On the Respiratory Pigments of Whale(Studies on vVhale Blood II.)
Tadashi Tawara
The diving method of whale is very regular and ordinarily this may
be divided into the two classes of short diving and lorig diving. That is,
it makes a relatively shallow dive, then breathes by raising its head andback. above surface of water and then makes a shallow dive again. Thediving and breathing is repeated several times. This repeated dives arecalled surface dives. After repeating this surface dives several times, itmakes a deep dive in the water. This long dive is called "sound".
After one " sound ", surface d~ves are repeated several time. The time'of " sound " is relaively constant, depending on kind of whale, in case ofbaleen whale, it is from 7 to 10 minutes, but this exceeds 30 minutes incase it senses danger. It is especially long in case of sperm whale, which
is an tooth whale. It is from 30 to 70 minutes and averages about 40minutes.
Several explanations are given on how oxygen is supplied during suchprolonged submergence.
OmmanneyCl) states that the vascular networks (retia mirabilia) functions as this oxygen supply. That is, a special structure called vascular
networks which are abundant in blood vessels and fats ac~ as accessory
lung and functions as strage for oxygen. However, Laurie(2) states that
this is impossible . and points out that from the relation between. basal
metabolism and lung's vital capacity, whale can be submerged for a longtime. That is he calculated the lung's vital capacity of a 122,000 kg blue
whale at not less than 3050 liters and that the air requirement per minuteat 178.75 liters. He states that according to these calculations, it is possible
for a whale to be submerged for at least 17 minutes.
As stated above, a whale makes several surface dives and then a longdive. "sound" From this fact, the author considers that the whale breathes
several times during the several surface dives, by this stores a large
quantity of oxygen in some part of the body and consumes it during a long
submer gence. One of the storage organ may be the vascular networks asproposed by Ommanney out the author thinks that it is rather the respir-'On the Respiratory Pigments of Whale97atory pigments. The muscles of mammals which carry on a swiming life
such as pinnipedias and cetaceas are darker in color than those of landmammals. Especially in the case of sperm whale, the muscles are almost
black in color.
The so-called respiratory pigments are haemoglobin, myoglobin (musclehaemoglobin) and various kinds of cytochromes. All of these are pigmentproteins containing iron and conbines readily with oxygen. Of the above,the molecular weight of myoglobine is 17,500(3) or about 1/4 of that of
haemoglobin, about the same as that of haemoglobin in iron content, or0.345%,(4) combining power with oxygen and carbon monoxide is muchstronger than haemoglobin and also, reaction is much faster.(5) There areseveral kinds of cytochrotnes and it is believed that they act as carriersof hydrogen by· the oxidation and reduction of iron within the molecules.
The representative ones are a, b and· c.
The author carried on quantitative analysis of iron .in haemoglobin,
myoglobin and cytochromes a and c of these respiratory pigments anddetermined the content of the different pigments. As control, the bloodand muscle of cow and horse, which are land mammals, were used.
To determine the haemoglobin content in blood, Sahli's method using
haemoglobinometer was used concurrently with the method in which ironcontent is quantitatively determined directly. The result of the determination shows that by Sahli's method, there is no great difference inhaemoglobin content between whale and land mammals.
However, thedirect determination of iron indicated that it is considerably greater inwhale than in land mammals. In the case of land mammals the resultsfrom Sahli's method and iron determination method coincides rather closely
but in the case of whale, the iron content is greater in each case. That· is, it .can be assumed that there is a greater quantity of iron compounds
other than in the haemoglobin state. By Barkan and Schales,(6) it wasdiscovered that in the blood. of ma,.mmals, desides haemoglobin, iron-containing organic compounds whose iron ion is readily liberated by dilutehydrochloric acid are also present. It is said that this substance has anaffinity to oxygen a,bout 4 to 10 times greater than that of haemoglobin.
It can be considered that perhaps the presence of these substances isanticipated.
In regards to cytochrome b, the quantity was so small that it was

98

Tadashi Tawara

impossible to determine so it will be omitted. Content in cytochrome aand c did not differ greatly from those of land mammals. Thus it 1s
assumed that it did not have any special· meaning relative to oxygenstorage.
Myoglobin content in whales is much greater in all cases when compared with land mammals and especially in the case of sperm whale which
ma.kes "sound", the content is about 8 times more.
From· the above result, it is assumed that one reason Why whales can~ithstand " sound" is that oxygen is stored by myoglobin within the muscles.
The author wishes to thin:k Dr. Tsutomu Naruyama and Mr. Tadashi
Nakai for guidance in this experiment and to Profs. Shichiro Akiya andTeizo Ogawa for their instructions. The author is grateful to Mr. Ryusuke
Fukazawa for assistance in the experiment and to the people of theAyukawa Station of the Taiyo Fishing Co., for assistance in providing
materials.
Experimental
Materials used in this experiment was a sperm whale and a sei whale
caught off shore of Kinkazan Island, the former 31 feet o and the latter
45 feet o . In both ·cases, they were 24 hours after death.*
Materials from cow and· horse used as control was obtained immediately
after slaughter from the Shibaura Slaughter House in the case of heart
and in the case of muscle, those on the market were used.
(1) Quantitative IX:ttirmination of Haemoglobin
Blood samples in each case was obtained from the heart.
(A) Sahli' s Method
According to Sahli's method, haemoglobin was transformed into acidhaematin by adding ~- HCl and colorimetric determination was madewith Sahli's Haemoglobinometer.
(B) Quantitative Determination of Blood Iron
In accordance with Pincussen's method,(7) blood was decomposed with·,sulphuric acid and 30% H202, and comparative colorimetric determination
was made of it, colored with KCNS, with standard concentration solutionof Iron Alum (FdS04)3 (NH4)2 S04•24RO) colored with KCNS, using* Materials were obtained in July, 1948. After this, there was a revision in law and cathing ofsperm wh&le les$ than 35 feet was prohibited,On the Respiratory Pigments of Whale99

Duboscq's colorimeter.
Table 1.

Sei Whale
Sperm Whale
Spem Whale foetus
Cow
Human(29 years old)

Quantity of Haemoglobin (in 100 cc of blood)
Haemoglobin

Fe (Hbx0.336)

Fe(Pincussen Method)

15.6g

52.4mg53.lmg19.Smg41.6mg43.7mg84.0mg95.0mg53;smg41.0mg46.5mg

i5.sg
5.8g
12.4g
13.0g

Quantity of haemoglobin determined by Sahli's method multiplied by
0.336 should give the quantity of iron in the haemoglobin state.
In the·case of cow and human the quantity of iron in the haemoglobin state andthe total iron content is about the same. However, in the case of whale,iron determined by Pincussen's method is much greater. The differenceis great especially in the case of foetus. It is believed that this is. becauseof the presence of larger quantity of iron other than in haemoglobin state.
(2) Quantitative Determination of Iron in Cytochrome State
In order to be aC"curate, the quantitative determination of cytochromeshould be made by spectral analysis but in the case, it was refined considerably and iron content determined. The determination figures· does notindicate absolute quantity but relative quantity determined under the sameconditions. For materials the above-mentioned heart and muscles of whale,
cow and horse were used.
(A) · Quantitat~ve Determination of Iron in Cytochrome a StateCS)
A solution containing 2% Na-cholat and -21Q- mol Na2HP04 is, forconvinience, designated as A-solution. Muscle is washed with water,
chopped with a meat chopper and 1 kg taken. 3 liter of A-solution isadded to this, extracted for one night at room temperature, strained withgauze and to 3 liter of the filtrate, 750 cc of a weakly ammoniac saturated
ammonium sulphate soltion is added (0.2 saturation). ·This liquid is· separated with a centrifugal separa!or and to the clear liquid is added
more saturated ammonium sulphate solution to make it 0.5 saturation.
Cytochrome a will precipitate out. The precipitate is placed in · a centrifugal separator, disrnlved in 200 cc of A-solution and made 0.2 saturation with saturated ammonium sulphate solution. This solution is left
standing for 2 days in an ice room, precipitate is removed with centrifugal separator, the clear liquid is mad~ 0.33 s;ituration with ammonium

Tadashi Tawara

100•sulphate, precipitate is dissolved with 100 cc of A-solution, precip:tateobtained by 0.2 saturat'on ammonium salphate is removed, precipitate
obtained by 0.33 saturation is collected and this operation is , repeatedonce more. Finally, precipitate obtained by 0.33 saturat:on becomes a
reddish brown, paste-like substance. This is dissolved in A-solufon, thetotal quantity brought to 100 cc., 10 cc. of this is taken, the entire precipitate obtained by saturation .with ammonium sulphate is decomposedwith HS04 and H202 and iron determined by the aforement'.oned Pincussen's method.
Table 2.

Quantitative Determination of Iron in Cytochrome a State (mg/kg)
Sei Whale

Sperm Whale

1.29
2.54

1.91
4.03/Cow··~-

Body muscle
Heart muscle!

2.28
0.27

In the case of CO\y, the figure for body muscle was larger than forheart. In this case, it was not possible to obtain body muscle ar:d heartfrom the same cow so the materials came from different cow.
(B) Quantitative Determination of Iron in Cytochrome c State(9)(10)The· muscle sample is washed with water, chopped with a meatchopper, 2.5% trichloracetic acid is added to 1 Kg of this, stirred andextracted for about 2 hours at room temperature. This is then strained.with gauze and 1 liter of the filtrate is brought to pH 7.0 with 6N-NaOH
solution. 500 g of ammonium sulphate is· added to this and left standingfor one night in an ice room. Thrs is centrifugally separated and theclear liquid brought to pH 3.7 by adding 5N-H2S04. Precip'tate preduced
from this is separated with centr;fuge, 20·cc. of water is added. to thisand· dialyzed (cellophane) against 1.% saline solut'on for 2 days. Theliquid. thus obtained is centlifugally separated and a dark red, clear
cytochrome c solution is obtained. This is diluted to 100 cc., 10 cc. of
this is saturated with ammonium sulphate at 60°C, the entire precipitate
is decomposed with H~so4 and 30% H202 solution and quantitative
determination of iron made by the aforementioned method.
Table 3.

Quantitative Determination of Iron Cytochrome c State (mg/Kg)

Sei Whale j Sperm Whale__B_o_d_y_m_u:C1-e---i--0-.4_3__
0.47

Blue Whale0.44

iIHorseCow--0-.3-0_ _ ,._ _ _ __1_.i!__ ____H_e_a_rt_m_u_sc_Ie______
1._29_~~-o_.5_7_~--~·-~!_ _ 0.70 _____

On the Respiratory Pigments of Whale101

Cytochrome c is obtained by multiplying this quantity of iron by ~~4~ .
(3) Quantitative Dete:rmination of Iron. in Myoglobin. StateC4)
Sample muscle is washed with physiological saline solution, chopped
with a meat chopper and l kg of this is taken. - l liter of water is added
to this, extracted for l night at room temperature and strained with gauze.
N-NaOH solution is added to l liter of this solution and brought to pH 7.0,
250 cc of concentrated basic lead acetate solution is added and the precipitate produced from this is separated with centrifuge. Na2HP04 solution
is added to the clear liquid while maintaining it neutral with NaOH andlead is removed. 5 cc. of the clear liquid obtained by centJ:ifuge is taken,
saturated with ammonium sulphate, the entire precipitate is decomposedwith H2S01 and 30% H202, and colorimetric determination of iron, in theform of Rhodan-Fe, is made.
Table 4.

Body muscle
Heart muscle

Quantitative Determination of Iron in Myoglobin State (mg/kg)
Sei Whale

Sperm Whale

Cow

I

31.35
7:62

151.00
23.12

18.08

IHorseI- - - - - - - - - - - - - - - - - - - - - - - - - - - -------------- ··------------

I

7.15----- ----- - - - - - - -Myoglobin is obtained by multiplying this iron quantity by ~~ .
5Compared to cow, the myoglobin content is 2 times more in sei whale, and8-9 times more in sperm whale.

0References( 1) Ommahey F. D.: Discovery Rep. V. 329 (1932).
( 2) Laurie A. H.: Discovery Rep. VII. 365 (1934).
( 3) Theorell H.: Biochem Z. 268. 46 (1934), C. A. 28. 2736 (1934).
( 4 ) Hugo H., Theorell H. : Ibid, 252. 1 (1932), C. A. 26. 5587 (1932).
( 5) Theorell H.: Ibid, 252. 1 (1932), 268. 64 (1934), C. A. 26. 5587 (1932), 28. 2737 (1934).
( 6) Barkan G., Schares, 0.: Z. Physiol. Chem., 244. 81 (1936), 244. 257 .(1936), 248. 96 (1937), C.
A. 31. 715 (1937), 31. 1051 (1937), 31. 7898 (1937).
( 7) Pincussen: Mikromethodik, 108 (1930). Sudo, K.:lgakuteki Biryo Sokuteiho 155 (1937).

( 8) Yakushiji E., Okunuki K.: Proc. Imp. Acad. 17. 38 (1941)•.
( 9) Keilin D., Hartree E. F.: Proc. Roy. Soc. (London), B 122. 278 (1937), C. A. 31. 5390 (1937).
(10) The~rell H. : Biochem Z. 298. 260 (1938), C. A. 32, 969 (1938).