XXX. BIOCHEMICAL STUDIES IN THE NITROGEN METABOLISM OF THE APPLE FRUIT.
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XXX. BIOCHEMICAL STUDIES IN THE
NITROGEN METABOLISM OF
THE APPLE FRUIT.
I. THE ESTIMATION OF AMINO-NITROGEN BY THE
VAN SLYKE METHOD IN PRESENCE OF TANNIN.
BY ALFRED CRESSWELL HULME.
Low Temperature Research Station, Cambridge, and
Ditton Laboratory, East Malling, Kent.
(Received December 1st, 1934.)
EARLY in a study of changes in the nitrogen fractions of the peel and pulp of the
apple fruit during development on the tree serious errors became apparent in the
determination of the amino-nitrogen fraction of the total soluble nitrogen by the
Van Slyke gasometric method. In certain cases-notably in the-peel at all stages
of maturity and in the pulp of young fruits-the amino-nitrogen figure obtained
was considerably higher than that of the total soluble nitrogen. This fact in-
dicated either that the total soluble nitrogen figures (determined by a modifica-
tion of the micro-Kjeldahl method to be described in a later communication)
were too low or that the "Van Slyke amino-nitrogen" figures were too high. On
carrying the investigations further it became apparent that the latter possi-
bility was the more probable. It appeared most likely that some substance
present in the extracts, other than amino-acids, was yielding gas in the amino-
nitrogen determinations. Various possibilities as to the nature of this substance
were examined without success until a paper by Wasteneys and Borsook [1924]
was noted in which it was shown that tannic acid, used by these investigators for
the precipitation of peptones, yielded large volumes of gas in the Van Slyke
apparatus.
Thus it appeared likely that tannin, present in the apple extracts, might be
leading to the anomalous amino-nitrogen results [Hulme, 1932].
Support is lent to this view by the results of Overholser and Cruess [1923]
who showed that there is considerably more tannin present in the peel than in
the pulp of the apple fruit. Thatcher [1915] gives figures varying between 0-132
and 0-208 % of the fresh weight for the tannin content of several varieties of
American apples. Wehmer [1929] gives the tannin content of apples as varying
between 0'1 and 0-3 % of the fresh weight.
Using the method of Spiers [1914] the present writer found the tannin con-
tent of the Bramley's Seedling apples used in this investigation to be of the order
of, peel 0-37-0-45 %, pulp 0-06-0-18 % (for criticism of the Spiers method see
later). This meant that in the dilutions used (see later) in the Van Slyke amino-
nitrogen estimations as much as 50 mg. of tannin might be present. It was
therefore decided to investigate the possibility of removing the tannin from
extracts prior to the amino-nitrogen determinations and the present paper is
concerned with the attainment of this object.
A brief description only will be given of the method of preparing the apple
extracts and of other details not directly concerned with the present problem since
it is proposed to present such details in full in a later paper in the present series.
( 263 )264 A. C. HULME
EXPERIMENTAL.
1. General Methods.
(a) Preparation of the apple extracts. The method of preparation consisted
essentially of extracting the frozen (-20°) and finely ground tissue with 60-
80 % alcohol at 400 in a special vacuum-extractor until all the nitrogen soluble
under such conditions had been removed. The alcohol was then evaporated off
from the extract at 500 under diminished pressure. The pulp extracts had a final
volume of 100 ml., contained 100-150 mg. of nitrogen and represented 400 g. of
fresh tissue; peel extracts had a final volume of 60 ml., contained 40-100 mg. of
nitrogen and represented 100-250 g. of fresh tissue depending on the size and
state of maturity of the fruit. The separation of the fruits into peel and pulp
tissue was achieved by means of a household potato peeler when the material
was in the hard frozen state.
(b) The Van Slyke amino-nitrogen determinations. The reaction chamber of
the apparatus used was intermediate in size between the macro- and micro-
forms originally suggested by Van Slyke, the burette used for the introduction of
the solution under examination having a maximum capacity of 5 ml. This re-
action chamber was used in conjunction with the micro-gas burette (3 ml.). The
shaking time for completion of the reaction was 15 min., this time being found
necessary for concordant results with apple extracts.
(c) Spiers's method for the estimation of tannin. It is necessary to outline the
essential points of this method as the method of removal of tannin used was
adapted to the present problem in preliminary experiments.
Briefly the method consists of titrating a known volume of the tannin-
containing solutions (Spiers was chiefly interested in cider), diluted with 750 ml.
of distilled water, with potassium permanganate in presence of indigo carmine (as
indicator), before and after treatment with fat-free caseinogen. The caseinogen,
treatment, which is supposed to remove all the tannin present, consists of shaking
50 ml. of the extract with two successive portions of 1 g. of fat-free caseinogen
with intermediate and final filtration through a fine filter medium. The method
was standardised by Spiers against Schering's "tannin leviss. puriss." and other
commercial tannin preparations.
In the present work Merck's "Caseinum-Natrium for bacteriological pur-
poses" was used after extraction with ether for several hours. Similar results
were obtained with a sterile solution of caseinogen prepared from milk by the
method of Van Slyke and Baker [1918] kindly supplied by Dr R. B. Haines of the
Low Temperature Research Station, who was using such a preparation for bac-
teriological work. The tannic acid used was Schering-Kahlbaum " acidum
tannicum pro analysi" and B.D.H. pure; both these samples gave identical
volumes of gas per unit amount in the Van Slyke determinations and their 1 %
solutions had approximately the same hydrogen ion concentrations (PH ca. 3-5).
2. Investigation of the amount of gas given by pure tannic acid
solutions in the Van Slyke estimation.
By "Van Slyke estimation" is meant here and elsewhere throughout the
present paper a determination carried out according to the Van Slyke technique
for the estimation of amino-nitrogen whether or not the solution under examina-
tion contains amino-groups.
Solutions containing various amounts of tannic acid between 5 and 100 mg.DETERMINATION OF AMINO-N IN FRUITS 265
per 5 ml. were prepared and 5 ml. of these solutions were taken for a Van Slyke
estimation. Within these limits a linear relation was found to exist between the
amount of gas evolved (at 750 mm. and 150; corrected for blank value) and the
amount of tannic acid present, as will be seen from Fig. 1.
10
10
410
xo
I0
0 10 20 30 40 50 60 70 80 90 100
Fig. 1. Tannic acid (mg.)
Thus it is clear that the amounts of tannin likely to be present in apple ex-
tracts are sufficient to interfere to an appreciable extent with the amino-nitrogen
determinations.
3. Removal of tannin prior to the Van Slyke estimations.
(a) Preliminary investigations. Spiers's method for the estimation of tannin
suggests that the treatment of extracts with fat-free caseinogen should bring
about a quantitative removal of tannin. If such a method were adopted it
would, of course, be essential to remove completely any excess caseinogen
before proceeding to an amino-nitrogen determination.
To examine the effect of the addition of caseinogen to pure solutions of tannic
acid, two successive portions of 0 5 g. of fat-free caseinogen were added to 50 ml.
lots of tannin solutions of various concentrations adjusted to various hydrogen
ion concentrations by phosphate or acetate buffers. Filtration through a What-
man No. 44 filter was carried out after each addition of caseinogen and 5 ml. of
the filtrates were taken for a Van Slyke determination. The results obtained may
be summarised as follows:
(i) The first treatment with caseinogen at PH 1 to 2 removed (as judged by
the reduction in the volume of gas given in the Van Slyke apparatus) about
35 % of the tannin; the second treatment at the same PH removed all but
19-20 % of the tannin. At this hydrogen ion concentration the caseinogen
formed a clot as soon as it entered the tannic acid solution.
(ii) The best results were obtained by two caseinogen treatments at PH 7 to 8
(which allowed some of the caseinogen to go into solution) followed by the addi-
tion of acid to bring the PH value of the filtrate from the second treatment to 4-6
(to precipitate excess caseinogen), and a further filtration. By this means 96 %
of the tannic acid was removed.
When, however, this latter method was applied to apple extracts alone and
to extracts to which tannic acid had been added, not more than 40 % of the
tannin was removed. Further modifications of the "caseinogen method," in-
cluding three caseinogen treatments, gave no better results. While this work was266 A. C. HULME
in progress a paper by Rahn [1932] appeared in which further emphasis was laid
on the serious interference of tannin with the Van Slyke method. Rahn says of
tannin " Es reduzierte im Vakuum des Apparates einen Teil des bei der Reaktion
entstehenden N203 zu elementaren N und verhohte so die vom xc-Amino-N
stammende N Menge auf das 3-7 fache "-although no evidence was adduced in
support of such a theory. Nevertheless Rahn showed that tannin could be
removed from plant extracts by precipitation with potassium dichromate. In
Rahn's case the tannin had been added for precipitation purposes during the
nitrogen fractionation of his extracts; the potassium dichromate solution was
allowed to remain in contact with the extracts for a day to bring about the pre-
cipitation of the tannin.
Preliminary results for the precipitation of small amounts of tannin from pure
tannin solutions by means of dichromate were very promising, so that a more
detailed examination of the method was initiated in relation to the present
problem. In particular it appeared necessary to ascertain whether the prolonged
contact of the dichromate with amino-acids and amides of the asparagine type
caused any destruction of these compounds.
(b) The precipitation of tannic acid by dichromate in solutions containing
glycine and asparagine. To 30 ml. of a 1 % solution of tannic acid 3, 4 and 6 ml.
quantities of 5 % potassium dichromate were added. The liquids were brought
to 50 ml., well shaken and allowed to stand for 18 hours. At the end of this time
they were filtered and 5 ml. of the filtrate taken for a Van Slyke estimation. The
results obtained are presented in Table I.
Table I.
Volume of "Van Slyke" gas
ml. dichromate (ml. at 750 mm. and 150)
3 0-23
4 0-17
6 018
The solution containing only 3 ml. of dichromate filtered extremely slowly
and the filtrate was opalescent. From Fig. 1 it is seen that the volume of gas
which would have been given if no tannic acid had been removed is 0x66 ml.
(equivalent to 30 mg. tannic acid). Since the water blank value was 0-16 it is
clear that 4 and 6 ml. of dichromate solution were sufficient to remove all the
tannic acid present.
To examine the effect of dichromate on glycine and asparagine and to ascer-
tain whether the dichromate effected a quantitative removal of tannin in the
presence of these compounds and also if such a removal were affected by the PH
of the solution, a series of experiments was carried out along the following lines.
To 5 ml. of a solution containing 0 5 g. of pure glycine and 0 5 g. of pure
asparagine per 100 ml. of solution was added phosphate or acetate buffer to give
the required PH. Five ml. of a 5 % solution of tannic acid were added together
with 2-4 ml. of 5 % potassium dichromate solution and distilled water to make
the volume to 45 ml. The mixture was well shaken and allowed to stand for 18
to 20 hours at 10 (to minimise any effect of the dichromate on the glycine and
asparagine). At the end of this time the liquid was filtered through a fine filter
and 5 ml. of the filtrate were taken for a Van Slyke estimation.
Similar mixtures were prepared with distilled water in place of the tannic acid
and also in the absence of dichromate. The results obtained are given in Table II.
The amounts of glycine, asparagine and tannic acid present and the ml. gas ob-
tained in the Van Slyke determination all refer to the 5 ml. of filtrate used in theDETERMINATION OF AMINO-N IN FRUITS 267
Van Slyke estimation. The dichromate volume refers to that initially present in
the whole solution. The gas volumes are all corrected to 750 mm. and 150.
Table II.
Tannic
Glycine Asparagine acid Dichromate ml. gas ml. gas
mg. mg. mg. ml. (theoretical) (observed) pH
(1) 2*767 - 4 0 94 1*14 4*6
(2) 2*767 2-767 4 1*45 1-61 3*8
(3) 2-767 - 28 1-58 1-66 39
(4) 2-767 28 4 0 94 1*00 3-2
(5) 2-767 2-767 - 2-09 2-06 39
(6) 2-767 2-767 28 1-45 1-42 3.4
(7) 2-767 2*767 28 4 As (6) assuming all 1-41 3-2
(8) 2-767 2-767 28 4 tannic acid to be 148 3.4
(9) 2-767 2-767 28 4 precipitated 1-44 50
(10) 2-767 2 767 28 4 , 1-18 5*5
(11) 2-767 2-767 6 2 ,, 1*16 5.7
(12) 2-767 2-767 28 4 ,, 1*19 6-4
(13) 2-767 2*767 28 4 1 10 6-8
(14) 2-767 2-767 28 4 ,, 120 7-5
Results (1) and (2) give the "blank" value (av. 0 18 ml.) which is applied to
all the subsequent results. The "theoretical" values are calculated from the
amino-nitrogen content of the glycine and asparagine present, assuming glycine
to give 103 % of its calculated nitrogen content in the Van Slyke apparatus
[Mitchell and Hamilton, 1929]; the "theoretical" value for tannic acid is ob-
tained from Fig. 1.
PH values were measured by means of a Morton glass electrode. Filtration
was extremely slow in the case of mixtures (12), (13) and (14) and the filtrates
were turbid.
From Table II it appears that all the tannin is removed from solution by the
dichromate treatment without destruction or adsorption of glycine or asparagine
between PH 3-2 and 5 0. Above this PH range the low values for amino-nitrogen
were probably due to some adsorption of the glycine and/or asparagine on the
tannin-dichromate precipitate. Between PH3-2 and 50 the precipitates of tannin-
dichromate complex were reddish brown and granular, changing to dark brown
and gelatinous with increase of the PH.
(c) The tannin-dichromate precipitation in presence of apple fruit extracts. The
effect of dichromate solution on the amino-nitrogen figure of apple extracts
(pulp and peel) alone and in the presence of added tannic acid, and on the
amino-figure for apple extracts plus added glycine-asparagine solutions with and
without added tannic acid was next investigated.
A brief description of the method of preparation of the apple extracts has
already been given. 10 ml. of each extract were nearly neutralised with NaOH
and 5 ml. of a 10 % suspension of magnesia added. The free ammonia was then
distilled off under diminished pressure at 400 in a modification of the apparatus
employed by Chibnall and Westall [1932]. The excess magnesia in the residue was
dissolved in a minimum of warm glacial acetic acid and the liquid transferred to
a 50 ml. standard flask. The requisite amount of 5 % dichromate solution was
then added, followed by distilled water to the 50 ml. mark. Where glycine-
asparagine solution (0 5 g. glycine and 0'5 g. asparagine per 100 ml.) and tannic
acid solution (5 %) were added, this was done and the requisite buffer added be-
fore the addition of the dichromate. After bringing to the 50 ml. mark the flask
was shaken vigorously for a few minutes and then placed at 10 for the times
stated. The tannin precipitation is a " time reaction." The solution first gradually
Biochem. 1935 xxix 18268 A. C. HULME
darkens and then the precipitate slowly forms, the whole process taking 10-30
seconds. The precipitate, under the most favourable conditions of hydrogen ion
concentration, does not settle for several hours. Shaking greatly accelerates the
process.
5 ml. of the filtrate from the tannin precipitation were taken for the Van
Slyke estimations. The results obtained are given in Table III. As before, the
figures for glycine, asparagine and ml. gas (corrected) refer to the 5 ml. of solu-
tion actually taken for the Van Slyke determination, and the volume of di-
chromate solution refers to that present in the 50 ml. of liquid before filtration.
Table III.
ml. gas* ml. gas
Material PH (expected) (observed)
Stood for 18 hours before filtration:
Pulp (E. 1)
Extract alone 4-6 - 1-31
+ 4 ml. dichromate 4-1 0-83
+ 1-5 mg. glycine + 1.5 mg. asparagine
+ 4 ml. dichromate f 41 1-60 1-60
+ 1-5 mg. glycine + 1-5 mg. asparagine t 4-0 1-60 1-57
+ 25 mg. tannic acid + 4 ml. dichromate
Stood for 42 hours:
Pulp (E. 3)
Extract alone 3-7 1-82
+ 2 ml. dichromate 40 1-11
+ 1-5 mg. glycine + 1-5 mg. asparagine 40 1-88 1.90
+ 2 ml. dichromate J
+ 1-5 mg. glycine + 1-5 mg. asparagine 1-88 1-91
+ 2 ml. dichromate i 3-6
+ 1-5 mg. glycine + 1-5 mg. asparagine
+ 4 ml. dichromate f 3.9 1-88 1-84
Stood for 18-20 hours:
Peel (E. 6)
Extract alone 4-4 2-39
, + 4 ml. dichromate 3-8 0-89
+ mg.
2 glycine + 2 mg. asparagine 1-92 1-95
+4 ml. dichromate i 3-7
Pulp (E. 5)
Extract alone 3-9 1-68
, + 2 ml. dichromate 3-9
-
1-30
,, + 4 ml. dichromate 3-8 1-29
* Assuming all the tannic acid to be removed by "dichromate-precipitation."
From the table it is clear that the dichromate treatment removes added
tannic acid from apple extracts without destruction or removal of added glycine
and asparagine. Presumably also the dichromate removes tannin from the apple
extracts themselves. The fact that a much larger proportional decrease in the
amount of gas given in the Van Slyke estimation follows the dichromate treat-
ment in the case of peel than of pulp extracts provides corroborative evidence
for such a suggestion, since, as has already been shown, the peel of apples con-
tains much more tannin than does the pulp. The hydrion concentration of the
medium does not appear to affect the precipitation of tannin at least between
PH 3-6 and 4-4. The precipitates in the case of apple extracts were very similar in
colour and texture to those obtained with pure tannic acid-glycine-asparagine
solutions. Even when 25 mg. of tannic acid were present in addition to that of
the extracts it is seen that 4 ml. of 5 % potassium dichromate per 50 ml. of
solution were sufficient to remove all tannin. It appears to be important not to
use a large excess of dichromate, for 5 ml. of undiluted 5 % dichromate solutionDETERMINATION OF AMINO-N IN FRUITS 269
gave a " Van Slyke blank " value which was about double the water blank. In
the above results the blank value obtained from results (1) and (2) in Table IA
was applied. This is the only satisfactory blank obtainable since the amount of
free dichromate after tannin precipitation in each individual case is unknown.
This blank value is only 0-02 ml. greater than the water blank and, as the
maximum variation in repeated estimations is 0 04 ml., this difference is
negligible.
Here may be given a typical result illustrating the relative effects on the Van
Slyke figure for an apple extract of the caseinogen and the dichromate treat-
ments. In Table IV are given the observed amounts of "amino-N" in 100 ml.
of an extract of a sample of young apples (after removal of free ammonia).
Table IV.
After After
caseinogen dichromate
Direct treatment treatment
mg. " amino-N" 39 0 35 0 26-2
This and similar results readily explain the high Van Slyke amino-nitrogen
figures mentioned in the introduction.
Finally it was necessary to ascertain whether there was any adsorption of
nitrogen (amino-groups) on the dichromate precipitates in the case of apple
extracts. With this object in view the residues from the estimation of free
ammonia in 10 ml. of extract, after neutralisation of the excess magnesia with
acetic acid, were washed into a 50 ml. flask, dichromate solution was added and
the liquid brought to 50 ml. After standing for 18-20 hours at 1° the precipitate
formed was filtered off. 5 ml. aliquot parts of the filtrate were taken for a Van
Slyke estimation. Other 5 ml. portions were evaporated nearly to dryness on a
water-bath with 2 ml. of concentrated sulphuric acid. The nitrogen contents of
these residues were then determined by a modification of the micro-Kjeldahl
method using hydrogen peroxide as catalyst. These were compared with the
total nitrogen content of an equivalent amount of original extract, after deduct-
ing the free ammonia-nitrogen. The results are given in Table V.
Table V.
ml. gas in
Total N Van Slyke
Pulp (V). Extract alone 1.53 2-10
,, +4 ml. dichromate 1*49 1*40
Pulp (IVc). Extract alone 2-17 3*20
+ 2 ml. dichromate 2-06 1-96
± 3 ml. dichromate 2-13 1-93
These results show that while the amino-nitrogen figure may be reduced by the
dichromate treatment by as much as 40 % the reduction is due to the removal of
a non-nitrogenous substance since, under the same conditions, the total nitrogen
is reduced by only about 2 %.
The final method adopted for the estimation of amino-nitrogen in apple pulp
and peel extracts is as follows.
The excess magnesia present in the residue from a free ammonia estimation
on 10 ml. of extract is dissolved in a minimum of glacial acetic acid and the
mixture transferred to a 50 ml. standard flask. 3 ml. of 5% potassium dichromate
solution are then added, and the liquid is brought to the 50 ml. mark with distilled
18-2270 A. C. HULME
water. After shaking well the flask is allowed to stand overnight at 10. The
dichromate-tannin precipitate is then filtered off, using a small Whatman No. 44
paper, and 5 ml. of the filtrate are taken for the usual Van Slyke determination.
No PH adjustment is necessary prior to the addition of dichromate as it was
found that the PH of the "neutralised" and diluted ammonia residues was
always in the region of PH 3-8 to 4-4.
No doubt the method is applicable to other tannin-containing plant extracts.
The fact that such small amounts of tannin give appreciable amounts of gas
in the Van Slyke estimation casts serious doubts on many of the results hitherto
reported for the amino-acid content of plant material. Also the data presented
make it necessary to question the accuracy of Spiers's method for the estima-
tion of tannin (depending as it does on the quantitative removal of tannin by
precipitation with caseinogen), especially when applied to complex solutions
such as cider and plant extracts generally.
Strange as it may appear on first consideration, it may be possible to make
use of the Van Slyke amino-nitrogen technique in the estimation of the tannin
content of plant material. For, since the amount of gas given by tannin in the
Van Slyke estimation is a linear function of the amount of tannin present, and
since dichromate appears to precipitate tannin quantitatively without affecting
the true amino-nitrogen figure, the reduction in the "Van Slyke gas" volume
brought about by the treatment with dichromate should be a quantitative
measure of the amount of tannin present. However a method based on these
facts would depend entirely on a calibration with oak gall tannin (the " purest "
standard at present available commercially), and it may be argued, since the
chemistry of the tannins is still somewhat obscure, that tannin from other
sources may not give the same volume of gas per unit amount in the Van Slyke
apparatus. Oak gall tannin is a mixture of pyrogallol tannins, whereas, for
example, apple tannin appears to belong to the group of catechol tannins
[Overholser and Cruess, 1923]. Nevertheless a similar objection applies to the
method of Spiers and to other methods except that relative (generally to oak
gall tannin) permanganate titration, relative weight of strychnine precipitate
etc., are used instead of the proposed Van Slyke gas volume. It is proposed to
investigate such a method as outlined above for the determination of tannin in
apples. Carried out in conjunction with amino-acid determination it should
prove convenient, rapid and valuable, at least as a means of determining the
relative tannin contents of a series of plant extracts.
One final suggestion which arises from the present results. It may be that
potassium dichromate could be used with advantage to replace lead salts and
other precipitants for plant extracts in certain cases.
SUMMARY.
1. Results are given which suggest that the abnormally high figures obtained
for the amino-nitrogen content of apple fruits by the Van Slyke method are due
to the presence of tannin.
2. It is shown that a linear relation exists between the amount of gas given
in the Van Slyke estimation in the case of solutions of pure commercial pre-
parations of tannic acid (from oak galls). As little as 10 mg. of tannic acid gives
0 20 ml. of gas in the "Van Slyke."
3. Attempts to remove tannin from pure tannin solutions and from apple
extracts by precipitation with caseinogen are described. While suitable conditions
can be found for thus achieving the removal of 96 % of the tannin from theDETERMINATION OF AMINO-N IN FRUITS 271
former, not more than 40 % can be removed from apple extracts by this
means.
4. Experiments are described which show that complete removal of small
amounts of tannic acid added to apple eixtracts can be achieved by precipitation
with potassium dichromate. Presumably, also, tannic acid present in the
extracts themselves is removed by this method which is shown not to interfere
with amino- or acid amide groups.
5. A method, based on this result, is described whereby the interference of
small amounts of tannin with the Van Slyke amino-nitrogen determination is
entirely overcome.
6. Tentative suggestions are advanced for a new method for determining the
tannin content of plant extracts.
REFERENCES.
Chibnall and Westall (1932). Biochem. J. 26, 122.
Hulme (1932). Report of the Food Investigation Board, p. 78.
Mitchell and Hamilton (1929). The biochemistry of the amino-acids. (New York.)
Overholser and Cruess (1923). Agric. Exp. Sta. Univ. Calif. Agric. Coll. Tech. Paper No. 7.
Rahn (1932). Planta, 18, 1.
Spiers (1914). J. Agric. Sci. 6, 77.
Thatcher (1915). J. Agric. Res. 5, 103.
Van Slyke and Baker (1918). J. Biol. Chem. 35, 127.
Wasteneys and Borsook (1924). J. Biol. Chem. 62, 1.
Wehmer (1929). Die Pflanzenstoffe. (Zweite Auflage, Jena.)You can also read
