[72] H U M A NPANCREATIC SECRETORY TRYPSIN INHIBITOR 813

taining 67 amino acid residues 12 (Table I I ) and comprising a sugar

moiety of more than 30% of its molecular weight. 2° The sugar is com-
posed of fucose, mannose, galactose, glucose, galactosamine, glucosamine,
and sialic acid. 2° Hog colostrum trypsin inhibitor has a similar composi-
tion. 1° As far as the multiple forms of both the cow and hog inhibitors
are concerned, some variations could be shown in the lysine ~,26,27 and
threonine ~ contents, and different forms containing 1, 2, or 3 lysine
residues could be isolated. Differences in content of other amino acids ~s,27
are also likely. The p r i m a r y structures of two inhibitor forms have al-
r e a d y been determined, 12,28 differing only by two amino acids: replace-
ment of threonine in position 3 by lysine. ~8 There is a 40% homology in
the p r i m a r y structure of the colostrum inhibitor with t h a t of the basic
pancreatic trypsin inhibitor, 12 and a comparison of the two structures
is given in Fig. 2. The disulfide bonds in the molecule of cow colostrum
trypsin inhibitor are shown in Fig. 3; their positions are identical with
the disulfides in the basic pancreatic trypsin inhibitor, s
26U. Kucich, Ph.D. Thesis, State University of New York at Buffalo, 1972.
27M. Laskowski, Sr., personal communication.
~8V. Jon~kov~. and D. ~echov£, Collect. Czech. Chem. Commun., in press.

[72] H u m a n Pancreatic Secretory Trypsin Inhibitor 1

B y LEWIS J. GREENE, MERTON H. PUBOLS,2 and DIANA C. BARTELT

Pancreatic juice, the exocrine secretion of pancreas, contains a poly-

peptide trypsin inhibitor 3-s in addition to hydrolytic enzymes and in-
active enzyme precursors (zymogens). The pancreatic secretory trypsin
inhibitor ( P S T I ) prevents the premature trypsin-catalyzed activation of
zymogens within the pancreas and the pancreatic duct. In the small in-
1Research carried out at Brookhaven National Laboratory under the auspices of
the U.S. Atomic Energy Commission.
~Visiting Biochemist at Brookhaven National Laboratory 1968 to 1969, supported
by the National Institutes of Health Special Fellowship GM-40285-01.
3M. H. Kalser and M. Grossman, Gastroenterology 29, 35 (1955)
4B. J. Haverback, B. Dyce, H. Bundy, and H. A. Edmondson, Am. J. Med. 29,
424 (1960).
5L. J. Greene, M. Rigbi, and D. S. Fackre, J. Biol. Chem. 241, 5610 (1966).
6H. Fritz, F. Woitinas, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem. 345, 168
(1966).
7L. J. Greene, J. J. I)iCarlo, A. J. Sussman, and D. C. Bartelt, J. Biol. Chem. 243,
1804 (1968).
8 M. H. Pubols, D. C. Bartelt, and L. J. Greene, J. Biol. Chem. 249, 2235 (1974).
 814 NATURALLY
OCCURRING PROTEASE INHIBITORS [72]
testine, enterokinase initiates the activation of trypsinogen, which is
responsible for the activation of all other zymogens. Enterokinase is not
inhibited by P S T I 2
K a z a l et al. '° isolated the first pancreatic secretory trypsin inhibitor ll
from bovine pancreas in 1948. I t could be distinguished from the bovine
t r y p s i n - k a l l i k r e i n inhibitor (Kunitz inhibitor) 1~ on the basis of its in-
ability to inhibit bovine chymotrypsin or porcine pancreatic kallikrein
and because the inhibition is t e m p o r a r y in the presence of excess
trypsin. Bovine and porcine P S T I , 13 as well as the bovine t r y p s i n - k a l l i -
krein inhibitor, 1~ have been described in this treatise. More recent infor-
mation m a y be found in review articles 1~,16 and reports of conferences. 1ms
The trypsin inhibitor activity present in human pancreas and p a n -
creatic juice was first demonstrated by H a v e r b a c k et al. ~ Partially
purified inhibitor was later identified as K a z a l - t y p e on the basis of its
inhibitory properties2 ,'J,2° This assignment is independently indicated
by the amino acid sequence of the h u m a n inhibitor, which is homologous
to the pancreatic secretory trypsin inhibitors of bovine, porcine, and
ovine origin. A comparison of these structures is included in this article.
The procedure s detailed here for the isolation of multiple
chromatographic forms of h u m a n P S T I utilizes inactive pancreatic juice
or tissue as the starting material. The inhibitor is not exposed to pro-
teolytic enzymes and is isolated by gel filtration and ion exchange
c h r o m a t o g r a p h y in the free form (not complexed with trypsin). The use
of trypsin-affinity c h r o m a t o g r a p h y for the isolation of inhibitor from
autolyzed h u m a n pancreas has recently been reported? t

S. Maroux, J. Baratti, and P. Desnuelle, J. Biol. Chem. 246, 5031 (1971).

~°L. A. Kazal, D. S. Spicer, and R. A. Brahinsky, J. Am. Chem. Soc. 70, 3034 (1948).
~lPancreatic secretory trypsin inhibitors are also referred to in the literature as
"Kazal4ype" inhibitors or specific inhibitors from pancreatic juice.
1~M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936).
13p. j. Burck, this series Vol. 19 [67].
~4B. Kassell, this series Vol. 19 [66b].
~5M. Laskowski, Jr., and R. W Sealock, in "The Enzymes" (P. D. Boyer, ed.),
3rd ed., Vol. III, p. 375. Academic Press, New York, 1971.
1~H. Tschesche, Angew. Chem. 13, 10 (1974).
17"Proteinase Inhibitors, Proceedings of the First International Research Conference
(Bayer Symposium V) Munich, 1970. de Gruyter, Berlin, 1971.
18Proteinase Inhibitors, Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder,
1973. Springer-Verlag, Berlin and New York, 1974.
~ H. Fritz, I. ttiiller, M. Wiedemann, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem.
348, 405 (1967).
~°P. J. Keller and B. J. Allan, J. Biol. Chem. 242, 281 (1967).
2~G. Feinstein, R. ttofstein, J Koifmann, and M. Sokolovsky, Eur. J. Biochem. 43,
569 (1974).
[72] HUMAN PANCREATIC SECRETORY TRYPSIN INHIBITOR 815

Assay Method
The inhibition of the trypsin hydrolysis of p-toluenesulfonyl-L-
arginine methyl ester (TAME) is used as the quantitative assay for the
trypsin inhibitor. The pH-stat method is recommended because it can
be used to assay trypsin inhibitor in tissue extracts where nucleotide
absorption interferes with the spectrophotometric determination. The
assay is carried out with a final substrate concentration of 0.008 M
T A M E in 0.005 M Tris-HC1 buffer, 0.1 M KC1, 0.02 M CaC12 at pH 7.8,
250. 5 Crystalline bovine trypsin (Worthington Biochemical Co., Free-
hold, New Jersey) is used. A description of the procedure used in our
laboratory has appeared in a previous volume of this treatise. 1~
Definition of Unit and Specific Activity. One inhibition unit is the
a m o u n t of inhibition t h a t has caused the reduction of T A M E hydrolysis
by 1 tLmole/min. Specific activity is defined as inhibitor units per a~ 280 l°m•

Preparation of H u m a n PSTI s
Individual samples of pancreatic juice and tissue are initially
screened for trypsin and trypsin inhibitor activity to ensure that no
tryptic activation has occurred. This precaution is taken so that the in-
hibitor can be separated in the "free" form from trypsin, trypsinogen, or
inhibitor-trypsin complex by gel filtration on Sephadex G-75. The in-
hibitor is then purified and separated into five chromatographic forms by
gradient elution chromatography on DEAE-cellulose and SP-Sephadex.
Pancreatic tissue is subjected to a preliminary fractionation by am-
monium sulfate precipitation and then processed in the same manner as
the pancreatic juice.
Step la. Collection of Pancreatic Juice. H u m a n pancreatic juice is
collected by catheterization of the pancreatic duct after related surgical
procedures. 2-0,23 The pancreatic juice is frozen as soon as possible after
collection. Trypsin activity is determined by the pH-stat method on an
aliquot of juice containing 250-500 t~g protein. Samples that do not have
demonstrable trypsin activity and contain inhibitor activity are used.
Pancreatic juice was stored at --22 ° after lyophilization.
Step lb. Extraction o] PSTI ]rom Pancreas. Postmortem human pan-
creas from approximately 100 individuals who did not have diagnosed
pancreatic disease were stored at --22 ° for up to 12 months. Minced
tissue in 50-100 g portions is suspended in 5 volumes (w/v) of chilled
0.1 mM diisopropyl phosphofluoridate ( D F P ) and homogenized in a

~"A. Morgan, L. A. Robinson, and T. T. White, Am. J. Surg. 115, 131 (1968).
23C. Figarella and T. Robeiro, Scan~d.J. Gastroenterol. 6, 133 (1971).
816 NATURALLY OCCURRING PROTEASE INHIBITORS [72]

Waring blender for 2 min at maximum speed. The suspension is ad-

justed to pH 4.5 with 6 N HC10~ and centrifuged at 13,000 g for 45 min
at 5 ° in a Sorvall GSA rotor. If the pH of the tissue suspension is
brought to pH 3 or less, large losses of trypsin inhibitor activity occur.
The sediment is reextracted, and the combined supernatants are brought
to 70% saturation by the slow addition of 472 g of solid ammonium sul-
fate per liter of supernatant at 5 °. After centrifugation at 13,000 g, 5 °,
for 45 min the supernatant is discarded. The precipitate is resuspended
in 3 volumes of 0.1 mM DFP and stored at --22 °. Each preparation is
assayed for trypsin and inhibitor activity before being combined with
others for gel filtration on Sephadex G-75. The DFP present does not
inhibit the trypsin under the conditions of the assay.
Step 2. Gel Filtration on Sephadex G-75. Two 7.6 X 180 cm columns
are prepared from 1.5 kg of Sephadex G-75 which has been swelled over-
night in 50 liters of 50% acetic acid. The fines are removed by suction
provided by a water pump. The acetic acid is replaced with distilled
water and then by the eluting buffer (0.5 M KCI, 0.01 M Tris-HC1, pH
8.1, and 0.1 mM DFP) by settling, followed by decantation with suction.
Equilibration of the gel is completed by storage at 4 ° for 4-5 days. Two
centimeters of glass beads are overlaid on top of the sintered-glass disks
fitted at the bottom of each column. The gel suspension is deaerated at
4 ° with a water pump before the columns are poured in sections. Thirty
liters of buffer are passed through each column before the columns are
connected in series. The flow rate is limited to 120 ml/hr by a peristaltic
pump attached to the outlet of the second column. Effluent is collected
in 60-ml fractions. The columns are operated in a cold room at 2o-4 °.
Lyophilized human pancreatic juice is suspended in 200-400 ml of
cold distilled water at 4 ° to give a final protein concentration of 1-2%.
One milliliter is removed for the determination of inhibitor activity later
to be used for the calculation of inhibitor recovery. The solution is then
made 1 mM with respect to D F P by the addition of 0.1 M D F P (in
isopropyl alcohol) and held at 4 ° for 2 hr with stirring before gel filtra-
tion on Sephadex G-75 (Fig. 1). The inhibitor activity, corresponding to
a molecular weight of ~6000, is retarded relative to most of the secre-
tory protein. Amylase activity is anomalously eluted after the inhibitor
in the effluent corresponding to fractions 240-270. Effluent with inhibitor-
specific activity greater than 12 (fractions 193-203, indicated by the
solid bar) is combined and lyophilized. This fraction is denoted "low
molecular weight fraction."
Step 3. Gel Filtration on Sephadex G-25. Sephadex G-25 is swelled
and degassed in 10 volumes of distilled wafer'by placing the beaker in a
boiling water bath for 2 hr. After settling, the supernatant and fines are
[72] HUMAN PANCREATIC SECRETORY TRYPSIN INHIBITOR 817

Z
~1 ,, , I , , ,I f , , I,, , i ,, , i ,, , i
)-

A F-

I 1.5 OA
I z~
Oo~
E -,5.0 E~
c rno

o 1.0
GO
o,J

i,1
0
zO.5
<
cn 5.0 E
n-
O
cD o
~0.1 E
::k
60 I00 140 180 220 260 300
FRACTION NUMBER, 60 ml/FRACTION
FIG. 1. Gel filtration of human pancreatic juice on Sephadex G-75. Sample,
34 A~so
1cm × 190 ml. @ - - - - 0 , Absorbance at 280 nm; O - - - - O , trypsin inhibition.
TAME, p-toluenesulfonyl-L-arginine methyl ester. From M. H. Pubols, D. C. Bartelt,
and L. J. Greene, J. Biol. Chem. 249, 2235 (1974).

decanted by suction. The eluting buffer (0.05 M ammonium bicarbonate,

pH 8.1) is then added to the swelled gel at 4 or 5 times the settled gel
volume. The Sephadex is gently stirred and allowed to settle; and fines
are removed by suction. Buffer addition, gel settling, and decantation are
repeated three times. The equilibrated gel is kept at 20-4 ° overnight be-
fore packing the columns as described for Sephadex G-75.
The lyophilized low-molecular-weight fraction from the Sephadex
G-75 column (Fig. 1) is dissolved in 400 ml of 0.1 m M D F P and de-
salted on a Sephadex G-25 (medium) column, 7.6 }( 72 cm, equilibrated
and developed with 0.05 M ammonium bicarbonate buffer, pH 8.1, con-
taining 0.1 m M D F P at 200 ml/hr, 4 °. Fractions of 30 ml are collected.
The effluent is monitored by measurement of absorbance at 280 nm, tryp-
sin-inhibitor activity, and effluent conductivity. Fractions with inhibitor
specific activity greater than 20 are pooled and lyophilized. Because the
inhibitor activity is partially included within the matrix of Sephadex
G-25, this preparation is then resubmitted to gel filtration on two col-
umns (1.8 X 160 cm) of Sephadex G-25 (Superfine) connected in series.
The columns are equilibrated and developed with 0.05 M ammonium
bicarbonate, pH 8.1, at 15 ml/hr. Effluent with a specific activity greater
than 90 is combined and lyophilized twice to remove the volatile buffer.
 818 N A T U R A L L Y OCCURRING P R O T E A S E I N H I B I T O R S [72]

Step 4. Chromatography on DEAE-Cellulose. To prepare resin for

two 1.8 }( 45 cm columns, 120 g DE-52 resin (Whatman) is suspended
in 2 liters of 0.280 Tris-HCl buffer, pH 9.0 (ten times the concentration
of the starting elution buffer). After the suspension has settled for 10
rain, the supernatant is removed by suction. The resin is resuspended
with one additional liter of buffer, and the pH of the slurry is adjusted,
if necessary. The resin suspension is degassed with a water pump for 30
rain and stored at 2o-4 °. The column is poured in the cold room at 20-4 °
using a final slurry volume which is 1.5 times of the wet settled volume.
The column is then equilibrated with the starting elution buffer (0.028 M
Tris-HC1, pH 9.0) by passing 2 liters or more of buffer through the
column, until the effluent pH and conductivity are exactly the same as
that of the starting elution buffer.
The lyophilized fraction containing inhibitor activity derived from
the Sephadex G-25 column is suspended in 25 ml of 0.014 M Tris-HC1
buffer, pH 9.0. The pH is corrected to pH 9.0, and the conductivity is
reduced, if necessary, by dilution to a value below that of the starting
elution buffer. After sample application, the column is operated with
starting elution buffer at 40 ml/hr with a piston pump until 1320 ml have
passed through the column (tube 66, indicated by the arrow in Fig. 2). A
linear gradient is then applied to the column. It is prepared from 2 liters
each of equilibrating buffer and 0.028 M Tris-HC1 buffer, 0.2 M potas-
sium chloride, pH 9.0, using mixing chambers of the same cross sec-
tional area. 2~ The inhibitor activity from both pancreatic juice (Fig. 2,
top) and tissue (Fig. 2, bottom) are each resolved into two chromatog-
raphic components, A and B, which are eluted with the same effluent
conductivity and present in similar proportions. The effluent correspond-
ing to each peak of inhibitor activity is combined, lyophilized, desalted
by gel filtration on Sephadex G-25 (Superfine), 1.8 by 72 cm, and de-
veloped with 0.05 M ammonium bicarbonate buffer, pH 8.1, at 4 °. After
gel filtration, tile active material, located by inhibitor activity and ab-
sorbance at 280 nm, is pooled and lyophilized.
Step 5. Gradient Elution Chromatography on SP-Sephadex. To pre-
pare resin for two 0.9 X 70 cm columns, 15 g of SP-Sephadex G-25 are
suspended in 1 liter of 0.5 M acetic acid overnight, and fine particles
are removed by settling several times in distilled water using suction to
remove the supernatant. The resin is equilibrated by suspending it in 1
liter of 0.1 M ammonium acetate, pH 4.5 (0.1 M in acetic acid) over-
night. The supernatant is removed and the equilibration process is re-
peated three times. The column is poured at 4 ° with glass beads over-
laying the sintered-glass disks at the bottom of the column.
~ R. M. B o c k a n d N.-S. Ling, Anal. Chem. 26, 1546 (1954).
[721 HUMAN PANCREATIC SECRETORY TRYPSIN INHIBITOR 819

I I I ' I i '
Z
PANCREATIC JUICE
B
0.4 6 ~o - 2 0 0 >_a-
D.-

0.5 2 150 ~
O~
0.2 2 o IOOz~
s
0. I 0 ~ 50No
'~
LIJ
0 u_ 0 ~
(.9 -PANCREATIC TISSUE W ~ T
z
<:[ 0.4 B
m
a0 : O . 5 4 I 150 ~
• C
GO I

m 0.2 2 E I00 ~
o ~,
0. I 0 = 50"5
E E
E 4.
60 80 I00 120 140
FRACTION NUMBER 2 0 m l / F R A C T I O N
FIG. 2. Chromatography of human pancreatic low-molecular weight fraction on
DEAE-cellulose. Top: Pancreatic juice low-molecular weight fraction, 4.3 A~0m X
3 ml. Bottom: Pancreatic tissue low-molecular-weight fraction, 2.4 A~sC0 m X 35 ml.
, Absorbance at 280 nm; O O , trypsin inhibition. T A M E , p-toluenesulfonyl-
L-arginine methyl ester. From M. H. Pubols, D. C. Bartelt, and L. J. Greene, J. Biol.
Chem. 249, 2235 (1974).

The sample, fractions A or B, derived from either pancreatic juice

or tissue, after desalting and lyophilization is dissolved in 2-5 ml of 0.05
M ammonium acetate buffer, pH 4.3, and is applied to the column (0.9 X
70 cm), which is equilibrated in 0.1 M ammonium acetate buffer, pH 4.5.
The pH and conductivity of the sample should be less than that of the
equilibrating buffer. After sample application, the column is developed
with a linear gradient prepared from 400 ml each of equilibrating buffer
and 0.1 M ammonium acetate buffer, pH 7.0, at 10 ml/hr, 4 °, with a pis-
ton pump. Effluent is collected in 5-ml fractions. The effluent is monitored
by measurement of inhibitor activity absorbance at 280 nm and effluent
pH. The active fractions of each inhibitor are pooled and lyophilized.
Figure 3 shows a comparison of SP-Sephadex chromatography elution
profilcs of the multiple chromatographic forms of human pancreatic
secretory trypsin inhibitor from pancreatic juice and tissue.
The table summarizes the purification of human pancreatic secre-
tory trypsin inhibitor from pancreatic juice and the ammonium sulfate
fraction derived from postmortem pancreatic tissue. The values given in
820 NATURALLY OCCURRING PROTEASE INHIBITORS [72]

I I I T I [ I I } I l [

A-FORM
1
o 500

400 5.50
500 4.90
200 . . . . 4.50T

o
~ b2OO - B-FORM I

I000

zi 800 5.30
T 600 4.90

g 4o0 4.50
$
~ 200
:t ol
20 40 60 80 I00 120
FRACTION NUMBER 5 m l / F R A C T I O N
FIG. 3 Comparison of SP-Sephadex chromatography elution profiles of the mul-
tiple chromatographic forms of human pancreatic secretory trypsin inhibitor isolated
from pancreatic juice and tissue. Forms A and B of inhibitor were prepared by
chromatography on DEAE-cellulose (cf. Fig. 2). O O, Trypsin inhibitor derived
from tissues; • @, trypsin inhibitor derived from pancreatic juice; - - - , effluent
pH, TAME, p-toluenesulfonyl-L-arginine methyl ester. From M. H. Pubols, D. C.
Bartelt, and L. J. Greene, J. Biol. Chem. 249, 2235 (1974).

the table are the averages for several preparations of human PSTI, each
requiring one or more chromatographic columns for each step. The over-
all yield for the preparation of the multiple chromatographic forms of
human PSTI is 55% from pancreatic juice and 38% from the ammonium
sulfate fraction derived from postmortem pancreases. Occasionally, dur-
ing the early stages of the preparation from tissue, trypsin activation
occurs, thereby reducing the yield of inhibitor.
The low concentration of inhibitor in the starting material necessi-
tates the use of large columns and thus large elution volumes at the be-
ginning of the procedure, as well as the repetition of several underloaded
analytical columns to achieve the separation of the closely chemically
related multiple chromatographic forms. As has been noted, the inhibitor
is isolated in the free form and is not exposed to active trypsin or acidic
conditions below pH 4.5. Recently, a procedure starting with autolyzed
[72] HUMAN PANCREATIC SECRETORY TRYPSIN INHIBITOR 821

human pancreas, utilizing trypsin affinity chromatography with elution

by 0.01 N NHC1 and isoelectric focusing, has been described for the iso-
lation of several forms of human PSTI from pancreas. 2° In the absence of
documentation of the homogeneity of these materials, it is not possible
to compare the efficacy of the isolation procedure nor the chemical prop-
erties of the inhibitors with those described here.

Trypsin Inhibitor Content o] Human Pancreatic Juice and Tissue

The average amount of trypsin inhibitor activity in more than 50
samples of pancreatic juice from 10 individuals was 0.3 mg/100 mg of
protein with a range of 0.1-0.6 mg. Unactivated human pancreas con-
tained, on the average, 3 mg inhibitor/100 g wet tissue (range 1-6 rag/
100 g). Approximately 40% of the 10 kg of postmortem human pan-
creases, examined in 50-100 g portions, contained active trypsin.

Properties
Multiple Chromatographic Forms. Pancreatic juice and tissue con-
tain the same multiple chromatographic forms of human PSTI (cf. Fig.
3). They could be distinguished on the basis of chromatographic behavior
on ion-exchange resins and by acrylamide electrophores at pH 8.3 and
4.5. The multiple chromatographic forms had the same amino acid com-
position after acid hydrolysis, specific activity for the inhibition of bovine
trypsin, and mobility in sodium dodecyl sulfate (SDS) acrylamide elec-
trophoresis. The three major forms, A3, B1, and B., had Asx-Ser as their
amino-terminal residues but could be distinguished on the basis of
asparagine/aspartic acid content and susceptibility to enzymic hy-
drolysis. The available chemical and physical evidence indicates that the
multiple chromatographic forms differ only in asparagine content.
Criteria o] Homogeneity. All forms of human PSTI are homogeneous
by acrylamide electrophoresis at pH 4.5 and 8.3, SDS acrylamide elec-
trophoresis, amino acid analysis, and on the basis of the stoichiometry
of their interaction with trypsin. Forms A3, B1, and B.., had Asx as the
only demonstrable amino-terminal residue. The only form examined by
high-speed equilibrium centrifugation, B1, behaved as a homogeneous
solute.
Physical Properties. The ultraviolet spectra of forms B1 and B2 were
identical with the molar absorptivity, e27551cm= 5950. The protein absorb-
ance index, -~s0Alc"(10 mg/ml) is 8.4. The spectra are consistent with the
absence of tryptophan and similar to those published for bovine 5 and
porcine ~ PSTI.
822 NATURALLY OCCURRING PROTEASE INHIBITORS [72]

Equilibrium sedimentation studies have been carried out with the

B1 form of inhibitor at 20 °, 56,000 rpm in 0.1 M KC1, 0.01 M Tris-HC1,
pH 7.8, with 3-ram column height. It behaved as a homogeneous, ideal
solute giving a measured molecular weight of 6300---+ 200. The specific
volume used, 0.71, was calculated from the amino acid composition.
Electrophoretic examination of human P S T I is conveniently carried
out in a vertical slab apparatus using Tris-glycine buffer, pH 8.3, and
20% acrylamide in the presence and absence of SDS. 2~ The fl-alanine-
acetic acid, pH 4.5, buffer system of Reisfeld et al. ~6 is also used in 20%
acrylamide gels. Inhibitor 2-5 t~g, was detected by staining at 40 ° for
1 hr with Coomassie blue, 0.25% in methanol-acetic acid-water (5: 1:4).
Gels were distained by diffusion.
A m i n o Acid C o m p o s i t i o n and Sequence. H u m a n P S T I exists as mul-
tiple chromatographic forms having identical amino acid compositions
but differing in asparagine content. All forms contain 56 amino acid
residues/molecule, which are arranged in a single linear polypeptide
chain. The major, most highly amidated form, A3, has the following
amino acid composition: Asp3, Ashy, Thr4, Ser3, Glu4, Gln~, Pro:~, Gly~,
Ala~, Cys6, Val2, Ile3, Leu4, Tyro, P h e , Lys4, and Arg3. It does not contain
methionine, tryptophan, histidine, glucosamine, or galactosamine. The
calculated minimal chemical weight, 6242, corresponds to that obtained
by equilibrium ultracentrifugation, SDS acrylamide gel electrophoresis
and the stoichiometry of the interaction with trypsin assuming a 1:1
molar ratio.
The amino acid sequence determination was carried out on the mix-
ture of chromatographic forms. Asparagine or aspartic acid were assigned
on the basis of the most highly amidated form A~ when both amino acids
were recovered in enzymic hydrolyzates of peptides. The amino acid se-
quence of human PST127 is compared with inhibitor from porcine, 2'~,'-'9
bovine, 3° and ovine 3~,32 pancreas in Fig. 4. Amino acids identical in all
four structures are indicated by the boxes. The structural homology of
the series is apparent. The reactive site residue P~ (residue 18) is either

~ F. W. Studier, J. Mol. Biol. 79, 237 (1973).

2oR. A. Reisfeld, V. J. Lewis, and D. E. Williams, Nature (London) 195, 281 (1962).
:~ D. C. Bartelt and L. J. Greene, ,l. Biol. Chem. submitted for publication.
~sH. Tschesche and E. Wachter, Eur. J. Biochem. 16, 187 (1970).
D. C. Bartelt and L. J. Greene, J. Biol. Chem. 246, 2218 (1971).
3oL. J. Greene and D. C. Bartelt, J. Biol. Chem. 244, 2646 (1969).
~1K. Hochstrasser, W. Schramm, It. Fritz, S. Schwarz, and E. Werle, Hoppe-Seyler's
Z. Physiol. Chem. 350, 893 (1969).
H. Tschesche, E. Wachter, S. Kupfer, R. Obermeier, E. Reidel, E Haenisch, and
M. Schneider, Proteinase Inhibitors, Proc. Int. Res. Con]. 1st Munich, 1970, p. 207.
de Gruyter, Berlin, 1971.
[72] HUMAN PANCREATIC SECRETORY TRYPSIN INHIBITOR 823

5 i0
Human Asp-Ser-Leu-Gly4AP~-Glu-Ala~Ly s ~ T y r - A s n ~ L e u - A s n
Porcine I Thr-Ser-Pro-Gln~Ars-Glu-Ala+Thr+Cys+Thr-Ser+Glu~Val-Ser
Bovine Asn-lle-Leu-Gly~Ar6-Glu-Ala+Lys+Cys+Thr-Asn~Glu~Val-Asn
Ovine A~n- n e- ~ u - ~ l , t A r g - G l u - ~ a t ~ sCYt._~'hr-Asn alt_~va~.-Asn

15 20 25
Human
Porcine I
Bovine
Ovine
~ Thr-Lys411e-Tyr-Asn-Pro-Val-Cys-Gly-Thr-Asp-Gly I
Pro-Lys~Ile-Tyr-Aen-Pro-Val-Cys-Gly-Thr-Asp-Gly[
Pro-Ar~Ile-Tyr-Asn-Pro-Val-Cys-Gly-Thr-Asp-Gly~
Pro-Ars111e-Tyr-Asn-Pro-gal-Cys-Gly-Thr-Asp-Gly [

3O 35 4O
Human Asp~--~--~Pro4Asn-Glu-Cy s~Val ~ P h e ~ A r E
Porcine I lle~Thr-Tyr~ Ser~Asn-Glu-Cy s~Val~Leu-Cy s+Ser~Glu-Asn~Ly s
Bovine Val~ Thr-Tyr~ Se r~Asn-Glu-Cy s$LeuSLeu- Cy sSMet+Glu-Asn~Ly s
Ovine Valt Thr- TY r~Al atAsn-Glu- Cy stLeu~Leu- Cy stMe ttGlu-AsnILy s

45 5O 56

Porcine I Ly s~Arg-Gln- Thr~ Pro-Val~Leu- Ile-Gln-Ly s-Ser-Gly-Pro-Cy s[

Bovine Glu~Ar6-Gln- Thr~Pro- Val~Leu- lle-Gln-Lys- Ser-Gly-Pro- Cy s[
ovlne OlutAr -Oln- rIPro-Va tLe.-I1e- n- .. r-G y-p . l.
FIG. 4. Comparison of the amino acid sequences of human, 2' porcine ~,:~ bovineff
and ovine ~'~-0 pancreatic secretory trypsin inhibitors. Superscript numbers refer to
text footnotes t h a t cite appropriate references.

lysine or arginine. Other amino acids near the reactive site (residues 15-
28) are identical, with the exception of the P2 position (residue 17) where
threonine occurs in human PSTI and proline in the other species.
Inhibitor Specific Activity (Bovine Trypsin). The calculated value
for the inhibition of bovine trypsin by human PSTI is 1790 t~moles
(TAME hydrolysis)/min/A~so. It is based on the specific activity of
trypsin (410 units/mg),5 the protein absorbance index of the inhibitor and
the molecular weights of the components, on the basis of a 1:1 molar
complex. The specific activity of the inhibitor in the SP-Sephadex column
effluents was 1500-1800 (cf. the table). A decrease of 10-20 in specific
activity was sometimes observed after lyophilization of ammonium bi-
carbonate buffer and ammonium acetate buffer solutions of human
PSTI.
Specificity. The initial studies of the specificity of partially purified
preparations of inhibitor showed that it inhibited bovine trypsin but not
a-chymotrypsin, pancreatic kallikrein, plasma kallikrein, plasmin, throm-
824 NATURALLY
OCCURRING PROTEASE INHIBITORS [72]
SUMMARY OF PURIFICATION PROCEDURE FOR HUMAN
PANCREATIC SECRETORY TRYPSIN INHIBITOR

Pancreatic tissue,
ammonium sulfate
Pancreatic juice fraction

Specific Yield Specific Yield

Fraction activity" (%) activity a (%)

Step 1. Juice (tissue) 3 (100) __b (100)

Step 2. Sephadex G-75 49 87 __b 70
Step 3. Sephadex G-25 200 87 b 70
Step 4. DEAE-cellulose 1350-1600 c 74 550-900 c 60
Step 5. SP-Sephadex 1500-1800 c 55 1500-1800 c 38

a Reduction of p-toluenesulfonyl-L-arginine methyl ester hydrolysis (bovine

trypsin) by 1 mole/min/A~o.
b Not reported because of interference by UV-absorbing materials in these fractions.
c Range of values found for all forms of inhibitor.

bin, or p a p a i n 2 ,19,2° T e m p o r a r y i n h i b i t i o n in t h e presence of excess t r y p -

sin w a s also d e m o n s t r a t e d . 19,2°
R e c e n t s t u d i e s w i t h h o m o g e n e o u s p r e p a r a t i o n s of h u m a n P S T I show
t h a t it effectively i n h i b i t s bovine, porcine, a n d b o t h h u m a n t r y p s i n s on
a 1:1 m o l a r basis. *,33,34 T h e i n h i b i t i o n of a - c h y m o t r y p s i n is m u c h less
effective, r e q u i r i n g a 4 - f o l d m o l a r excess to o b t a i n 3 0 % i n h i b i t i o n . 23 I n -
hibitors prepared from autolyzed pancreas by trypsin-affinity chroma-
t o g r a p h y h a v e been r e p o r t e d to i n h i b i t b o v i n e a n d b o t h h u m a n t r y p s i n s ,
to give p a r t i a l i n h i b i t i o n of b o v i n e a - c h y m o t r y p s i n , a n d to h a v e no in-
h i b i t o r y a c t i v i t y t o w a r d t w o h u m a n c h y m o t r y p s i n s . 35
M o s t s t u d i e s of t h e s p e c i f i c i t y of t r y p s i n i n h i b i t o r s h a v e used e n z y m e
t u r n o v e r a s s a y s to d e t e r m i n e free e n z y m e c o n c e n t r a t i o n . T h i s m e t h o d
c a n d e t e c t i n t e r a c t i o n s w i t h l a r g e a s s o c i a t i o n c o n s t a n t s b u t is n o t
s u i t a b l e for m e a s u r i n g w e a k e r i n t e r a c t i o n s (cf. L a s k o w s k i a n d co-
workers15,~). Since m o s t of t h e r e p o r t s in t h e l i t e r a t u r e w h i c h d e s c r i b e
e i t h e r w e a k or no i n t e r a c t i o n s b e t w e e n h u m a n P S T I a n d o t h e r serine

C. Figarella, G. A. Negri, and O. Guy, Protelnase Inhibitors, Proc. Int. Res. Conf.,
2nd (Bayer Syrnp. V), Grosse Ledder, 1973, p. 213. Springer-Verlag, Berlin and
New York, 1974.
34L. J. Greene, D. E. Roark, and D. C. Bartelt, Proteinase Inhibitors, Proc. int. Res.
Con]., 2nd (Bayer Symp. V)," Grosse Ledder, 1973, p. 188. Springer-Verlag, Berlin
and New York, 1974.
G. Feinstein, R. Hoffstein, and M. Sokolovsky,'Proteinase Inhibitors, Proc. Int.
Res. Conf., 2nd (Bayer Syrnp. V), Grosse Ledder, 1973, p. 199. Springer-Verlag,
Berlin and New York, 1974.
[731 GUINEA PIG SEMINAL VESICLES INHIBITORS 825

proteinases have used turnover assays, these data should be considered

as tentative and deserve to be examined by burst titrant ~6 and other ap-
propriate methods. Some of the differences in specificity reported between
bovine and porcine PSTI (see Table III of Burck 13) and human PSTI
probably reflect differences in the type of assay used or in the assay
conditions.
A discussion of the evolution of the specificity of protein-proteinase
inhibitors is given by Laskowski et al. 3G
3~M. Laskowski, Jr., I. Kato, T. R. Leary, J. Schrode, and R. W. Sealock, Proteinase
Inhibitors, Proc. Int. Res. Conf., 2nd (Bayer Syrup. V), Grosse Ledder, 1973,
p. 597. Springer-Verlag, Berlin and New York, 1974.

[73] Proteinase Inhibitors from Guinea Pig Seminal Vesicles

B y EDWIN FINK a n d HANS FRITZ

Studies on a trypsin inhibitor in guinea pig seminal vesicles were

initiated by Haendle et al. 1 Furthermore investigation of inhibitor prep-
arations obtained by affinity chromatography on trypsin resin revealed
the presence of two inhibitors, distinguishable on the basis of inhibition
characteristics and physical and chemical properties: 2-4 the one was
characterized as a trypsin inhibitor, TI; the other as a trypsin-plasmin
inhibitor, TPI. Their physiological role was unknown.
In 1968 Stambaugh and Buckley ~ described a proteinase, localized in
the acrosome of mammalian spermatozoa, which is responsible for pene-
tration of the sperm through the zona pellucida of the egg. Following the
suggestion of Zaneveld, this enzyme was named acrosin. * Both inhibitors
from guinea pig seminal vesicles turned out to be potent inhibitors of
acrosins from various species. 6-s This finding, which suggested an impor-
1H. Haendle, H. Fritz, I. Trautschold, and E. Werle, Hoppe-Seyler's Z. Physiol.
Chem. 343, 185 (1965).
2 E. Fink, Ph.D. Thesis, Faculty of Science, University of Munich, 1970.
3H. Fritz, E. Fink, R. Meister, and G. Klein, Hoppe-Seyler's Z. Physiol. Chem.
351, 1344 (1970).
E. Fink, G. Klein, F. Hammer, G. Miiller-Bardorff, and H. Fritz, Proteinase Inhi-
bitors, Proc. Int. Res. Conf., 1st, Munich, 1970, p. 223. de Gruyter, Berlin, 1971.
5 R. Stambaugh and J. Buckley, Science 161,585 (1968).
6L. J. D. Zaneveld, K. L. Polakoski, R. T. Robertson, and W. L. Williams, Pro-
teinase Inhibitors, Proc. Int. Res. Conf., 1st, Munich, 1970, p. 236. de Gruyter,
Berlin, 1971.
7H. Haendle, H. Ingrisch, and E. Werle, Kiln. Wochenschr. 48, 824 (1970).
s R. L. Stambaugh, in "Biology of Mammalian Fertilization and Implantation"
(K. S. Moghissi and E. S. E. Hafez, eds.), p. 185. Thomas, Springfield, Illinois, 1972.