A COLORIMETRIC METHOD FOR THE ESTIMATION

OF AMINO-ACID a-NITROGEN.

BY VICTOR JOHN HARDING AND REGINALD &I. MAcLEAK.

(Prom the Biochemical Laboratory, McGill University, Afontreal.)

(Received for publication, January 14, 1915.)

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Two methods are at present in use for the determination’ of
amino-acid nitrogen. The first, that of Sorensen,’ is a rapid,
easy method, but is neither very delicate nor accurate. The
second, that of Van Slyke,2 is the method now usually accepted
for the determination of nitrogen in amino groups. In it,s latest
development, the micro chemical form: the method will estimate
0.5 mgm. of nitrogen with an accuracy of 1 per cent, and its
application to the determination of amino-acid nitrogen in blood
and tissues has yielded in its author’s hands very valuable and
interesting results. It was felt, however, that a third and inde-
pendent method for the estimation of the a-nitrogen of the amino:
acids, especially if the sensitiveness of the reaction could be
increased beyond that of the Van Slyke method without loss of
accuracy, would not only be valuable but necessary as the time
approached for a study of the chemistry of the single cell.
It soon became apparent that such a hope could be fulfilled
only by the quantitative application of a color reaction of amino-
acids, and of all such the most probable seemed the reaction with
triketohydrindene hydrate.
This reaction was discovered by Ruhemann4 who found that
all acids containing a free amino group in the LYposition reacted
with triketohydrindene hydrate with the production of an in-
tense blue color. p-, y-, and &amino-acids only gave small amounts
of color, and a-amino-acids substituted on the amino or carboxyl

1 S. P. L. Sijrensen: Biochem. Ztschr., vii, p. 45, 1908.

2 D. D. Van Slyke: this Journal, ix, p. 185, 1911; xii, p. 275, 1912.
3 Van Slyke: ibid., xvi,‘p. 121, 1913-14.
4 S. Ruhemann: Z’r. Chem. Sot., xcvii, pt. ii, p. 2025, 1910.
217
218 Estimation of Amino-Acid a-Nitrogen
group did not react at all. This discovery was confirmed and
extended by Abderhalden, who applied it to the det.ection of
pregnancy and cancer, triketohydrindene hydrate now being a
commercial product under the name of “ninhydrin.” The
sensitiveness of the reaction is unquestioned; for, according to
Abderhalden and Schmidt,6 it will detect one part of glycine in
G5,OOOparts of water, though the other amino-acids will not react
in quite so dilute a solution.
The constitution of the blue coloring matter was investigated
by Ruhamann,6 who isolated from the interaction of alanine
and triketohydrindene hydrate a body which was found to be
identical with the ammonium salt of diketohydrindylidene-diketo-

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hy&indam.ine,

Thus the chemistry of the coloring matter was weil known and
the problem became the determination of the conditions under
which the grouping
RCH (NHZ) COZH
common t.o all a-amino-acids would react quantitatively with
triketohydrindene hydrate to produce the blue colored ammon-
ium salt of diketohydrindylidene-diketohydrindamine, which could
then be compared calorimetrically with a definite amount of
coloring matter as standard.
The reaction has already been made use of in a roughly quan-
tit,ative way by Abderhalden and Lamp6’ in studying the fate of
amino-acids during absorption, but they made no attempt to
place their results on a strictly quantitative basis. Herzfeld,*
however, devised a method for the estimation of the group

6 E. Abderhalden and H. Schmidt: Ztschr. f. physiol. Chem., lxxxv, p.

143, 1913.
6 Ruhemann: Tr. Chem. Sot., xcix, p. 1486, 1911.
7 E. Abderhalden and A. E. LampB: Ztschr. f. physiol. Chem., lxxx& p.
473, 1912.
* E. Herzfeld:Biochem. Ztschr., lix, p. 249, 1914.
 V. J. Harding and R. M. MacLean

NH2
-CH
< COOH

using the ninhydrin reaction as a basis. The method was to

evaporate the amino-acid and excess of triketohydrindene hydrate
t’o dryness on a water bath, dissolve the purple colored residue in
a little alcohol with a drop or two of ammonium hydroxide, make
up to a known volume, and determine the amount of coloring
matter by measuring its extinction coefficient in a spectrophoto-
meter. The present authors have repeated this method of pre-
paring the coloring matter in a quantitative way, estimating it,
however, by a Duboscq calorimeter instead of measuring the

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extinction coefficient in a spectrophotometer. The former
method is much more rapid, and at present is the only one which
could be successfully applied in hospital laboratories to the study
of amino-acid excretion in pathological conditions. Working in
this way, the results were very unsatisfactory. It was found
by heating varying amounts of an amino-acid with an excess of
ninhydrin that the different amounts of the amino-acid could be
estimated with moderate accuracy, using a fixed amount of the
same amino-acid as standard. The following figures illustrate
this point.

cc. OF 0.1 PER CENT COLORIXIETER READING

AMINO-ACID SOLUTION ~- .-
Found: Calculated:
~___~ ~~~____~_ __.--
I
Glycine.. . .. . . . . . .! 1.0 Standard 1 Set at 2.00 cm.
1.5 1.51 1.33
2.0 1.00 I 1.00
Alanine. .. .. . . . . . . . . . . 1 .O Standard Set at 2.00 cm.
2.0 0.99 1.00
4.0 0.48 0.55
Aspartic acid.. . . 1.0 Standard / Set at 3.00 cm.
1.51 1.50
/ 28 i 0.95 1.00
Glutaminic acid.. . . . . ./ 1 .O Standard Set at 2.00 cm.
1.5 1.33 1.33
3.0 0.69 / 066
I .

Thus it will be seen that varying amounts of glycine can be

estimated by using the color produced by a known amount of
 220 Estimation of Amino-Acid a-Nitrogen
glycine as standard. The same is t’rue for alanine, and aspartic
and glutaminic acids.
When, however, we attempted to estimate alanine, or aspartic
or glutaminic acids, using glycine as a standard, the method gave
totally erroneous results.
, h‘t PER cc
.4YINO-ACID
Found: Calculated:
.-
mgm. nlgm.
Alanine............................I 0.016 0.036
Glutaminic acid.. . . . . . .. . . .’ 0.152 0.095
-. --__.

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In the case of aspartic acid the color produced by the reaction
was of such a pronounced reddish shade that it was found impos-
sible to match it against the bluish violet color of the standard.
Thus it will be seen that this method of estimating amino-acid
nit:ogen fails completely.

Reaction of amino-acids and triketohydrindene hydrate in

slight excess.
In our earlier experiments on this subject, attempts were made
to study the reaction between various amino-acids and the tri-
ketone, with strictly molecular amounts of the two substances, but,
owing to causes at present unknown, these experiments failed to
give any concordant results. Neither of us, working alone or
together, could be certain of obtaining any two series of experi-
ments which agreed within 5 per cent. What we desired to do
was to heat a known amount of an amino-acid, usually glycine 01
alanine, with the molecular equivalent of triketohydrindene hy-
drate and determine the time at which the maximum development
of the blue coloring matter took place. This could be accom-
plished by immersing a series of test-tubes containing the equivn-
lent amounts in a boiling constant-level water bath, removing them
one by one at stated intervals (usually five minutes), diluting them
to a known volume (100 cc.) with distilled.water, using the color
produced in the test-tube first removed as standard, and comparing
the others against that in a Duboscq calorimeter. At first the com-
parisons were made by daylight, but it was soon found that much
 V. J. Harding.and R. M. MacLean 221

more consistent readings were obtained by. the use of a 25 watt

tungsten lamp as a source of illumination in a dark room. The lamp
was placed in a conical, semi-opaque shade, the outer end of which
was covered with a sheet of tissue paper to diffuse the light; and
the whole was placed about a foot away from the calorimeter.
In this way a strongly and evenly illuminated field was obtained
on the white reflector of the calorimeter only, and thus the eye was
free from any other disturbing sources of light during the deter-
minations. A small electric bulb on a convenient switch enabled
one to take the vernier readings. In artificial light the bluish
colored solution of the coloring matter changes to a red violet,
resembling very much the appearance of, a dilute solution of

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potassium permanganate when viewed in daylight.
Even under these conditions, which greatly improved our
ability to make concordant readings, the results continued to be
very irregular and capricious, when using equimolecular amounts
of the two reagents. The presence of traces of impurity, the
rapid fading of the coloring matter, and the sensitiveness of the
reaction to heat, probably were all factors, not under these con-
ditions properly controlled, which contributed to the failure of
our experiments from a quantitative point of view. However,
under more stable and more accurately defined conditions, it is
hoped at a later date to return to the study of the reaction in
equimolecular solution and to endeavor to obtain from it some in-
sight into the mechanism of what is without doubt a complicated
series of reactions.
It was soon found, however, that a slight excess of triketo-
hydrindene hydrate enabled us to approach a solution of our
problem of the time of the maximum development of the coloring
matter in dilute solution. The same method of procedure was
adopted as that previously described. The coloring matter in the
test-tube first removed was used as standard and the others were
read against it. The series of curves in Figure 1 shows the relative
amounts of coloring matter plotted against the time, each amino-
acid being taken separately.
It will be noticed that the time of the development of the
maximum amount of color is different for each amino-acid, thus
precluding the use of this technique for a method of estimating
amino-acid a-nitrogen in a mixture of amino-acids. No attempt
TREJOcxiNAL
OFBIOLOGIClL
CHEMISTRY,
VOL.xx. NO.3
222 Estimation of’ Amino-Acid a-Nitrogen
was made to compare one ,amino-acid against another, as such a
series of experiments would have served no useful purpose at
that time. Moreover, the actual amounts of color produced in
the cases of aspartic and glutaminic acids and asparagine were so
small that the reaction liquid was only diluted to 50 cc. instead of
100 cc. Also the color produced by aspartic acid and asparagine
was of a pronounced yehowish tint and could not be compared
with the reddish violet given by alanine or glycine.

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’ i j / I I
h I I I I

FIG. 1.

Reaction of amino-acids and triketohydrindene hydrate in

large excess.
In our next series of experiments we determined the time of the
maximum development of color between the different amino-acids
and the triketone, using a large excess of the latter in high con-
centration. The necessity of a high concentration to produce
quantitative amounts of color’in this reaction had been pointed
out by Herzfeld,g and a series of experiments of our own had con-
firmed this conclusion. We heated together in a boiling constant-
level water bath a series of 1 cc. of0.1 per cent solution of amino-
acid and 0.5 cc. of 1 per cent solution of triketohydrindene hydrate

* Herzfeld: Zoc cil.

 V. J. Harding and R. M. MacLean 223

for periods of 5,10,15, and 20 minutes. The amount of color pro-

duced in the test-tube heated for five minutes was taken as
standard, and the results were plotted as before. Each amino-
acid was considered separately (Figure 2).
The effect of the higher concentration of the triketone was at
once readily apparent. The amounts of color produced in the
same time were much larger, and concordant experiments were

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FIG. 2.

easily obtained except in the case of asparagine. It will also at

once be noticed that the times of maximum development of color
with the different amino-acids are much shorter than in the first
series of experiments and lie closer together. Thus, in ihe first
series, the times vary from twenty minutes (tyrosine) to seventy
minutes (alanine), whereas in the second series the variation is
only from ten minutes (glycine) to twenty minutes (aspartic acid
and tyrosine). The other amino-acids possess the time of maxi-
224 Estimation of Amino-Acid a-Nitrogen

mum development of color at fifteen minutes. The colors with

aspartic acid and asparagine, however, were still very weak and
still possessed a strong yellowish tinge which rendered their com-
parison with the’ other acids extremely difficult. Other irregu-
larities, too, were noticeable. The colors produced by aspartic
and glutaminic acids faded very much more rapidly than those
produced by the other amino-acids; that produced by aspart.ic
acid being discharged completely in a dark room at the end of
twelve hours. As these two acids differed from the others only
in the presence of a second carboxyl group, thus rendering them
more acidic in character, the disappearance of the coloring matter
on standing was put down to this cause. Ruhemann had pointed

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out that long contact with acids causes a decomposition of the
coloring matter, giving a colorless solution, and experiments by
us had proved the strong inhibitory effect of small amounts of
organic acids on the production of the ammonium salt of diketo-
hydrindylidene-diketohydrindamine from the interaction of ala-
nine and the triketone.
In order to t.est this assumption a third series of experiments
was carried out in presence of a base which would neutralize the
acidity of the second carboxyl group of aspartic and glutaminic
acids. Such a base should not be strong enough to hydrolyze the
triketohydrindene hydrate, as happens with the hydroxides of
the alkali metals, and should not interfere with the reductions
and condensations which take place in the reaction. The base
chosen was pyridine.

Interaction of amino-acids and triketohydrindene hydwte in large

excess, in presence of pyridine.
One cc. of a 0.1 per cent solution of the amino-acid was added
to 0.5 cc. of a 1 per cent soIution of triketohydrindene hydrate,
and 0.2 cc. of pure, freshly distilled pyridine was added, and the
mixture heated in a boiling water bath for varying intervals of
time, as in the two previous series of experimenbs. The estima-
tions of the relative amounts of coloring matter were carried out
in a manner similar to the former experiments, each acid being
compared with itself onIy. The curves in Figure 3 show the
results obtained. They show quite clearly that the addition of
 T(. J. Harding and R. M. MacLean 225
the pyridine had achieved the desired result. They show that
the reaction between amino-acids and triketohydrindene hydrate
in presence of pyridine takes place rapidly, and reaches a maximum
amount of coloration which remains constant for some minutes,
except in the case of aspartic acid. This time of maximum devel-
opment of color is constant at about twenty minutes. Moreover,
the addition of the pyridine had increased enormously the actual
amounts of coloring matter produced, and the colors now did not
fade appreciably in a short period of time. In the curves obtained
with glycine and alanine 0.5 cc. of pyridine was used.

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FIG. 3.

A determination was now made of the relative amounts of

coloring matter produced by four of the amino-acids, of bvhose
purity we were quite certain, by dissolving equivalent amounts
of each of them in water and using one as a standard. The fol-
lowing table will illustrate the experiment and the result. Being
in equivalent amounts, they should produce the same amount of
coloring matter and thus give the same calorimeter reading.
It will be seen that aspartic acid was still a little low in amount
of coloring matter produced when compared with the other acids.
As a result of further experiments, it was decided to reduce the
amount of amino-acid nitrogen per cc. to 0.05 .mgm., to reduce
 226 Estimation of Amino-Acid Q-Nitrogen

TRIKETONE:
nfmw-ncrD iv2 PEll cc. 1.0 PER CENT

~ mgm. cc. cc.

Alanine ...............
Glycine ................
.i
;
0.1
a.1
0.2
0.2
0.5
0.5
1.00
0.99
Aspartic acid ................ I 0.2 0.5 1.17
Glutaminic acid ./ 8:: 0.2 0.5 1.01

the pyridine to 1.0 cc. of a 10 per cent solution in water, and to

increase the triketohydrindene hydrate to 1.0 cc. of a 2 per cent
solution.
Method of estimation of amino-acid a-nitrogen by use of triketo-

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hydrindene hydrate.
One cc. of the solution to be estimated, containing not more than
0.05 mgm. of amino-acid a-nitrogen and neutral to phenolphtha-
lein, is mixed with 1 cc. of a 10 per cent aqueous solution of pure
pyridine and 1 cc. of a freshly prepared 2 per cent solution of t.ri-
ketohydrindene hydrate and heated in a rapidly boiling constant-
level water bath for twenty minutes. At the end of that time the
test-tube is removed, cooled, and diluted to a suitable volume,
usually 100 cc., but if the amino-acid a-nitrogen is very small in
amount, a correspondingly smaller dilution can be used. The solu-
tion of coloring matter thus obtained is compared with the stand-
ard. color in the usual way, in a Duboscq calorimeter.

Preparation of a standard color.

The standard solution is prepared by dissolving 0.3178 of a
gram of pure freshly crystallized alanine in a liter of distilled
water. Such a solution contains 0.05 mgm. of nitrogen per cc.
To prepare the standard color 1 cc. of the standard alanine solu-
tion is heated for twenty minutes in a boiling water bath with
0.5 cc. of a 10 per cent solution of pyridine and 1.0 cc. of a 1 per
cent solution of triketohydrindene hydrate.‘O At the end of that
time the contents of the tube are cooled and diluted to 100 cc.
This solution of coloring matter is used as a standard and will
keep for twenty-four hours. The standard solution of alanine is
lo The employment of a more concentrated solution of the ketone gives
no further increase in the amount of coloring matter in the case of alanine.
 V. J. Harding and R. M. MacLean 227
st,able for three months. Attempts were made to prepare a per-
manent standard color without success. A preparation of the
pure coloring matter was made according to the directions of
Ruhemann, and a dilute solution of it made in water. It was
found, however, that when the dried coloring matter was used,
it was not as freely soluble in water as the freshly prepared sub-
stance, and that an aqueous solution of the pure coloring
matter faded rapidly; it was much more stable in the presence of
pyridine, but even then at the end of three months it had faded
completely. There was thus no advantage to be gained in using
a solution of the coloring matter prepared per se. The standard
color prepared as directed is little or no trouble to prepare along

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with the determinations themselves.

Determination of amino-acid a-nitrogen in various amino-acids and

comparison with the method of Van Slylce.
A series of determinations was then made of the amino-acid
nitrogen in various amino-acids, and the results were compared
with theory in the case of the pure acid. A parallel series of
determinations was made by the method of Van Slyke and the
results were compared with the calorimetric determinations. In
the case of the acids whose purity was doubtful, the method of
Van Slyke served as a standard. The results are expressed in
mgm. of nitrogen per cc.

*MINO-AmD -
Calorimeter T Van Slyke
~- _ ~~~.~~~~- ~~. - - ~__ I- ~- .-
mgm. mgm. mgm.
Glycine............................ 0.186 0.189 0.189
Alanine............................ 0.100 0.101 0.102
Valine............................. 0.201 0.201 0.196
Leucine........................... 0.104 0.102
cu-Amido-n-caproic acid.. . . . 0.160 0.164 0.161
Phenyl alanine.. . . . 0.128 0.126 0.126
i-Tyrosine......................... 0.084 0.087
Tryptophane. . . . . .. . . 0.090 0.092
Histidine.......................... 0.085 0.089
Bspartic acid.. . . . 0.105 0.103 0.104
Asparagine. .. .. . 0.17.5 0.178
Glutaminic acid.. .. . .. 0.15s 0.157 : 0.157
.-__- i
228 Estimation of Amino-Acid a-Nitrogen

The results show quite clearly the excell&t agreement of the

calorimetric method with theory and with the method of Van
Slyke. It must be pointed out that the solutions of the amino-
acids were made of just sufficient concentration to be estimated
in the Van Slyke micro apparatus. To determine the amount,
calorimetrically, it was necessary to dilute those solutions from
two to four times, so that our actual error of determination is less
than appears in the table.
The estimation of cystine, however, by this method gives
results which are much too low. Moreover, the coloration devel-
oped has an intense red color, which makes it almost impossible
to compare it with the standard alanine. As a consequence of

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this it, would be impossible to estimate the amino-acid a-nitrogen
in a mixture of amino-acids containing a large proportion of
cystine without a large error. Such cases, however, are not very
common, and the authors do not think that the amount of cystine,
occurring in the hydrolysis’mixture obtained from the majority
of proteins, will introduce any serious error. To determine this
point and to see if the method would estimate the a-nitrogen in a
mixture of amino-acids in the proportions in which they occur in
a native protein, an estimation of the amino-acid a-nitrogen in
ereptone was made and compared with the Van Slyke method.

r-it PER cc. Ii*

- . ..------- -
mym. per cent
Calorimeter....................... 0.132 8.5
Van Slyke.. . . . . ... .. ... . 0.139 9.0
__~_

It will be seen that the calorimetric method gives figures

slightly lower than the Van Slyke method, but not, low enough to
invalidate seriously the result. The difference, however, is ca-
pable of easy explanation. The coloritietric method gives too
low a result in the presence of cystine. The Van Slyke method
gives a result, a little too high in the case of glycine and cystine.
These three facts are sufficient,, we think, to explain the difference
of 0.5 per cent.
A series of determinations of the amino-acid a-nitrogen in
peptones from various sources was next, carried out in order to
 V. J. Harding and R. M. MacLean 229

test the efficacy of the method when dealing with partial hydrol-
ysis products of proteins. The following table shows the results
to be in good agreement with those obtained by the Van Slyke
method.

7-72PER cc.
I --.--~-.__.
ORIOIIV OF PErnOHE I-
~- Calorimeter Van Slyke

Meat (Merck). . . . . . . . . . . . . . . . . . .I 0.062 0.061

“Ecarne” (Schuchardt). , . . . . . . ., 0.035 0.031
Precipitated by alcohol (Schu-l
chardt). . . . . . . . . . . . . . . . . . . . . . . 0.025 0.021
Witte.. . . . . . . . . . . . . . . . . . . . . . . . 0.067 0.067

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Detemzination of the maximal and minimal amounts of amino-acid
a-nitrogen capable of estimation by this method.
The early experimental results described in this paper (page
226) had shown that although some of the amino-acids could be
accurately estimated when the solution contained 0.1 mgm. of
nitrogen per cc., yet other amino-acids could be estimated only
when the nitrogen was only 0.05 mgm. per cc. This value is
taken as the maximal value. To determine the minimal value
two series of experiments were performed, one on an aIanine
solution, the other on an ereptone solution. A standard solution
of alanine and a solution of ereptone were diluted by, known
amounts of water and calorimetric estimations made of the amino-
acid a-nitrogen present in the diluted solutions. The coloring
matter produced was diluted to a suitable volume so that the
calorimetric readings did not differ from the standard by large
amounts. The theoretical amounts given against the results
obtained for ereptone solutions were obtained by calculating from
the mean result of the calorimetric and Van Slyke determinations
given on page 228.
?ngm. 9n!pz. mpm.
Alanine. . . . . . . . Found 0.050 0.025 0.012 ctJ5
Per cc. . . .. .. . Calculated 0.050 0.025 0.012 0.006
Ereptone. . .. . Found 0.065 0.032 0.016 0.008
Per cc.. . . . Calculated 0.067 0.033 0.016 0.008
230 Estimation of Amino-Acid a-Nitrogen

It will thus be seen that the method is accurate over a range

of 0.05 mgm. to 0.005 mgm. per cc. Whether amounts less than
0.005 mgm. can be estimated, we have not determined. At that
concentration the amount of coloring matter produced is so small
that it can only be measured with difficulty. Qualitatively, how-
ever, it is possible to detect as little as 0.001 part of a mgm. of
amino-acid a-nitrogen in 1 cc. of solution. This, in the case of
alanine, means the detection of one part of the amino-acid in a
little over 1,500,OOO parts of water.
The question naturally arises as to whether the reaction will
proceed quantitatively in presence of other substances. From
the results obtained with ereptone (page 228) and the various

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peptones (page 229), the method can be used for the determination
of the amino-acid a-nitrogen set free in the hydrolysis of proteins.
Its application to the analysis of urine and blood, however, is still
attended with difficulties, ammonium salts and urea causing
disturbances in the reaction. These points are being investigated,
and the results will be reported shortly.

SUMMARY.

1. A method has been devised for the estimation of amino-acid

a-nitrogen by the use of triketohydrindene hydrate and, pyridine.
2. The method possesses an accuracy equal to that of the Van
Slyke method.
3. It will estimate within the ranges of 0.005 mgm. to 0.05 mgm.
amino-acid a-nitrogen per cc.
4. The method is inaccurate, however, for cystine.
5. The method is applicable to the determination of amino-
acid a-nitrogen (in neutral solution) set free in protein hydrolysis.
A COLORIMETRIC METHOD FOR THE
ESTIMATION OF AMINO-ACID α
-NITROGEN
Victor John Harding and Reginald M.
MacLean
J. Biol. Chem. 1915, 20:217-230.

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