors were assayed against the same preparation of trypsin, it seems justified to assume that the ratio found in this experiment may be trans- ferred to any other preparation of trypsin. Kunitz (2) found that 1 y of soy bean inhibitor neutralized 1 y of his standard trypsin; therefore 1 y of the inhibitor from colostrum would be equivalent to 2.3 y of the stand-

4 We are indebted to Dr. M. Kunitz for this preparation.


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ard trypsin of Kunltz. Of course, this reasoning is valid only on the assumption that both inhibitors are (or are not) reacting to the same degree with the inactive trypsin.

DISCUSSION

It has been known from the time of Ehrlich (5) that the new-born mammal can directly absorb the immune bodies from colostrum. The mechanism of this process, however, remained obscure. It may be worth

2.5 5.0 z5 IO 12.5 I aOF INHIBITOR PER TUBE

FIG. 10. Comparison of the inhibiting potencies of soy bean inhibitor and the inhibitor from colostrum. Each tube contained 25 y of trypsin and the indicated amounts of one of the inhibitors. After subtraction of the values of the blanks the activity was read from the standard curve of Kunitz (2) and expressed in his units.

while to quote Howe (6) verbatim: The function of colostrum has not been well understood; the most common explanation is that it acts as a purgative. In the twenties, due to the work of Smith and Little (7) and Orcutt and Howe (8), at least one side of this phenomenon was elucidated; namely, the absence of immune globulins in the blood of the new-born, their presence in colostrum, and their appearance in the circulation of the new-born as early as 3 hours after feeding colostrum. The immune globulins of colostrum have been recently investigated by Smith (9), and the entire problem has been reviewed by McMeekin and Polis (10).

The discovery of a powerful trypsin inhibitor in colostrum offers an explanation for the next obscure point in the mechanism of transmission


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of immune globulins. It explains how the immune globulins can escape the proteolytic digestion, particularly if one takes into account the low gastric acidity of the new-born and consequently an impaired peptic digestion. An observation made by Smith and Little (11) for a different purpose can be used as evidence that a considerable amount of protein actually escapes digestion: The distended fourth stomachs frequently encountered at the autopsies, filled with colostral milk, and the presence of cougulable protein in the contents of the ileum5 suggested the hypothesis that the protein in the urine may be associated with colostrum.

Our thanks are due to Miss M. L. Trautmann for assistance, to Miss V. Kubacki for electrophoretic patterns, to Mr. L. C. Massopust for photo- graphs and drawings, and to the Bloehowiak Dairy Company and their producers for generous supplies of colostrum and for splendid cooperation.

SUMMARY

The presence of large amounts of trypsin inhibitor in bovine colostrum has been discovered. Much smaller quantities were found in human colostrum.

Trypsin inhibitor from bovine colostrum has been purified. A crystalline compound composed of trypsin and trypsin inhibitor has been obtained. This compound was inactive either as trypsin or as trypsin inhibitor. After the compound was split into trypsin and inhibitor, both fractions were found active. From the latter fraction crystalline inhibitor was obtained. The details of the methods of crystallization have been presented, and some of the properties of the inhibitor have been described.

BIBLIOGRAPHY

1. Laskowski, M., Jr., and Laskowski, M.? Federation Proc., 9, 194 (1959). 2. Kunita, M., J. Gen. Physiol., 30, 291 (1947). 3. Kunitz, M., and Northrop, J. H., J. Gen. Physiol., 19, 991 (1936). 4. Goodwin, T. W., and Morton, R. A., Biochem. J., 40,628 (1946). 5. Ehrlich, P., 2. Hyg. u. Infektionskrankh., 12, 183 (1892). 6. Howe, P. E., J. Biol. Chem., 49, 115 (1921). 7. Smith, T., and Little, R. B., J. Exp. Med., 36,181,453 (1922); 37, 671 (1923). 8. Orcutt, M. L., and Howe, P. E., J. Esp. Med., 36, 291 (1922). 9. Smith, E. L., J. BioZ. Chem., 164, 345 (1946); 166, 665 (1946).

10. McMeekin, T. L., and Polis, B. D., Advances in Protein Chem., 6, 291 (1949). 11. Smith, T., and Little, R. B., J. Exp. Med., 39, 303 (1924).

6 Italics ours.


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M. Laskowski, Jr. and M. Laskowski

FROM COLOSTRUMCRYSTALLINE TRYPSIN INHIBITORARTICLE:

1951, 190:563-573.J. Biol. Chem.

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crystalline trypsin inhibitor from colostrum

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