STUDIES ON WHALE BLOOD. I.
On the separation of histidine from whale blood.

Tadashi Tawara
Histidine is an amino acid of the following formula :HC=C-CH2-CH-COOH

IIN NH'\,/INH2cH

This amino acid was discovered in 1896 by Kossel and is found invarious proteins, being most abundant in hemoglobin which contains about
11% histidine.
Histidine is one of the most important amino acids for nutrition and it
is contained comparatively in a large amount in fish meat. Putrefactionof meat turns histidine into a poisonous histamine. Histidine is used forgastric ulcer.
Two methods of synthesis have been worked out by Pymann.1m The
most common material now being employed for hislidine manufacture isblood. There are two principal manufacturing methods, one by precipitating
the silver salt in alkaline medium, 3)4)5) and the other using the mercuricsalt.6>7 )3>
The author used dried blood of a sperm whale (Physeter catodon L.)
as material for his experiments.
Cystin, methionine, ornithin, tryptophane and histidine are some ofamino acids that precipitate by corrosive sublimate (mercuric chloride).
Cystine almost precipitate out in neutral media, while ornitine and tryptophane are not obtained by hydrolysis with hydrochloric acid. Methionine
precipitates almost wholly in saturation of sodium chloride around pH 6.0.
The author, therefore, undertook the experiments with a special consideration for the removal of methionine.
Experimental
1 kg. Dried blood of sperm whale was hydrolysed with 4 L. 30%
hydrochloric acid for 20 hours. After cooling, pigment that precipitated

On the separation of histldine from whale blood

39out (melanine) was removed by filtration and the filtrate was neutralized topH 2.5 with concentrated sodium hydroxide solution. After standing overnight, the solution was filtered, and the precipitate was washed with a small
amount of water. The filtrate and washings were neutralized to pH 6.0
with sodium hydroxide solution and concentrated untill sodium chloride
precipitated out. The mixture was cooled and filtered. Methionine canbe obtained from this precipitate.
A hot saturated solution of 400 g mercuric chloride was added to thefiltrate with stirring and after being left overnight, was filtered. A concentrated solution of .sodum carbonate was added slowly, with stirring, until
the pH of the solution reached 7.2. After stirring overnight, the supernatant liquid was decanted and the flask filled with water to the original
volume. This procedure was repeated several times, and the mixture was
finally filtered by a Buchner funnel. From the washings, mercury can be .
recovered by passing hydrogen sulfide gas.
This precipitate (mercuric salt of histidine) was suspended in lL. ofwater and decomposed by passing hidrogen sulfid gas. This was filtered
to remove mercuric sulfide, which was then boiled with water several time
to recover histidine adsorbed in the precipitate.
•The filtrate and washings from mercuric sulfide were concentrated toabout 150 cc, mixed with 300 cc 95% alcohol. and stood overnight ii:l an ice
chamber. Crude crytstals of histidine monohydrochloride were thus obtained.
The crude product was dissolved in five times its weight of water, andthen diluted with twice its volume of 95% alcohol. The yield of recrystallized histidine monohydrochloride was 39 g. This compound melts at 252°C
with decomposition.
In conclusion, the author wishes to express his sincere thanks to Dr.
T. Maruyama, Mr. H. Okuda and Mr. T. Nakai for their kind guid andadvice in this study.
His thnks are also due to prof. Dr. S. Akiya and prof. Dr. T. Ogawa,Tokyo University, for their valuable suggestions.
He is also indebted to Mr. R. Fukazawa for kind assistance during tl;Iework.
References
1)

Pyman: J. Chen. Soc., 99, 668, 1386 (1911).

•Tadashi Tawara

402)
3)
4)

Pyman: J. Chen. Soc., 103, 159 (1941).
A. Kosse! F. Kutscher: Z. physiol. Chem., 31, 165 (1900-1).
H. B. Vickery, Leayenworth : J. Biol. Chem., 72, 403 (1927) ; 75, 115 (1927) ; 76, 707(1928) ; 79. 377 (19'.:8).

••5)

H. B. Vickery, K. J. Block : J. Biol. Chem., 93, 105 (1931).

6)7)Org. Syntheses : 18, 43 (1.938).
Brig! : Z. physiol. Chem., 64, 337 (1910).

8)

M. T. Hanke, K. K. Koessler: J. Biol. Chem., 43, 521 (1920).