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DISTRIBUTION OF AMINO ACID IN PROTEINS FROMVARIOUS PARTS OF WHALE BODYTADASHI NAKAI*(DIRECTED BY PROF. SHICHIRO AKIYA**)INTRODUCTION

Numerous studies have been already made on amino acid distribution
in tissue and organ proteins of terrestrial mammals, and most of theirresults have been taken in the Block and Weiss' handbook (1956). As
regards whales, however, there have been only a few works. It is not
only a contribution to our biochemical knowledge of whales to clarify
the amino acid distribution in tissue and organ proteins of whales but
also necessary in order to find out better utilization of whales. The
present study was undertaken with the author's such intention. Proteins obtained from muscle meats of fin, sei, sperm, and baird beaked
whales, muscular tissues of diaphragm and heart, mucous membranes offirst stomach and small intestine, pancreas, liver, cerebrum, lung, spleen,
testis, crystalline lens, aorta and epidermis (black skin) of baird beaked whale, and also gum and baleen plate of whalebone whales were
quantitatively analyzed for their constituent amino acids, and results
obtained were discussed.
AMINO ACID DETERMINATIONIn the present study, the author intendecj to carry out the determination of amino acids chiefly by means of the direct photometry on
ascending one-dimensional paper chromatograms. Many solvent systems
which had been recommended in literature for quantitative separationof each amino acid on one-dimensional paper chromatograms were traced
in the present work, but unsatisfactory results were obtained in thesemany cases. Probably it was due to circumstances that the experimental
conditions in the present work, especially the kind of filter paper, themethod of development and temperature during developing were unable
to be brought into line with those in the literature. On the basis ofresults of the author's many experiments, a method for direct estimation of 18 amino acids on paper chromatograms was devised, which wassummarized in Table 1. Estimation of amino acids on the chromatograms that were revealed with color reagents was carried out by theauthor's modification of the procedure of measuring maximum color* Pharmaceutical Faculty, Toho University.

** Tokyo Medical and Dental University.

305

306

T. NAKAI

densities of individual spots (Block, 1950; McFarren and Mills, 1952)
except the case of methionine where the method of measuring area ofthe spot (Block et al. 1958) was employed.
Histidine estimation was not mainly made by this method, but by theelution method, i.e., aqueous eluate of the histidine area of the finished
one-dimensional chromatogram was subjected to the Pauly's diazo-reaction and estimated colorimetrically.
By means of these methods, 18 kinds of common, naturally occurring
amino acids except tryptophan and hydroxyproline were easily andeconomically determined with satisfactory results.
EXPERIMENT AL

I. Direct Estimation on Paper Chromatograms
1. Filter Paper. Toyo No. 51 filter paper sheets 30 cm. wide and 30
cm., 40 cm. and 43 cm. long respectively are used for 24 cm., 30 to 35 cm.
and 36 to 38 cm. of solvent run (cf. Table 1).
TABLE 1.

OUTLINE OF THE PRESENT METHOD FOR DIRECT ESTIMATIONOF AMINO ACIDS ON PAPER CHROMATOGRAMS

Solvents
1.

n-Butanol · acetic acid · water(4:1:1 v/v)

2.

n-Butanol · acetic acid . water(25:6:25 v/v) (Block and Weiss,
1956)

Lengths of solventColor
run beyond
reagents* Amino acids estimatedoriginal spots
33-35 cm.
Arginine, cystine, hydroDevelop 3 times
xylysine, phenylalanine
35 cm.
Lysine, histidine, argiI
Develop 3 times
nine, alanine, tyrosine{
Proline24cm.
Methionine{~

n-Butanol · acetic acid . water
Phenylalanine, tyrosine(40:5:7 v/v) (Rao and Wadh35cm.
wani, 1955)
Leu cine
4. n-Butanol ·ethanol· water (20:36-38cm.
Pro line
Develop 3 times
1:4 v/v)
5. Phenol saturated with pH 12
phosphate buffer of 0.067 M.
Threonine, glycine, se(Treat filter paper with therine, glutamic acid, as30-32cm.
Nsame buffer before sample appartic acidplication)(McFarren, 1951,
1952)
6. Pyridine·isoamyl alcohol.wa35cm.
Isoleucineter (1:2:1 v/v)
Develop 3 times
7. Pyridine·isoamyl alcohol.water (10: 10:7 v/v) (Giri, 1953)(Treat filter paper with 0.05 M
34-35 cm.
Valinesodium acetate solution before
sample application)* Color reagents:
I =0.4% (w/v) ninhydrin in acetone.
II =0.2% (w/v) isatin in acetone.
III=4cc. of 0.002M PtK2Cl6 , 0.25cc. of lM Kl, 0.4cc. of 2N HCl, 76cc. of acetone(Toennies and Kolb, 1951; Block and Weiss, 1956).
N=0.4% (w/v) ninhydrin, 4% (v/v) acetic acid in acetone.
3.

DISTRIBUTION OF AMINO ACID IN VARIOUS PARTS OF WHALE BODY307

The buffered paper sheets (No. 5 and No. 7 in Table 1) are preparedby dipping filter paper sheets, 30 x 60 cm., into the buffer solutions,
air-drying by suspending by one end and cutting into the desired size.
pH 12 phosphate buffer solution is prepared according to McFarren(1951).
2. Preparation of Protein Hydrolyzate. 200 mg. of protein is hydrolyzed with 2 cc. of 6 N HCl in sealed glass tube in an oven at 110°C
for 24 hrs. The contents of the tube are filtered through a glass filter,
and after thoroughly washing the precipitate thus obtained with water,
the combined filtrate and washings are concentrated to dryness ona steam bath. The residue is then dissolved in 10 cc. of water, again
evaporated to dryness, and placed in a vacuum desiccator over KOH for
24 hrs. The residue is finally taken up in 2.0 cc. of water (Original
solution).
For estimations of leucine and hydroxylysine, this original solution isused as it is, and for estimations of the others, four or two times dilution of the original is used in most cases.
3. Preparation of Standard Solution. 50 mg. glycine, 50 mg. DLalanine, 70 mg. DL-valine, 80 mg. L-leucine, 60 mg. DL-isoleucine, 50 mg.
DL-serine, 50 mg. DL-threonine, 30 mg. L-methionine, 10 mg. L-cystine,
50 mg. L-phenylalanine, 50 mg. L-tyrosine, 65 mg. L-proline, 70 mg. Laspartic acid, 120mg. L-glutamic acid, 40mg. L-histidine·HCl·ff,0, 70mg.
L-arginine·HCI and 100mg. L-lisine·HCI are dissolved in water under
addition of an adequate quantity of 6 N HCl, and the solution is made up
to a final volume of 10.0 cc. 2.0 cc. of the solution is evaporated to dryness
on a steam bath. The residue is dissolved in 10 cc. of water, again
evaporated to dryness, and placed in a vacuum desiccator over KOH for
24 hrs. The residue is finally dissolved in 2.0 cc. of water (Original
standard solution).
For estimation of leucine, this original solution is employed as it is,and for estimation of the others, four or often two times dilution of
the original is employed.
As the standard of hydroxylysine, a gelatin hydrolyzate in whichhydroxylysine content was already determined by periodate oxidation(Block and Bolling, 1951) is employed.
4. Application of Solutions. On a line 5 cm. from the lower edge ofthe paper, seven points are marked at 2.5 cm. or 3.0 cm. intervals. The
standard and the test sample are applied at alternate points with a selffilling micropipette. The desired concentration on the paper is built up
by repeated sequence of spotting and drying. Every spotting must be
Amino acid specimens employed as the standards were manufactures of Ajinomoto Co.,
Ltd. and Tokyo-Kasei-Kogyo Co., Ltd. These were paper-chromatographically pure.

308

T. NAKAI

made in the same quantity of the solutions. In the author's experiment, one spotting was 77 x 10- 5 cc. in quantity. The standard solution
is applied 1, 2, 3, and 4 times at the range of four respective points,
and the test solution, in most cases, 2 to 3 times at the range of threerespective points.
5. Developing Solvents. The solvent mixtures in Table 1 are preparedin the usual fashion.
6. Development of Chromatograms. Being spotted, the paper sheet
is suspended in air at room temperature for more than one hour. Then,the sheet is formed into cylinder, and subjected to the ascending development.
In the case that one of the solvents No. 1, 2, 4, and 6 is used, themultiple development technique (Jeanes et al., 1951) is adopted, i. e.,
the sequence of developing and air-drying is repeated for a total of 3
times. In the case of 35 cm. solvent run using one of the solvents No.
2, 3, and 6, it is recommended that the paper is allowed to remain in
the developing chamber for several hours more after the solvent front
has reached the upper edge of the paper.
The development on the pH 12 buffered paper should be commenced
within 20 hrs. from the time when the air-drying of the paper moistenedwith the buffer solution was commenced. If not so, the resolution of aminoacids will grow worse owing to decline of the buffer action of the sheet.
7. Color Formation of Amino Acids. At the completion of the development, the paper is dried in a gentle current of air at room temperature. The sheet is then dipped in a color reagent and again driedat room temperature. When one of the ninhydrin solutions in Table 1
is employed as the color reagent, the color is developed by heating inan oven at 60° to 63° C. for 15 minutes. When the isatin solution isused to reveal proline, the color is developed by heating at 70° to 73°C.
for 10 minutes. Proline gives a blue spot on a yellow background.
The oven should be saturated with water vapor in every case.
Platinum reagent for methionine must be prepared with acetone purified with use of KMn0 4 and K 2C0 3 (Block and Weiss, 1956). On treating the sheet with this reagent, methionine gives a white spot on a redpurple background without heating.
8. Measurement of Maximum Color Densities of Amino Acids except
Methionine. After the revealed chromatogram has been allowed to keep
at room temperature for more than one hour, the color densities of the
spots are determined with a photoelectric colorimeter as in thefollowing.
Each spot is cut out of the chromatogram and fastened with the aidof clips to a sample holder which contains an aperture of suitable size.

DISTRIBUTION OF AMINO ACID IN VARIOUS PARTS OF WHALE BODY

309

In order to reduce the experimental error which may be derived fromunevenness of thickness of paper in the same sheet, it is advisable thatthe size of the aperture is made as large as possible within the size ofthe spot to be estimated, provided that the separation of the spot allows. Shape and size of the aperture should be properly changed according to shape, size, and separation of the spot to be estimated.
However, when a series of spots of the same acid is estimated on thesame chromatogram, the same aperture must be used for every spot.
In the author's experiment, circular apertures 7 mm., 9 mm., 11.5 mm.,and 14 mm. in diameter and rectangular apertures 3x14 mm., 5x14 mm.,and 10x14 mm., have been employed.
9. Measurement of Areas of Methionine Spots. Circumferences ofwhite spots revealed with the platinum reagent are marked in pencil,
and the areas are determined with a planimeter.
10. Calculation. The standard curve for each amino acid is preparedfrom the values at individual concentrations of the standard. The aminoacid concentration in each spot of the test solution is estimated byinterpolation from the curve, and then the content of each amino acidin the sample of protein is calculated. The result obtained at every
spot of each amino acid is averaged. An example of calculation formula
is as follows :If A=quantity (mg.) of protein before hydrolysis contained in 2.0 ce.
of the hydrolyzate (original),
B=the number of times of spotting the hydrolyzate (4 times dilution of the original),
C=quantity (mg.) of each amino acid in 10.0 cc. of the standard solution (original), andD=the number of times of spotting the standard solution (4 times
dilution of the original) being equivalent to B (' D' is estimatedfrom the standard curve), theneach amino acid content in protein (g. per 100 g. of protein)=20·C·D/A·B
In order to employ this formula, concentrations of the amino acidand test sample in every spot should be expressed in numbers of timesof spotting the standard solution and hydrolyzate respectively, as mentioned above.

II. Determination of Histidine by the Elution Method0.025 to 0.1 cc. of a solution containing 25 to 200 r histidine is applied
as a streak, 18 cm. long and about 3.0 mm. wide, on a line 5 cm. fromthe bottom of a sheet 30 cm. square of Toyo No. 131 filter paper. One
drop of the same solution is also placed at each point 1 cm. from eachend of the same line, with the object of preparing guide strips. After

310

T. NAKAI

the solvent, n-butanol·acetic acid·water (4: 1: 1 v/v), has upwards run
about 17 cm. past the line, the guide strips are cut off and sprayed witha diazo-reagent (Block et al., 1958, p. 133) to reveal histidine spots.
The location of histidine area in the remaining part of the sheet is presumed from the results of the guide strips. A strip about 2.4 cm. wide
which contains the whole histidine area is cut out of the remaining
sheet and irrigated with water by the same technique as descending
paper chromatography. The lower end of the strip should be cut ina wedge form. First 2 cc. of the eluate is subjected to the Macpherson's
modification (1946) of the Pauly-reaction to estimate histidine, excepting
the electrodialysis in his method.

III. Determination of Tryptophan and Hydroxyproline
Tryptophan was determined by Spies and Chambers' ' procedure H '(1949). Test solution for the determination was prepared as in the following: About 0.5 g. of each protein was weighed accurately, boiled
with 10 cc. of 5 N NaOH in an oil bath at 125° C. under reflux for 5
hours, and then the solution was made up to a whole volume of 50.0 cc.
with adding water.
Hydroxyproline was determined by Neuman and Logan's method(1950). The preparation of the test solution was as follows: 200 mg.
of protein was hydrolyzed with 2 cc. of 6 N HCl in a sealed glass tube
in an oven at 110° C. for 24 hours. The contents of the tube were
filtered through a glass filter, and after washing the precipitate with
10 cc. of water, the combined filtrate and washings were concentrated
to dryness on a steam bath. The residue was then taken up in 10 cc.
of water, again concentrated to dryness, dissolved in 30 cc. of water,
and after neutralized with N NaOH, evaporated to dryness. The residue
was finally taken up in 10.0 cc. of water. At the time of the determination the solution was diluted so as to contain 10-40 r of hydroxyproline per cc.
DISCUSSION

The previously published methods of amino acid estimation using onedimensional paper chromatography have adopted mostly the descending
developing technique. With the descending technique, the desirable
prolongation of the length of solvent run is easily achieved by properly
extending the length of development time, and consequently, there is
the possibility of promoting the resolution of amino acids in this way(Roland and Gross, 1954). With the ascending development technique,there is a limit to the length of solvent run as is generally known, andDISTRIBUTION OF AMINO ACID IN VARIOUS PARTS OF WHALE BODY

311

consequently the resolution of amino acids are restricted in some measure by this fact. However, the ascending technique has been exclusively adopted in the present work because of its merits of simplicity
of apparatus and the ease with which a large number of analyses may
be made (Williams and Kirby, 1948).
I. The Direct Estimation on Paper Chromatograms
The resolution patterns (Fig. 1) is scarcely distorted by considerable
variations of experimental conditions, especially temperature during thedevelopment. In choosing the solvents, the author paid attention tothis point also, because all the developments in the present work had
to be made at room temperature. Conditioning the filter paper in thedeveloping chamber before development is unnecessary.
Solvent front

0
QPHE

8TYR

0

()

CJ
8PRO

B
8ARG

8~~~ 00

QP!lE 0

0

0

0

0

OTYR

CJ

Starting line
Solvent No.

0

0

0

0

0

QASP

0

~-0-

_o_

Fig. 1.

Qiso

8SER SPRO
GLU

0
3

OPHE
LEU

OGLY

PRO

0

,.--,-----

0
OTHR

QARG
8HIS
LYS

HIS
LYS

QHO-LYS
0 GYS

8LEU
ISO
PHE

4

0

Q~
OVAL

Jl
·....
..,

Abbreviations
Glycine
GLY
Alanine
ALA
VAL
ValineLeu cine
LEU
Isoleucine
ISO
SerineSER
Threonine
THR
Cystine
CYS
Methionine
MET
Pheny !alanine
PHE
Tyrosine
TYR
Pro linePRO
Aspartic acid
ASP
Glutamic acid
GLU
Arginine
ARG
Histidine
HIS
LysineLYS
HO-LYS Hydroxylysine

Resolution Patterns

In most cases of paper chromatography with the ordinary solvents,
leucine and isoleucine as well as methionine and valine overlap eachother. It is these amino acids that the author took pains in resolving.
The solvent No. 4 used for separation of leucine is the author's modification of one of the Rao and Wadhwani's solvents (1955) used in theirquantitative circular paper chromatography. Leucine and isoleucine aswell as phenylalanine still overlap slightly, but the estimation of leucine
is completely achieved with use of a narrow aperture at the time of
measuring color densities of the spots. In the present experiment,
apertures 3 mm. and 5 mm. wide were employed for estimation of leucine.

312

T. NAKAI

Isoleucine is sharply separated from other amino acids with the aidof the solvent No. 6.
With the use of the solvent No. 7, valine is separated even on unbufferedpaper, but in such a case, the color of the spot is uneven and consequently the estimation is unable to be made. The unevenness of the color ispresumably caused by hydrochloric acid contained in the protein hydrolyzate and the standard solution. The author could remove this hindrance by means of buffering the paper sheet with sodium acetate solution before the sample application.
A portion of methionine is autoxidized into methionine sulfoxide on
the paper during development and air-drying, and so, applying themultiple development technique to the estimation of methionine (Blockand Weiss, 1956, p. 78) is unadvisable. Thus, some amino acid andmethionine sulfoxide often overlap each other on the chromatograms
made by the multiple development technique. The author, therefore,
paid attention to this point when this technique was adopted for resolution of amino acids.
In descending (McFarren, 1951, 1952; Reidel and Bienenfeld, 1956;
Lange, 1956; Schnuchel, 1956 ; Roberts and Kolor, 1957) and circular(Krishnamurthy and Swaminathan, 1955) paper chromatography, the pH
12 buffered phenol has been favorably used for separation of aspartic
acid, glutamic acid, serine, glycine, threonine and alanine, but in ascending chromatography, according to the author's experience, alanine isincompletely separated. In the author's method, therefore, this solvent
is used only for separation of the five amino acids other than alanine.
The location of hydroxysine on .the chromatogram was experimentally
determined basing on the author's investigation of chromatogram ofa hydrolyzate of gelatin which contains a comparatively large amountof this amino acid, the fact that this amino acid is precipitated withphosphowolframic acid, and Inskip's study (1951).
Hitherto, determination of hydroxylysine has been made exclusively
by periodate oxidation (Block and Bolling, 1951; Akabori and Mizushima,1954), but its procedure is complicated and a relatively large amount of
the test sample is required for one estimation. By use of the abovementioned technique, the determination of this amino acid is simplified
and enabled to be made in a micro- or submicro-scale.
The present method was successfully applied to determination of 17
amino acids in casein.
Casein (E. Merck) desiccated in vacuo over H 2S04 was hydrolyzed with
6 N HCl, as above described, for 24 hrs. for estimation of methionine,phenylalanine and tyrosine, and for 48 hrs. for all the other amino acids.
The values obtained in the present experiment and also the values

DISTRIBUTION OF AMINO ACID IN VARJOUS PARTS OF WHALE BODY

313

reported in literature are listed in Table 2. There is a good agreement
between the values in both cases. The values for serine and threonine
in the literature include corrections made for decomposition of these
acids during acid hydrolysis of protein, but the values obtained in thepresent experiment include no correction. In the literature, tyrosine wasestimated on alkaline hydrolyzates, and in the present experiment, on
the acid hydrolyzate. This is presumably the reason of the fact thatthe present value for tyrosine is somewhat lower than the values in
the literature.
TABLE 2.

RESULTS OF AMINO ACID DETERMINATION ON CASEIN
Values expressed in g. per
100 g. protein

Values expressed in g.
amino acid N per 100 g.
protein N

Constituent

Present
Work

Gordon et al.
(1949, 1950)

Sundararajan
et al. (1957)

Present
Work

Gordon et al.
(1949, 1950)

Total N
Glycine
AlanineValineLeu cine
Isoleucine
Pro line
Phenylalanine
Tyrosine
SerineThreonine
Cystine
Methionine
Arginine
Histidine
LysineAspartic acid
Glutamic acid

15.02
2.0
3.4
7.2
9.7
6.1
10.9
4.9
5.2
5.0
4.3
0.31
2.9
3.8
2.9
7.6
6.5
21.4

15.63
2.00
3.20
7.2
9.2
6.1
10.6
5.0
6.3
6.3
4.9
0.34
2.8
4.1
3.1
8.2
7.1
22.4

15.56
1.90
3.38
6.94
9.54
6.13
11. 72
4.98
6.32
6.27
5.28
0.35
2.93
3.92
3.03
8.18
7.66
21.62

2.5
3.6
5.7
6.9
4.3
8.8
2.8
2.7
4.4
3.4
0.24
1.8
8.1
5.2
9.7
4.6
13.6

2.39*
3.22*
5.5
6.3
4.2
8.3*
2.7
3.1
5.4
3.7
0.3
1. 7
8.4
5.4
10.l
4.8
13.6

* The author's calculation from figures in the literature expressed in g. per 100 g.
protein (Gordon et al., 1950).

Because accuracy of the quantitative paper chromatography using the
maximum color density technique was exhaustively discussed already(Salander et al., 1953; Roland and Gross, 1954; Block and Weiss, 1956;
Roberts et al., 1957, 1958), no attempt on it has been made in thepresent study.
Block (1958, p. 117) pointed out that cystine was decomposed whena hydrolyzate was allowed to stand. In the present experiment thesame tendency was observed with methionine.

314

T. NAKAI

II. Determination of Histidine by the Elution Method.
In the Macpherson's method (1946) for histidine estimation, histidine isseparated from tyrosine, which also presents the Pauly-reaction, bymeans of electrodialysis. In the author's procedure, the separation iseasily achieved by means of the one-dimensional paper chromatography.
There is such satisfactory reproducibility as the results of two times
determination on 3 samples of protein are shown in Table 3.
TABLE 3.

HISTIDINE CONTENT IN THREE SAMPLES OF PROTEIN
DETERMINED BY ELUTION METHOD
Values are expressed in g. per 100 g. of dry prot~in.
Total N

Histidine

Meat, fin whale

15.29

Liver, baird beaked whale

14.59

2.69
{ 2.63
2.81{2.88

Pancreas, baird beaked whale

15.23

2. 79
{ 2.42After the present experiment of determining histidine contained intissue and organ proteins of whales was finished, Frank and Petersen(1956) published a similar technique in their study on estimation of histidine concentration in blood-plasma.
MATERIALS AND PREPARATION OF SAMPLES OF PROTEIN

All the muscle meats used in the present work were dorsal muscles.
Muscle Meat of Fin Whale (Balaenoptera physalus). The material was
taken from a male fin whale 61 feet in body length which was caught
at the position of 54° 14' N, 166° 40' W on Sept. 19th 1954. About 2.5hours elapsed from the time of killing to the commencement of thetreatment. A lump of meat was taken out of the carcass by Mr. K.
Fujino of the Whales Research Institute and refrigerated until the commencement of the experiment (Oct. lOth 1954).
4 kg. of minced meat was boiled with water twice as much as themeat for 5 minutes and then filtered under pressure. The residue wasboiled again with water and filtered as above. The final residue was
air-dried. 50 g. of the air-dried matter was treated with tepid water,
boiling water, alcohol, and ether successively as muscle meat of baird
beaked whale mentioned below.
Muscle Meat of Sei Whale (Balaenoptera borealis). The material was
taken from a male whale 44 feet in body length which was killed at
the position of 51° 44' N, 173° 00' E on Aug. 4th 1952. About 4 hours
elapsed from the time of killing to the commencement of the treatment.
'Ihe material was taken by Mr. K. Fujino and refrigerated until theDISTRIBUTION OF AMINO ACID IN VARIOUS PARTS OF WHALE BODY

315

commencement of the experiment (Oct. lst 1952). 16.4 kg. of mincedmeat was treated similarly as fin whale meat.
Muscle Meat of Sperm Whale (Physeter catodon). A piece of muscle
meat was taken from a male whale 41 feet in body length which was
killed off the coast of Kinkazan Island on Dec. 6th 1948. Presumably
about 18 hours elapsed from the time of killing to the arrival at thelandstation. The grade of freshness of the carcass (Nakai, 1948) at thecommencement of treating was about 70. Without delay, the material
was minced, treated with acetone and ether successively and then airdried. Thus treated material was further treated with tepid water,
boiling water, alcohol, and ether as muscle meat of baird beaked whalein the following.
Muscle Meat of Baird Beaked Whale (Berardius bairdii). A lump ofmeat was taken from a male whale which was killed off the coast of
Kinkazan Island in the summer of 1953. Presumably about 10 hours
elapsed from the time of killing to the arrival at the landstation. The
carcass at the commencement of treating was in good fresh condition.
60 g. of minced and then thoroughly mixed material was mixed with
60 cc. of acetone to prevent subsequent decay and brought to the laboratory. Acetone was distilled off on a steam bath. The residue was extractedwith 120 cc. of tepid water for 30minutes under occational stirring and thenfiltered. The filtrate yielded no proteinous coagulation when it was boiled
for a short time, and so it was abandoned. The extraction was repeated
twice. The meat residue was treated 3 times with succesive 120 cc.
portions of boiling water for 5 minutes each on a boiling water bathunder occational stirring and filtered with suction. 120 cc. of alcohol
was added to the residue, boiled in a water bath for ten minutes andsucked off. The extraction was repeated twice with renewal of alcohol ateach time. Alcohol which was included in the final meat residue was
evaporated on a water bath. Then, the meat residue was submitted tothe ether extraction using the Soxhlet's apparatus and then air-dried.
Protein thus obtained was desiccated over sulfuric acid under a reducedpressure.
Muscular Tissues of Diaphragm and Heart of Baird Beaked Whale.
The whales from which the materials were taken, were killed off thecoast of Kinkazan Island in the summer of 1953 as in the foregoing.
The muscular tissue of ventricle of the heart wi;i.s taken from a male
whale and the phrenic muscular tissue from a female one. These materials were similarly treated as muscle meat of baird beaked whale.
Various Tissues and Organs. Cerebrum, aorta, epidermis (black skin)and mucous membrane of small intestine were sampled from male baird
beaked whales. Pancreas, lung and mucous membrane of first stomach

316

T. NAKAI

were sampled from female ones. Lenses were taken from male andfemale baird beaked whales, and mixed. Sex of baird beaked whales
from which spleen and liver were taken and also sex of the sei whalefrom which gum* was taken, are unknown. These whales were killed
off the coast of Kinkazan Island in the summer of 1953 and were ingood fresh condition until the commencement of the treatment at thelandstation. A baleen plate which had been taken from a whalebone
whale (probably, Balaenoptera edeni) caught in the adjacent waters of
Ogasawara Islands and then air-dried was used as a material for thepresent experiment.
From mucous membranes loose connective tissue under the membranes(tela submucosa) and others were removed as perfectly as possible bymacro-observation, and then the membranes were washed with waterto remove mucos.
From pancreas, liver, cerebrum, lung, spleen, and testis, were removed
capsules, connective tissues, and blood vessels as much as possible.
From epidermis the outermost thin layer, namely, horny layer(stratum corneum) being apt to peel off was removed. Thus treatedmaterial, therefore, consists mainly of germinal layer (stratum germinativum).
The aorta was free from surrounding connective tissues.
The lenses taken from the whales were slightly washed with waterto remove the remainder of vitreous body and others.
The minced materials, except the baleen plate, were mixed with thesame amount of acetone to prevent subsequent decay and brought tothe laboratory. Acetone was distilled off on a steam bath. The residue
was treated with tepid water, boiling water, alcohol, and ether succesively in the same manner as the baird beaked whale meat. The baleen
plate was cut in thin slices and then treated with tepid water andothers as above. Samples of protein thus prepared were desiccated oversulfuric acid under a reduced pressure.

RESULTS
Results obtained are listed in Table 4. The blank means that the corresponding amino acid was not determined because its existence in thetest sample was not confirmed by means of qualitative paper chromatography. For comparing with whale meat, values of beef reported inliterature are also listed in Table 4.

* The 'gum', in the present paper, means a elastic tissue which occupies the gaps be·
tween roots of adjacent baleen plates and is grayish white in color.

DISTRIBUTION OF AMINO ACID IN VARIOUS PARTS OF WHALE BODY

311

DISCUSSION

The whale meat is an important proteinous food-stuff in Japan. The
result of the feeding experiment with albino rats carried out by Suzuki
et al. (1919) revealed that its protein seems to be not inferior to proteins of other food-stuffs in nutritive value. However, very few studies(Okuda and Okimoto, 1919; Yamakawa and Shibuya, 1926; Schulze,
1949; Carpenter et al., 1955; Ogawa et al., 1958) have been made of
its amino acid composition by which the nutritive value should be substantiated, and Ogawa and co-worker's study (1958) on fin whale meatis the only one which achieved neary complete amino acid analysis onwhale meat. In the present work, amino acid composition of muscle
proteins of four important species of whales in the adjacent waters ofJapan was revealed.
As shown in Table 4, there is a close resemblance of the amino acidcomposition of the first five voluntary muscles. Especially, muscle
meats of the two species of toothed whales (sperm and baird beaked
whales) resemble very closely each other. It seems that the resemblance
between muscle meats of the two species of whalebone whales (fin andsei whales) is somewhat inferior to that between the muscle meats ofthe toothed whales. It is interesting that in the amino acid compositionof protein of muscle meat, sperm and baird beaked whales which areclassified into different families (Physeteridae and Ziphiidae respectively) resemble each other rather more closely than the two species ofwhalebone whales which are classified into the same family (Balaenopterydae).
In comparing the whalebone whales with the toothed whales on eachamino acid content in protein of muscle meat, it is noticed that theformer is richer in arginine, methionine, cystine, aspartic acid andvaline, and the latter in histidine, phenylalanine, tryptophan and glycine. It can readily be imagined that histidine content in muscle protein ispretty affected by myoglobin content in muscle because of the large
content of histidine in myoglobin (Schmid, 1949). The relatively large
content of histidine in muscle meats of the two species of toothed
whales and in the phrenic muscular tissue of baird beaked whale ispresumably due to the large content of myoglobin in these tissues. It
is already known that the myoglobin content in muscles of sperm whale
is much larger than that in muscles of sei whale (Tawara, 1950). Muscle
meat and phrenic muscular tissue of the beaked whale are almost blackin color as sperm whale meat, and so myoglobin content in these tissues
must be also very large. From the results in Tawara's work (1950),
it is probable that the amount of myoglobin contained in muscles of318

T. NAKAITABLE 4.

AMINO ACID COMPOSITION OF
Values expressed inSpecies ofwhale

Protein ofMuscle meat
Muscle meat
Muscle meat
Muscle meat
[Beef (Bigwood, 1953)
Phrenic muscular tissue
Cardiac muscular tissue
Mucous membrane of first stomach
Mucous membrance of small intestine
Pancreas
Liver
Cerebrum
Lung
SpleenTestis
Lens
Aorta
Epidermis
GumBaleenTotal
GLY ALA VAL LEU
N

Fin
Sei
Sperm
Baird beaked

15.29
15.01
15.78
15.52

Baird beaked

"
"
"
"
"
"

"
"
q

v

"
"

Sei
Balaenoptera
edeni?

ISO

14.87
15.26
15.90
16.11
15.23
14.59
13.42
15.32
15.34
14.34
15.44
15.48
15.45
15.38

3.8 6.4
4.0 6.1
4.4 6.2
4.3 5.8
4.5 6.4
4.5 6.0
5.1 6.0
11.8 6.1
12.6 6.8
7.8 6.1
5.6 6.8
4.4 5.7
7.2 7.4
6.5 7.5
8.5 6.3
4.4 3.3
17.8 11. 7
5.8 4.5
5.2 4.6

5.3
5.6
5.1
4.4
5.0
4.2
5.0
4.4
4.3
5.3
5.7
5.3
6.4
6.3
4.9
5.1
9.3
4.5
4.9

9.9
10.4
10.1
11.4
8.2
11.1
10.2
7.3
6.5
8.7
9.5
9.2
11.2
10.7
7.5
9.2
6.5
9.0
10.2

5.5
6.5
5.4
5.1
5.2
5.3
3.9
3.5
2.9
4.5
4.4
4.0
2.8
2.8
3.4
4.6
2.9
4.1
4.5

14.69

6.2

4.1

4.3

8.3

3.5

Abbreviations:
GLY Glycine
ALA Alanine
VAL Valine

TRY Tryptophan
PHE Phenylalanine
TYR Tyrosine

LEU Leucine
ISO Isoleucine
PRO Proline

TABLE 5.

AMINO ACID COMPOS!

Amino acid content is expressed in
Investigator
Ogawa et al.*Nakai

SampleTotal N

GLY

ALA

VAL

LEU

ISO

PRO

Fresh meat
Protein

3.85
15.29

4.8
4.6

5.1
6.6

3.5
4.1

5.1
6.9

3.2
3.8

2.6
3.0

* Values of amino acids were calculated from the figures in the original paper ex-

DISTRIBUTION OF AMINO ACID IN VARIOUS PARTS OF WHALE BODY319

TISSUE AND ORGAN PROTEINS OF WHALES
g./100 g. protein
PRO TRY PHE TYR SER THR CYS MET ARG HISL YS ASP

GLU HO- HOPRO LYS

3.8
3.6
3. 7
3.4
3.6
4.4
4.7
8.7
8.6
5.8
6.1
4.0
5.7
4.9
6.5
4.6
12.4
6.5
4.1

1.06
1.08
1.23
1.22
1.1
1.05
0.83
0.57
0.57
0.94
0.87
0.89
0.82
0.92
0.81
2.58

2.66
3.6
4.3
5.6
3.8
4.5
2.7
1.7
1.7
2.6
2.84
2.5
4.2
3.6
1.9
4.1
0.62
2.0
1.9

9.4
9.5
9.8
9.8
9.3
9.4
7.8
5.8
5.1
7.1
6.7
6.9
7.3
8.6
6.0
4.8
2.3
7.8
5.9

9.1
9.9
8. 7
8.5
9.8
8.2
9.1
7.9
7.3
7.5
8.3
7.5
9.4
8.3
7.3
9.2
3.6
7.8
8.9

16.0
13.6
16.2
15.1
15.9]
15.1
12.9
10.9
10.5
10.4
10.2
10.0
9.8
8.4
11.2
13.0
5. 7
14.3
16.0

6.1

0.99 7.7 1.8

4.5

7.6 12.3

0.46
0.47

4.2
3.8
4.8
4.6
4.5
4.7
4.0
3.3
3.3
4.2
4.3
4.4
4.5
5.0
3.6
6.6
3.7
3.4
3.5

3.8
3.5
3.4
3.4
4.1
3.3
2.9
2.7
3.2
3.6
3.1
4.0
2.7
3.1
2.9
8.0
3.0
3.5
3.9

4.4
4.2
4.5
4.5
4.5
3.8
4.9
4.7
4.7
4.2
4.2
4.9
4.1
4.2
4.2
6.9
2.5
6.2
6.8

5.0
5.4
5.2
5.2
4. 7
5.2
5.1
4.1
4.0
4. 7
5.5
5.0
4.7
6.3
4.6
3.4
2.8
4.6
5.2

0.47

2.7

6.3

7.4

5.0 6.5

SER Serine
THR Threonine
CYS Cystine

0.97
1.0
0.77
0.83
1.3
0.75
0.99
1.0
1.0
1.4
0.86
1.4
1.0
0.97
1.1
2.0
0.58
3.0
3.2

MET Methionine
ARG Arginine
HIS Histidine

2.5 7.2
2.4 6.6
2.2 5.9
2.1 5.9
2.9 5.4
2.1 6.0
2.4 6.0
1.4 7.4
1.3 7.6
1.8 7.1
1.6 6.3
2.0 6.0
1.3 5.0
1.5 5.3
1.3 6.1
2.2 11.7
0.34 4.6
1.8 6.2
1.3 7.3

LYS Lysine
ASP Aspartic acid
GLU Glutamic acid

0.24

0.86
±
6.3 0.37
7.2 0.43
2.7
+
1.2
±
2.0
0.95
2.94

+
±

+

4.84 0.23

HO-PRO Hydroxyproline
HO-L YS Hydroxylysine

TION OF FIN WHALE MEAT
g. amino acid N/100 g. total N

TRY

PHE

TYR

SER

THR

CYS

MET

ARG

HIS

L YS

ASP

GLU

0.89
0.95

2.0
2.3

1.5
1.9

3.7
3.8

3.2
3.8

0.31
0.74

1.5
1.5

9.7
15.2

6.9
4.72

8.5
11.8

6.1
6.3

7.6
10.0

pressed in g. amino acid/100 g. total matters.

320

T. NAKAI

whales is closely associated with their habitual lengths of time of diving, which are relatively constant depending on whale species. As isgenerally known, both sperm and baird beaked whales are long and deep
divers, and their chief foods are both alike and mostly consist of squids.
Thus, the author believes that the similarity of the amino acid composition of muscle proteins of these animals is closely connected with thesimilarity of their ecology.
As compared with beef, whale meat seems to be inferior to it in contents of the sulfur-containing amino acids, namely, methionine andcystine. However, in contents of the so-called essential amino acids
other than methionine, whale meat is not always inferior to beef.
The heart muscle protein contains larger amounts of hydroxyproline,
proline, and glycine, and smaller amounts of isoleucine, tryptophan,tyrosine, histidine, lysine, and glutamic acid than the voluntary muscle
proteins. The existence of hydroxylysine in the heart muscle protein was
also proved. As described below, hydroxyproline and hydroxylysine arenearly peculiar to collagen, and so the collagen content in heart muscle
must be much larger than that in voluntary muscles. Presumably, thisfact suggests that heart muscle contains the intercellular substance moreabundantly than voluntary muscles. Assuming that whole amount ofhydroxyproline in proteins of muscle meat and heart muscle exists as collagen and that collagen contains hydroxyproline at the ratio of 13.5% (Buddecke, 1958), the collagen content in proteins of muscle meat and heart
muscle of baird beaked whale comes to 1.8% and 6.4% respectively.
In Table 5, the present results of protein of muscle meat of fin whale
are compared with the Ogawa and co-workers' values of fin whale meat
(1958). There is considerable disparity of each amino acid content between both cases. It is probably due to the facts that in Ogawa andco-workers' study, fresh meat was itself hydrolyzed, but in the author's
experiment, the protein free from nitrogenous extractive, fat, etc. washydrolyzed and also both cases differed from each other in the techniqueof protein hydrolysis.
Striking similarity of the amino acid composition of mucous membranesof the first stomach and small intestine should be noticed.
It is worth noticing that all four samples of protein of digestivesystem have relatively large contents of hydroxyproline and hydroxylysine. (The author found also that protein of parotid gland of a rabbit contained hydroxyproline at the ratio of 3.5% .) Especially, themucous membranes contain much greater quantities of these aminoacids than all the other samples. The distribution of both amino acids
in nature seems to be nearly confined to collagen. The hydroxyproline
content in collagen is about 14% (Akabori and Mizushima, 1954-5 i

1l

DISTRIBUTION OF AMINO ACID IN VARIOUS PARTS OF WHALE BODY

321

Buddecke, 1958; Block and Weiss, 1956). According to results of someinvestigators' experiments, hydroxyproline seems to be contained also
in elastin, but the content in this protein is only about 1.5% (Block andWeiss, 1956). As shown in Table 4, the hydroxyproline content in proteinsof mucous membranes and pancreas is much greater than the content inelastin. Accordingly, it is evident that the greater part of hydroxyproline inproteins of mucous membranes and pancreas exists as collagen or collagen-like substance. The existence of hydroxylysine in proteins otherthan collogen and gelatin hasn't been confirmed. From these facts, it
is inferred that each of the analyzed tissues and organs of digestivesystem contains a large quantity of collagen or collagen-like substance.
The much larger content of glycine in proteins of mucous membranes
as compared with the alanine content is also an evidence for this inference, because collagen contains a very large quantity of glycine anda slightly large quantity of alanine while elastin contains very great
quantities of both glycine and alanine (cf. Table 6.). Assuming that
TABLE 6.

APPROXIMATE AMINO ACID COMPOSITION OF COLLAGENSAND ELASTINS
Calculated to 16.0 g. of Nitrogen (Block & Weiss, 1956, p. 343)

Glycine
AlanineValineLeucineIsoleucine
Pro line
Tryptophan
Pheny !alanine
Tyrosine
SerineCollagens
21. 9
9.1
2.8
3.2
1.6
12.8
0.0
2.2
0.9
4.0

Elastins
24.2
21.3
15.6
7.6
3.4
13.0
0.1
4.3
1.9
0.9

Collagens
Threonine
2.6
Cystine0.2
Methionine
0.9
Arginine
7.7
Histidine
0.8
Lysine4.0
5.7
Aspartic acid
Glutamic acid 10.1
Hydroxyproline 12. 4

Elastins
1.1
0.2
0.2
1.2
0.1
0.5
0.7
2.8
1. 5

whole amount of hydroxyproline in proteins of mucous membranes exists
as collagen and that collagen contains hydroxyproline at the ratioof 13.5% (Buddecke, 1958), the collagen content in proteins of
mucous membranes of first stomach and small intestine comes to
46.6% and 53.3% respectively, namely about half of the whole quantityof each sample. It is supposed that such a large content of collagenin mucous membranes has some important meaning concerning their
physiological functions. Collagen is a kind of scleroprotein and noteasily affected by proteolytic enzymes. It is, therefore, presumable thatthe large content of collagen (or collagen-like substance) in mucous
membranes of stomack and small intestine may be one of the reasons
why these mucous membranes can withstand the action of their own
proteolytic enzymes.

322

T. NAKAIThe nutritive value of mammalian liver has been often discussed fromold times. When liver is regarded as a kind of protein food-stuff, theliver of baird beaked whale is probably inferior to muscle meat in nutritive value because its contents of a number of essential amino acids,
namely, methionine, lysine, tryptophan and isoleucine are considerably
smaller than the contents of these amino acids in muscle meat.
The cerebrum protein, as compared with other tissue and organ proteins, contains a remarkably small amount of total nitrogen. The similarfact is also observed with other kinds of animals (Block and Weiss,
1956, p. 284). In the amino acid composition, this protein shows nonoteworthy special feature.
The amino acid composition of crystalline lens protein is considerably
characteristic. In contents of arginine, tryptophan, phenylalanine andtyrosine, the lens protein is the largest among all the samples of protein treated in the present work, and the smallest in the alanine content.
The amino acid composition of aorta protein is specific. The value ofnearly every amino acid contained in this protein is either always larger
or always smaller than values of the same amino acid in all other kindsof protein analyzed in the present work. As is generally known, elastin
is principal constituent protein of arteries (Akabori and Mizushima, 1954,
Vol. II, p. 2 and 7). As shown in Table 4, the aorta protein contains
a pretty large quantity of hydroxyproline and also an appreciable quantityof hydroxylysine. It is, therefore, undoubted that collagen is ·also an
important constituent of the aorta. The amino acid composition of aorta
protein shows that the protein consists mainly of elastin and collagen,which is easily understood when the aorta protein is compared withelastin and collagen in amino acid composition (Table 6).
Epidermis and gum resemble closely each other in amino acid composition. From their good similarity in amino acid composition, elasticity, appearance and others, the author believes that both tissue proteins
are substantially the same. From their relatively high content of
cystine, it is probable that these proteins contain some amounts ofkeratin.
The baleen is regarded as a kind of crude keratin. Its high content
of cystin is an evidence for that.
From the results obtained in the present work, generally, it is foundthat internal organ proteins, as compared with protein of muscle meat,
contain larger quantities of glycine, proline, hydroxyproline, and hydroxylysine and smaller quantities of isoleucine, tryptophan, methionine,lysine, and glutamic acid. This fact shows that internal organs are muchricher in quantity of collagen than muscle meat. That is easily understoodDISTRIBUTION OF AMINO ACID IN VARIOUS PARTS OF WHALE BODY

323

from the amino acid compositfon of collagen and from the fact thathydroxyproline and hydroxylysine are almost peculiar to collagen as
stated before. In other words, the aforesaid fact shows that internalorgans, as compared with muscle meat, are much richer in quantity ofconnective tissue, because collagen is the principal constituent proteinof connective tissue.
Internal organ proteins are probably inferior to protein of muscle
meat in nutritive value on account of their smaller contents of theabove-mentioned essential amino acids, namely, isoleucine, tryptophan,methionine and lysine.
SUMMARY

1. In order to carry out the present study, a simple and economical
method for direct estimation of 18 common amino acids except tryptophan and hydroxyproline. on ascending one-dimensional paper chromatograms was devised and Macpherson's method for dermination of histidine
was modified. By use of the former, determination of hydroxylysine
was also enabled to be made in a micro- or submicro-scale.
2. Amino acid distribution in proteins obtained from muscle meats offin, sei, sperm, and baird beaked whales, muscular tissues of diaphragmand heart, mucous membranes of first stomach and small intestine,
pancreas, liver, cerebrum, lung, spleen, testis, lens, aorta, and epidermisof baird beaked whale, and also gum and baleen plate of whalebone
whales was investigated.
3. Proteins of voluntary muscles, especially muscle proteins of sperm
and baird beaked whales resemble very closely each other in amino acidcomposition. It seems that the similarity of the amino acid compositionof muscle proteins of these animals is closely associated with the similarity of their ecology.
4. The whale meat seems to be inferior to beef in contents of sulfurcontaining amino acids, but not always inferior to beef in contents ofessential amino acids other than methionine.
5. Heart muscle differs considerably from voluntary muscles in aminoacid composition. Collagen content in heart muscle is much larger thanthat in voluntary muscles. From this fact it is presumed that heart
muscle contains the intercellular substance more abundantly than voluntary muscles.
6. Large content of collagen in each digestive organ protein wasinferred from its amino acid composition. The collagen content in proteins of mucous membranes of first stomach and small intestine reaches
about half of the whole quantity of each sample. The large content

324

T. NAKAIof collagen (or collagen-like substance) in the mucous membranes may
be one of the reasons why these membranes can withstand the actionof their own proteolytic enzymes.
7. Lens protein is characterized by its large contents of tryptophan,
phenylalanine, tyrosine, and arginine.
8. Aorta protein consists mainly of elastin and collagen. The valueof nearly every amino acid contained in this protein is either always
larger or always smaller than values of the same amino acid in all othersamples of protein analyzed in the present work.
9. Gum and epidermis without horny layer are substantially thesame.
10. Generally, internal organ proteins, as compared with protein ofmuscle meat, contain larger quantities of glycine, proline, hydroxyproline, and hydroxylysine and smaller quantities of isoleucine, tryptophan,methionine, lysine, and glutamic acid. This fact shows that internalorgans are much richer in quantity of connective tissue than muscle
meat, and that internal organ proteins are probably inferior to proteinof muscle meat in nutritive value.
ACKNOWLEDGMENTS

The author wishes to express his deepest gratitude to Prof. Dr. Shichiro
Akiya of Tokyo Medical and Dental University for his kind guidanceand encouragement given throughout the course of the present workand for his inspection of the manuscript.
Further, the author wishes to express his sincere thanks to Prof. Dr.
N. Kishi, Dean of the Pharmaceutical Faculty of Toho University, Dr.
T. Maruyama, Chief of the Research Laboratory of Taiyo Gyogyo K. K.,
and the late Mr. Hisashi Okuda for their cordial considerations given for
execution of the present work, and to Dr. H. Omura, President of the
Whales Research Institute and Prof. K. Hisauchi of Toho University
for their kind instructions. Hearty thanks are due to Messrs. K.
Fujino and S. Ohsumi of the Whales Research Institute and to Mr. H.
Hasegawa of Taiyo Gyogyo K. K. who helped the author in obtaining
the materials. Thanks are also due to Miss E. Shiraishi and Miss R.
Moriyama, Assistants of Toho University, for their co-operation.
REFERENCESAKABORI, S. and MIZUSHIMA, S. (Editors) (1954-5). "Chemistry of Proteins" (Kyoritsu
Shuppan Co., Ltd., Tokyo), Vol. I, II and III (In Japanese).
BIGWOOD, E. J. (1953). Cited from BLOCK and WEISS (1956) p. 270-1.

DISTRIBUTION OF AMINO ACID IN VARIOUS PARTS OF WHALE BODY

325

BLOCK, R. ]. (1950). Estimation of amino acids and amines on paper chromatograms.
Anal. Chem., 22: 1327-1332.
BLOCK, R. ]. and BOLLING, D. (1951). "The Amino Acid Composition of Proteins andFoods" 2nd ed. (Charles C Thomas, Springfield, Illinois), p. 69.
BLOCK, R. ]., DURRUM, E. L. ali.d ZWEIG, G. (1955). "A Manual of Paper Chromatography
and Paper Electrophoresis" (Academic Press Inc., New York).
- - - (1958). Ibid. 2nd ed., Chapter IV-V.
BLOCK, R. J. and WEISS, K. W. (1956). "Amino Acid Handbook" (Charles C Thomas,
Springfield, Illinois).
BUDDECKE, E. (1958). Untersuchungen zur Chemie der Arterienwand. I. Z. physiol. Chem.,
310: 171-81.
CARPENTER, K. ]., ELLINGER, G. M. and SHRIMPTON, D. H. (1955). Cited from Block andWeiss (1956) p. 272-3.
FRANK, H. and PETERSEN, H. (1956). Ein Verfahren zur Bestimmung der Histidin-Konzentration im Blutplasma. Z. physiol. Chem., 303: 276-281.
GIRi, K. V. (1953). Cited from Block, Durrum and Zweig (1958), p. 145.
GORDON, w. G., SEMMETT, w. F., CABLE, R. c. and MORRIS, M. (1949)_ Amino acid composition of a-casein and $-casein. J. Am. Chem. Soc., 71: 3293-3300.
GORDON, w. G., SEMMETT, w. F. and BENDER, M. (1950). Alanine, glycine, and proline
contents of casein and its components.J. Am. Chem. Soc., 72: 4282.
INSKIP, L. W. (1951). The occurrence of hydroxylysine in proteins. J. Am. Chem. Soc.,
73: 5463-5464.
JEANES, A., WISE, C. S. and DIMLER, R. J. (1951). Improved techniques in paper chromatography of carbohydrates. Anal. Chem., 23: 415-420.
KRISHNAMURTHY, K. and SWAMINATHAN, M. (1955). Quantitative determination of aminoacids by circular paper chromatography. Anal. Chem., 27, 1396-1399.
LANGE, K. (1956). Aminosaurezusammensetzung Kristallisierter Alkohol-Dehydrogenase aus
Backerhefe. Z. physiol. Chem., 303: 272-275.
MCFARREN, E. F. (1951). Buffered filter paper chromatography of the amino acids. Anal.
Chem., 23:168-174.
MCFARREN, E. F. and MILLS, J. A. (1952). Quantitative determination of amino acids onfilter paper chromatograms by direct photometry. Anal. Chem., 24: 650-653.
MACPHERSON, H. T. (1946). Biochem. J., 40: 470-481, cited from AKABORI, S. and MIZUSHIMA, S. (1954), Vol. I, p. 237.
NAKAI, T. (1948). Chemical studies on the freshness of whale meat. I. Sci. Rep. Whales
Res. Inst., No. 1: 17-26.
NEUMAN, R. E. and LOGAN, M. A. (1950). The determination of hydroxypro!ine. J. Biol.
Chem., 184: 299-306, cited from AKABORI and MIZUSHIMA (1954), Vol. l, p. 254.
OGAWA, T., TSUNODA, T. and OSAWA, M. (1958). Amino acid composition of whale meat.
Sci. Rep. Whales Res. Inst., 13: 303-7.
OKUDA, Y. and OKIMOTO, T. (1919). Hydrolysis of whale muscle protein. (In Japanese).
Tokyo Kagakukai-shi, 40: 60-67.
RAO, N. A. N. and WADHWANI, T. K. (1955). Quantitative estimation of amino acids bycircular paper chromatography. J. Indian Inst. Sci., 37A: 130-140, cited from C.A.
49: 13349 (1955).
REINDEL, F. and BIENENFELD, W. (1956). Untershiede in den freien Aminosauren in Blattpreszsaften und blattrollkranker Kartoffelpflanzen. Z. physiol. Chem., 305: 123-131.
ROBERTS, H. R. and KOLOR, M. G. (1957). Accuracy of quantitative paper chromatography
in amino acid determination using direct photometry. Anal. Chem., 29: 1800-1802.
ROBERTS, H. R., KOLOR, M. G. and BUCEK, W. (1958). Rapid paper chromatographic
method for quantitative determination of tryptophan. Anal. Chem., 30: 1626-1627.

326

T. NAKAIROLAND, J. F. and GROSS, A. M. (1954). Quantitative determination of amino acids using
monodimensional paper chromatography. Anal. Chem., 26: 502-505.
SALANDER, R. C., PIANO, M. and PATTON, A. R. (1953). Accuracy of quantitative paper
chromatography in amino acid analysis-addendum. Anal. Chem., 25: 1252-1253.
SCHMID, K. (1949). Zusammensetzung des Wal-myoglobin. Helv. Chem. Acta, 32: 1198-1208.
SCHNUCHEL, G. (1956). Untersuchungen an Rinderleberkatalase II. Mitteil.: Die Aminosauren des Proteins. Z. physiol. Chem., 303: 91-93.
SCHULZE, G. (1949). The use of whale meal for human nutrition. Deut. Lebensm.-Rundshau,
45: 149-53, cited from C.A., 46: 8783.
SPIES, J. R. and CHAMBERS, D. C. (1949). Chemical determination of tryptophan in proteins. Anal. Chem., 21: 1249-66.
SUNDARARAJAN, T. A. and SARMA, P. S. (1957). Preparation and amino-acid compositionof enzymically dephosphorylated casein. Biochem. J., 65: 261-266.
SUZUKI, u., OKUDA, Y., OKIMOTO, T. and NAGASAWA, T. (1919). Nutritive value of various
proteins (I). (In Japanese) Tokyo Kakagakukai-shi, 40: 385-404.
TAWARA, T. (1950). On the respiratory pigments of whale. &i. Rep. Whales Res. Inst.,
No. 3: 96-101.
TOENNIES, G. and KOLB, J. J. (1951). Techniques and reagents for paper chromatography.
Anal. Chem., 23: 823-826.
WILLIAMS, R. J. and KIRBY, H. (1948). Science, 107: 481-483, cited from BLOOK, DURRUM
and ZWEIG (1955), p. 23.
YAMAKAWA, M. and SHIBUYA, Y. (1926). Chemical studies of marine animals (II). (In Japanese) Suisan Koshusho Shiken Hokoku 22: 68--77, cited from Complete Abstracts ofJapanese Chemical Literatures, I, 7 (1926): 373.