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of the extreme ultra-violet rays, the so-called Schumann rays,
and points out that the photolysis of protein and of protoplasm
increases with the diminution in the wave length of the incident
light; in the extreme ultra-violet region, the amount of
chemical change is proportional to the product of the light
intensity and the time of exposure.

HYDROLYSIS OF PROTEIN ON GERMINATION.

The protein content of a seed is a reserve food which must
be hydrolyzed before it can be translocated and made available
for the growing parts. The hydrolysis of proteins yields amino
acids, such as leucine, asparagine, and tyrosine, both in the
organism and in the test tube. From the facts of animal
physiology there is no doubt that amino acids, the products of
the hydrolysis of protein food in the alimentary tract, are recon-
structed to form protein in the various tissues after their passage
through the walls of the intestine into the blood stream. There
is reason to suppose that the same sequence obtains in the
plant. Thus Zaleski f found that during the ripening of pea
seeds there was an increase in the amount of the protein at the
expense of the amino acids and organic bases, as indicated by
nitrogen determinations of these compounds.





Control.


After Five Days.


N of proteins
N of am no acids
N of organic bases
N of other compounds


79-2 per cent of total N

87
10-8

i'4


89-2 per cent of total N
4*6

5'6

8



The results were not so well marked for all seeds ; thus under
similar conditions but little protein synthesis took place in the
maize, whilst in the sunflower there was a diminution of protein.

Of these dissociation products of proteins, asparagine is
amongst the more conspicuous in the plant. It occurs in the

* Bovie : " Bot. Gaz.," 1916, 61, i.

f Zaleski: " Ber. deut. hot. Gesells.," 1905, 23, 126; " Bern. hot.
Zentrbl.," 1911, 27, 63,



ASPARAGINE 59

developing parts* in greater abundance than in the members
where the reserve proteins are stored ; Schulze f found that only
7*62 per cent of asparagine occurred in the cotyledons, whilst
3 1 -8 1 per cent obtained in the axis of the lupin. Also the
relative amounts of asparagine and aspartic acid show consider-
able variation during germination and, in the last stages, the
amount of asparagine formed is in a proportion greater than the
amount of protein decomposed. In the instance of Cicer
arietinum, there is a marked increase in the amount of amino
acids and their amides during germination, which increase is at
the expense of the proteins. J Similarly there is an accumula-
tion of amides in the shoots of germinating peas ; the amount
of amide nitrogen in the seed leaves fluctuates much at first but
in the later stages of germination there is a marked increase,
whilst, concurrently, the a-amino acids decrease in amount and
and finally disappear. From these and such like facts there
is no doubt that in the plant asparagine, and possibly also
other amides, have their origin in the dissociation of the already
elaborated protein.

With regard to the formation of asparagine, this compound
may possibly be produced by the combination of ammonia and
aspartic acid || to form ammonium aspartate which gives origin
to the asparagine by the loss of a molecule of water. Prianich-
nikov,*F whilst recognizing in ammonia the end product of
protein dissociation, considers that it also is an initial stage in the
formation of proteins and, according to him, asparagine is the
form in which it is stored.

Sure and Tottingham, from the relationship obtaining
between the amides and amino acids during the germination
of the pea, consider that the amino acids serve for the produc-
tion of amides in the plant.

Asparagine lends itself to condensation more or less readily,

* This may easily be shown by germinating lupin seeds in the dark until the
hypocotyl is a few inches in length. On mounting a section of the hypocotyl in
strong alcohol and examining under the microscope, a large number of crystals
of asparagine will be seen.

t Schulze : " Landw. Jahrb.," 1878, 411.

JZlataroff : " Biochem. Zeitsch.," 1916, 75, 200.

Sure and Tottingham : " Journ. Biol. Chem.," 1916, 26, 535.

|| Schulze: loc. cit. Prianichnikov : " Ber. deut. bot. Gesells.," 1904, 22,
35. Treboux: " Ber. deut. bot. Gesells.," 1904, 22, 570.

II Prianichnikov ; " Bull. Agric. Intell.," 1917, , 204,



60 THE SYNTHESIS OF PROTEINS

and by simply boiling a solution, the dipeptide of aspartic acid
is formed.*

With regard to other views concerning the synthesis of
proteins, Treub,f from his investigations on the distribution of,
the periodic variation in the amount of, and cognate observations
on the cyanogenetic glucosides,J concluded that hydrocyanic
acid is the first recognizable product of nitrogen assimilation
and possibly is the first organic nitrogen compound formed.
Whilst on purely chemical grounds it is not impossible that
acetone cyanhydrin, CH 3 COHCNCH 3 , may be a stage in
protein synthesis, Treub's conclusions are not convincing : free
hydrocyanic acid has not been identified in plants and its
compounds may equally well result from the oxidation of
amino acids : in other words, the known facts regarding com-
pounds of hydrocyanic acid in the plant neither prove nor
disprove Treub's hypothesis.

* Ravenna and Bosinelli : " Rend. R. Acad. Lincei.," 1919, 28, ii. 113.
tTreub: "Ann. Jard. Bot. Buitenzorg," 1895, J 3 * J I 94 I9>86; 1907,
21, 107.

$ See Vol. I., p. 173.

Rosenthaler : " Schweiz. Apoth. Ztg.," 1920, 58, 137.



CHAPTER V.

RESPIRATION.

THE maintenance of life is impossible without a supply of
energy, the motive power in the absence of which activities
must come to an end. This is more obvious in an animal
than in a plant, which generally is less obtrusive in its move-
ments and various activities and may make use of radiant
energy, more particularly in the production of food.

That energy may be produced by the combustion of a suitable
fuel is a commonplace and is illustrated in the steam engine, the
boilers of which are heated by fires fed with fuel which varies
according to local circumstances. The heat-producing power,
or calorific value, of fuels varies and the most efficient material
in this respect is the one which produces the maximum number
of heat units or calories for a given weight of substance.

In all cases the heat produced in the combustion of a fuel
is that due to the chemical reaction of oxidation, or, in other
words, the heat given out when the constituent elements of the
fuel severally combine with oxygen to form the corresponding
oxides. The heat of combustion of a compound will, therefore,
depend upon the heat of combustion of its constituent elements
and is greater the richer the compound is in elements possessing
a high heat of combustion.

Now it is a principle in physical chemistry that the heat of
any chemical reaction depends solely upon the initial and final
products, and the total heat evolved is the same by whatever
method the final products are obtained, i.e., whether in one
single process or by a series of intermediate stages, and also
whether the reaction proceeds rapidly or so slowly that there is
no perceptible rise in temperature. In view of these facts it
will be clear that the same laws hold for the low temperature of
oxidation of various oxidizable substances in the living cell as
for the combustion of these substances in air or oxygen.

61



62 RESPIRATION

The heat of combustion of an element is determined by caus-
ing it to combine with oxygen in a closed chamber and measuring
the heat evolved calorimetrically. The two equations

C + O 2 = CO 2 + 94-8K
H 2 + O = H 2 O + 6gK

indicate that by the complete oxidation of 12 grams of carbon

or 2 grams of hydrogen 94-8 and 69 kg. calories* are evolved.

Expressing the above two equations in another form

i gram of hydrogen on combustion yields 34'sK
i carbon ,, y-gK

From this it will be seen that hydrogen on combustion
yields relatively much more heat than carbon ; consequently
compounds rich in hydrogen have a high calorific value.
Moreover, since oxygen itself has no calorific value, it follows
that the presence of this element in compounds reduces their
calorific value. Thus it comes about that fats which contain
only about 1 1 per cent of oxygen have a considerably higher
heat of combustion than carbohydrates which contain as much
as 5 3 per cent of this element. The actual values of the heat
obtainable by the combustion of some of the more important
fuel substances of the living cell are as follows :

i gram of carbohydrate = 4'! calories
i alcohol = 7-1 ,,

i ,, fat = 9-1

i protein = 5-8

In the procurement of energy the plant exhibits a wider
range than does the animal, and this to a larger extent than is
often thought ; thus Ramann and Bauer f have estimated that
young saplings of deciduous trees may show a loss of 20 to
45 per cent of their dry weight during the burst of activity
which follows the winter's sleep.

The term respiration here is used to include all those
processes which involve a liberation of energy employable by
the organism in its various activities. Respiration is not
merely the absorption of oxygen and the excretion of carbon

* A kilogram calorie is the amount of heat required to raise the temperature
of i kg. of water through i C. A statement to the effect that the complete
oxidation of glucose, for example, liberates 709 kg. calories therefore means that
the heat energy liberated during the combustion of 180 grams of glucose is
sufficient to heat 709 kgs. of water through i C.

f Ramann and Bauer: "Jahrb. Wiss. Bot.," 1911, 50, 67.



OXIDATION 63

dioxide as is too often supposed, an idea having its origin in
the lungs of an animal being termed the organs of respiration.
Respiration is essentially a catabolic process, and any organ
of a plant or of an animal which is doing work is an organ
of respiration in that it cannot accomplish its task without
the energy obtained by the exertion of appropriate mechanisms.
The lungs and the respiratory tract, on the one hand, and the
stomates, lenticles, " respiratory chamber," and the intercellular
space system on the other, are strictly comparable : they are
organs of breathing ; structures, reservoirs, and surfaces for a
preliminary of respiration, the conveyance and initial absorption
of oxygen, and for the ultimate elimination of the gaseous
waste of physiological combustion.

This motive power commonly is obtained by the physio-
logical combustion of carbohydrate, by which, theoretically, a
molecule of sugar is completely oxidized by 6 molecules of
oxygen giving origin to 6 molecules each of carbon dioxide
and water with the liberation of a considerable quantity of
energy.

But oxidation is not quite the simple operation as might
be supposed from the foregoing statement. Thus the expres-
sion CO + O = CO. 2 may be a true statement of the result of
the complete oxidation of carbon monoxide to carbon dioxide,
but it does not necessarily represent the mechanism by which
the change is effected ; as a matter of fact it has been shown
by Wieland * and others that water is an essential in the pro-
cess and the first phase is an hydratiori which leads to the
formation of formic acid.

CO + H 2 = H C

Interaction between the formic acid and oxygen then takes
place, leading to the production of water and carbon dioxide,
HCOOH + o = H 2 o + co a

It will, however, be seen that the resultant of these two reac-
tions is correctly expressed by the equation CO + O = CO 2 .
From considerations such as these, together with the fact
that oxidative processes enter largely into the energy-obtaining

* Wieland, " Ber. deut. chem. Gesells.," 1912, 45, 679, 2613, where earlier
literature is also quoted.



64 RESPIRATION

processes of plants, it is desirable to examine the mechanism
of oxidation somewhat closely.

THE MECHANISM OF OXIDATION.

Oxidation may be effected in one of two ways ; either by
the addition of oxygen to, or by the removal of hydrogen
from, a given compound. Similarly reduction is regarded as
the addition of hydrogen to, or the removal of oxygen from, a
given compound.

The change from hydroquinone (I.) to quinone (II.) by the
removal of two atoms of hydrogen may be regarded as oxi-
dation.

OH o

/\ /\

| | + 0->H,0 + | I

OH O

(I.) (II.)

Likewise the conversion of alcohol into aldehyde, which in-
volves the removal of two atoms of hydrogen, is an example
of oxidation

CH 3 CH 2 OH + O = H 2 O + CH a CHO.

This conversion of alcohol into aldehyde can be catalytically
effected by shaking the alcohol with palladium black in the
absence of oxygen.* This is due to the affinity of palladium
for hydrogen, resulting in the formation of palladium hydride,
PdH 2 . The reaction, however, soon comes to an end unless
the hydrogen is removed ; this may be effected by means of a
substance, such as quinone or methylene blue, which will act
as an hydrogen acceptor; if quinone is employed, this sub-
stance takes up the hydrogen, becoming reduced to hydro-
quinone, a colourless substance ; by the use of methylene blue,
however, the change is made visible since the reduction of the
quinonoid methylene blue involves a loss of colour with the
formation of a colourless, or leuco, compound. A more appro-
priate example, in that it recalls a once not uncommon labora-
tory experiment, is found in the changes brought about in an
aqueous solution of methylene blue by the living plant : a

* Wieland : " Ber. deut. chem. Gesells.," 1912, 45, 488, 2606.



MECHANISM OF DEHYDROGENATION 65

healthy twig of Elodea^ placed in a test tube of cold well-
boiled water coloured blue with the dye, is placed in the dark ;
after a time the water is quite colourless but quickly assumes
its blue colour on exposure to sunlight, owing to the evolution
of oxygen by the plant. This may be explained on the as-
sumption that during the respiration of the plant hydrogen is
absorbed by the methylene blue, the hydrogen acceptor, which
is thereby converted into the leuco compound. The subse-
quent presence of oxygen, on exposure to light, brings about
the re-oxidation of the leuco compound into methylene blue
and water.

This mechanism of dehydrogenation is, according to Wie-
land, the one underlying many, if not all, oxidations and
especially those associated with enzymes.

Reverting once more to the dehydrogenation theory of
oxidation, it has been shown by Wieland that the oxidation
of aldehyde to acid may be explained on the same principle,
since palladium black has no action whatever upon the dry
aldehyde. This is explained by the fact that the dry aldehyde
has not in itself any hydrogen for activation but that in the
presence of moisture it forms the hydrate

.0 OH

+ HOH = C



H H

Palladium black can then activate two hydrogen atoms in
this latter compound and these are then removed by a suitable
hydrogen acceptor :

/OH /OH

CH 3 C^-0;H! -> CHjC/ + H 2

^Hj ^Q

This view is supported by the fact that whereas chloral,
CC1 3 CHO, is unacted upon by palladium and methylene blue,
that well defined substance chloral hydrate, CC1 3 CH(OH) 2 , is
at once oxidized. Further corroborative evidence concerning
the essential part played by water is provided in the fact that
even such a well marked aldehyde-oxidizing agent as silver
oxide has no action upon dry aldehyde but oxidizes it readily
when wet.

//* / OH

CH 3 C;f + HOH = CH 3 C^-OH + Ag 2 O = CH 3 COOH + aAg + H 2 O

\H \H

VOL. II. 5



66 RESPIRATION

Wieland * also effected the oxidation of glucose to carbon
dioxide by shaking it with palladium black and methylene
blue at room temperature. He similarly effected the oxida-
tion of phenolic substances such as hydroquinone and pyro-
gallol to quinone and purpurogallin respectively in the entire
absence of oxygen, oxidations which, according to the Bach
and Chodat theory ,f are effected by atmospheric oxygen acti-
vated by an oxidase system.

Wieland J also investigated the so-called Schardinger reac-
tion in milk. This reaction, designed by Schardinger to dis-
tinguish boiled from unboiled milk, depends upon the fact that
unboiled milk when warmed with methylene blue and a drop
of acetic aldehyde decolorizes the dye, whereas boiled milk
produces no such change. According to Wieland this action
is due to an enzyme, for which he proposes the name dehydrase ;
this enzyme dehydrogenates the aldehyde hydrate in the same
way as the palladium black, the methylene blue again acting
as hydrogen acceptor. The same enzyme is also able, by
slightly varying the conditions, to produce from two mole-
cules of salicylic aldehyde one molecule of the corresponding
acid and one molecule of the alcohol ; the latter being pro-
duced by one of the molecules of salicylic aldehyde itself
acting as the hydrogen acceptor.

xOH~ /OH

C 6 H 4 OH . CHO + H 2 -> C 6 H 4 OH . C^-OH -> C 6 H 4 OH . C^ + 2H

\OH ^O

A second molecule of salicylic aldehyde then acts as
hydrogen acceptor forming a molecule of salicylic alcohol

C 6 H 4 OH . CHO + 2H -> C 6 H 4 OH . CH 2 OH.

This change effected by enzyme activity of two molecules of an
aldehyde into one molecule each of the corresponding alcohol
and acid was originally thought by Parnas to be due to a
special enzyme to which he gave the name aldehyde mutase ;
Wieland's work, however, shows that this substance is none
other than dehydrase. This same enzyme dehydrase can be

* Wieland : " Ber. deut. chem. Gesells.," 1913, 46, 3331.
tVol. I., p. 396.

J Wieland: " Ber. deut. chem. Gesells.," 1914, 47, 2085.
Parnas : " Biochem. Zeitsch.," 1910, 28, 274.



DEHYDRASE ACTION 67

invoked to explain the reducing action of an aqueous extract of
potato. The existence of this was first demonstrated by Bach
in the following simple experiment. One gram of freshly
pounded potato is heated in a test tube to 60 C. with 10 c.c.
of 4 per cent aqueous solution of sodium nitrate together with
3 drops of 10 per cent solution of acetic aldehyde. After
two minutes the solution gives a strong reaction for nitrite by
the Griess Ilosvay reagent. The reduction of the nitrate to
nitrite was thought by Bach to be due to the activity of a per-
hydridase existing in the potato together with a per-
oxidase system. According to Wieland, however, this
is simply explained by the dehydrase activating the hydrogen
of the aldehyde hydrate whilst the sodium nitrate acts as
the hydrogen acceptor.

^O x>H

1. CH 3 cf + HOH = CH 3 C^-OH

\H \H

/OH yOH

2. CH 3 C^-OH -> CH 3 C<f + 2 H

\H ^O

3. NaN0 3 + 2 H = NaN0 2 + H 2 O.

Even the biological conversion of alcohol into acetic acid
by the vinegar plant, Bacterium aceti^ was shown by Wieland
to be a dehydrase action which could be effected in an atmos-
phere of nitrogen by leaving freshly washed cultures of the
plant in contact with alcohol and methylene blue in a flask
from which air had been displaced by nitrogen. In a com-
paratively short time the methylene blue was decolorized and
after some days a measurable quantity of acetic acid had been
produced.*

There is thus considerable ground for regarding biological
oxidations as being primarily due to enzymes such as de-
hydrase which activate hydrogen in the oxidizable substances
so that it may be removed by a suitable hydrogen acceptor,
which may be atmospheric oxygen. Wherefore the oxidizable
substance is to be regarded as a potential hydrogen donator,
and, in fact, Thunberg f goes so far as to regard hydrogen as
the essential fuel of the living cell and regards only those

* Wieland : " Ber. deut. chem. Gesells.," 1913, 46, 3336.
t Thunberg: Skand. Archiv. Physiol.," 1920, 40, i.

5*



68 RESPIRATION

substances as possible intermediate metabolites which when
left in contact with methylene blue in the absence of oxygen
decolorize this substance and therefore act as hydrogen
donators. His technique consists in placing the material
under examination in a test tube with freshly washed frog's
muscle, to supply the dehydrase, and methylene blue, filling
the tube with boiled water, and leaving the whole in a ther-
mostat and examining at intervals. If the methylene blue is
decolorized, the substance in question is a hydrogen donator
and consequently a possible intermediate metabolite.

A highly important contribution to the mechanism of
oxidation in the living cell is furnished by the discovery and
isolation by Hopkins * of a dipeptide composed of cystein and
glutamic acid to which he gives the name of glutathione.
This substance can act alternatively as an hydrogen acceptor
or as an hydrogen donator, according as it exists in the
oxidized or the reduced form. This will be intelligible from
an examination of the accompanying formulae :

CH . SH



()


2 CHNH 2


- 2H -> CHNH 2


CHNH 2




COOH


COOH


COOH




CH 2 . S .


S.CH 2


CH 3 SH


(*)


CHNH 2


CHNH 2 + 2H -5


> 2 CHNH 2




COOH


COOH


COOH



From these formulae the glutamic acid residues have been
omitted for the sake of simplicity; the mode of action of
the dipeptide as hydrogen donator in the first instance and
as hydrogen acceptor in the second is obvious ; in other words,
this substance may act alternately as an oxidizable reducing
substance and as a reducible oxidizing agent. Glutathione is
not uncommon in plant tissues although the coloration by
which it is recognized usually is far less intense in vegetable
than in animal tissue. Its presence in yeast may be de-
monstrated by grinding the cells in a mortar with a little sand
and some saturated solution of ammonium sulphate. On
pouring off and adding to the supernatant liquid a few drops of

* Hopkins: " Biochem. Journ.," 1921, 15, 286.



CATALASE 69

5 per cent solution of sodium nitroprusside and a little strong
ammonia, a pink colour is produced. This colour reaction, which
also is given by a number of other reducing substances such
as aldehydes, acetone, hydrogen sulphide, etc., is only given
by the reduced or cysteine form of the dipeptide and not by
the cystine or oxidized modification. The isolation of the
pure substance from yeast in a yield of about O'l to 0*15
gram per kilo has also been effected by Hopkins.

The existence of a somewhat similar system of alterna-
tively oxidized and reduced materials in the plant have been
postulated by Palladin and termed by him respiratory pig-
ments ; these will be considered later. (See p. 103.)

The existence in organic tissues and fluids of the enzyme
catalase, whose characteristic property is the destruction of
hydrogen peroxide with the evolution of oxygen, has been
known for some time. Wieland* recently has drawn atten-
tion to observations by Lesser,f Rywosch,J and Jorns that
anaerobes such as Bacillus tetanus and B. botulinus are deficient
in catalase whilst aerobic organisms such as Pneumococcus and
Sarcina, and facultative aerobes such as yeast contain catalase
in quantity. It is considered that the occurrence of catalase
in those cells and tissues which require oxygen is necessary
for the two-fold purpose of preventing the accumulation of
hydrogen peroxide which is toxic to the cell, and also for the
liberation of oxygen. Hydrogen peroxide is regarded as the
first product of the oxidation of hydrogen by oxygen which
acts as the hydrogen acceptor in the cell,

2 + 2H = H a 2

The catalase then acts upon the resulting hydrogen peroxide,
breaking it up into water and oxygen, which latter is then
available for further oxidation,

H 2 o 2 = H 2 o + o

This cycle of changes does not occur in anaerobic oxidations,
which may explain the absence of catalase under these con-
ditions.

* Witland: " Ber. deut. chem. Gesells.," 1921, 54, [B], 2353.

t Lesser: "Zeitch. Biol.," 1906, 48, i.

% Rywosch : " Zentr. Bact.," 1907, 44, 295.

Jorns: " Archiv. d. Hyg.," 1908, 67, 134.



70 RESPIRATION

Reductive processes also may result in the liberation of
energy fit for use by the plant ; thus the fermentation of sugar
by yeast, in which process the carbohydrate is converted into
alcohol and carbon dioxide without the agency of atmospheric
oxygen,* is a case in point.

The energy freed in these processes is derived from the
molecular energy of the substances disintegrated and is gener-
ally expressed in terms of heat units. Thus the complete oxi-
dation of i gram molecule of maltose liberates 1351 kg.
calories.

The fermentation of sugar by yeast also is accompanied by
the evolution of heat, although to a much lesser extent, a gram


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