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calcium chloride is maintained although each of these salts in
these concentrations and acting alone will accelerate respira-
tion.* With regard to respiration, the chlorides of magnesium
and sodium, of sodium or potassium and calcium exhibit a
conspicuous antagonism, whilst the chlorides of magnesium and
calcium and of sodium and potassium are antagonistic only to
a slight degree.

ACIDITY. The degree of acidity of the cell sap shows
much variation and is an expression of the physiological con-
dition and of particular metabolism. Thus the acidity of
anthocyanin containing leaves may be double that of green
leaves of the same species, the acidity of fleshy plants is greater
by night than by day.

In particular cases the hydrogen ion concentration may
profoundly modify the respiration intensity ; Nitrosomonas, for
example, shows the greatest rate of respiration in a medium
in which the P H value is between 8-4 and 8 '8, beyond the
limits of P H 9*4 - 7'6 the process comes to a standstill.! In
Penicillium chrysogenum variations in the value of P H between
4 and 8 do not affect the normal rate of respiration, that is, the
rate at neutrality, P H = 7 ; an increase of the value to 8 '8
results in the respiration decreasing to 60 per cent of the
normal, at which level it remains ; a decrease, on the other
hand, in the value of P H to 2-65 causes a gradual rise in the
respiration rate followed by a gradual fall to the normal. At
P H i'io to 1-95 the preliminary rise, amounting to 20 per
cent, is followed by a fall to below normal. The depression
brought about by a concentration P H = I -95 or less is irrever-
sible, whilst the similar decrease effected by a P H value of 8 '8
is reversible, the respiration rate returning to normal after the
plant is placed in a neutral solution. In acid solutions there
is an increase in the production of carbon dioxide and a de-
crease in alkaline solutions, a phenomenon which may be
paralleled in the test-tube : a neutral solution of dextrose and

* Gustavson : " Journ. Gen. Physiol,," 1919, 2, 217,
fMeyerhof: loc, cit f



INFLUENCE OF LIGHT 93

hydrogen peroxide show an increase in the evolution of carbon
dioxide on the addition of acid but not on the addition of
alkali.* Witzemann f also has demonstrated the oxidation of
sugar by hydrogen peroxide in the presence of disodium
hydrogen phosphate. Whether the phosphate here plays any
part in the formation of a hexose phosphate such as is known
to occur in alcoholic fermentation + has yet to be demon-
strated.

LIGHT. That plants respire both by day and by night is
a well-known fact from which it would appear that radiant
energy as such is not a conditioning factor in respiratory
activity ; its action is indirect in providing through its photo-
synthetic activity a supply of respirable material. Thus an
isolated green leaf in darkness shows a continuous fall in
respiration ; in the light, on the other hand, the respiratory
values during a carbon assimilation experiment are continually
changing by virtue of the carbon assimilation and may be
doubled by an exposure of four or five hours to light and
carbon dioxide. This is possibly due to the production of new
carbohydrate although the observed increase is not proportional
to the amount of carbon dioxide decomposed. The high in-
tensity of respiration sometimes observed in green plants in
bright sunshine || is due, at any rate in part, to the copious
supply of sugar. Under such conditions, however, other factors
are operative : the higher temperature, for instance, would in-
crease the respiration intensity ; whilst if the illumination, the
temperature and humidity conditions of the atmosphere were
operative all in the same direction to cause undue loss of
water, the flaccid leaves would ultimately show a respiratory
depression.

In addition to this indirect action of light, there appears
to be some direct action, for Spoehr 1T finds that the respiration
of the plant is regularly higher under conditions which vary
only as regards daytime air and night-time air. The differ-
ences observed are, however, not considerable ; in the case of

* Gustavson : " Journ. Gen. Physiol.," 1920, 2, 617 ; 3, 35.

t Witzemann : " Journ. Biol. Chem.," 1920, 45, i.

Vol. I., p. 379. Matthaei : loc. cit.

II See Rose" : " Rev. ge"n. Bot.," 1910, 22, 385.

IT Spoehr : " Bot. Gaz.," 1915, 59, 366.



94 RESPIRATION

the wheat the ratio day rate/night rate was found to be i -042
in normal air and 1*010 in deionized air. The reason for the
difference is not obvious : it is known that during daylight the
air becomes ionized to various degrees under conditions of low
relative humidity. Ionized oxygen is more potent than deion-
ized oxygen so that the ionized oxygen of the daytime air,
according to Spoehr, possibly accelerates the purely oxidative
process of respiration, not the initial disruption of the respir-
able material. But until it can be said with certainty at what
stage in respiration oxygen becomes operative, an adequate
explanation is not possible.

THE MECHANISM OF RESPIRATION.

The catabolic processes of plants may be directly referable
to specific enzymes, zymase for instance in alcoholic fermenta-
tion ; but in the respiratory activities of higher plants, their
role is not defined with that precision and degree of complete-
ness which is desirable, although it is generally agreed that
enzyme action plays an important part in the process. En-
zymes associated with the common end products of respiration
are subjects for first consideration and of them most attention
has been given to oxidase, peroxidase, catalase, zymase, and
carboxylase.

Oxidase, to use the generic term, has remarkable properties
of effecting with rapidity the oxidation of various substances
in the presence of oxygen.* They are of wide occurrence in
the vegetable kingdom, as should be the case if they are prim-
arily concerned in aerobic respiration, but whether they are
present in all living cells is doubtful. According to Atkins f
they are absent or inactive in tissues markedly acid in reaction
or containing large amounts of reducing substances. Bunzel J
also has shown that the activity of these enzymes is inhibited
by acids, their greatest activity being at or near the point of
neutrality ; the limits of the P H value corresponding to com-
plete inhibition io^the various subjects of experiment are
narrow and tljejngure of acid sensitiveness is almost invariable
in a partic^fer




Vol. I., p. 392.

kins: " Proc. Roy. Dublin Soc.," 1913, 14, 144.,
unzel : " Journ. Biol. Chem.," 1916, 28, 153.



OXIDASES 95

In view of the importance ascribed to this class of enzyme
in respiration processes, it is desirable here to reconsider
them.* A freshly prepared I per cent alcoholic solution of
guaiacum resin, made by dissolving pieces of the resin from
which the outer layer has been scraped off, forms a delicate
reagent for a certain type of oxidizing system which turns
the solution blue on being brought into contact When
properly prepared, the guaiacum solution is not turned blue
on exposure to the air, nor by the addition of hydrogen
peroxide. If, however, a mixture of guaiacum and hydrogen
peroxide be brought into contact with the juice of certain
plant tissues, either the expressed sap or a freshly-cut surface,
such as a slice of horse radish, a blue colour is immediately
assumed. From this it may be concluded that the tissue
contains a material capable of setting in motion some oxidizing
mechanism which causes the oxidation of guaiacum to the
blue compound. Horse radish and like substances exhibiting
a similar action are said to contain a peroxidase, that is, an
enzyme which can activate hydrogen peroxide so as to make
it turn guaiacum blue. Peroxidase is, in fact, assumed to
act in much the same way as does ferrous sulphate upon a
mixture of hydrogen peroxide and tartaric acid : if a little
hydrogen peroxide be added to a solution of tartaric acid, no
change is observed ; on the addition of a few drops of ferrous
sulphate, however, a yellow coloration is immediately pro-
duced which changes to violet on the addition of caustic soda.
The ferrous sulphate activates the hydrogen peroxide to
oxidize the tartaric acid to dihydroxymaleic acid. Two
kinds of oxidizing enzymes are now generally recognized
in the plant world, namely, the direct acting oxidases
which turn blue an alcoholic solution of guaiacum with-
out the addition of hydrogen peroxide, and the indirect
acting peroxidases which are unable to produce a change
in colour until hydrogen peroxide has been added. To
account for these facts, it was assumed that whereas the
peroxidase required the addition of a peroxide in order that
it might have something from which to liberate active oxygen,
the oxidase contained not only the peroxidase but also a

* See Vol. I., p. 392.



96 RESPIRATION

second enzyme, an oxygenase,* whose function was to generate
with the help of atmospheric oxygen the requisite peroxide.
The oxidase system therefore was direct acting owing to
the fact that it contained the necessary mechanism in the
oxygenase for generating its own peroxide and so making
it independent of any added hydrogen peroxide. This view
was supported by the fact that by the fractional precipitation
with alcohol of an aqueous extract of Lactarius> the contained
oxidase could be separated into its two constituents :

(a) A portion soluble in alcohol, the peroxidase constitu-
ent, which was without direct action on guaiacum.

(b} A portion insoluble in alcohol, the oxygenase con-
stituent, which acted but feebly upon guaiacum, but showed
a much stronger action on the addition of the alcohol soluble
fraction.

The presence of an oxidase in a plant tissue may also be
revealed by bruising the surface or by exposing to chloroform
vapour. Under such treatment the cell contents are some-
how disturbed, with the result that the oxidase contained
therein causes the formation of a coloured substance in the
substrate.

The observation of Wheldale Onslow f that the oxidase
contained in the pea and in the potato are able to oxidize
pyrocatechol and allied substances,! suggest that the brown-
ing on injury is due to the action of an oxidizing enzyme
upon some such substance contained in the tissue injured.
In support of this view, the presence of a substance of a
pyrocatechol nature may be demonstrated as follows. By
grinding thin slices of potato under 96 per cent alcohol, the
oxidase system may be divided into its two constituents, an
alcohol soluble portion and the peroxidase which owing to

*Bach and Chodat: " Ber. deut. chem. Gesells.," 1903, 36, 606; 1904,
37, 36, 1312.

f Wheldale Onslow : "Biochem. Journ.," 1919, 13, i.

The substances to which reference is made are protocatechuic and caffeic
acids.

COOH CH = CH . COOH



JOH I JOH \X OH

OH OH OH

Pyrocatechol. Protocatechuic acid. Caffeic acid.



PYROCATECHOL GROUPING 97

its insolubility in alcohol is precipitated on the potato residue.
This potato residue no longer turns blue guaiacum directly,
but does so on the addition of hydrogen peroxide. In order
to demonstrate the presence of the pyrocatechol grouping in
the alcohol soluble extract, the following procedure may be
adopted. Boil 500 grams of slices of freshly-peeled potato
for a quarter of an hour in 250 c.c. of 96 per cent alcohol
over a water bath. After filtering, evaporate off the alcohol
and extract the residue with a little warm water and filter.
To the filtrate add a concentrated solution of lead acetate
until no further precipitate is formed. Filter off the precipitate
and after washing, suspend it in a little water and add 10
per cent sulphuric acid drop by drop until the yellow colour
of the precipitate turns white owing to the formation of lead
sulphate. Filter and carefully neutralize the filtrate with I
per cent caustic soda. The presence of the pyrocatechol
grouping may be demonstrated by the addition of a drop
of ferric chloride which produces a green colour; on adding
a few drops of I per cent sodium carbonate, the colour changes
to blue and then to reddish purple. According to Wheldale
Onslow it is the presence of this pyrocatechol group in the
substrate which provides the peroxide for the peroxidase to
activate and there is consequently no need to postulate the
existence of a second enzyme or oxygenase as is assumed by
Bach and Chodat. The existence of an oxygenase also is
disputed by Moore and Whitley.*

In addition to guaiacum, other reagents have been used
in the investigation of oxidizing enzymes ; amongst these may
be mentioned benzidene, a-naphthol, /-phenylene diamine,
and pyrogallol ; none of these, however, produce any colour
with plant tissues or extracts unless hydrogen peroxide be
added. This is a very significant fact, inasmuch as all oxidiz-
ing enzymes found in plants would appear to be peroxidases
as tested by these reagents and the distinction between oxidase
and peroxidase would therefore appear to be dependent upon
the reagent employed. Contrariwise the discovery is within
the bounds of possibility of a reagent of sufficient delicacy
to be changed in colour by direct action not only by plant

* Moore and Whitley : " Biochem. Journ.," 1909, 4, 136.
VOL. II. 7



98 RESPIRATION

material containing so-called oxidases but also by material
which at present only gives the indirect action. If this
substance were known, the distinction between peroxidase
and oxidase would no longer be necessary, as indeed it would
seem even now to be, inasmuch as this distinction is entirely
dependent upon the external reagent, guaiacum, employed
and disappears when another reagent is used. Moreover,
if in order to activate a peroxidase it is necessary to add
hydrogen peroxide, since the plant itself does not supply
the necessary peroxide, unanswerable questions arise regarding
the employment of these enzymes in those plants which con-
tain only peroxidases.

It would, in fact, appear to be more likely that the difference
between oxidase and peroxidase is one of degree rather than
of kind, and that the so-called peroxidase is really only a
slightly less powerful oxidizing enzyme than the so-called
oxidase ; the lack in power may be due to a lower concentra-
tion, as has already been suggested by Ewart,* or to some
slight difference in the molecular complex, rather than to a
different mechanism involving the necessity for the presence
of a third substance.

An alternative explanation of the working of oxidizing
enzymes has been formulated by Wieland and has been con-
sidered in previous pages.

Catalase, which has the property of setting free gaseous
oxygen from hydrogen peroxide, although of common occur-
rence, is not universally present in living cells.

Zymase is the enzyme particularly associated with yeast
and is responsible for the alcoholic fermentation of sugar. It
has been described as occurring in higher plants such as the
beetroot, potato, lupin, and others. f

Carboxylase has the property of removing carbon dioxide
from carboxyl groups, thereby converting pyruvic acid, for
example, into carbon dioxide and acetic aldehyde ; J it occurs
in yeast, where it is associated with zymase, and, according to
Bodnar, in the beetroot and potato tuber.

* Ewart : " British Ass. Rep.," 1915.

t Palladia and Kostytschev: " Ber. deut. bot. Gesells.," 1906, 24, 273;
Stoklasa and Chocensky : Id., 1907, 25, 122.

t See Vol. I., p. 383-

Bodnar: " Biochem. Zeitsch.," 1916, 73, 193; see also Zaleski : "Ber.
deut. bot. Gesells.," 1913, 31, 349.



OXIDASE AND CATALASE 99

If respiratory processes are esentially enzymatic, it is
natural to suppose that the respiration intensity would increase
in the presence of accelerators : various substances have been
so described, amongst them being phosphates * which are im-
portant accelerators in zymase action, f

Palladin looks upon lipoids as being of the nature of co-
enzymes, since the more they are extracted from the plant
tissue, the greater is the reduction in the intensity of respiration
although not in an exact proportion. J

Galitsky and WassiljefT observed that the addition of
boiled water extracts of bean seeds and wheat grains increased
the output of carbon dioxide in living and in dead seedlings to
a degree depending on the acidity of the culture medium : in
neutral cultures the increase reached 117 per cent, whilst in a
slightly alkaline medium an increase of 86 per cent obtained
as compared with an increase of 60 per cent in a slightly acid
medium.

Of the enzymes mentioned, oxidase and catalase are the
more prominent, numerous observations having been made on
their distribution and relation to the oxidative aspects of
respiration. Appleman || found that the oxidase content of
the expressed juice of the potato is not indicative of the in-
tensity of the respiration of the tuber, whilst the catalase
activity shows a striking correlation. Similarly for sweet corn,
in which instance the respiration in the milk stage is high
when first collected but in storage rapidly decreases, the de-
crease being accompanied by a nearly proportional fall in cata-
lase activity. In the pine-apple, Reed 11 found that oxidase and
catalase are independent ; the amount of the former remains
constant during ripening of the fruit whilst the catalase in-
creases. In the wheat and certain allied plants, the embryo
shows a twenty-eight to twenty-nine-fold greater catalase and
oxidase activity than the endosperm, and this also holds for
the intensity of respiration. In air-dry fruits of Andropogon

*Iwanoff: " Biochem. Zeitsch.," 1910,25, 171; 1911,32,74; Zaleski and
Reinhard : id., 1910, 27, 450.
t See Vol. I., p. 379.

J Palladin : *' Ber. deut. hot. Gesells.," 1910, 28, 120.
Galitzky and Wassiljeff : Id., 182.

|| Appleman: " Amer. Journ. Bot.," 1916, 3, 223 ; 1918, 5, 207.
IT Reed : " Bot. Gaz.," 1916, 62, 409.

7*



ioo RESPIRATION

halepensis, catalase activity runs parallel to respiration, a corre-
lation which does not obtain in the seeds of Amaranthus* In
Acer saccharinum, Jones f found that the intensity of respiration
of seeds during the process of desiccation at 25 C. at first de-
creases, then rises to a maximum and finally a gradual decline
to zero : with regard to the catalase activity, there is a slight
initial increase, then a decrease as desiccation proceeds, during
which process there is also a gradual decrease in peroxidase
activity. In the early stages of germination, when respiration
is high, there is a large increase in catalase activity. From
these results it would appear that catalase and respiration are
more closely correlated than are oxidase and respiration. It
is to be remembered, however, that catalase and oxidase are
closely related enzymes and that agreement has not been
reached regarding the interrelation of these enzymes in the
oxidizing complex, an aspect of the subject which has before
been considered.^

There appears to be no general concurrence of opinion re-
garding the precise mode of action of these respiratory
enzymes. Palladin is one of the few who have formulated a
more or less complete scheme of the mechanism, but before
considering his thesis, it is desirable briefly to draw attention
to the salient phenomena presented in the anaerobic respira-
tion of higher plants. It has been mentioned that the higher
plants, although normally aerobic, may be facultatively anaerobic :
the anaerobic respiration of germinating pea seeds, for example,
is a commonplace of the laboratory. In such instances the car-
bon dioxide evolved certainly has no relation to initial gaseous
oxygen ; it represents a dissociation product of the substances
physiologically consumed. Comparison with the alcoholic fer-
mentation of sugar is an obvious pursuit and the two phe-
nomena show remarkable resemblances. Anaerobic respiration
is an extravagant method of obtaining energy and gives rise to
carbon dioxide and ethyl alcohol which in a sense is wasted un-
less further oxidized. The amount of alcohol and carbon
dioxide produced is a variable quantity depending on the

* Crocker and Harrison : " Journ. Agric. Res.," 1918, 15, 137.

t Jones: " Bot. Gaz.," 1919, 69, 127.

JVol.iL, p. 396.

See Kostytschev: "Journ. Russ. Bot. Soc.," 1916, I, 182.



ANAEROBIC RESPIRATION 101

amount of carbohydrate available for reduction in the plant
and on the experimental conditions employed.*

Thus the amount of carbon dioxide evolved from etiolated
bean leaves, in an atmosphere free from oxygen, is negligible,
but if kept with their petioles immersed in a solution of sugar
for some time previous to their being placed under anaerobic
conditions much carbon dioxide is produced and their life is
more prolonged. With regard to alcohol, a similar correlation
obtains ; in a specific instance etiolated bean leaves under
anaerobic conditions gave 256-8 mgs. carbon dioxide and 68-3
mgs. of alcohol in thirty hours, a ratio of 100 : 26-5, whereas
leaves previously given sugar yielded under precisely similar
conditions 782-4 mgs. of carbon dioxide and 724-6 mgs. of
alcohol a ratio of 1 10 : 92 -6. f

Whether zymase and carboxylase are of general occur-
rence in all those normally aerobic plants or members of
plants which are capable of living for a time under anaerobic
conditions remains to be discovered.

This survey, brief though it be, is sufficient to indicate the
close connexion between, if not the identity of, anaerobic
respiration and fermentation. Amongst the lower plants
studied, this parallelism is not so obvious, thus Kostytschev J
found that mushrooms containing no sugar give origin to
much carbon dioxide but no alcohol when grown under an-
aerobic conditions, possibly because the alcohol is oxidized
almost as soon as it is formed : but however this may be,
more information is necessary before an adequate attempt can
be made to correlate the catabolic processes of these and like
plants possessed of such plastic metabolic methods.

Reference may now be made to Palladin's ideas on respira-
tory processes. With regard to the origin of carbon dioxide,
he considers that there are three possible sources : that arising
from the activity of enzymes more or less closely associated

* Palladia : Rev. gn. Bot.," 1894, 6, 201.

t Palladin and Kostytschev : " Abderhalden's Handbuch," 1910, 3, 479.

Kostytschev : " Ber. deut. hot. Gesells.," 1908, 25, 188 ; 26a, 1674 ; " Zeit.
physiol. Chem.," 1910, 65, 350.

Glucose has been described as occurring in the mushroom and allied plants.
The amount varies greatly, according to the conditions under which the plants
are grown, and varies, not only in different batches, but also in individual plants
of one batch. These fungi also contain carbohydrates capable of yielding
sugars.



102 RESPIRATION

with the protoplasm and which may or may not be found in
the expressed sap ; that produced directly from the protoplasm
under stimulation such as wounding, which aspect of the sub-
ject has already been considered ; and that produced as a
result of oxidase activity.* With respect to the production of
carbon dioxide by oxidase enzymes, Palladin accepts Bach and
Chodat's hypothesis regarding the constitution of these en-
zymes and considers that this phase in respiration depends on
the presence of an oxidizable substance which under the action
of the oxygenase yields an organic peroxide which transfers
oxygen, when stimulated by the peroxidase.

Palladin further accepts Bach and Chodat's opinion that
oxygenases, if they exist, are very unstable and are quickly
used up ; it is for this reason that the presence of oxygenases
is difficult to demonstrate. This idea of the action of oxidases
in respiration is bound up with the tenability of Bach and
Chodat's opinion regarding these bodies. There is, however,
much to be said for the thesis that respiration is not neces-
sarily one single operation but consists of at least two, of
which one is the oxidation of substances by enzymes only
indirectly related to the protoplasm and is not therefore a
concomitant of life. Palladin and Kostytschev, for instance,
showed that germinated peas which had been killed without
injury to the enzymes may give off more carbon dioxide than
during life, and so also may the bulb of an onion, killed by
exposure to a temperature of - 20 C. on thawing, although
in this instance the amount of oxygen absorbed is less than in
the living condition. Haasf found that plants of Laminaria
poisoned by various substances such as ethyl bromide, acetone,


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