AMINO ACID COMPOSITION OF ,&LACTOGLOBULIN AND

BOVINE SERUM ALBUMIN

BY WILLIAM H. STEIN AND STANFORD MOORE
(From the Laboratories of The Rockefeller Institute for Medical Research, New York)

(Received for publication, October 8, 1948)

The experiments described in this communication are concerned with

the use of starch columns (l-3) for the chromatographic determination of
the amino acid composition of protein hydrolysates. The techniques out-
lined in the preceding paper (3) have been employed to analyze hydrolysates
of @actoglobulin and bovine serum albumin. In conjunction with the

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results of earlier chromatograms run with the butyl-benzyl alcohol solvent
system (l), the analyses appear to have accounted for all the amino acids
present in acid hydrolysates of these two proteins. Since numerous amino
acid determinations have already been reported for both P-lactoglobulin
and bovine serum albumin, an opportunity is afforded for comparison of
the chromatographic data with the results obtained by other methods.
The experimental procedure used for the chromatographic analyses was
identical with that employed in the preceding studies with synthetic mix-
tures of amino acids (3). The sample of p-lactoglobulin used in this work
was prepared by Dr. G. Haugaard according to the method of Palmer
(4,5), and was one of the samples recently analyzed by Brand and cowork-
ers (6). The bovine serum albumin, prepared according to the method of
Cohn et al. (7), was obtained through the kind cooperation of Dr. Erwin
Brand, and was the same preparation (Armour, Lot 18) analyzed in his
laboratory. Hydrolysis was performed in the manner already outlined
(l), with 200 volumes of 6 N HCl twice distilled in glass. For convenience
in manipulation, 250 to 500 mg. of protein were hydrolyzed. Since the
chromatographic analyses require only 2.5 mg. per experiment, the pro-
cedure for hydrolysis and for addition of the sample to the column can be
scaled down, if desired, to permit the series of chromatograms to be com-
pleted with 25 to 50 mg. of protein.

Analyses of Hydrolysates of j3-Lactoglobulin

A hydrolysate of p-lactoglobulin was chromatographed with 1:2: 1 n-
butyl alcohol-n-propyl alcohol-O.1 N HCl and 2:l n-propyl alcohol-O.5 N
HCl as solvents. The resulting effluent concentration curve, given in Fig.
1, is similar in all respects to the curve obtained in experiments with a
synthetic mixture containing seventeen amino acids and ammonium chlo-
ride (3). The general pattern and the approximate positions of the peaks
79
 80 /.hACTOGLOBULIN AND SERUM ALBUMIN

are the same in the two curves. There are no peaks in Fig. 1 which cannot
be ascribed to the common amino acids. No evidence has been obtained,
therefore, for the existence in p-lactoglobulin hydrolysates of unsuspected
amino nitrogen-containing constituents. In order to confirm the positions
assigned to the peaks in Fig. 1, threonine, serine, and histidine were added
to the hydrolysate. The designated peaks rose without loss of symmetry.
Since /3-lactoglobulin is a protein, the composition of which has already
been explored rather fully by a variety of methods, the identification
of the peaks in the chromatogram can be made with a relatively high degree
of certainty (1, 3). Characterization of the components of the peaks by

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Leucine +
9 , Mleucine I

Alanine +

Solvent change
Effluent cc.
FIG. 1. Chromatographic fractionation of a hydrolysate of fi-lactoglobulin. Sol-
vents, 1:2:1 n-butyl alcohol-n-propyl alcohol-O.1 N HCI and 2:l n-propyl alcohol-
0.5 N HCl. Column dimensions, 0.9 X 30 cm. Sample, an amount of hydrolysate
corresponding to about 2.5 mg. of protein.

isolation of the amino acids from the effluent has not been attempted in
these analytical experiments.
Integration of Ejluent Curves-The quantitative data yielded by the
experiment shown in Fig. 1, and replicates thereof, are given in Table I.
The total nitrogen recoveries of 98.9 and 99.7 per cent indicate that the
chromatogram has almost completely accounted for the nitrogenous com-
ponents of the hydrolysate. There are several approximations involved,
however, in this calculation. As already mentioned (3), the peak for
valine plus methionine plus tyrosine yields only an approximate value for
the per cent of the total nitrogen of the protein attributable to these com-
bined amino acids. The peak is integrated by using a color yield of 1.00,
whereas the respective color yields for the individual components are 1.01,
1.00, and 0.86. The recoveries obtained for the first six fast moving com-
 W. H. STEIN AND S. MOORE 81

ponents in Fig. 1 are useful in the preparation of a nitrogen balance sheet for
the chromatogram, but are replaced by the results from a butyl-benzyl
alcohol column in later calculations. The glutamic acid plus alanine in-
tegration also yields a slightly low figure, since a correction for losses of
glutamic acid as a result of esterification is not included. Tryptophan is
not present in the acid hydrolysate, as evidenced by the absence of nin-
hydrin-positive material in the valley between phenylalanine and leucine

TABLE I
Chromatographic Analyses of Hydrolysates of fl-Lactoglobulin
Solvents, 1:2:1 n-butyl alcohol-n-propyl alcohol-O.1 N HCI and 2:l n-propyl alco-
-

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hol-0.5 N HCI (cf. Fig. 1).

Constituent
I Chromatogram 412 Chromatogram
-
491 Chromato-
gram 486’

h. amino N as per ( h. amino N as per Gm. amino

cid per 100 cent of .%,:id per 1W cent of acid per 100
m. protein protein N n n. protein protein N gm. protein

Leucine-isoleucine ............ 22.6 .5.47 22.4 15.34

Phenylalanine................ 3.85 2.09 3.89 2.12
Valine-methionine-tyrosine. ... 8.38 ca. 8.30 ca.
Proline. ...................... 5.29 4.13 5.16 4.03 4.96
Glutamic acid-alanine ......... .8.60 cu. 19.35 cu.
Threonine.................... 4.85 3.65 4.75 3.58 4.42
Aspartic acid. ................ 11.46 7.74 11.64 7.86 11.47
Serine ........................ 3.56 3.04 3.76 3.21 3.37
Glycine ....................... 1.35 1.62 1.48 1.77 1.34
Ammonia. .................... 1.44 7.61 1.47 7.76 1.45
Arginine ...................... 3.07 6.34 2.74 5.66 2.91
Lysine ........................ 12.25 .5.05 12.70 15.60 12.80
Histidine ..................... 1.49 2.59 1.61 2.80 1.78
Cystine ....................... 3.52 2.63 3.15 2.35 3.09
I
Total nitrogen recovery. . .I j8.9 39.7

* A 40 cc. fore fraction was collected and the column placed on the fraction col-
lector before the emergence of the proline peak.

in the earlier butyl-benzyl alcohol chromatograms (1). Since tryptophan

represents 1.71 per cent of the protein nitrogen, as determined by Brand
and coworkers (6) who published the first approximately complete analysis
of p-lactoglobulin, the theoretical recovery for Table I should be 98.3 per
cent. For somepurposes, such as comparative studies on different protein
preparations, the type of data afforded by Table I may be adequate without
the performance of additional chromatograms. Quantitative figures are
obtained for ten of the individual components, and an approximate total
nitrogen recovery may be derived from the single chromatogram.
82 &LACTOC~LOBULIN AND SERUM ALBUMIN

The hydrolysates have given a negative nitroprusside test. In addition,

there was no evidence of interference from cysteine in the valleys on either
side of the histidine peak. Apparently any cysteine originally present is
oxidized during the preparation of the hydrolysate for analysis.
In the present experiments the data from Table I have been combined
with the results from additional columns to give a more complete picture
of the composition of the hydrolysate. Glutamic acid and alanine have
been determined with 2: 1: 1 tert-butyl alcohol-set-butyl alcohol-O.1 N HCl
(3). A fore fraction was collected and the chromatogram placed on the
fraction collector prior to the emergence of proline. Duplicate determina-
tions gave values of 19.30 and 18.85 per cent for glutamic acid, and 7.10

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and 7.08 per cent for alanine. The results obtained in the experiments
with n-butyl alcohol-benzyl alcohol columns have already been reported
(1).
Composition of /3-Lactoglobulin- The amino acid composition of /3-lacto-
globulin indicated in Table II is based in major part upon the analytical
results from the three types of chromatograms discussed above. From
the standpoint of protein composition, data of the kind given in Table II
are always subject to uncert.ainties arising from possible decomposition of
various amino acids during hydrolysis. Significant amounts of threonine
and’ serine, for example, are known to decompose during acid hydrolysis,
with the formation of ammonia. In the present work, the values of 4.67
and 3.56 for threonine and serine, obtained from the hydrolysate, have been
divided by 0.95 and 0.90, respectively, in accordance with the estimates
of Rees (17) for the decomposition of these amino acids during a 20 hour
period of acid hydrolysis. Although the hydrolytic conditions employed
in the present experiments differ from those used by Rees, the parallelism
between his results and the present figures indicates that the same correc-
tion factors are probably applicable in both cases. For example, Rees
obtained uncorrected values of 4.84 and 3.64 per cent for threonine and
serine in hydrolysates of P-lactoglobulin. He found the total ammonia
content of the hydrolysate to be 1.49 per cent. The corresponding chroma-
tographic value is 1.45 per cent (Table I).
A maximum possible value for the amide ammonia of the protein can be
obtained from the chromatographic data by subtracting from the total
ammonia content of the hydrolysate the amount of ammonia estimated to
be formed by the decomposition of threonine and serine. The resulting
figure, 1.35 per cent of ammonia, is slightly higher than the amide ammonia
value of 1.31 per cent as determined by Warner and Cannan (20), Brand
et al. (6), and by Rees (17).
Essential data in Table II derived from other sources include the pho-
tometric value of Brand et al. (6) for tryptophan and the sulfur partition
results obtained by the same authors. The independent value for me-
 .tv. R. STEIN AND s. MOORE 83

TABLE II
Amino of &Lactoglobulin
Acid Composition
The values for phenylalanine, leucine, isoleucine, tyrosine, and valine are from
chromatograms carried out with 1: 1:0.288 n-butyl alcohol-benzyl alcohol-water (1).
Glutamic acid and alanine were determined with 2:l:l tert-butyl alcohol-set-butyl
alcohol-O.1 N HCl. The remaining chromatographic values are the average figures
from Table I. The nitrogen content of the protein was 15.6 per cent, on an ash- and
moisture-free basis.
-7 -
h. amin<
hn: amine acid resi-
acld per Literature values, gm. amino acid
Constituent 100 gm. due per %A “d;’ per 100 gm. protein
protein 100 gm. orotein N
protein

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Phenylalanine...... 3.78 3.37 2.06 3.54 (6), 4.3 (8), 4.2 (9)
Leucine............ 15.50 13.38 10.61 15.7 (7), 15.4 (lo), 15.9 (11)
Isoleucine.......... 5.86 5.05 4.01 8.4 (6), 7.0 (S), 6.1 (12)
Methionine. ........ 3.22* 2.84 1.94
Tyrosine ........... 3.64 3.28 1.81 3.78 (6), 3.6 (13)
Valine ............. 5.62 4.76 4.31 5.8 (6), 5.5 (8), 5.8 (9)
Proline. ............ 5.14 4.33 4.01 4.1 (6), 5.5 (9), 4.84 (14)
Glutamic acid. ..... 19.08 16.75 11.64 19.5 (6), 19.1 (15), 21.51 (16)
Aspartic “ ....... 11.52 9.98 7.80 11.4 (6), 11.2 (15), 9.9 (16)
Alanine ............ 7.09 5.67 7.15 6.2 (6), 6.1 (9), 7.05 (14)
Threonine.......... 4.921 4.18 3.71 5.85 (6), 5.11 (17)
Serine .............. 3.961 3.28 3.39 5.0 (6), 4.07 (17)
Glycine ............ 1.39 1.06 1.66 1.4 (6), 1.5 (15), 1.56 (14)
Arginine ........... 2.91 2.61 6.00 2.87 (6), 2.89 (16), 2.91 (18)
Lysine ............. 12.58 11.02 15.45 11.4 (6), 11.4 (15), 10.55 (19)
Histidine. .......... 1.63 1.44 2.83 1.58 (6), 1.50 (8), 1.55 (16)
Cystine + cysteine. 3.401 2.89 2.54
Tryptophan. ....... 1.94E 1.77 1.71
Amide-NH3 ........ 1.311 6.93 1.31 (6), 1.30 (17)

Total. . .. . .. ... ... 97.7 99.6

-
* The value for methionine is that determined by Brand et al. (6). An independ-
ent check on this figure was not obtained with the butyl-benzyl alcohol chromato-
grams in this case, since the experiments were carried out before the introduction of
thiodiglycol as an antioxidant.
t The average threonine and serine values of 4.67 and 3.56 from Table I have been
divided by 0.95 and 0.90, respectively, in accordance with the estimates of Rees (17)
for decomposition of these amino acids during hydrolysis.
1 The cystine + cysteine value is that determined by Brand et al. (6). The
chromatograms gave an average value of 3.25.
$ The tryptophan value is that determined by Brand et al. (6).
11The amide-NH8 value is that determined by Warner and Cannan (20). From
the chromatographic data a maximum amide-NH3 value of 1.35 can be calculated.

thionine is necessary in this case, since the butyl-benzyl alcohol chromato-

grams were performed before the incorporation of thiodiglycol in the solvent
as an antioxidant (1). Brand et al. (6) obtained a value of 3.40 per cent
84 @LACTOGLOBULIN AND SERUM ALBUMIN

for cysteine plus cystine, which, in conjunction with the methionine content,
accounted for 99.6 per cent of the sulfur of the protein. The average
chromatographic value in Table I is 3.25 per cent. It should be noted that
the hydrolytic conditions employed for the chromatographic experiments
were not the same as those, recommended as optimum for cysteine and
cystine determinations by Brand and Kassell (21). Values for cystine in
the present hydrolysates have run from 5 to 10 per cent below the accepted
figures for cysteine plus cystine. The quantitative recovery of cystine
from synthetic mixtures (3) indicates that the chromatogram reflects the
true cystine content of the hydrolysate, and that the values are low as a
result of some decomposition of cysteine or cystine during the preparation

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of the hydrolysate. In the present experiments, an attempt has been made
to obtain as much information as possible from an HCl hydrolysate.
Adequate chromatographic values for the special case of cystine would
require studies on hydrolysates prepared by alternative procedures.
The fact that the calculated cystine sulfur from the chromatogram is
slightly less than enough to account, together with methionine, for the
total sulfur of the protein leaves no room for the presence of ornithine or
hydroxylysine as possible overlaps in the cystine range (3).
The total nitrogen recovery of 99.6 per cent in Table II rests primarily
on the chromatographic data. The weight recovery is 97.7 per cent.
For many purposes this is a satisfactory balance sheet for the protein.
The 2 per cent unaccounted for on a weight basis is a discrepancy which
may arise from one or more of several causes. There may be some decom-
position of amino acids other than those for which corrections have been
made. Some of the amino acid analyses may be in error in such a manner
as to compensate on a nitrogen basis but not on a weight basis. There
may be amino acid constituents present which have not been determined
by the chromatograms. Hydroxyproline, for example, would fall under
the aspartic acid peak, and would not be detected. Negative calorimetric
tests for this amino acid were obtained by Brand et al. (6). Recently,
Keston, Udenfriend, and Cannan’ have demonstrated by their isotopic
derivative method, using radioactive p-iodophenyl sulfonyl chloride, that
hydroxyproline is completely absent ( < 0.05 per cent) in p-lactoglobulin.
It is also possible that the sample of protein contains small amounts of
non-nitrogenous constituents or impurities in addition to the ash and
moisture. The latter have been corrected for in the assignment of a nitro-
gen content of 15.6 per cent to the protein. In the present state of accu-
racy of the chromatographic methods, however, it is not possible to base
any conclusions on the observed small difference between the weight and
nitrogen recoveries.
1Keston, A. S., Udenfriend, S., and Cannan,R. K., personalcommunication.
 W. H. STEIN AND S. MOORE 85

Comparison of Chromatographic Results with Other Values-Analytical

results obtained by other methods are included in Table II. A more com-
plete summary of literature values has been given by Brand (22). On
synthetic mixtures of amino acids the chromatograms have yielded re-
coveries of 100 f 3 per cent for individual components (1, 3). The
microbiological assays, and some of the other methods, have a potential
error at least this large. Thus, for purposes of comparison, conformity
to within 5 per cent has been considered as reasonable agreement.
For leucine, tyrosine, valine, arginine, and histidine, the several literature
values in Table II are all in agreement with the chromatographic results.
Glycine also falls in this group if the variation from 1.39 to 1.56 per cent is

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considered satisfactory for an amino acid present to so small an extent.
The threonine and serine values are in excellent agreement with those of
Rees (17) obtained by periodate oxidation, as already mentioned, but are
about 20 per cent below the results of Brand et al. (6). The glutamic and
aspartic acid figures are both checks with the isotope dilution values of
Foster (15) and the microbiological results of Brand et al. (6). They are
not in line with the analyses of Chibnall, Rees, and Williams (16). The
isoleucine result has been discussed previously (1) and is in agreement with
the most recent microbiological determination (12). For phenylalanine,
the two microbiological values (6, 8) are more than 5 per cent above and
below the chromatographic value from starch columns. The figure ob-
tained by Tristram (9), who employed chromatography of the acetylated
amino acids on silica gel, is also higher than the present phenylalanine
determination.
Alanine has previously been a difficult amino acid to determine. The
present value is higher than the microbiological figure (6) or that of Tris-
tram (9), but is in agreement with the recent results of Keston, Udenfriend,
and Cannan (14), obtained by the isotopic derivative method. The proline
content is 20 per cent above the microbiological value of Brand et al. (6),
but in fairly good agreement with the isotopic derivative data (14).
The value for lysine given in Table II is 10 per cent higher than the
microbiological (6) and isotope dilution (15) figures. References to addi-
tional lysine determinations, all of which fall between 10.5 and 11.4 per
cent, are reviewed by Brand (22). The chromatographic value in this
instance may be in error. For bovine serum albumin, discussed later,
the chromatographic determination of lysine is in good agreement with
the isotope dilution data. In the P-lactoglobulin hydrolysate, there may
be a small amount of some component other than lysine traveling in the
same range on the column, although none of the additional substances
studied to date (3) fall in this category. It is also possible that the chro-
matographic figure may be correct, if there is some racemization of lysine
during hydrolysis. It has been shown previously (3) that the sum of the D
86 @ACTOGLOBULIN AND SERUM ALBUMIN

and L isomers in the hydrolysate is determined by the chromatographic

method. As usually employed, the microbiological (6), isotope dilution
(15), and lysine decarboxylase (23) methods permit the determination of
only the L isomer.
A calculation of a minimum molecular weight for the protein from
analytical figures has been made by Brand et al. (6), by adjusting the
molar ratios to integers within 3 per cent. For the amino acids present
in the smallest amounts in the protein, the attainment of this degree of
accuracy by the chromatographic methods, at least, cannot be assured.
For purposes of molecular weight calculations, an absolute accuracy of 5
per cent is about as close a limit as can be placed on the smaller figures in

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Table II. For example, the deviations from the mean in the triplicate
analyses for arginine and histidine in Table I emphasize the need for caution
in the mathematical interpretation of the results. The determination of
histidine (1.63 per cent) to within 5 per cent of its value is equivalent to
determining about 0.1 per cent of the total weight of the protein. This is
about the limit of accuracy of the chromatographic procedure. The value
of any detailed mathematical treatment of the analytical figures in this
case is subject to the further limitation that /3-lactoglobulin, although it is a
prote‘n which can be prepared in a fairly reproducible form, may not be
homogeneous (ct. (24, 25)).

Analyses of Hydrolysates of Bovine Serum Albumin

The effluent concentration curve obtained upon chromatography of a
hydrolysate of bovine serum albumin is given in Fig. 2. The curve parallels
that obtained with the synthetic mixture (3) in all respects. No new
peaks are present. When threonine, serine, and histidine were added to a
sample of the hydrolysate, the designated peaks rose without loss of sym-
metry, and the added amino acids were recovered in yields of 105, 99, and
99 per cent, respectively.
Integration of E$uent Curves-The quantitative data obtained from the
experiment shown in Fig. 2, and replicates thereof, are given in Table III.
As was true in the case of P-lactoglobulin, the nitrogen of the hydrolysate
has been almost completely accounted for in each experiment. The trypto-
phan content of bovine serum albumin, according to the data of Brand et
al. (26, 22), corresponds to only 0.50 per cent of the total nitrogen of the
protein, and the theoretical recovery for Table III is thus 99.5 per cent.
The observed recovery is subject to the same approximations which were
discussed for p-lactoglobulin.
Duplicate chromatograms with 2 : 1: 1 tert-butyl alcohol-set-butyl alcohol-
0.1 N HCI gave values of 16.7 and 16.3 per cent for glutamic acid and 6.32
and 6.17 per cent for alanine.
 W. H. STEIN AND S. MOORE 91

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FIG. 2. Chromatographic fractionation of a hydrolysate of bovine serum albumin,
Solvents, 1:2:1 n-butyl alcohol-n-propyl alcohol-O.1 N HCl and 2:l n-propyl alcohol-
0.5 N HCl. Column dimensions, 0.9 X 30 cm. Sample, an amount of hydrolysate
corresponding to about 2.5 mg. of protein.

TABLE III
Chromatographic Analyses of Hydrolysates of Bovine Serum Albumin
Solvents, 1:2:1 n-butyl alcohol-n-propyl alcohol-O.1 N HCI and 2:l n-propyl alco-
hol-0.5 N HCl (cf. Fig. 2).
Chromatogram 480 Chromatogram 481 Chromatogram 488

Gill. GItl. Gm.

Constituent amino amino amino
:e,s %
acid per :enas s acid per :es %
acid per
100 ml. 100 gm. “‘“C 100 gm.
protein proiin protein protein proc
~ -.- -~
Leucine-isoleucine ........... 14.4 9.58 14.75 9.80 14.3 9.51
Phenylalanine............... 6.32 3.34 6.44 3.40 6.52 3.44
Valine-methionine-tyrosine ... 7.42 ca. 7.37 cu. 7.20 ca.
Proline. .................... 4.65 3.52 4.75 3.60 4.85 3.67
Glutamic acid-alanine. ...... 14.90 cu. 15.25 cu. 15.40 cu.
Threonine................. 5.44 3.98 5.72 4.19 5.50 4.05
Aspartic acid. ............... 10.91 7.15 10.86 7.12 10.96 7.19
Serine ....................... 3.91 3.24 3.76 3.12 3.77 3.13
Glycine ..................... 1.85 2.15 1.75 2.03 1.85 2.15
Ammonia. .................. 1.03 5.28 1.08 5.54 1.06 5.44
Arginine .................... 5.96 11.92 6.03 12.07 5.72 11.45
Lysine ..................... 12.70 15.15 12.62 15.05 13.15 15.68
Histidine. ................... 4.29 7.24 3.67 6.18 4.04 6.82
Cystine ..................... 5.83 4.23 5.81 4.22 6.08 4.41

Total nitrogen recovery. . 99.1 98.9 99.5

88 @-LACTOGLOBULIN AND SERUM ALBUMIN

TABLE IV
Amino Acid Composition of Bovine Serum Albumin
The values for phenylalanine, leucine, isoleucine, tyrosine, and valine are from
chromatograms carried out with 1:1:0.288 n-butyl alcohol-benzyl alcohol-water con-
taining 0.5 per cent thiodiglycol (1). Gl u t amic acid and alanine were determined
with 2: 1: 1 tert-butyl alcohol-set-butyl alcohol-O.1 N HCl. The remaining chromato-
graphic values are the average figures from Table II. The nitrogen content of the
protein was 16.07 per cent, on an ash- and moisture-free basis.

GIlI. Gm.
amino N as
amino xid resi- er cent
Constituent acid pa of Literature values, gm. amino acid per
100 fgn. due per wotein 100 gm. protein
protein 100 gm. N
protein

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Phenylalanine ... 6.59 5.87 3.48 6.1 (22), 6.48 (27), 6.05 (28)
Leucine ......... 12.27 10.58 8.17 13.7 (22, 26), 11.8 (27), 13.2 (28)
Isoleucine ....... 2.61 2.25 1.74 2.9 (22), 2.97 (27), 2.7 (28)
Methionine. ..... 0.81’ 0.71 0.47 0.86 (27), 0.80 (28)
Tyrosine ........ 5.06 4.56 2.44 5.5 (22, 29), 5.2 (30), 5.3 (13)
Valine .......... 5.92 5.01 4.41 6.5 (22), 5.4 (30), 6.6 (27)
Proline. ......... 4.75 4.00 3.60 5.6 (22), 5.1 (27), 5.5 (28)
Glutamic acid. .. 16.50 14.49 9.78 16.9 (22), 16.6 (27), 16.95 (29)
Aspartic “ ... 10.91 9.44 7.15 10.6 (22), 11.1 (27), 10.25 (29)
Alanine ......... 6.25 4.99 6.12
Threonine. ...... 5.831 4.95 4.27 6.5 (22, 26), 6.2 (27), 6.3 (30)
Serine ................ 4.231 3.51 3.52 4.5 (22,26), 4.9 (28)
Glycine .............. 1.82 1.38 2.11 1.9 (22), 1.96 (29), 2.0 (28)
Arginine ............. 5.90 5.29 11.80 6.2 (22, 26), 5.9 (27), 6.1 (30)
Lysine ............... 12.82 11.25 15.30 12.4 (22), 12.4 (29), 12.3 (30)
Histidine. ............ 4.00 3.54 6.75 3.8 (22, 26), 3.35 (31), 4.1 (30)
Cystine + cysteine. .. 6.52# 5.54 4.73
Tryptophan .......... 0.585 0.53 0.50
Amide-NH3 .......... 0.9511 4.87 1.05 (22, 26)

Total .............. 97.9 01.2

* The value for methionine is that determined by Brand et al. (22,26). The chro-
matograms gave a figure of about 0.92 (1).
t The average threonine and serine values of 5.55 and 3.81 from Table III have
been divided by 0.95 and 0.90, respectively, in accordance with the estimates of Rees
(17) for decomposition of these amino acids during hydrolysis.
$ The cystine + cysteine value is that determined by Brand et al. (22, 26). The
chromatograms gave an average value of 5.91.
$ The tryptophan value is that determined by Brand et al. (22, 26).
I] This figure is a maximum value for amide-NH8 calculated from the total NH3 of
the hydrolysate corrected for the approximate amount of NH8 formed on the decom-
position of serine and threonine.

Composition of Bovine Serum Albumin--The data on bovine serum al-

bumin have been summarized in Table IV. The chromatograms gave a
figure of 0.92 per cent for methionine (1) which is close to the value of 0.81
 W. H. STEIN AND S. MOORE 89

per cent as determined by Brand et al. (26). The same authors obtained a
figure of 6.52 per cent for cysteine plus cystine which, in conjunction with
the methionine content, accounted for 99.0 per cent of the sulfur of the
protein. The average chromatographic value for cystine in the hydrolysate
is 10 per cent below the above figure.
The total ammonia in the hydrolysate of bovine serum albumin (Table
III) averages 1.06 per cent. Corrected for 0.068 per cent ammonia formed
by the decomposition of serine and 0.040 per cent from threonine, the
maximum possible figure for the amide ammonia of the protein becomes
0.95 per cent. This result, uncorrected for additional ammonia that might
be formed as a result of the decomposition of other amino acids, has been

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used in Table IV. The amide ammonia determination of Brand (22) on
bovine serum albumin gives the apparently high figure of 1.05 per cent.
The total nitrogen recovery in Table IV is 101.2 per cent. The weight
recovery is 97.9 per cent. Possible causes for the discrepancy between the
weight and nitrogen recoveries have already been mentioned.
Comparison of Chromatographic Results with Other Values-The results
for the first six amino acids in Table IV have already been discussed (1).
Additional values in Table IV include microbiological assays reported by
Henderson and Snell(27)) Gunness, Dwyer, and Stokes (13), Hier, Graham,
Friedes, and Klein (30), and Velick and Ronzoni (28). As in the case of
P-lactoglobulin, hydroxyproline has been shown to be absent (<0.006 per
cent) in bovine serum albumin by Keston, Udenfriend, and Cannan.
For glutamic acid, arginine, and lysine, several values from the literature
and the chromatographic results are all in agreement. The glycine figure,
considering the small amount present, checks with the isotope dilution
value of Shemin (29) and the microbiological assays (26,28). The chroma-
tographic value for aspartic acid is in agreement with the microbiological
results (26, 27) and within 6 per cent of the isotope dilution value (29).
The figure for proline is 5 to 15 per cent below the microbiological deter-
minations (26-28). The values for threonine and serine are also lower
than the earlier results. The chromatographic figure for histidine is in
agreement with the results of the microbiological (30) and photometric
(26) methods, but is higher than the value obtained by the isolation method
of Vickery and Winternitz (31). Alanine has not previously been deter-
mined in bovine serum albumin.
SUMMARY

Chromatography of amino acids on starch columns has been applied

to the determination of the composition of P-lactoglobulin and bovine serum
albumin. A single chromatogram run with 1: 2 : 1 n-butyl alcohol-n-propyl
alcohol-O.1 N HCl distributes the amino acids to give an effluent curve
which yields quantitative values for ten of the components of the hydro-
90 P-LACT~GLOBULIN AND SERUM ALBUMIN

lysate. When the integrations of the overlapping peaks were included, a

nitrogen distribution was obtained in each case which accounted for 100 f
2 per cent of the total protein nitrogen. A combination of chromatograms
run with three solvent systems is required for quantitative estimation of
essentially all the components of the acid hydrolysates. About 2.5 mg.
of protein are used per chromatogram. The complete seriescan be carried
out in triplicate with a hydrolysate prepared from 25 to 50 mg. of protein.
To estimate the composition of the protein from data obtained on the
hydrolysate, the values for serine and threonine must be corrected for the
decomposition undergone by these amino acids during hydrolysis. Values
for tryptophan and cysteine plus cystine determined by other methods are

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also required. The final tabulation of the results on p-lactoglobulin and
bovine serum albumin has given total nitrogen recoveries of 99.6 and 101.2
per cent respectively, and weight recoveries of 97.7 and 97.9 per cent.
The individual amino acid analyses have been compared with values
previously obtained by chemical, microbiological, and isotopic methods.

The authors wish to acknowledge the assistance of Miss Enid Mellquist

and Mr. Anton Hornicek in the performance of this work.
BIBLIOGUPHY

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 AMINO ACID COMPOSITION OF β
-LACTOGLOBULIN AND BOVINE
SERUM ALBUMIN
William H. Stein and William H. Stein
J. Biol. Chem. 1949, 178:79-91.

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