CRYSTALLINE

BY

(From

the

TRYPSIN

INHIBITOR

M. LASKOWSKI,

Department

of

Biochemistry,
Milwaukee,

(Received

JR.,t

AND

Marquette
Wisconsin)

for publication,

FROM

COLOSTRUM*

M. LASKOWSKI
University

December

School

of

Medicine,

18, 1950)

EXPERIMENTAL

The inhibitor was determined by the spectrophotometric

method of Kunitz (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 administered by the Marquette University
School of Medicine.
A preliminary
note describing 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.
563

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A chance observation was made in this laboratory that raw cream possessed 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.

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

trypsin.

Solution

relationship
of inhibitor
after

INHIBITOR
step

between
the inhibitor
from
(2) of purification
procedure.

colostrum

and

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 huThe colostrum of ten human subjects has been investigated.
man colostrum contained less inhibitor; the highest figure obtained was
60 y of trypsin inhibited per ml. The maximum individual content occurred 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
ing

1 We are
us with

indebted
to Dr.
these samples.

W. A. D.

Anderson

and

Dr.

H. B. Benjamin

for

supply-

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ml.OF

FIG. 1. Stoichiometric

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 introducing 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

of trypsin

inhibitor

in human milk during

the

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
It is then cooled
and allowed to stand at that temperature for 5 minutes.
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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FIQ. 2. Variation
in the content
first 5 days after delivery.

566

TRYPSIN

INHIBITOR

FROM

COLOSTRUM

* 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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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 combined 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 (Waring 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-Manville) 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

M.

LASKOWSKI,

JR.,

AND

M.

567

LASKOWSKI

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 completely is accurately determined on an aliquot.
The calculated amount of
recrystallized trypsin (Armour and Company) is dissolved in a small vol-

3. Crystalline
4. A different

trypsin-trypsin
form of crystalline

FIG.

inhibitor
compound
trypsin-trypsin

(usual
inhibitor

form),
X 240.
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
If a precipitate appears at that time, it should
to pH 5.5 with 1 N HCl.
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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FIG.
FIG.
FIG.

568

TRYPSIN

INHIBITOR

FROM

COLOSTRUM

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

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
(Fig. 5). The solution is
appears. Crystals form almost immediately
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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FIG, 5. Crystalline

M.

LASKOWSKI,

JR.,

AND

M.

569

LASKOWSKI

TABLE

Balance
Purification

step No.

Sheet

of Experiment
Total

with

amount of trypsin

Gallons of Colostrum

inhibited

potency

trypain

inhibited
lzlso

(7)

Sm.

43

1.7

2
3
4
5

1.1
1.0

130
220

0.6
0.4

550
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 difference between acid and alkaline spectra, which is characteristic for predominance 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
pound will be published separately.

study

of the trypsin-trypsin

inhibitor

com-

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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 Table I. The purification was also followed by an occasional electrophoretie 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~Ution 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

570

TRYPSIN

INHIBITOR

FROM

COLOSTRUM

tyrosine and tryptophan was calculated according to Goodwin and Morton (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

AmAASCENDING

ASCENDING

DESCENDING

preparation

after step (5) in 0.1

-DESCENDING

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

DESCENDING-

inhibitor

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.
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

the limits

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 neutralization experiment
would have been significant
only in respect to the prepa-

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FIG. 6. Electrophoretic
pattern of the inhibitor
p acetate buffer at pH 5.5, after 180 minutes.

M.

LASKOWSKI,

JR.,

AND

M.

571

LASKOWSKI

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
1700
IGOO

1200
>
!I
g? 1000

;:
u
k
0

800

GO0

400

200

240
FIG.

Solution

9. Ultraviolet
of inhibitor

ZGO

280
WAVELENGTH

320

3~0

340

irIp

absorption spectra of recrystallized

inhibitor
from colostrum.
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 transferred 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 stand4 We are indebted

to Dr. M. Kunitz

for this preparation.

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1400

572

TRYPSIN

INHIBITOR

FROM

COLOSTRUM

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

aOF

5.0
INHIBITOR

z5

IO

12.5

PER TUBE

FIG. 10. Comparison of the inhibiting

potencies
inhibitor
from colostrum.
Each tube contained 25
amounts of one of the inhibitors.
After subtraction
activity was read from the standard curve of Kunitz

of soy bean inhibitor

and the
y of trypsin and the indicated
of the values of the blanks the
(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
In the twenties, due to the work of Smith and Little (7) and
purgative.
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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2.5

M.

LASKOWSKI,

JR.,

AND

M.

LASKOWSKI

573

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.

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.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.

Laskowski, M., Jr., and Laskowski, M.? Federation Proc., 9, 194 (1959).
Kunita, M., J. Gen. Physiol., 30, 291 (1947).
Kunitz, M., and Northrop, J. H., J. Gen. Physiol., 19, 991 (1936).
Goodwin, T. W., and Morton, R. A., Biochem. J., 40,628 (1946).
Ehrlich, P., 2. Hyg. u. Infektionskrankh.,
12, 183 (1892).
Howe, P. E., J. Biol. Chem., 49, 115 (1921).
Smith, T., and Little, R. B., J. Exp. Med., 36,181,453 (1922); 37, 671 (1923).
Orcutt, M. L., and Howe, P. E., J. Esp. Med., 36, 291 (1922).
Smith, E. L., J. BioZ. Chem., 164, 345 (1946); 166, 665 (1946).
McMeekin, T. L., and Polis, B. D., Advances in Protein Chem., 6, 291 (1949).
Smith, T., and Little, R. B., J. Exp. Med., 39, 303 (1924).
6 Italics ours.

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Our thanks are due to Miss M. L. Trautmann for assistance, to Miss V.

Kubacki for electrophoretic patterns, to Mr. L. C. Massopust for photographs and drawings, and to the Bloehowiak Dairy Company and their
producers for generous supplies of colostrum and for splendid cooperation.

ARTICLE:
CRYSTALLINE TRYPSIN INHIBITOR
FROM COLOSTRUM
M. Laskowski, Jr. and M. Laskowski

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