Reprinted from Tas JousNan or Brovoaican CHEMISTRY
Vol. XC, No, 1, January, 1931

ON THE FUNCTION OF HEXURONIC ACID IN THE
RESPIRATION OF THE CABBAGE LEAF

By ALBERT SZENT-GYORGYI
(From the Division of Chemistry, The Mayo Foundation, Rochester, Minnesota)

(Received for publication, June 5, 1930)

It has been shown (3) in a previous paper (1928) that plants
which contain a peroxidase system and the suprarenal cortex of
animals contain a relatively high concentration of a substance
called hexuronie acid. This substance is characterized by its
high reducing power and its reversible oxidizability. It has been
suggested that hexuronic acid is involved as a catalyst in the
respiration of the cell. The subject of this paper is the inquiry
into the part this substance plays in the respiration of the cabbage
leaf.

If a part of the leaf is placed in the respirometer! in the presence
of potassium hydroxide, it shows a vigorous oxygen uptake of
between 2.5 and 6 c.mm. for each gm. of tissue each minute.
This rate of oxygen uptake remains constant for more than
an hour.

If, however, the leaves are ground in a meat mincer, which
injures most of the cells and releases the intracellular fluid, and the
pulp is placed in the respirometer, it shows a small oxygen uptake,
between 1 and 2 c.mm. for each gm. each minute. This shows
that mincing the leaves greatly injures the normal mechanism of
respiration.

In experiments in the respirometer, the first 5 minutes are lost
for observation because of the time necessary for the respirometer
to reach equilibrium as well as the few minutes required to weigh
the pulp and fill the respirometer. If at the beginning of the
readings a sample is taken, it will be found to contain practically
no reduced hexuronic acid. Since the acid is present in the
intact leaf chiefly in the reduced condition, it is evident that the

1 The thin marginal parts of inner leaves were used for these experiments.
385
386 Hexuronic Acid in Leaf Respiration

reduced form disappears between the time of mincing and the
beginning of the readings.

The disappearance of the reduced hexuronic acid can be followed
by color reactions. 15 cc. of the Folin phenol reagent are placed
in a series of beakers. The leaves are minced and the pulp
quickly spread on a glass plate. At intervals of 2 minutes,
samples of about 2.5 gm. of the pulp are placed in the reagent.
After the first eight samples have been taken, the content of the
beakers is filtered through muslin into test-tubes. The reduced
hexuronie acid reduces the reagent without addition of alkali.

In the sample taken immediately after mincing, a deep blue color
indicates the presence of a high concentration of reduced hexuronic
acid. Inthe sample taken 2 minutes later, the color is moderately
deep. In the third sample the color is rather faint. The fourth
and fifth samples indicate only a trace; the remainder are negative.

This experiment shows that the relatively large amount of
reduced hexuronic acid in the intact leaf disappears within the
first 5 minutes after mincing. Since this disappearance does not
take place if the pulp is kept in a vacuum, it is evident that the
disappearance of the substance is due to its oxidation. During
this interval, the pulp thus takes up oxygen at a rate of about
the same order as that of the normal respiration. The absorption
of oxygen lasts as long as reduced hexuronic acid is present. The
pulp does not reduce oxidized hexuronic acid, or does so to a small
extent only and indicates that the mechanism which brings about
the reduction of hexuronic acid is damaged by the processof minc-
ing to a greater extent than is the mechanism of the oxidation of
the acid.

If the original concentration of reduced hexuronic acid is
restored to the pulp by addition of this substance, a vigorous
uptake of oxygen again takes place which lasts until the theoretic
quantity of oxygen is taken up which is required for the reversible
oxidation of the added acid. Fig. 1 shows the result of such an
experiment.

Hexoxidase

If the pulp is quickly boiled, and then cooled, it will not be
able to oxidize hexuronic acid. This shows that the oxidation
of the acid in the fresh pulp is due to the presence of a thermola-
A. Szent-Gyorgyi 387

bile catalyst. For the sake of brevity this catalyst will be referred
to as hexoxidase.

The first question to be studied was whether the hexoxidase is
bound to the formed elements of the cell or is, in part at least,
also present in the cell sap in solution. It was found that the
hexoxidase is present to a large extent in the juice.

If the pulp is pressed through muslin, placed in the respirom-
eter, and hexuronic acid is added, a great increase of oxidation
shows the presence of the hexoxidase. The oxygen uptake, on

 

Hexuronie acid added

 

 

 

emm agen uptake tr five minutes
3

Minutes 35 40 15 20 20 30 35 40 45 50

 

Fig. 1. The effect of hexuronic acid on the uptake of oxygen by minced
cabbage leaves.

addition of the acid, will be the same if the juice is freed from all
suspended material by centrifugation.

In Fig. 2 are shown the results of an experiment in which the
pulp was pressed out at once and placed quickly in the
respirometer.

If barium acetate is added to the juice a heavy precipitate is
produced which can be separated in the centrifuge. The super-
natant fluid shows undiminished activity.

If an equal quantity of saturated solution of ammonium sulfate
ig added to the juice, about half of the hexoxidase is precipitated.

If the juice is saturated with ammonium sulfate, all of the
388 Hexuronic Acid in Leaf Respiration

hexoxidase will be found in the precipitate, which can be easily
separated on the Buchner funnel. If the precipitate is dissolved
in water or a phosphate buffer, it can be reprecipitated repeatedly
practically without loss of activity. Such a preparation obtained
by precipitation with ammonium sulfate does not show spon-
taneous uptake of oxygen in the respirometer.

If the ammonium sulfate precipitate is suspended in m/15
phosphate solution of pH 5.9 and is allowed to stand overnight in
the ice box, an inactive precipitate will settle, which can be

 

Hexuronic acid added.
60Fr

40r

nN
Oo
T

omm. oxygen upteke in five minutes
o
7

 

 

1 1 1 L 1 1

Minutes 10 20 50 40 sO 60 70

 

Fic, 2. The effect of hexuronic acid on the uptake of oxygen by juice
pressed from cabbage leaves.

separated in the centrifuge, leaving a limpid, highly active enzyme
solution.

All further observation will deal with the enzyme which had been
precipitated from a solution saturated with ammonium sulfate
and redissolved in m/15 phosphate buffer of pH 5.9 (corresponding
to the pH of the cabbage juice).

The hexoxidase is resistant to many different chemical and
physical agents. The preparations can be kept for approximately
a week in the ice box without loss of activity. The wet ammonium
A. Szent-Gyérgyi 389

sulfate precipitate can be dried in the air or ina vacuum. The
solution can be frozen in solid carbon dioxide. A solution of 1
per cent acetic acid, or of sodium carbonate or formalin, will not
destroy its activity in 5 minutes. The dried preparation can be
extracted with ethyl ether without loss of activity or by absolute
alcohol or alcohol-ether with but little loss of activity.

Rapid heating to boiling will completely destroy the enzyme.
Solutions of the enzyme or the wet ammonium sulfate precipitates
are very sensitive to solvents like aleohol or acetone. Treatment
with three times its own volume of methyl alcohol destroys 80
per cent of the activity. Acetone destroys it almost completely.

The enzyme is not sensitive to the cyanide ion, showing that in
its action a mechanism usually classified as “oxygen activation”
is not involved. Sodium cyanide 0.005 per cent has no effect
on the rate of oxidation; 0.01 per cent has a small inhibitory
action. High concentration, as 0.1 per cent, inhibits oxidation to a
great extent. It has been shown by Dixon (1927) (1) and by me
(1926) (2) that such a high concentration of cyanide inhibits
enzymes in a non-specific way. Shardinger’s enzyme is inhibited
by such high concentration, although oxygen activation is not
involved in its action. As emphasized by Dixon (1927) (1) only
the inhibition by small concentrations of cyanide is characteristic
for oxygen activation.

As criterion for hydrogen activation the reduction of methylene
blue is usually applied. It is easy to demonstrate that the reduc-
tion of methylene blue by hexuronic acid is not enhanced by the
presence of hexoxidase. Also narcotics, such as 5 per cent of
urethane or chloroform, have no inhibitory influence.

The insensitivity of the enzyme to cyanide gives further evidence
of the significance of this enzyme, together with hexuronic acid,
in the normal respiration of the cabbage leaf. It was found that
the respiration of intact leaves is inhibited only to 6 per cent by
the presence of 0.01 per cent of sodium cyanide. The respiration
of thin slices of a plant containing a phenol-oxidase, the potato,
was found to be inhibited in an identical experiment up to 60 or
75 per cent by the same concentration of cyanide.

The results show the mechanism of activation of hexoxidase
to be different from that of all other oxidizing enzymes. So do the
kineties of its activity. If the rate of oxidation in varying con-
390 Hexuronic Acid in Leaf Respiration

centrations of hexuronic acid is plotted, a straight line is obtained,
which slopes but gently, and the rate of oxidation tends to become
parallel to the abscissa in low concentrations of the acid. The
results of two of the several experiments performed are shown in
Fig. 3. The same result can also be obtained by a different
method. If hexuronice acid is mixed with the hexoxidase and
samples are taken at regular-intervals and the quantity of the
unoxidized acid is plotted against time, an almost straight line is

 

a

 

 

 

0,5 10 1s 2.0 20 30
Yoo Mol concentration of hexuronic acid

Qydation in ca of 001 N jodine Ga 30 minutesl

Fra. 3. The effect of concentration on the velocity of oxidation of hexu-
ronic acid by hexoxidase.

obtained, until oxidation of the acid is almost complete. The
experiments were carried out at room temperature in beakers
with continuous mechanical shaking. The quantity of unoxidized
hexuronic acid was followed iodometrically.

No other substance has been found besides hexuronic acid on
which the hexoxidase will act. Pyrogallol, catechol, quinol
p-phenylenediamine, the diamine plus naphthol, leucoindophenol,
all remain unoxidized?

* It should be mentioned that the pulp of cabbage leaves or the pressed
juice oxidizes dibrom-indophenol white at a high rate. This reaction is
sensitive to cyanide. By precipitation with ammonium sulfate the juice
loses this activity.
A. Szent-Gyorgyi 391

Also glutathione’ remains unoxidized in the presence of hex-
oxidase. If, however, hexuronic acid is present, the glutathione
is oxidized; the hexuronic acid plays the rdle of catalyst; it is
oxidized by the enzyme and reduced by glutathione.‘

A study of the velocity of oxidation of hexuronic acid shows that
the oxidizing enzyme is of a complex nature. The probable
mechanism by which the enzyme brings about its effeet has been
discussed in a recent publication (4). The curves which represent
the velocity of oxidation of hexuronic acid in the presence of the
enzyme support the hypothesis that an intermediate substance, z,
is involved. If the rate of oxidation of hexuronic acid is rapid
compared to the rate of oxidation of 2, the line in Fig. 3 would be
parallel to the abscissa. The gentle slope indicates that the rate
of oxidation of x to the rate of oxidation of hexuronic acid by x
stands as 1 to 3.

EXPERIMENTAL

Experiment 1. Uptake of Oxygen by Minced Cabbage Leaves—
The outer leaves of the cabbage are rejected. The thick main ribs
of the inner leaves are cut out with a sharp knife, and the leaves
are quickly minced in a meat mincer, which reduces them to a
relatively fine, juice pulp. A sample of 813 mg. is quickly weighed
and transferred to a respirometer of the Warburg type and
suspended there in 2 cc. of am/15 phosphate buffer of pH 5.9.

The side arm of the respirometer is filled with 0.5 ec. of a solution
of hexuronie acid, brought to pH 5.9 with disodium phosphate.
This sample reduces 3.4 cc. of 0.01 N iodine and requires thus for
its reversible oxidation about 200 c. mm. of oxygen.

0.4 ee. of potassium hydroxide solution is placed in the central
tube of the respirometer to absorb carbon dioxide. The respirom-
eter kept at 24° is closed and the readings which are begun after
5 minutes, are repeated at intervals of 5 minutes.

* Crystalline glutathione was prepared by the method of Kendall.

* Formaldehyde in the presence of the enzyme preparation leads to
rapid disappearance of hexuronic acid (iodometrically) and of cysteine
and glutathione. This is, however, not an oxidative process since it
occurs anaerobically at the same rate. Acetaldehyde is only slightly
active. Propionic aldehyde, heptylic aldehyde, crotonic aldehyde, ben-
zaldehyde, cinnamic aldehyde, anisic aldehyde, vanillin, and dimethyl
p-aminobenzaldehyde are inactive.
392 Hexuronic Acid in Leaf Respiration

After four readings, the side arms are dumped. Since the
side arms cannot be washed out, the dumping is not quantitative.
The horizontal dotted line in the curve (Fig. 1) gives the oxygen
uptake of the same weight of intact cabbage leaves.

Experiment 2. Uptake of Oxygen by Pressed Cabbage Leaves—
Cabbage leaves are minced quickly. The pulp is wrapped in
muslin, pressed by hand, and 2 cc. of the juice pipetted into the
respirometer. Further details are the same as those given in
Experiment 1. The curve obtained is shown in Fig. 2.

Experiment 3. Effect of Concentration on the Velocity of Oxida-
tion—A 0.01 m solution of hexuronic acid is prepared by the
addition of 8.9 mg. of hexuronic acid to a solution of hexoxidase
in 5 ce. of phosphate buffer pH 5.9. The solution is gently shaken
for 30 minutes and is then acidified with acetic acid and titrated
with a solution of 0.01 N iodine. Similar experiments were made
with solutions which contained 0.015 m, and with 0.03 mM, con-
centrations of hexuronic acid. The influence of the concentration
of hexuronic acid is shown in Fig. 3.

Preparation of Hexuronic Acid

The hexuronic acid used in the experiments was prepared in the
chemical laboratory of The Mayo Foundation from suprarenal
glands of beef. Since (for the purpose of constitutional analysis)
greater amounts of hexuronic acid had to be prepared, the methods
of preparation described elsewhere (1928) were applied for quanti-
ties of 25 kilos of glands. It was found, however, that the original
method was inadequate for work on such a large scale. The
original method was thus modified in several respects. By
this modified procedure, I have obtained 4 gm. of crystalline
hexuronic acid from 25 kilos of glands. The same yield has been
obtained by my assistant, Miss Bair. More than 20 gm. of
crystalline hexuronic acid have been prepared in this way.

The steps of the preparation were the following. The glands
were cut from the freshly killed animals at the Swift packing
plant at South St. Paul, Minnesota. They were at once trimmed,
and packed in solid carbon dioxide, in which they were transferred
to the clinic, where they were minced still in frozen condition.
Then to every kilo of the pulp, 1 liter of 5 per cent trichloroacetic
acid was added, and the whole mass was thoroughly mixed.
A. Szent-Gyorgyi 393

After half an hour, the fluid was separated in the Sharples centri-
fuge. 40 gm. of neutral lead acetate for each kilo of glands were
dissolved in a small quantity of hot water. To every liter of the
solutions of the glands 0.1 ce. of 20 per cent sodium cyanide was
added. Two-thirds of the lead acetate solution was added to the
solution; then a 10 N solution of sodium hydroxide was added with
rapid agitation until the fluid distinctly turned the indicator
brom-thymol blue. The rest of the lead acetate was then poured
in. The mixture was cooled with ice for half an hour; the precipi-
tate was separated in the Sharples centrifuge.

The precipitate was suspended in about 2 liters of water and 25
per cent sulfuric acid was added, until the fluid was distinctly acid
to brom-thymol blue. Then the maximal amount of 20 per cent
phosphotungstic acid was added which just did not leave any
excess of this acid in the solution after being cooled with ice.
(The necessary quantity was estimated on a small sample, sodium
hydrosulfite being used as indicator for free phosphotungstie
acid after the precipitate had been separated.) The solution was
filtered on a suction filter.

The further steps of the method were similar to those described
in the previous paper (1928).

SUMMARY

Evidence is given that hexuronic acid plays an important part in
the respiration of the cabbage leaf. It connects, as hydrogen
carrier, the system in which the molecular oxygen enters into
reaction with the system which supplies hydrogen and is involved
in the oxidation of the foodstuff. There is in the cabbage leaf a
highly active enzyme which in the presence of oxygen rapidly
oxidizes hexuronic acid to its reversible oxidation product. The
name hexoxidase is used for this enzyme, the properties of which
are described.

BIBLIOGRAPHY

1. Dixon, M., Biochem. J., 21, 841 (1927).

2, Szent-Gyérgyi, A., Biochem. Z., 178, 275 (1926).
3. Szent-Gydérgyi, A., Biochem. J., 22, 1387 (1928).
4. Szent-Gyoérgyi, A., Sctence, 72, 125 (1930),