THE JOURNAL OF RIOLOGICAL CHEMISTRY

Vol. 243, No. 8, Issue of April 25, pp. 1718-1724, 1963

Printed in U.S.A.

Bovine Pepsinogen and Pepsin

I. ISOLATION, PURIFICATION, AND SOME PROPERTIES OF THE PEPSINOGEN*

(Received for publication, December 6, 19G7)

ROSIE B. CHOW AA-D BEATRICE KASSELL

From the Department of Biochemistry, Marquette School of Medicine, Inc., Milwaukee, Wisconsin 53233

SUMMARY The molecular weight of the bovine pepsinogen is 38,900

by amino acid analysis and 37,500 by ultracentrifugation.
As the first step in the investigation of the structure and

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action of little known gastric zymogens and enzymes, pep-
sinogen has been isolated from the mucosa of the fourth
stomach (abomasum) of the cow. The pepsinogen was
purified by ammonium sulfate fractionation, batch absorption
on diethylaminoethyl (DEAE) cellulose, recycling gel filtra- Bovine pepsin was isolated from gastric juice and crystallized
tion on Sephadex G-100, and finally chromatography on by Northrop in 1933 (1). At that time, solubility studies showed
DEAE-cellulose. The preparation appears to be homoge- that it was a different protein from swine pepsin (2)) although both
neous by chromatography, amino acid analysis, ultracen- enzymes had equal specific activities on several protein substrates.
trifugation, and amino-terminal analysis, and nearly homo- Other than the studies of Fruton and Bergmann on specificity
geneous by disc electrophoresis. (3) and on the pH dependence of the activity (4), which were
Amino acid analysis by ion exchange chromatography both strikingly similar to swine pepsin, the bovine enzyme has
indicates the presence of 362 f 2 amino acid residues, which received little attention. To our knowledge, bovine pepsinogen
account quantitatively for the total nitrogen of the prepa- has never been isolated.
ration. Compared to swine pepsinogen (RAJAGOPALAN, In contrast, a great deal of work has been done on the structure
T. G., MOORE, S., AND STEIN, W. H., J. BioZ. Chem., 241, and properties of swine pepsinogen and pepsin. Sequence studies
4940 (1966)), which has 363 amino acids, the bovine pep- are approaching completion in several laboratories (e.g. Ref-
sinogen shows considerable similarity in composition, es- erences 512).
pecially with respect to the large number of acidic and small A second well characterized zymogen and enzyme of this
number of basic residues. However, there are at least 22 important group would aid in identifying the portions of the
amino acid substitutions. In both proteins there are five molecule essential for activity and for maintenance of conforma-
amino acids that occur in equal numbers of residues per tion. It would supplement chemical modification studies in
molecule; these are 4 methionine, 6 half-cystine, 6 tryp- elucidating the requirements for zymogen activation and for the
tophan, 15 phenylalanine, and 35 glycine. In addition, the action of the enzyme. We have therefore undertaken to study
sum of the residues of certain similar amino acids is constant the primary structure of bovine pepsinogen and pepsin. The
for bovine and swine pepsinogens; this is true for the pairs present paper describes the isolation, purification, and amino
lysine and arginine (14 residues), aspartic and glutamic acids acid composition of the pepsinogen. It has proved to be
(72 residues), and leucine and isoleucine (57 to 58 residues). sufficiently different from the swine zymogen to be of considerable
Significant differences occur in some other amino acids, interest.
notably histidine (2 residues in bovine pepsinogen, 3 in
EXPERIMENTAL PROCEDURE
swine) and proline (15 and 19, respectively).
Materials-The fundic portion of the fourth stomach of the
* This investigation was supported by Public Health Service cow (abomasum) was obtained partly through Wilson Labora-
Research Grant. AM-09826 from the National Institute of Arthritis tories, Chicago, andpartly from theLiebmannPackingCompany,
and Metabolic Diseases and by Public Health Service General
Research Support Grant 5-Sol-FR-5434. An abstract of part of Green Bay, Wisconsin. Swine pepsin (twice crystallized, Lot
this work has been published (Abstracts Seventh International PM 6HE) and swine pepsinogen (crystalline, Lot PG1117) were
Congress of Biochemistry, 1967, IUB Vol. IV, p. 782, F-135). This obtained from Worthington. Bovine hemoglobin (twice
work has been taken from a thesis to be submitted by Rosie B. crystallized) was purchased from Pentex. Sephadex G-25 and
Chow to Marquette University Graduate School in partial ful-
fillment of the requirements for the degree of Doctor of Philos- 1 We wish to thank Mr. Leonard C. Liebmann for supplying part
ophy. of the abomasum used in this investigation.

1718
Issue of April 25, 1968 R. B. Chow and B. Kassell 1719

G-100 (both bead form) were Pharmacia products. DEAE- accordingly. A solution was considered to be free of active
cellulose was No. 71, type 20, from Schleicher and Schuell. pepsin if it did not clot in 2 min when it contained at least 25 pg
Deionized distilled water was used throughout. Chemicals were of pepsinogen.
reagent grade. Analytical Methods-Amino acid analysis was carried out
Pepsin Assay-The potential pepsin content of the pep- essentially as described by iMoore and Stein (16). Duplicate
sinogen was routinely determined by a modification* of the samples of about 15 mg were dissolved and diluted to 10 ml with
hemoglobin method of Anson (13). The substrate solution was water. From these solutions, l-ml samples were taken for
prepared by dissolving 1 g of hemoglobin in 32 ml of water, then hydrolysis in 5.7 N HCl for 16, 24, 48, and 72 hours at 110 in
adding 8 ml of 0.3 M HCI; the solution was filtered through a boiling toluene, and 1.5-ml samples were removed for total
coarse, sintered glass funnel and kept in the refrigerator for up nitrogen determination by the Kjeldahl method (17). A blank
to 1 week. The amount of 0.3 M HCI required to bring 0.1 ml with HCl alone was used to correct for ammonia in the reagents.
of each unknown solution to pH 2.0 was predetermined. Poly- A Beckman/Spinco model 120B amino acid analyzer was used
carbonate centrifuge tubes (100 x 16 mm) were placed in a 37.6 for ion exchange chromatography (18, 19). The absorbance of
water bath. The required amount of 0.3 M HCl plus a solution the protein at 280 rnp was determined on appropriate dilutions
of 0.01 M HCl in 0.1 M NaCl to bring the volume to 0.9 ml were of the same solutions.
placed in the tubes; 0.1 ml of the solutions to be assayed was A moisture determination (100 in vacuum) was made on a
added to each. With timing, 1 ml of previously equilibrated sample taken at the same time. Inorganic phosphate was
hemoglobin solution was added, the solution was mixed, and determined (20) after removal of the protein from solution by
after exactly 5 min the reaction was terminated by adding 2 ml precipitation with trichloracetic acid. The content of phosphate

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of 5% trichloracetic acid. The mixture was cooled in ice for 10 in the sample, which had been dialyzed at pH 7.0, was calculated
min and centrifuged in a Sorvall SS-4 centrifuge at 28,000 x g as the sum of NaH2P04 and NazHPOI, determined from the pK,
for 30 min. Cellulose nitrate tubes, 17 x 95 mm, were used as value of 7.28 at 5. The weights of the samples were then
adapters for the polycarbonate tubes in the SM.24 rotor. The corrected for moisture and salts.
absorbance of the clear supernatant solution was measured at Cystine was determined as cysteic acid (21), and tryptophan
280 rnp, usually against a blank which contained all the reagents was measured spectrophotometrically (22) on separate samples.
but the solution to be assayed. With the crude, dark solutions Methionine sulfoxide was determined after alkaline hydrolysis
of the early purification steps, blanks were prepared (12) by (23). The Sanger method as described by Fraenkel-Conrat,
using the same amount of assay solution, and reversing the order Harris, and Levy (24) was used for amino-terminal analysis.
of addition of trichloracetic acid and hemoglobin solutions. Disc electrophoresis on 3 to 5 pg of the purified protein was
Standard solutions of swine pepsin, containing 3 to 10 pg/O.l carried out with a Canalco instrument, model 66. Standard
ml, were prepared in 0.1 M sodium acetate buffer of pH 5.3. A procedures were followed except for buffer modifications (25).
standard curve was prepared by assaying these as described A diethylbarbituric acid Tris buffer of pH 7.0 was used during
above. The potential specific activity of the bovine zymogen electrophoresis.
was expressed as microgram equivalents of swine pepsin per AZW Conductivity of chromatographic effluents was measured on a
of the bovine pepsinogen solution. Yellow Springs Instrument Company conductivity bridge, model
Milk-clotting Test-Milk-clotting activity was used to estimate 31.
the amount of active pepsin (if any) present in the pepsinogen
RITSULTS
solution. The method was a modification of the procedure of
Seijffers, Segal, and Miller (14). The stock milk solution was PuriJicatim of Bovine Pepsinogen
prepared by dissolving 16.8 g of nonfat dry milk (Carnation) in
Bovine abomasa were removed immediately after slaughtering
100 ml of deionized water. The diluted milk solution contained and were washed thoroughly to prevent activation of the zymogen
0.1 M sodium acetate buffer of pH 5.3, milk stock solution, and
by the highly acidic gastric juice (26) The fundic portion was
0.1 M CaClz solution in the proportions 5:5: 1. The solution for cut out; the mucosa was separated, trimmed and frozen for
testing consisted of 2 ml of the acetate buffer and 0.5 ml of the storage. All subsequent operations were carried out at about 5.
diluted milk solution in a glass test tube (15 X 125 mm) in a Step 1: E&rc&i&-The hashed mucosa (9.1 kg) was ex-
water bath at 37.6. The solution was mixed by means of an tracted with 18 liters of 0.1 M sodium phosphate buffer of pH 7.3
air-driven magnetic stirrer immersed in the water bath. The clot for 15 min. Solca Floe BW-20, 4.5 kg, was added, and the
was detected more readily by observing the solution as it moved
mixture was filtered on three al-cm-diameter Buchner funnels.
in a siphon (15), made by inserting a small piece of tubing (0.5- The filtered cakes were re-extracted with two 18-liter portions of
mm inside diameter) into one end of a 35-mm length of trans- the pH 7.3 buffer.
parent Tygon tubing of 1.5-mm inside diameter. This siphon Step z?: Ammonium Sulfate Fractionation-The extracts were
tube was filled with buffer and clamped. After 0.1 ml of the combined. Solid ammonium sulfate was added to 23% satura-
enzyme solution, containing about 5 pg of pepsin, had been added tion, with NaOH introduced as needed to keep the pH close to
to the milk-buffer mixture, the siphon was inserted and the clamp 7.3. Again 4.5 kg of BW-20 were added, and the mixture was
was released. The time required to show the first flock in the filtered. The cakes were washed with 10 to 15 liters of buffer
tubing was taken as the milk-clotting time, and was about 30 solution, 23% saturated with ammonium sulfate. The filtrate
set for 5 pg of pepsin. For unknown solutions, up to 0.5 ml
could be used, and the volume of acetate buffer was adjusted 3 We are grateful to Wilson Laboratories, especially to Dr.
Conrad de Fiebre and Mr. Edward P. Smith, for carrying out the
2 We are grateful to Dr. Robert J. Peanasky for a part of the extraction and ammonium sulfate fractionation, and also for
modification described. supplying part of the abomasum.
1720 Bovine Pepsinogen and Pepsin,. I Vol. 243, No. 8

TABLE I several liters of the same buffer until the absorbance of the
Purification steps and yield of bovine pepsinogen effluent at 280 rnp fell to about 0.3. The pepsinogen was then
eluted with 0.45 M NaCl in the same buffer. The elution profile
otal pepsinogen a
each step is shown in Fig. 1.
- pecific poten. Elution dia-
step tial activity* gram in Fig. The portion of the peak containing potential pepsin activity
At start 1 over 40 I.cgper A283 unit was pooled and concentrated to about 750
ml by ultrafiltration in a DiaFlo model 400 ultrafiltration cell
w with reservoir (Amicon Corporation, Cambridge, Massachusetts).
Solution of ammonium The membrane used was No. UM-1. The solution was stirred
sulfate cake. 18 very slowly during ultrafiltration to avoid denaturation. The
3. First batch absorption ultrafiltrate contained a small amount of material which absorbed
on DEAE-cellulose. 1433 68 63 1 at 280 rnp but did not have potential peptic activity.
5. Second batch absorp- Step 4: Desalting on Sephadex G-%5-A mixture of coarse and
tion on DEAE-cellu-
fine Sephadex beads in the proportion of 9: 1 was used to give a
lose 910 38 228 2
6. Sephadex G-100, re- flow rate of about 180 ml per hour. The, column, 5 X 100 cm,
cycling. 114 83 430 4 prepared as recommended by the manufacturer (28), was
7. Chromatography on equilibrated with 0.01 M Tris-phosphate buffer. Up to 250 ml
DEAE-cellulose 95 72 514 5B of the concentrated solution from Step 3 could be placed on the
column at one time. The protein was eluted with 0.01 M
a Recovery at each step is based on the center portion of the Tris-phosphate buffer. The column was used repeatedly after

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peak actually pooled. The yield could be improved by combining washing out the salt. The desalted protein was pooled.
and rechromatographing the edges of the peaks from several runs. Step 5: Second Batch Absorption on DEAE-cellulose, with
At Step 5, the yield based on the three potentially active peaks
Gradient Elution-The desalted solution, containing 15,180 Azso
was 59%.
b Contamination with active pepsin, which amounted to 5 to units and 910 mg of pepsinogen (Table I), was treated as in
15% of the total potential activity of the ammonium sulfate cakes, Step 3 with 100 g of DEAE-cellulose in a total volume of 5 liters.
decreased to zero by Step 6. The suspension was filtered as before; the filtrate contained 2,100
AQgOunits, but no pepsinogen. The filter cake was suspended in
and washings were combined. Solid ammonium sulfate was 1,200 ml of 0.05 M Tris-phosphate buffer, and the slurry was
added to 63% saturation. Perlite 4106 (500 g) was added, and transferred to a column (5 x 60 cm) containing a 15-cm packed
the mixture was allowed to settle overnight. The supernatant bed of fresh DEAE-cellulose, equilibrated with the same buffer.
was siphoned off and discarded. The precipitate was collected The gradient described by Arnon and Perlmann (5) for the
on three Buchner funnels and was shipped frozen to the labo- purification of crystalline swine pepsinogen was adjusted to the
ratory. size of the column used. The first constant volume bottle
Step 3: First Batch Absorption on DEAE-cellulose-The contained 2,250 ml of equilibrating buffer. The gradient solu-
ammonium sulfate cake was divided into 600-g batches. Each tion was 0.45 M NaCl in the same buffer. The elution profile is
batch was suspended in 500 ml of 0.01 M Tris-phosphate buffer of shown in Fig. 2. Fractions with potential peptic activity of
pH 7.0 and dialyzed against two changes of the same buffer. over 100 pg per Azso unit were pooled in three portions, as
(The molarity of the Tris-phosphate buffers was determined by indicated by the arrows. The center portion, used for the
the concentration of Tris; all were adjusted to pH 7.0 f 0.05.) subsequent steps of purification, contained 348 mg.
Centrifugation of the suspension for 3 hours at 13,000 X g in a Step 6: Recycling Gel Filtration on Sephodex G-100--Initially,
Sorvall refrigerated centrifuge sedimented the Perlite, which was a single gravity flow filtration was tried, but imperfect separation
resuspended in an equivalent volume of buffer and centrifuged
again. The combined supernatant solutions were then centri-
fuged at 36,000 X g in a Spinco L-2 ultracentrifuge for 3 to 4
hours. The sediment was washed as before. An almost clear
solution was obtained, which was filtered through Schleicher and
Schuell No. 588 folded paper.
The DEAE-cellulose was precycled with 0.5 M NaOH and 0.5
M HCl (27) and equilibrated with 0.05 M Tris-phosphate buffer.
After removal of fine particles, it was allowed to settle overnight
or longer.
An amount of the crude pepsinogen solution, equivalent to
80,000 Azso units and containing 1.0 to 1.5 g of pepsinogen (see
Table I for a typical experiment), was diluted to 7,900 ml with
0.05 M Tris-phosphate buffer and mixed with 2,100 ml of settled EFFLUENT, L
DEAE-cellulose (equivalent to about 190 g). The suspension FIG. 1. Stepwise elution of pepsinogen with 0.1 and 0.45 M
was gently stirred mechanically for 1 hour and filtered through NaCl in 0.05 M Tris-phosphate buffer of pH 7.0, after the first batch
cloth on a 24-cm Buchner funnel. The moist cake was resus- absorption on DEAE-cellulose (see the text, Step 3). The effluent
was collected in large fractions, indicated by boxes, during elution
pended in fresh 0.05 M Tris-phosphate buffer and transferred to a
with 0.1 M NaCl. The column was 7 X 31 cm; sample weight, 1433
chromatographic column 7 X 60 cm. After settling, the height mg; flow rate, 120 ml per hour. O--O, Az~o; A- - -A, specific
of the resin was 30 to 35 cm. The column was washed with activity.
Issue of April 25, 1968 R. B. Chow and B. Kassell 1721

of inactive from potentially active material was obtained (see

1
below). Therefore, a recycling device (29) was used for subse-
quent runs. The column (3.2 x 105 cm), fitted for upward 3.5-
flow, and the recycling valve were obtained from LKB Instru-
ments. The column, prepared in the normal way (28), was $X0-
equilibrated with 0.05 M Tris-phosphate buffer in the upward
direction, with a diastolic pump. The sample of 50 ml was %5-
introduced through the pump at a flow rate of 10 ml per hour. Q20-
The pump and tubing were washed at the same rate with three
2-ml portions of the buffer. The flow rate was then increased to g1.5 -
17 ml per hour; no back-pressure developed in the column. A
single separation is shown in Fig. 3 for comparison with the &.O-
recycling experiment of Fig. 4. For recycling, the effluent was 8 200 %
collected in the fraction collector until the first inactive peak had s-5 -
emerged. The next fraction of 120 ml, shown by the vertical
0
Zincs of Fig. 4, was recycled. Additional material of low activity 100 300 500 700 900 1100
appeared both before and after the pepsinogen peak. Fractions EFFLUENT, ML
with potential peptic activity greater than 400 pg per A280unit
FIG. 4. Recycling gel filtration on Sephadex G-100 (see the
were pooled and concentrated by ultrafiltration to 35 ml. text, Step 6). The sample size was 114 mg in 50 ml. The fraction

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between the vertical lines (120 ml) was recycled. Other conditions
were the same as in Fig. 3.

if 1.0

z 0.8 z
3
- 0.6 6005
P
$ 0.4 400:
cI4 12 2.0 2.8 3.6 44 5.2 6.0
EFFLUENT, L 8 aw
g a2 2OOcn
FIG. 2. Gradient elution of pepsinogen after the second batch Q
absorption on DEAE-cellulose (see the text, Step 5). The column 0
was 5 X 50 cm; sample weight, 910 mg. The closed mixing cham- 60 180 300 420 15 300 600 900
ber contained 2250 ml of 0.05 M T&phosphate buffer of pH 7,.0. EFFLUENT, ML.
The reservoir contained 0.45 M NaCl in the same buffer. The flow
rate was 85 ml per hour at the start; then it slowed and was main- FIG. 5. Chromatography on DEAE-cellulose (see the text,
tained at 20 ml per hour by a peristaltic pump. O-O, ⅈ Step 7). A, sample from Fig. 3; 7.7 mg in 19 ml; column, 1 X 49
A- - -A, specific activity; -. .-, conductivity expressed as re- cm; flow rate, 22 ml per hour. Buffers were the same as in Fig. 2,
ciprocal ohms X 1c3. with 90 ml in the mixing chamber. B, sample from Fig. 4; 95 mg in
40 ml; column, 2 X 49 cm; flow rate, 54 ml per hour. Buffers were
the same as in Fig. 2, with 360 ml in the mixing chamber.
O-O, A2so; A- ---A, specific activity.

Step 7: Chromatography 072DEAE-cellulose-The solution

from the recycling gel filtration of Step 6, 40 ml, including
washings from the ultrafilter, was placed on a column (2 X 49 cm)
of DEAE-cellulose equilibrated with 0.05 M Tris-phosphate
buffer. The gradient wasthe sameasfor Step 5, with 360 ml of
the equilibrating buffer in the constant volume bottle. Fig. 5B
showsthat a small amount of inactive material was separated,
and that the main peak obtained wasalmost symmetrical, with
uniform potential activity.
The samplewaspooledand dialyzed against 3 liters of 0.01 M
Tris-phosphatebuffer, 3 liters of 0.01 M sodiumphosphatebuffer
of pH 7, and, finally, three 4-liter changesof deionized water
O 100 300 500 700 900 1100
adjusted to pH 7. After dialysis, the sample was lyophiliaed.
EFFLUENT, ML.
No active pepsin was detected.
FIG. 3. Single gel filtration on Sephadex G-100 (see the text, For comparison, rechromatography of the material from Fig.
Step 6). The column was 3.2 X 105 cm; sample size, 174 mg in
50 ml; buffer, 0.05 M Tris-phosphate of pH 7.0; upward flow rate,
3 is shownin Fig. 5A. For this experiment, a column (1 X 49
17 ml per hour; void volume of the column, 225 ml. O-O, cm) of DEAE-cellulose was used with an equivalent gradient,
A 280; A- - -A, specific activity. i.e. 90 ml of eauilibrating
A buffer in the constant volume bottle.
1722 Bovine Pepsinogen and Pepsin. I Vol. 243, No. 8

TABLE II
Amino acid composition of bovine pepsinogen
Values are based on a molecular weight of 38,943 and a total nitrogen content of 14.560/,.
i-
Amino acid residues per molecule Calculated residues Residues per molecule
to nearest integer

I-
Amino acid - - Nitrogena
16 hrs 24 hrs 48 hrs 12 hrs No. per molecule Per cent0 Bovine Swine (6Jb
--
% of iOld
Lysine 8.44 7.94 7.87 8.07 8.03c f 0.16 2.43 3.64 8 10
Histidine 1.98 1.98 2.01 2.02 1.99 f 0.03 0.64 1.35 2 3
Arginine 5.92 5.83 5.82 5.88 5.85 f 0.05 2.16 5.31 6 4
Aspartic acid .. 42.24 39.21 38.47 40.65 40.14c f 1.67 10.89 9.09 40 44
Threonine 25.83 25.87 24.22 23.67 2G.G2d f 0.28 6.34 G.03 27 26
Ferine 46.49 46.49 40.12 37.19 49.84d f 0.90 10.23 11.29 50 46
Glutamic acid... 35.17 29.78 30.31 33.03 32.0ic f 2.51 9.75 7.27 32 28
Proline. 15.42 15.83 15.29 14.95 15.37c 3z 0.38 3.44 3.48 15 19
Glycine 3G.12 34.01 34.14 35.09 34.84c f 0.98 4.89 7.90 35 35
Alanine. 18.75 15.50 15.50 15.96 15.920 f 0.59 2.67 3.GO 16 19
Half-cystine. 5.86e 1.44 1.36 B 6
Valine 22.90 22.96 24.47 24.91 24.911 5.81 5.64 25 23

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Methionine . 4.20 3.78 3.52 3.67 3.79 f 0.29 1.17 0.80 4 4
Isoleucine. 29.39 30.46 30.78 31.53 31.53/* 0 8.41 7.14 32 25
Leucine. 25.33 24.93 24.73 25.12 25.03 f 0.26 8.68 5.07 25 33
Tyrosine 17.59 17.04 16.74 16.64 17.59d f 0.21 G.76 3.98 18 17
Phenylalanine. 15.26 14.84 14.88 15.04 15.0C*hf 0.20 5.20 3.40 15 15
Tryptophan.. 5.75 2.75 2.84
NH,... .:::.: 41.76 42.93 50.73 56.79 36.93d f 0.53 10.17 (3;) (4
Total. 91.46 100.0 362 f 2 363
-
a Based on sample weights. 1 Seventy-two-hour hydrolysate.
b Molecular weight 38,944 (F). 0 Includes alloisoleucine.
c Average of all four hydrolysates. h Phenylalanine was taken as 15 residues to calculate the resi-
d Intercept of a straight line calculated by the method of least dues of amino acids per mole.
squares. i Tryptophan was determined spectrophotometrically (22).
* Determined as cysteic acid (21).

FIG. 6. Sedimentation pattern of the purified pepsinogen, 10 mg per ml in 0.05 w Tris-phosphate buffer of pH 7.0. Sedimentation was
from left to right, and photographs were taken at a phase plate angle of 70 at 8, 16,32,48, and 64 min after reaching 59,780 rpm. The
temperature was 20; ~~0,~ = 3.24.

A large, inactive peak overlapped the desired peak of pepsinogen, pepsinogen,and mentionedthe possibility that someninhydrin-
indicating again the advantage of recycling. negative constituents could be present. Methionine sulfoxide
Yield-Table I summarizes the yields and specific activities present in the alkaline hydrolysate accounted for 7 y0 of the total
during purification. methionine.
The absorbanceof the purified pepsinogenat 280rnp was1.305
Properties of Bovine Pepsinogen for 1 mg of protein per ml.
Amino Acid Composition-Table II showsthe completeamino Molecular Weight-Molecular weight was determined by
acid composition. The 362 amino acids account for 100% of sedimentation and diffusion with a Spinco model E ultracentri-
the nitrogen determinedby the Kjeldahl method. On a weight fuge.4 The initial concentrationswereapproximately lye, 0.5%,
basis, the amino acids account for 91% of the sample. No amino and 0.25%. The sedimentation pattern is shown in Fig. 6.
sugarswere detectedon the amino acid analyzer, Kajagopalan, 4 We are indebted to Dr. C. J. Van Oss of the Milwaukee Blood
Moore, and Stein (6) found an identical 91% recovery with swine Center for carrying out the determinations.
Issue of April 25, 1968 R. B. Chow and B. Kassell 1723

the same (362 and 363). There are at least! 22 substitutions in

3.1 a actual amino acid composition. Nevertheless, certain char-
$ acteristics already well known for swine pepsinogen are also
a.1 4 t apparent in bovine zymogen, especially the large number of
acidic residues and the small number of basic ones. In both,
2 methionine, cystine, tryptophan, phenylalanine, and glycine
w .I o-
occur in equal numbers, and histidine, threonine, and tyrosine
Y
differ by only 1 residue. The large differences are compensated
$ .06-
in numbers by similar amino acids. For example, bovine pep-
5 sinogen has 32 isoleucine and 25 leucine residues, while swine
$ .02- pepsinogen has 25 isoleucine and 33 leucine residues. Similar
inversions occur with lysine and arginine and with aspartic and
glutamic acids. Notable differences occur in proline and histi-
EFFLUENT, ML
dine content; the bovine zymogen has 15 instead of 19 prolines,
FIG. Elution profile of bovine
7. and swine pepsinogens from and 2 instead of 3 histidine residues.
the same column, 155 X 0.9 cm, of Sephadex G-100. The sample
sizes were 1 mg in 0.5 ml; fractions, 1 ml; flow rate, 8 ml per hour; Based on amino acid composition,6 the 22 substitutions in the
eluting buffer, 0.05 M Tris-phosphate of pH 7.0. l - - -0, bovine; pepsinogens show a greater difference than the 16 to 17 which
O-O, swine. occur in the corresponding bovine (32) and porcine (33) tryp-
sinogens or chymotrypsinogens (34). It is worthy of note that
The molecular weight calculated by this method was 34,600. bovine and porcine heart cytochromes c have identical primary

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W7hen a sample from the same batch was used and the molecular structures (35).
weight was determined according to Archibalds approach to Three or four pepsinogens have been found in porcine (36)
equilibrium ultracentrifugation method (30), the result calculated and human (14) gastric mucosa. The existence of more than
was 40,400, giving an average value for the two methods of one pepsinogen in bovine mucosa appears likely. In the ex-
37,500. Since these were each single determinations, the amples of chromatography on DEAE-cellulose (Figs. 1,2, and 5),
e&mated error is 10% for each. As a further check, a com- there is evidence of separation of peaks or shoulders of potentially
parison of the elution profiles of bovine and swine pepsinogens on active material from the main fraction. These portions have
the same Sephadex G-100 column was made. Fig. 7 shows that been stored separately for future investigation.
both proteins were eluted within 1 ml, i.e. at the same position Although all other criteria indicate homogeneity, it would
within experimental error. This is in agreement with the equal appear from the evidence of disc electrophoresis that the purified
molecular weights for bovine and swine pepsinogen calculated pepsinogen is slightly heterogeneous. The minor contaminant
from amino acid analysis (38,943 and 38,944). is probably a second pepsinogen, which differs only slightly in its
liTomogeneity--Five criteria of purity were used. (a) At the amino acid composition from the major pepsinogen. Such small
final step of purification (Fig. 5B), the potential peptic activity variations would not have been detected if they were in the same
was uniformly distributed across the peak. (b) The molar ratios amino acids as those present in large amounts in the major
of amino acids present in amounts less than 10 residues per mole- pepsinogen.
cule were close to integral values (Table II). (c) The ultra- Another possible explanation of the second band seen in disc
centrifuge showed a single symmetrical peak (Fig. 6). (d) No electrophoresis is the presence of 7% of the methionine as
dinitrophenylamino acid spots, other than dinitrophenylserine, methionine sulfoxide. Sulfoxide-containing ribonuclease sep-
were detected by amino-terminal analysis (24). (e) Disc arated during chromatography from the same protein containing
electrophoresis showed a slight amount of heterogeneity; a methionine only (23).
second band, less negatively charged than the main band, was Preliminary experiments on the activation of bovine pep-
present, but by visual estimation it did not account for more than sinogens have indicated that the main fraction of active pepsin is
5y0 of the total in the preparation used for analysis (see Dis- easily separated from other small fractions, some of which are
cussion). Thus, we can conclude that the pepsinogen was active and some inact,ive. Since our intention is to study first
almost, if not absolutely, homogeneous. the sequence of the main pepsin (a smaller molecule than the
Pepsinogen Content of Bovine Abomasum-The total pepsinogen pepsinogen), we hope that the problem of slight heterogeneit,y,
of the fundic portion of a single mucosa varied from 0.4 to 1 g. if any, will be resolved during the activation of the pepsinogen
Mikhalenko (31) reported 0.42%. and the purification of the resulting pepsin.
Stability-Pepsinogen solutions in 0.05 M Tris-phosphate
Acknowledgments-We are grateful to Miss Rita K. Smyth
buffer of pH 7.0, with and without 0.45 M NaCl, were stored for
and Miss Janet Mak for technical assistance, and to Dr. Pat,ricia
9 days at 5 without loss of potential activity. At pH 5.0,
A. Meitner for helpful suggestions during the final stages of this
bovine pepsinogen was slowly activated overnight; swine
work.
pepsinogen was stable under the same conditions5
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Bovine Pepsinogen and Pepsin: I. ISOLATION, PURIFICATION, AND SOME
PROPERTIES OF THE PEPSINOGEN
Rosie B. Chow and Beatrice Kassell
J. Biol. Chem. 1968, 243:1718-1724.

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