Studies on Tryptase from a Whale(Balaenoptera Borealis L.)
Shichiro Akiya, Yasutaro Ishikawa, Setsuzo Tejima
and Toyohiko Tanzawa
In order to investigate the presence of protein decomposing enzyme
in the pancreas of a whale (Balaenoptera borealis Lesson), the pancreas
was dehydrated and degreased with acetone and other, dried and preparedinto a powder. Following results were obtaind.
- 1) The powder obtained by grinding the dried powder in the mortar andputting it through No. 5 sieve can pass the test for pancreatin describedin Par. 6 of Japanese Pharmacopoeia.
2) The most suitable pH value for gelatine is in the range of pH 8-9(Fig. 1) and the most suitable temperature (for 1 hour duration of activity
is 50°C.)
3) When the amount of base material is constant and the amount ofenzymes varied, the activity curve (Fig. 2) shows that the amount ofenzyme and actiyity are proportionate.
4) The activity curve (Fig. 3) obtained by holding the amount of enzyme,material, pH and the temperature of activity at a constant and varying thetime of activity, shows it increasing up to 4-hour period.
5) Distribution of tryptase in various parts of pancreas did not vary
greatly between those in tail, body and head parts, but the amount in thetail part was 110% of the total of other two parts.
The purification of pancreatic tryptase is to follow the preparative
methods for chymotrypsin and trypsin crystals irom bovine pancreas asreported by Northrop. Prepartions are now being made for carrying out theexperiments. Although no definite report can yet be made, the authors areconvinced that if bovine pancreas is used, crystals of chymotrypsinogen andtrypsinogen can be prepared comparatively easily by the Northrop method.
EXPERIMENTAL1) Preparation of enzyme powder
Pancreas obtained from a whale (Balaenoptera borealis Lesson) dissected at Ayukawa in Miyagi Prefecture was imnersed in twice its volume ofStudies on Tryptase from a Whale

9acetone for 10 days, taken out, finely cut with acissors and put.' through amincing machine. To this was added 2 to 3 volumes of acetone, drained,
and the process repeated 3 to 4 ti:nes to dehydrate and degrease. Finally,
the residue was shaken with an equal mixture of acetone and ether, filterd, inthe open and then dried in a vacuum desiccator over calcium chloride. From
2500 g of pancreas, 300 g of dried substance were obtained. This substancewas ground in a mortar and put through a No. 5 sieve (J.P.). Yield of thepowder to the dried substance, ea 40%.
2)

Panereatin Test, Par. 6, Japan Pharmacopoeia
0.1 g Enzyme powder prepared as in the above (1) was taken in a
500 cc measuring bottle distilled water added to make the total volume
500 cc and filtered. 2 cc of the filitrate was dissolved in 1 cc N/10 KOH
and distilled water added to make the total volume 50 cc, 3 cc of this solution was added to 2 cc distilled water, reacted for 1 hour at 40°C, after which
3 drops of acetic acid alcohol solution (1 parts glacial acetic acid, 9 parts
water, 10 parts alcohol were added. Only slight opalescence could be seen.

3) Determinnation of suitable pH of adivity
3 g Enzyme powder as described in (1) was added to 30 cc 80%
glycerine and left standing over-night at room temperature. A clear uppersolution was separated centrifugaly, 9 times its volumeo of distilled water
was added and 1 cc of this solution was used-as enzyme sample. For the base material,'.Z
0.5 g of gelatine was added to 10 cc each of ---.._•>- 1.500glycocoll-NaOH buffer solution (S:)rensen·s) 0/.l/ ~Qlof varying pH values, and melted at 50°C. [r;· 1.25/
To this was added 1 cc enzyme sample at :r: 1.0
040°C, held there for 2 hours and the amino .::i: 0.75
enacids thus formed titrated, in Willstatter's 0 .
ft0.50alcohol solution, with N/5 alcoholic KOH " 0.25
solution with thymo~-phthalein as an indicator. f>
1098
7
On the other hand, the base with 1 cc of disPH
tilled water instead of the enzyme powder
Fig. 1
was titrated and the difference between
the two values were taken as the degree of digestion at that pH value .
. Results are shown in Fig. 1.
4) Activity curve by the change in the amount of enzyme/

S. Akiya, Y•.Ishikawa:, S. Tejima · and T. Tanzawa

0.5 cc, lee, 2 cc, 3 cc, 4 cc and 5 cc of the glycerineextract of pancreas,as described in (3), were added to solutions in which 0.5 g gelatine wasdissolved in 10 cc NaOH-glycocoll buffer solution of pH 8.1 and water added
to make the total volume 15 cc. This was reacted for 2 hours at 40°C andfinally titrated as in (3). Results are shown in Fig. 2.
4~

3/

2/
v

.

/

glycerin

0extrac~ c.c.

//v/

3Fig. 217J/

I/2-i~~ ~/,__ v

z'CJl/

12
3
Dried pancreas(g)(0.03) (Q.06) (0.()9)

I!

I:i>;:;- 1I0~04(0.12)5(0.15~iI

1~I:i:~012345

6~Hours.

Fig. 35) Activity curve for varied action time
2 cc glycerine extract of pancreas as described in (3) was dissolved in
10 cc solution of NaOH-glycocoll buffer solution of pH 8.1 in which a baseof 0.5 g gelatine was dissolved. The reaction temperature was kept constant at 40°C and time varied at 15 minutes, 30 minutes, 1, 2, 3, 4, 5 and 6hours. Titration was performed as in (3). Results are shown in Fig. 3.
(Pharmaceutical department, the Facluty of Medicine, University of Tokyoand Whales Research Institute.)..