Isolation of Histidine from Whale Blood using 3,4-Dichlorobenzene Sulfonic AcidSHICHIRO AKIYA and 0TOMATSU HOSHINO

Isolation1) of histidine from hydrolyzed product of proteins used silver ormercury salts method, and a sublimate method in industry. However, the latter
necessitates the use of hydrogen sulfide gas, a large amount of mercuric chloride,
and results in a variety of yield on histidine according to the condition of precipitation, as well as the process being very complicated.
Stein and Bergmann2J.3l, in 1938-1940, made a wide ressearch to find aromatic
sulfonic acids which would form insoluble salts with amino acids and found threekinds which made insoluble salts with histidine. These acids were:Cl

3,4~Dichlorobenzene sulfonic acidI-)-c1HOas--(-II2,6-Diiodophenol-4-sulfonic acidHo-<_~SOaH
I

I/="'-/="'-.~

0-(2,4-Dinitrophenyl)-p-phenol sulfonic acid 02N -,_._/-o-,I/ ~OaH--

N02

Vickery >, in 1941, employed one of the salts, 3,4-Jichlorobenzene sulfonic4acid (hereafter designated 3,4-D), and obtaine::I good results in isolating histidine
il'from the hydrolyzed solution of horse red blood cells. Further, in 1942, heapplied 3,4-D method for the quantitative estimation of histidine. 5 )The features of histidine isolation by 3,4-D are as follows:
1) Preparation of 3,4-D is very easy and 3,4-D is the specific precipitation
agent for histidine. ·2) Histidine precipitates gradually as a disulfonate from acid solution whereas precipitation as silver or mercury salts occurs from neutural to weak alkaline
solutions.
3) Isolation of the disulfonate of histidine is complete and easy since it iscrystalline and can easily be purified by discoloration and. recrystallization. The. crystals also have far higher decomposition point (275-280°C) than 3,4-D salts ofother amino acides (generally m.p. around 200°C) which makes it easy to prove.
4)

Although the process is not quantitative, it is much easier than the silver

or mercury salt metho::I.

42

S. AKIYA, 0. HOSHINO

Tawara6l separated histidine from dried whale blood powder. The a~thors
examined isolation of histidine from whale and cattle blood by the use of 3,4-D
and the results were compared with that of Tawara's.
It should be noted . that the dried and powdered whale blood contains farlarger amounts of moisuture anl salts than those of cattle and horse blood, sothat ·a correction must be· made of the values. If this crude whale blood powder
is hydrolyzed and 3,4-b used, isolation does not proceej smoothly due to obstruction by salts, resulting in the decrease in the amount of histi1ine disulfonate,
so that the yield cannot be made better by adjusting the content of moisture andsalts.
In order to remov:e salts from whale blood p)wder prior to hydrolysis, apretreatment is made by adding water to the crude blood powder, stirring welland filtering. The residue is washed well with water and air-dried. This bloodpowder (hereafter designated as the pretreated blood powder) contains almost nosalts so that, when the hydrolyzed solution is concentrated, only the hydrochloridesof glutamic acid precipitates out ... The yield of histidine also increases remarkably, being better than that obtained from cattle blood carried out as a control.
On the other hand, isolation of glutamic acid hydrochloride is very difficult from
untreated whale blood powder owing to occlusion of sodium hydrochloride.
Addition of water to cattle blood powder results in its swelling which makes
it hard to' wash with water. This elimination of wa~hing with water al.lows someinclusion of sal.ts. The bl.ood cells used by Vickery was washed well with water
so that the yield in final ·product is very high. It can be assumed., therefore, thatthe content of ·sait in blood powder controls the formation of histidine disulfonate
when using 3,4..:D method-.
Monosulfonate of leucine is obtained in a considerable amount from the motherliquor after separating histidine disulfonate.
Recently, Takagi, Suzuki and Asahina7l, used acid clay in the hydrolyzed
solution of cattle blood to adsorb basic amino acids, then eluted this by weakalkali. This alkaline solution was electrolytically dialyzej, then 3,4-D added, andhistidine was isolated with a good yield as its disulfonate. As was explained in theirreport, majority of 3,4-D can be recovered as a barium salt.
Acids used for hydrolysis were hydrochloric and sulfuric acids but the yieldof histidine disulfonate was not so good when 30-35Yo l:)Ulfuric acid was used.
The method of isolation and the results are given in Tables I and II.

Isolation of Histidine from Whale Blood

Table I.

43

Method of Isolation(Pretreated) Whale hlood (100 g.)

Ij cone.
HC1(300~cc.)
w .iter (200 cc.)

Heating for
20-24 hrs.

Filtration

I·I

I

Filtrate(Ca. 500 cc.)
Discoloration[ (Charcoal 10 g.)
Low-pressure·Concentration

III

Crude glutamic acidhydrochloride

RemoveHCl asmuch as! Add water.

(Ca. 9-10 g.)

po~sibleI

DiscolorationI Recrystallization

IGl.utamic addhydrochloride
m.p. 208-9°C(ea. 5-5.5 g.)

IIRepeat
low-pressure concentration 2-3 times

Syrup
Add .

·I Add water to make

3,4-D-~> 1400 cc.

(45 g.)

Discolor

ILeave in ice-box
II
I

I

Histidine disulfonate(20-21 g.)
Recrystalized(18-19 g.)

II

I

Histidine
Histidinehydrochloride decompn. 270-2°C
m.p. 252-4° (ea. 4-4.5 g.)(ea. 6.0 g.)

IFiltrate

Ij Ba(OH)2I~--~~~~~~~~-I

Iconcentration
Low pressure,---,

I

Leucinemonosulfonate(ea. 20-25 g)

I

Filtrate. HClFiltrate

Barium saltof 3,4-D.

: decomposed: by H2S04

3,4-D
recovered

44

S. AKIYA, 0. HOSHINO

Table II. Amount of Histidine Disulfonate isolated
from 100 g. (moisture corrected) of Various
Blobd Powder (salts uncorrected)
Kinds ofBlood
Untreated Whale: No. I
bloodi No. 2f No. 3
j No. I
Cattle blood
No. 2I

Pretreated whale: No. I
I No. 2blood!
\BloodHorse bloodcells
TechnicalhemoglobinI(Xot€:II-:Moisture j Histidine jRemarks(%)
disulfonate(g)
12.3
28.6
' Glutamic acidCcntained
28.6
13.3
jchorides/ hydrochloride
2 8.6
13.6
(as NaCl) 9.4~~ ~ not isolated
due to salt
12.0
14.5
precipitation
12.0
17.3
Hydrolyzed
18.6
Gr~amic acid l~~fu~l~l~ri~1e i~Pd
after
19.0
11
not isolated
corection
25.7)

II

5.5

25.7

7.7

18.1

Taken from. the dataof Vickery 4) for cJmparison

The yield on histidine disulfonate does not differ much even whenseparation of glutamic acid hydrochloride is carried out as in No.
I of pretreated whale blood. The amount of leucine monosulfonate
decreases slightly).

EXPERIMENTAL1)

Preparation of 3,4-Dichlorobenzen sulfonic acid:A mixture of 65 cc. of o-dichlorobenzen and 140 cc. of cone. H2S04(d= 1.84) isheated in a flask in an oil bath at 185±5°C for 241'ours under air cooling. Aftercool, 700 cc. of water is added and discolored with activated charcoal. The discoloredsolution is concentrated to one-half of its volume on a water bath, cooled, andfiltered through a glass filter. The residue on the filter is washed with cone.
HCl, H2S04 removed, suck and dried. In order to remove cone.. HCl and water,
this residue is place:! in an evaporating dish, melted on a small flame andheated gradually to 130°C at which the heating is continued for a ·few minutes.
After cooling and before solidification, the mass is stirred into small pieces and is
kept in the dessicator since it is hygroscopic.
3,4-Dichlorobenzen sulfonic acid (3,4-D) herewith obtained contains a small
amount of water aiid is a white, crystalline mass which melts at around 70°C.
This can suitably be employed for the isolation of histidine.

Hydrolysis of Whale Blood Powder:
In order to examine the effect of salts, hydrolysis were carried out with crude
whale blood powder, washed and unwashed with water.
(a) Untreated blood powder-Small pieces of crude whale blood powder iSground in a mortar, sieved to remove all occluded substances, and prepared intoa fine powder.
Moisture-I g. of untreated blood powder is dried at 100-l10°C to con2)

Isolation of Histidine from Whale Blood45

stant weight and the residual powder was found to weigh 0.714
g. Therefore, moisture is 28.6 %.
Chloride-Sufficient water is added to 10 g. of untreated blood powder tomake 100 cc., filtered, and 1 cc. of the filtrate is titrated with 0.1

N AgN03 to determine the amount of chloride which, as NaCl,came out as 9.4,%.
It follows, therefore, that the protein portion amounts to 62 %.
(b) Pretreated blood powder-The untreated blood powder is stirre::I into 5
times its volume of warm water of around 50°, filtered, washedwell with water and dried. By this process, majority of haemoglobin remains in the residue while albumin and globulin dissolve
into the aqueous solution together with salts and water-soluble
proteins.
Untreated blood powder possesses a saline taste while such is not the case intreated blood in which the odor peculiar to whales has-also disappeared.
Method of isolation using pretreated blood powder is given as follows:
A mixture -of.100 g. of pretreated whale blood powder, 300 cc. of cone. HCland 200 cc. of water is heated for 2-3 hours in a water bath, and then hydrolyzed
by heating over an open flame for 20 hours with reflux attachment. After separating by filtration, the solution is discolored by a small amount of activatedcharcoal* and is concentrated under reduced pressure on a water bath of 50-60"C.
The solution turns syrupy when reduced to one-third its original volume. Whenthe solution becomes cool, the hydrochloride of glutamic acid precipitates out. If
the untreated blood has been used, a large amount of salts precipitate out here.
The syrup is left overnight in an ice-chamber after which it is filtered, the ·residue
washed with a small amount of cone. HCl and drie::I. A viscous, crystalline massof crude glutamic acid hydrochloride is ·obtained to the amount of 9-10 g. Thisis discolored and recrystastallized from hydrochloric acide to white crystals of m.
p. 208-9~C. Yield, ea. 5-5.5 g.
The filtrate obtained after separation of glut<!mic acid hydrochloride and thewashings"'* from activated charcoal are brought together, sufficient water added tomake the total amount about 300 cc, and is again concentrated under reducedpressure to a syrupy state. Addition of water and concentration under reduced* The pretreated whale blood powder No..1 in Table 11 was discolored before concentration and No. 2·was not treated. When not discolored, the solution, during concentration, isvery hard to bring down the volume, and the concentrated liquid is very dark in color.
Glutamic acid hydrochloride cannot be sep::irated from such a liquid.
** ActivatEd charcoal used for discoloration is' extracted twice with boiling water.

s. AKiYA, o. HOSHINO

46

pressure is repeated again to remove as much HCI as possible. Water is thenadded to the syrup to bring the whole volume to 400 cc:, and discolored until thesolution becomes clear and yellow. The hot washings from activated charcoal isconcentrated, added to the main· syrup and the whole is again concentrated toabout 400 cc. The pH of the solution becomes 1.0 to 2.0.
3) Isolation of Histidine:
To the hydrolyzed solution, 45 g. of 3,4-D is dissolve1, a minute amount ofhistidine disulfonate crystals is added as a seed, and the whole is left in an icech:imber for a few days. The disulfonate precipitates out in the bottom of theflask as a crystalliiie mass.
The crystals . of histidine disulfonate are prismatic while monosulfonate ofleucirie is fine needles. The latter easily precipitates out by slight swirling of thehydrolyzed solution so that it must be decanted its supernatent liquid, quietly andcarefully.
If the sulfonates of histidine and leucine percipitates out at the same time, thesolution is heated to dissolve both crystals and then left for a few days in an
ice-chamber so as to allow histidine disulfonate to precipite out. In general, histidine salt. crystals precipitate out first, and separation of leucine monosulfonate is
not difficult. · Yield of crude leucine monosulfonate, 20-25 g.
The crude crystals of histidine disulfonate, decompn. 260-270°, is di~solved inwater and disc~lored, an1 then recrystallized to white needle crystals of decompn.
275-280°C. The yield decreases by about 5-8 % when recrystallized_.
To obtain histidine from its disulfonate, the latter is dissolved in eight times.
'.
its volume of boiling water, cold1 saturated solution of Ba(OH)2 added to pH 7.2,
and the barium salt of 3;4-D that separates out on cooling is filtered off. Thebarium salt is washe1 · with water, the washings added to the mother liquid andthe whole volume condensed to less than one-half of its volume l'inder reducedpressure. In order to remove 3,4-D still present in the solution, the solution ofBa(OH) 2 is added until the pH is 8.5, and the small amount of barium salt that
separates out is filtered off. Barium hydroxide is quantitatively removed by
H2S04 , the solution brought to pH 7.2, and concentrated under reduce1 pressure
by which free histidine separates out.
After removal of histidine, the filtrate is further concentrated and a small
amount of histidine precipitates out upon addition of alcohol. These are recrystallized by dissolving in a small amount of water and adding alcohol. Histidine has
a decomposition point of 270-2°C. Yield is about one-quarter of the disulfonate.
Histidine hydrochloride is obtained by the addition of equimolecular amountof HCl to the solution of pH 7.2 obtained after removal of 3,4-D by Ba(OH)2I

Isolation of Histidine from Whale Blood

47

which brings the pH of the solution to 2.8. By concentration under reducedpressure, and allowing the whole to stand overnight after addition of alcohol,
white crystals of histidine hydrochloride, m .p. 252-4°, can be obtained, These
crystals did not show any depression of melting point when fused with histidinehydrochloride of Takeda Pharmaceutical Industries. Some histidine disulfonate
can be · recovered from the mother liquor by concentration and addition of anexcess of 3,4-D. The yield of hydrochloride is about one-third of histidine disulfonate.
CONCLUSION
Isolation of histidine from whale bloo:l powder using 3,4-dichlorobenzene
sulfonic acid was carried out. Whale blood cotains a lal'ge amount of moistureand salts and the latter is responsible for the obstruction of isolation process.
Therefore, the blood was pretreated to warm and cold water washings by which
the salt& were removed and a result approximately equal to those from cattle
and horse blood was obtained. The hydrochlorides of ·glµtamic acid can also beobtained easily from the pretreated blood whereas its separation is very difficultfrom untreated blood owing to the precipitation of salts.
The authors wishes to acknowledge their indebtedness to Mr. Tawara of theWhales Research Institute for his kindnesses in SUJilplying the material and formany valuable advices. The authors' thanks are also due Mr. Motohashi, cooperation, and to Mr. Nambara who undertook part of the experiments.
1) Org. Syntheiffi: 18, 43 (1938).
2) Doherty, D.G., Stein, W.H., and Bergmann, M.: J. Biol. Chem., 135, 487 (1940).
3) Stein, W.H., Niemann, C., and Bergmann, M., and Stein, W.H.: J. Biol. Chem.
3) Stein, W.H., Niemann, C., and Bergmann, M.: J. Am. Chem. Soc., 60, 1703 (1938);
Bergmann, M., and Stein, W.H.: J. Biol. Chem., 128, 217 (1939); ibid., 129, 609 (1939);
Ing, H.R., and Bergmann, j\f.: J. Biol. Chem. 129, 603 (1939).
4) Vickery, H.B.: J. Biol. Chem. 143, 77 (1942).
5) Vickery, H.B.: J. Biol. Chem. 144, 719 (1942).
6) Tawara, T.: The Scientific Rep0rts of the Whales Research In~t., No. 1, 38 (1948).
7) Takagi, Suzuki and Asahina: J. Pharm. Soc., Japan. 69, 355 (1849).

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