Protein Digestive Power of Sperm Whale
Pancreatic Enzyme. II.
"'Yasutaro Ishikawa
IIn the lst report, protein digestive power of pancreatic enzyme from
sperm whales was compared with that of a cow and was found much
weaker than the latter. Casein was used as the substrate and the digestive powers were measured by the method of Michaelis given in the, Japanese Pharmacopoeia.
Even after being activated in various manners, enzyme ·from whales
digested only 2.5 times of casein of its own weight while that from a cow
digested 125 times. This large difference was almost unbelievable.
However in the experiment previously reported the samples werecollected from too few bodies to discuss generally. And the time-lapse
from the caching to the dismembering of these whales was so long underhigher temperature of the summer, that there was a suspision that a partof proteases may have been destroyed by autolysis.
The present experiment was undertaken in order to eliminate these
suspicions and .to determine the true protein digestive power of a sperm
whale- pancreatic enzyme. The samples, therefore, were collected at thecoldest season during whaling (December_ 1948) and only the very freshones were used. It had been desired to collect s.amples from as many
animals as possible in order to eliminate individual differences and samples were collected from six fresh sperm whales during the two weeks at
the landstation. Ayukawa, Miyagi Prf.
EXPERIMENTAL
1) Whales Used,No.

KindSex

Body length(ft.)

Time elapsed from·catching to dismember (hrs.)

Freshness

1

Sperm
Sperm
Sperm
Sperm
Sperm
Sperm

male
male
male
male
male
male

45
46
46
50
4251

25252318222085
8590858585

23456%

The freshness is the value evaluated by the whaling experts on thestation by macro-observations with considerations to the elasticity and color of

Yasutaro Ishikawa

72

the whale meat and others. The freshest state, i. e. that right after death,is taken as 100 and deterioration from that is shown by a rough percentage. Various scientific methods for the determination of freshness havebeen forwarded but none is compl~ted and procedures are too complicatedso that it has to rely on these 'empirical method.
2) Treatment a,nd Preparation of Samples
As soon as the whale body has been cut open, the pancreas taken out,
adipose and connective tissues removed as much as possible· and mincedby a hand chopper, From this were preparad 3 kinds of samples forreasons as described in (3). These samples were:a) One dried immediately with acetone.
b) ·The minced pancreas left standing overnight at room temperature
with small amount of toluen in a flask, stoppered, which was thendried with acetone.
c) The minced pancreas mixed with 30% aqueous acetone solution in
an equal amount and stored.
Samples (a) and (b) were prepared by adding anhydrous acetone tominced pancreas in an amount about 5 times its volume, well stirred andthe supernatant solution decanted. The same amount of acetone is thenadded and filtered With suction. The residue on the funnel' is washed, firstwith acetone and then with ether, and is dried by spreading it out in theroom. This is then well dried in a dessicator over Sulfuric ac:d. Procedures
up to this point were carried out at the statiori and this was ,then brought
to the laboratory for testing. The dried pancreas was first powdered bya boat-shaped mortar; sieved through a 60 mesh sieve and divided into
powders of gland substances and connective tissues. Special note shouldTable I.
No.

·powder (g)Sieve residue (g)Amount sieved (%)

IA
2A
3A
4A
4B
5A
5B
6A

2.8
1.9
3.0
3.2

5.4
11.6
10.7
9.4
4.0
9.0
5.1
7.5

34
14
22
25
26
37
55
41

1.4

5.3
6.3
5.3

A denotes samples dried immediately
B denotes samples dried after being left overnight.

. .-•.!.~Protein Digestive Power of Sperm Whale Pancreatic Enzyme. ll.

abe taken of the f(tct that the pancreas ofsperm whale contains muchlarger percentage of connective tissues compared to that of bovines, hogs
and baleen whales, so that the yield of gland substance is very poor. The
resultant yield of sieving is given in Table I.
The samples immersed in 30% acetone were brought back to the laboratory and stored at room temperature. And the supernatant solution
which might contain protease, was taken from time to time to test thedigestive power.
The kinds of samples prepared were as follow :No. 1-A,
No. 4-A,
BcNo. 2-'A,B,
No. 5-A,cc
No. 3-A,
No. 6-A, ·B (lost), CcA; denotes samples dried immediately,B, samples dried after being left overnight, andC, samples immersed in 30% acetone.

Method of Activation
As in other kind of animals, pancreatic protease from sperm whales
do not show digestive action in its fresh state. It follows, therfore, that
this protease must in some way be activated and the subsequent testingof digestive powers must be carried out at the same degree of activation,in order that a compaison can be made ; if possible, at -a maximal activatedstate. For this reason, activation was carried out in following three ways
and the digestive powers determined and compared.
a) The pancreas was dried in the most fresh condition, powdered as has
been described before and this was activated by enterokinase from asperm whale.
b) Minced pancreas was left overnight at a room temperature and treated in the same way as before. This was then activated with enterokinase.
The above two were made in order to find the effect of autolysis in
the fresh condition.
c) Minced pancreas was dispersed in the same amount of 30% aqueous
acetone solution. and its supernatant solution, assumed to contain protease from the pancreas, was used as a sample for testing.
The reason that this experiment was carried out was that Kleiner andTauber reports2) that the protease from hog pancreas is best extractedwith 33% alcohol and is well activated by a long-time storage.
Tejima,3)3)

74

Yasutaro Ishikawa

also obtained a successful result by extracting protease from a sei whale
pancreas by the use of 30% acetone.
4) Meth0,d of Digestive Power Determination
The method of determining digestive power followed the same one as
described in the previous report1>, i. e. the Item 6 of the Japanese Pharmacopoeia for pancreatin which uses Michaelis' method of protease testing.
The substrate used was casein purified by Hammesten's method andstored in a dessicator over sulfuric acid. 5 cc. each of 0.2% solution ofthis casein (containing 2 cc. 1/10 N-KOH in 100 cc. of this solution) is taken
in test tubes and these tubes are stood in a row. A definite amount ofenzyme is dispersed in a definite ~quantity of water to extract the enzyme
and a definite amount of this enzymatic solution is added step-wise to thecasein solution and the total volume is then brought to 10 cc. each. Thissolution is kept for 1 hour at 40° to effect digestion of casein. After exactly
1 hour, 3 drops of a mixture of 1 cc. glacial acetic acid, 9 cc. water and 10cc. alcohol is added to such test tube. One that remains clear is taken as
digested and the one showing least opalescence is taken as the limit ofdigestion. Digestive power of enzym-containing powder is found from theamount of the powder used to digest 5 cc. of 0.2% casein solution (i. e.
0.01 gm. casein) in the limit case and is then shown by the ratio of caseinto the enzymatic powder.
In the case of 30% acetone extracted solution, a definit of its supernatant solution is taken, suitably diluted and used in determination asabove.
5) Results of the Experimentsi} Acetone dried powdera) Digestive power when enterokinase is not added
Digestive power was determined with No. lA, No. 2A and No. 4B.
None of these had the power to digest casein equal to their own weight.
It follows, therefore, that the acetone dried powder either lacks digestive
power totally or has a very weak action.- This in turn may mean that theenzyme is in an inactivated state due to freshness of pancreas or thatenzyme had been destroyed altogether. 'b) Digestive power when enterokinase is added
0.2 g. each dried powder of No. lA and No. 2A was taken, 0.05 g. ente·rokinase added end dispersed in 100 cc. of water. This was warmed at 40°

Protein Digestive Power of Sperm Whale Pancreatic Enzyme. II.

75

for 15 minutes, 30 minutes and 1 hour and activated. The result of thedetermination of digestive power using these extracts was as follows :No. lA (30min.)Digest 10 times its weight of casein but
cannot digest 12.5 times its weight.
ditto

No. 1 A (15 min.)No. 1 A (1 hour)ditto

No. 2 A (15 min.)ditto

No. 2 A (30 min.)ditto
Digests 7 times its weight of casein but
cannot digest 10 times its weight.

No. 2 A (1 hour)

From these results, it can be seen that the time of warming for activation does not differ much between 15 minutes and 1 hour but is slightly
inferior at 1 hour.
Therefore, the conditions . were set for an addition of a 1/4 amount ofenterokinase and warming for 30 minutes at 40° to effect proper activation~
The result of determination of digestive power of various samples were as
follows:
No.1 A

Digests 10 times its own weight but not 12.5 times.

No. 2 Aditto·

No. 3 A

ditto

No. 4 A

ditto

No 4 B

Digests 8.3 times iots own weigh~ but not 10 times.

No. 5 A

ditto

No. 5 B

Digests 10 times its own weight but not 12.5 times.
Digests 8.3 times its own weight but not 10 times.

No. 6 A

Digestive power of each sample made from above results is shown in
Table II, which indicates that there are hardly any difference between thesamples:
Table II. Digestive Power of Acetone Dreiecl. Powder 'Sample No.

Digestive Power(cas./powder)

Sample No.

Digestive Power(cas./powder)

IA102A
3A
4A

104B
5A
5B
6A

881081010'30% Acetone Extracted Solution
The solution was stored at room temperature for about 2.5 months
during which a definite amount of the clear, supernatant solution was taken
and ·its digestive power determined. Table III, shows the relationshipbetween the digestive power and the time lapsed after immersion in acetone.
The figures denote the amount of acetone solution (in cc.) necessary toii)•Yasutaro Ishikawa

76

digest 0.01 g. of casein (shown by the least opalescence manifested) so thatthe smaller the figures, greater the digestive power.
Table Ill. Relationship between digestive power and lapse of time.
Lapse of time(days)

1 c5791/100

11

1/100

1315
17
21
2326
34
54
75

2 c

3 c

SC

6C

2/100

6/100

1/100
1/100

10/100

1/100

4 1100

1/100

2/100

1/100
11100
0.8/100
11100

1/100
1/100
0.8/100
1.5/100

15/100
1/100

3/100

15/100

1/100
1/100
1/100
1/100
1.5/100

2/100
1/100
1/100
1/100
1.5/100

12/10012/100
9/100
9/100
9/100

As can · be seen from Table III, each sample differs greatly in its
digestive power at t?e start of experiment but after about one month, allsamples, except No. 3 C, show constant vaiues of 1/100. This level is kept
until about 2 _months have elapsed after whi~h the power gradually decreases·slightly.
DISCUSSIONS
1) Direct comparison cannot be made between the value of 8-10 fordigestive power obtained by the determination of dried powder and thevalues obtained with 30% aceton solution.
However, if the amount ofwater included in minaed pancreas is considered and if the whole amountof enzyme present in the pancreas were to have transferred to the solution,then in the case of 30% acetone solution, the digestive power of dry pancreas as a whole can easily be calculated.
If the amount of the dried matter is assumed to be 25% of the mincedpancreas, and since the minced pancreas was added with an equal amountof 30% acetone, then 1 g. minced pancreas + 1 cc. ~0% acetone· . 0.25 gdry pancreas + 1.75 g extracted solution. i. e. 0.25 g dried matter froni
pancreas should have the same digestive power as 1.75 g. of the extracted
solution. Therefore, 1/100 cc of this extracted solution is equal to10·25. d ma tt er of pancreas.
1.
xg dne75 100Protein Digestive Powar of Sperm Whale Pancreatic Enzyme. II.

77

Since this amount had digested 0.01 g. of casein, then the digestive powerof the dried matter of pancreas is/ 0.25
170·01 '1.75
xToo =i. e., it has digested 7 times its own weight of casein. Of course, this .
calculated amount shows the digestive power of pancreas as a whole but inthe case of acetone dried power, it has been put through the sieve during
which purification of the enzyme may have been efected so that a precise
comparison cannot be made as to which is larger. However, it is consideredthat the order is well adapted in this case.
2) When enterokinase is not added to the dried powder of pancreas,· nodigestion occurred. From this fact, it can be assumed that these pancreas
was stored in a comparatively fresh state until treatment and that protease
had not been destroyed and stayed in its natural state.
The digestive power of the dried powder of pancreas when activatedby the addition of enterokinase was found to be about the same in eachindividual and digested 8_:__10 times its weight of casein. The protein
digestive power of 30% acetone extracted solution varies greatly at first
but becomes most activated about one month after immersion at whichtime, with the exception of one sample, the values are exactly the samefor all the samples. Ih this oi1e exception, i. e. 3 C the supernatant solution
was found to be considerably opalescent due to the inclus:on of grease (theothers were almost clear). This is apparently the error due to the inclusionof a large amount of adipose tissues at the time of sampling.. The value
when this same level had been reached in all the sample, i. e. 1/100, showsthat the dry matter of the original pancreas would digest ~bout 7 times
its own weight of casein.
From these results, it is considered that the digestive power of proteasefrom sperm whale is considerably inferior to that from bovines and hogs.
However, this is true only when casein has been used as a substrate andit is doubtful whether the same tendency will be manifested when otherproteins are chosen as a substrate.
From the results of. these experiments, it can be assumed that there
is very little difference between individuals.

78

Yasutaro Ishikawa

SUMMARY
Protein digestive power of pancreas from 6 sperm whales 'Yas determined and following results were obtained :1) Acetone dried powder does not show digestive action as it is.
2) When acetone dried powder is activated by the addition of 1/4 its
amount of enterokinase from a sperm whale and warmed for 30 minutes
at 40°C, it digests 8-10 times its own weight of casein and there were
no differenc between individual animals.
3) 30% Acetone extracted solution of minced pancreas, reached its maximal activated state after storage of about one month at room temperatureand the digestive power was the same for all the samples, i. e. 1/100 cc.
of this. solution digested 0.01 g. of casein. Calculated from this value, thedry matter of the pancreas used would have digested about 7 times its
weight of casein.
References·1) Ishikawa: Bulletin of the Japanese Society of scientific Fisheries Vol. 14, No, 2, 107 (1948);
Ishikawa and Tejima: Sc. Report of the Whale Reserch Institute, No. 2, (1949).
2) I. S. Kleiner and H. Tauber: J. Biol. Chem., 104, 267 (1934).
3) Tejima a1t1d Ishikawa: Lecture read before the 3 rd Meeting of the Whale Reserch Institute in October, 1948, unpublished.