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Willstatter and Stoll's views regarding the mechanism of
carbon assimilation may now be summarized.

They agree with Baeyer that formaldehyde is the inter-
mediate link between the carbon dioxide supplied to the plant
and the carbohydrate synthesized with the help of chlorophyll.
It has already been pointed out that their chief argument in
support of this view is based upon the constant value, I,
obtained for the so-called assimilatory ratio; this ratio is
obtained by dividing the volume of carbon dioxide supplied to
the plant by the volume of oxygen evolved ; according to
theory, the complete reduction of carbon dioxide, i.e. the
removal of both oxygen atoms, might proceed through succes-
sive stages of oxalic, formic and glycollic acids down to the
formaldehyde stage, but in that case the assimilatory ratio
would be respectively 4, 2, or 1-33 for each of the acids men-
tioned, whereas formaldehyde alone requires the value I which
is the figure actually obtained in a number of experiments
under most varied conditions. This argument is not based
upon the actual experimental demonstration of formaldehyde
isolated from assimilating leaves, and, with regard to this
particular question, these authors have revised many of the
statements made by previous workers. It will be remembered
that repeated attempts have been made to demonstrate the
formation of formaldehyde by the action of chlorophyll upon
carbon dioxide outside the plant. The earlier work of Usher
and Priestley, demonstrating the formation of this substance in



46 THE SYNTHESIS OF CARBOHYDRATES

films of chlorophyll exposed to an atmosphere of carbon dioxide
in sunlight, was shown by Wager* and Warner f to be faulty,
inasmuch, as Wager showed, that no formaldehyde was pro-
duced if oxygen was excluded, whilst Warner showed that
carbon dioxide was unnecessary and took no part in the pro-
duction of the formaldehyde. Warner concluded that the
formaldehyde was in fact an oxidation product of the chlorophyll
since oxygen was actually absorbed in the process. This view
has, however, since been superseded by the experiments of Will-
statter and Stoll who showed that no formaldehyde at all was
formed if pure chlorophyll in colloidal solution was employed,
the colloidal solution being considered to approximate most
closely to the condition of the chlorophyll in the chloroplast.
The formaldehyde described by the earlier workers is attri-
buted to the oxidation of impurities accompanying the samples
of chlorophyll used by them.

The failure to obtain any trace of formaldehyde from pure
chlorophyll in vitro is attributed by Willstatter and Stoll to
the absence of the essential enzyme which these authors postu-
late in the green leaf, and this brings us to the consideration
of the mechanism of the action of chlorophyll upon carbon
dioxide as visualized by these authors.

Experiments in vitro have shown that carbon dioxide can
form with chlorophyll (I.) in colloidal solution an additive
compound of the type of a bicarbonate (II.) as expressed by
the equation




>N \

R<>>Mg-O.C'

1>NH



This compound (II.) cannot be imagined to be capable of
parting with two atoms of oxygen with regeneration of chloro-
phyll, so that some intramolecular rearrangement must first
take place, and this, according to Willstatter and Stoll, involves
the absorption of energy which is supplied by the sunlight.

* Wager: "Proc. Roy. Soc.," Lond. B., 1914, 87, 386.
t Warner: id. 378.



REACTION OF CHLOROPHYLL WITH CO 2 47

This change to a formaldehyde peroxide compound (III.) may
be illustrated as follows :





It is true that experiments in vitro have entirely failed to
demonstrate the formation of a peroxide compound by means
of horseradish peroxidase, but this is considered to demonstrate
the essential difference between test tube experiments and the
activity of the living cell. There is no doubt that chlorophyll
in the chloroplast is protected from photo-oxidation or
decomposition by carbon dioxide in a way that chlorophyll in
colloidal solution in vitro is not, since the chloroplast will
tolerate concentrations of carbon dioxide which decompose
chlorophyll in colloidal solution to the magnesium free com-
pound phaeophytin (V.) with precipitation of magnesium
carbonate as illustrated by the equation

C M H n O B N 4 Mg + H,0 + C0 2 = C 55 H 74 5 N 4 + MgCO 3

V.

Such a peroxidic compound (I II.) as is postulated above should
be fairly easily capable of losing oxygen either in one or in
two stages with regeneration of unaltered chlorophyll and
formation of formaldehyde (IV.)




f>N

R ;:v-Mg.O.C^ - R Mg+0+HCHO
1>NH



Within the living cell the decomposition of the peroxide
formaldehyde compound (III.) is assumed to be brought about
by an enzyme, the existence of which enzyme is supported by
the following experimental evidence.



48 THE SYNTHESIS OF CARBOHYDRATES

The assimilation number is the ratio of the number of milli-
grams of carbon dioxide assimilated to the number of milligrams

CO

of chlorophyll in the assimilating leaf surface, i.e.



chlorophyll

In leaves in which the chlorophyll content is small, the ratio
will be higher than in those in which it is large. In such
leaves the proportion of enzyme will be comparatively large,
with the result that conditions which favour enzyme activity,
such as temperature, will have but little effect upon the
assimilation number ; increased illumination, however, should
assist the chlorophyll which is comparatively deficient ; on the
other hand, in cases where the chlorophyll is in excess, only
those conditions which can assist enzyme activity will have any
effect upon the assimilation number. Experimental results
entirely bear out these theoretical considerations and so support
their correctness.

In this connection further reference to the views of Baly and
Heilbron upon the formation of carbohydrates in the plant
may be made. The authors point out that to effect the change
represented by the equation

CO 2 + H 2 O = HCHO + O 2

energy must be supplied since the energy contents of the
products are greater than those of the reactants. Since an
aqueous solution of carbon dioxide is unable to absorb visible
light but will absorb ultra-violet light of short wave length, it is
the latter kind of radiation which will normally be required to
effect the change. Since, however, ordinary sunlight contains
but little of such short wave-length light, it is assumed that the
function of the chlorophyll is to absorb the visible light and to
radiate infra-red energy of a frequency identical with that of
the carbon dioxide, which can then absorb it ; thus the photo-
synthetic process can proceed in the absence of the ultra-violet
light normally required for the process. In this capacity the
chlorophyll is said to act as a photocatalyst. The authors
have shown experimentally that while formaldehyde is synthe-
sized from carbon dioxide and water by short wave-length
ultra-voilet light, it is polymerized to carbohydrate by ultra-
violet light of long wave length. By adding to a solution of
carbon dioxide some substance such as sodium phenpxide. or



PHOTOCATALYTIC ACTION OF CHLOROPHYLL 49

paraldehyde which absorbs long wave-length ultra-violet light,
the formaldehyde formed by the short wave-length is protected
from polymerization. To photocatalyse the production of
formaldehyde from carbon dioxide and water, Baly and
Heilbron employ malachite green which has the same infra-red
frequency as carbon dioxide and forms an additive compound
with this substance ; methyl orange and /-nitrosodimethyl aniline
may be used in place of malachite green.

A coloured substance which can photocatalyse the poly-
merization of formaldehyde to carbohydrate has not yet been
found. It is assumed that chlorophyll is an ideal substance for
photocatalysing both reactions; it absorbs visible light from
the sun and possesses the power of forming an additive com-
pound with carbonic acid, as shown by Willstatter and Stoll ; by
absorbing those rays which tend to decompose carbohydrate, the
chlorophyll will, moreover, shift the equilibrium towards the side
of the carbohydrate and the plant will thus be able to synthesize
carbohydrate even when the concentration of carbon dioxide is
low. In the opinion of Baly and Heilbron the formaldehyde
peroxide compound of Willstatter and Stoll can have only a
very transient existence. The formaldehyde as it is split off is
instantly polymerized to a sugar which would account for the
inability to identify formaldehyde in active leaves.



VOL. II. 4



CHAPTER IV.
THE SYNTHESIS OF PROTEINS.

IN view of our limited knowledge of the chemistry of proteins,
the degree of our ignorance respecting their synthesis in plants
is not surprising.

It is generally agreed that the leaves are the important
centres of protein formation, and they show a periodicity in
their nitrogen content. Otto and Kooper* and Le Clerc du
Sablonf found that there is a gradually decreasing amount of
nitrogen from the spring to the autumn, and that leaves of
several different plants, even in different stages of development,
exhibit a greater nitrogen content in the morning than in the
evening.

The supply of nitrogen is an essential factor, and this
element must be presented in a form suitable for the nutritive
processes of the plant. Thus amongst the non-green plants,
Saccharomyces is unable to make use of nitrates and but little
use of simple amines ; urea can be assimilated under certain
conditions, but the best results obtain from the employment
of peptone, a culture solution of peptone and sucrose giving
177*4 P er cent increase in dry weight after the completion of
fermentation. |

There is, however, much variation shown by the lower non-
green plants in this respect, hardly a matter for surprise in such
metabolic gymnasts ; Bacillus coli will flourish on a diet contain-
ing salts either of ammonia, of simple amides or of amino acids,
whilst the cholera bacillus can apparently make no use of
ammonium salts but is able to utilize amino acids. For the
Fungi, Boas considers that in general ammonium salts, and
especially the ammonium salts of organic acids such as quinic

* Otto and Kooper : " Landwirthsch. Jahrb.," 1910, 39.
f-Le Clerc du Sablon : " Rev. Gen. Bot.," 1904, 16, 341.
Bokorny: " Chem. Zeit.," 1916, 40, 366.
Boas: " Biochem. Zeit.," 1918, 86, no.

50



NITROGEN SUPPLY 51

acid, are better than amino acids as a source of nitrogen in
protein synthesis. He points out that such feeding experi-
ments should be brief, in order to avoid secondary, especially
proteolytic, changes, and that the reaction of the culture
medium is all-important. Acidity may inhibit the growth of
many fungi, so that if ammonium salts of inorganic acids be
used as a source of nitrogen, the observed results may be due
not to the inability of the plant to employ the particular salt,
but to the acidity which develops as the nitrogen is as-
similated.

For ordinary green plants the supply of nitrogen is found
in the simple nitrogen-containing salts of the soil water. Thus
the fertility of the soil, not only with respect to nitrates but
also in regard to other substances,* is an important factor,
conditioning the amount of protein found in the plant. In
addition to nitrates, some plants can make use of ammonium
salts. Hutchinson and Miller f found this to be true under
conditions of culture which precluded the presence of nitrates
in the soil. In this respect, however, all plants do not be-
have alike ; whilst some will grow equally well whether
supplied with nitrates or ammonium salts, others flourish
best when supplied with the former, and others seemingly
prefer ammonium salts to begin with and then nitrates.

Mention has just been made of the importance of soil
substances other than nitrates in the protein synthesis of
plants : potassium may be taken in illustration. Species of
bacteria grown in the dark in culture media containing the
requisite organic food materials and salts but lacking potas-
sium show but poor development and no protein synthesis.
Under similar cultural conditions seedlings of the beet showed
seven times less protein and eighteen times less sugar, when
grown without potassium, as compared with the controls ;
when grown under sterile conditions in a culture medium
containing a sugar and supplied with known amounts of
carbon dioxide, it was found that those grown in the light
were independent of potassium as regards the synthesis of
protein, and that the addition of sugar to the culture medium

* Whitson and Stoddart : "Ann. Rep. Wisconsin Exp. Sta.," 1904, 193.
t Hutchinson and Miller: " Journ. Agric. Sci.," 1909, 3, 179.

4"



52 THE SYNTHESIS OF PROTEINS ]

resulted in an increase of protein. In darkness, on the other
hand, a less vigorous development obtained and only those
plants supplied with potassium salts showed protein formation.

It would therefore appear that the action of potassium is
partly indirect, and only in darkness does it play an important
role in the production of proteins. Since potassium is mildly
radioactive, it may possibly serve as a source of energy in
promoting the analysis of carbohydrate.*

These observations introduce the problems connected with
the light factor. Earlier opinions were that light was an
important and a direct factor in the synthesis of proteins :
radiant energy is certainly of great importance in that it
is a necessity for the green plant in the making of carbo-
hydrate, which is in its turn a requisite in the formation of
proteins.

The obvious essentiality of nitrogen in the building of
proteins and the fact that in general the element is absorbed
by the plant in the form of nitrate has been remarked upon :
but nitrate as such is a relatively inert substance and does
not readily lend itself to chemical change; nitrite, on the
other hand, is a more labile substance.

That the plant is able to convert nitrate into nitrite was
first observed by Laurent,f and later Irving and Hankinson,J
working on Sagittaria> came to the conclusion that nitrite
must be an intermediate compound in the metabolism of
nitrates.

With regard to the occurrence of nitrites in the plant, Aso
has established their occurrence in etiolated potato shoots.
In this connexion it is of interest to note that so long ago
as 1888 Schimper,|| experimenting with cut leaves of Sambucus
and with potted plants of Pelargonium zonale, found that
nitrates were destroyed in green leaves exposed to daylight
but were not so destroyed if the leaves were kept in the dark,
and in agreement with this, shade leaves were found to be
richer in nitrates than sun leaves. Furthermore, no destruc-
tion of nitrate occurs in etiolated leaves exposed to sunlight.

* See Stoklasa: " Biochem. Zeitsch.," 1916, 73, 107.
tLaurent: "Ann. Inst. Pasteur," 1890, 4, No. n.
% Irving and Hankinson: " Biochem. Journ.," 1908, 3, 87.
Aso : " Beih. Bot. Zentr.," 1903, 15, 208, and 1914, 32, 146.
HSchimper: " Bot. Zeit.," 1888,46, 128.



REDUCTION OF NITRATE AND NITRITE 53

It was first shown by Thiele* that potassium nitrate, on
exposure to the rays from a quartz mercury vapour lamp,
was reduced to potassium nitrite with evolution of oxygen.
With a view to the further study of the photochemical de-
composition of nitrites, Baudisch f exposed mixtures of potas-
sium nitrite and methyl-alcohol in aqueous solution to diffused
daylight and to ultra-violet light and found that the methyl-
alcohol became oxidized to formaldehyde at the expense of
the nitrite which was reduced to hyponitrite, and this latter,
at its moment of formation, reacted with the formaldehyde
to produce the potassium salt of formhydroxamic acid (I.)

KNO 3 + CH 3 OH = KNO + HCHO + H a O
KNO + HCHO = H . C . OH
II

NOK
(I.)

This reduction of nitrate or nitrite in presence of alcohol is
a purely photochemical reaction since no such change could
be produced in the dark even if the solutions were boiled.

More recently Baly and his collaborators, to whose work on
carbohydrate synthesis reference has already been made, have
investigated the photosynthesis of nitrogen compounds from
nitrates and carbon dioxide by passing this gas through
aqueous solutions of potassium nitrate or nitrite exposed to
ultra-violet light. In these circumstances activated formalde-
hyde is produced for which the formula H . C . OH is suggested,
the activity being due to the divalent carbon. According to
Baly, Heilbron and Hudson J this activated formaldehyde
reacts with the nitrite to produce the potassium salt of
formhydroxamic acid (I.), an atom of oxygen being evolved
which oxidizes a further quantity of formaldehyde to formic
acid. These changes may be represented by the following
equations :

H . C . OH + O : NOK -> H . C . OH -> H . C . OH

II II + o

0:NOK NOK

and O + H . C . OH = HCOOH

* Thiele: " Ber. deut. chem. Gesells.," 1907, 40, 4914. See also Moore
and Webster : " Proc. Roy. Soc.," Lond., B., 1919, 90, 158.

f Baudisch: "Ber. deut. chem. Gesells.," 1911, 44, 1009. See also loc.
cit., 1916, 49, 1176, and 1918, 51, 793.

, Heilbron and Hudson : " Journ. Chem. Soc.," 1922, 121, 1078,



54 THE SYNTHESIS OF PROTEINS

Under the conditions of the experiment, the potassium salt
is completely hydrolyzed to the free acid

H.C.OH

II

N.OH

The latter compound can readily lose oxygen to produce
a compound of the formula

H.C.OH*

II

N.H

which could condense with more activated formaldehyde to
produce a labile ring compound (III.)

H.C.OH H.C.OH

NH
(HI.)

which by intramolecular rearrangement would give glycine
CH 2 NH 2 COOH.

Evidence in support of the correctness of these views is
furnished by the actual production of a amino acids on expos-
ing aqueous solutions of formhydroxamic acid and formalde-
hyde to ultra-violet light, whereby the earlier claims made by
Baudisch f to have obtained substances of this nature are con-
firmed.

In addition, the authors claim to have produced alkaloids.
The formation of these substances is explained by assuming
formhydroxamic acid to condense with three or four molecules
of activated formaldehyde to produce compounds (IV.) and (V.).

H.C.OH

/\

H.C.OH H.C.OH HCOH HCOH

iOH HC.OH HCOH HCOH

\/

(IV.) (V.)

* This compound is a hydrate of hydrocyanic acid which is of significance
with regard to the formation of cyanogenetic glucosides.
f Baudisch: " Zeit, angew, Chem.," 1913, 26, 612,



HC(



FORMATION OF NITROGEN COMPOUNDS 55

which by loss of oxygen and water should give pyrrole* or
pyridine compounds respectively.

By the condensation of two molecules of formhydroxamic
acid with one molecule of formaldehyde, the compound (VI.)
would be produced which by loss of oxygen and water would
yield glyoxaline (VII.)



H . COH NH CH N

II II I!

HCOH HCOH CH CH

\/

NH

(VI.) (VII.)



NrfT



and again evidence of the actual formation of this substance is
adduced, as well as of histidine,f the sustituted a-amino acid
derived from glyoxaline.

The authors' interpretation of their results is best given by
quoting their own words : " Our results leave no doubt that the
activated formaldehyde photosynthetically produced in the
living chloroplast reacts with potassium nitrite with extra-
ordinary ease to produce formhydroxamic acid, which at once
proceeds to condense with more of the activated formaldehyde
to give various nitrogen compounds. It follows from this that
the synthesis of the nitrogen compounds found in the plant is
not photosynthetic except in so far as the production of the
activated formaldehyde by the chlorophyll is concerned. The
various amino-acids, proteins, alkaloids, etc., are natural and
indeed inevitable results of the photosynthesis of formaldehyde
in the presence of potassium nitrite. Their formation has been
considered by some to savour of the mysterious, the mystery
being found in the question as to how a plant succeeds in
synthesizing the very substances it requires for its existence.

" The life and growth of a plant consists in the utilization of
the products formed in its leaves. There is no real mystery in

CH

s\

* CH CH CH CH

II II II I

CH CH CH CH



V



Pyrrole Pyridine

t See Vol. I., p. 325.



56 THE SYNTHESIS OF PROTEINS

the formation of these products, the plant has no choice in the
matter, since with given conditions of chlorophyll, carbonic
acid, light energy and potassium nitrite the synthesis must
follow its natural course just as we have found to take place
in vitro.

" A further conclusion of importance is that the region where
the synthesis occurs must necessarily be restricted to the
leaves. Since it must not be forgotten that the synthesis of
hexoses is taking place concurrently, the conditions are perfect
for the formation of glucosides and we believe that the products
of the nitrogen synthesis are translocated as soluble glucosides.
The fact that nitrogen derivatives are found in other parts of
the plant cannot be accepted as an argument that they must
have been synthesized in those parts. There can be no doubt
that the synthesis takes place in the leaves and that the com-
pounds are subsequently distributed as soluble glucosides by
the normal translocatory processes."

Commenting on the last statement, it should be borne in
mind that no evidence is adduced in support of the existence of
such soluble protein glucosides.

The authors summarize their ideas in the following scheme.

Potassium nitrate Carbonic acid

* *

Potassium nitrite Activated formaldehyde



Formhydroxamic acid



*

Hexoses



1 1

Nitrogen base? a Amino-acids



* *

Alkaloids and xanthine I



derivatives Substituted a-amino-acids

(Histidine, etc.)

*.

Proteins

Whilst the foregoing pages have dealt with the synthesis of
proteins in daylight or ultra-violet light it must be borne in mind
that the synthesis of proteins can also take place in the dark
and in tissues free from chlorophyll, provided that an adequate



PROTEIN FORMATION IN DARKNESS 57

supply of carbohydrate be at hand.* Zaleski and Suzuki f
found that the leaves of the sunflower floating upon a solution
containing sugar and nitrate produced considerable quantities of
proteins in the dark, from which it appears that nitrogen as-
similation is not a photochemical process, and that light is only
of indirect importance in providing one of the means for the
formation of carbohydrates. J This opinion also is held by
Loew.

The synthesis of proteins is conditioned by the available
supply of carbohydrate, and since photosynthesis is a daylight
process, it is not surprising to find that the production of pro-
teins may be four or five times as great in the light as in
darkness.!! BaudischlF is of the opinion that the formation of
protein under abnormal conditions in the dark is no proof
that the process is not a photochemical one under normal con-
ditions ; he considers that the synthesis may in this case be
due to some abnormal chemical processes which reduce the
nitrates and so aid in the production of proteins. It has also
been stated that if but small quantities of carbohydrate are
available, the synthesis of proteins, in darkness, may stop at
the formation of amides,** which some plants, e.g., Algae such
as Pleurococcus and Raphidium> and the Fungi Eurotium and
Penicillium, can directly assimilate,ff an aspect of the subject
to which allusion has already been made.

There is available but little exact knowledge regarding the
specific action of different wave lengths of radiant energy
which may be concerned in the different phases of protein
metabolism. Schanz JJ finds that ultra-violet light renders
proteins less soluble, possibly an account of its deleterious
action on enzymes, and in this is found a reason for the

* Jost: "Biol. Centrlbl.," 1900, 20, 625.

f Zaleski and Suzuki: " Ber. deut. hot. Gesells.," 1897, 15, 536; " Bot.
Centrlbl.," 1901, 87, 281 ; Suzuki : ' Bull. Coll. Ag. Tokyo," 1898, 2, 409 ; 3,
241,

Zaleski : " Ber. deut. hot. Gesells., 1909, 27, 56.

Loew: id., 1917, 50, 909.

|| Montemartini : " Atti. R. Inst. Bot. Pavia," 1905, II., 10, 20.

IT Baudisch : " Zentr. Bakt. Parasit.," 1912,32, 520.

** Jakobi : " Biol. Centrlbl.," 1898, 18, 593.

ttLutz : " Bull. Soc. Bot. France," 1902, 48, nS.

t Schanz : " Ber. deut. hot. Gesells,," 1918, 3$, 619.

SeeVolI,,p. 353,



58 THE SYNTHESIS OF PROTEINS

dwarf habit of alpine plants which are subjected to a high
degree of insolation. Bovie * draws attention to the efficiency


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