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Albert P. Mathews.

Physiological chemistry: a text-book and manual for students online

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Pehling's solution until it is decomposed. [arjy= — 60.52.

Guanylic acid has also been separated from ox liver and Jones suc-
ceeded in getting it from yeast nucleic acid by a quick digestion by an
enzyme, tetra-nucleotidase, found in the pig pancreas. Guanylic acid
is dextro-rotatory.

Inosinic acid. — This is an acid similar to guanylic acid, but it is



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172



PHYSIOLOGICAL CHEMISTRY



composed of a molecule of hypoxanthine, a pentose and phosphoric acid.

It was isolated from Liebig's beef extract and is supposed to occur in

muscle. Whether it does pre-exist in the muscle is probable, but not

certain. It was the study of this acid by Neuberg which really gave the

key to the structure of the nucleic acids. Neuberg thought it had the

formula O

II
Hypoxanth ine — — P — — pentose

OH
But Levene and Jacobs isolated from it a compound called inosine, a
union of pentose and hypoxanthine, showing that inosinic acid must have
a formula similar to guanylic acid. It is not, however, identical in its
structure. From yeast another pentoside was isolated, an adenine pen-
toside called adenosine. Ouanosine had already been isolated by Schulze.
from plants and called by him vernin.

Nucleic acid. — Levene and Jacobs have also isolated other fragments
of the molecule of yeast and thymo-nucleic acid. They conclude from
their work and that of Steudel that the structure of thymus nucleic acid
is probably

. OC— NH

H H O H H



H



_ b_ C — C — C — C — C — C<f



,NH-



^ <L:



NH



.fc_ C— C — C — C — C — CI l| l|

I X I I J i N -o-to

I OH H OH OH H



HO— P=0



O OH
0-1I6*



H — C— C — C — C — C — C —

HUnH



Thymine.



H H H OH
H— — C — C — C — C — C — Cytosine.



i k



I
H O



O 0— POH

1 <A*

HO— P =



HN-C = N

2

NH C C— H

II II



OH H OH OH H
H-C— A— C— A— i—fc-c/

k A i i i I ^ N — C - N

Thymo-nucleic acid (Levene and Jacobs).



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THE PROTEINS



173



Such



This would correspond with Steudel's formula, C 48 H 57 N l5 8 oP 4 .
a nucleic acid would be a tetra-nucleotide.

While the facts seem to bear out this formula, in its main features
at any rate, it cannot be said that it is as yet conclusively established
The exact point of attachment of the phosphoric acid to the sugar is still
obscure. The great difficulty of hydrolyzing the di-nucleotide, thymic
acid, seemed to indicate that the union between the pyrimidine nucleo-
tides was not through phosphoric acid, but was an ether-like union. It
will be noticed that the molecule as written in the Levene- Jacob's formula
is hexabasic.

Another possible formula would be the following:



HO

H

= t>— 0— c



u T

H I H H I

.b-U-t-L

H OH ft OH OH fl



NH 2 C = N
.NH— C CH
C <N-U



H H 6 H H
= P— O— C — fc — 6 — C — C — C— Thymine
H OH k OH OH ft



H- H 6 H H

= P— O— i — c — C — C — C — C — Cytosine

H OH H OH OH H

= C— NH



H H



f



H H






H OH H
Nucleic acid.



I I
OH OH



A possible formula.




CNH



Does nucleic acid exist outside the nucleus? — There are several very
interesting questions as yet unsolved concerning the location in the cell
of the nucleic acid. It seems probable, though there is nothing really
known about it, that guanylic and inosinic acid may be in the cytoplasm
of the cells in which they occur, though they may be in the nucleus. It
is possible that they do not exist free in the cell, but are united with the
true nucleic acid and are set free by endocellular enzymes. Nothing is
really known about their function or location. Their staining reaction
will probably resemble that of the real nucleic acids. Guanylic acid
gelatinizes much as the nucleic acids, and it was this property that caused
Bang to maintain that it must be more complex than a single nucleotide.
Inosinic acid is probably the source of the hypoxanthine of muscle and



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174 PHYSIOLOGICAL CHEMISTRY

it is very interesting that this substance is increased during muscular
contraction.

There can be little doubt that the true nucleic acids, that is the poly-
nucleotides, like thymus nucleic acid, are found only in the nucleus. This
was first indicated by the work of Kossel, who determined the amount
of purine bases obtainable from different tissues. The amount ran pro-
portional to the amount of nuclear material present; it was high in
embryonic tissue; in the thymus; and low in muscle. It is shown also
by the fact that no nucleic acid is found in some unfertilized eggs where
the nuclei are very small in proportion to the cytoplasm, and none in the
mammalian red blood cells which lack nuclei. On the other hand, nucleic
acid is found wherever nuclei occur, as in the red corpuscles of bird's
blood which are nucleated. It has never been shown positively to be
a constituent of the cytoplasm, but it is certain that it is found in the
nucleus. It is probable, therefore, that it is confined to the nucleus, but
there are some facts which may be urged against this conclusion. For
example, some believe that nucleic acid is found in the cytoplasm, because
not all the cytoplasmic phosphoric acid in organic union is split off from
its union by sodium hydrate. If the substance in the cytoplasm was a
vitellin, or casein-like compound, it would presumably have been split
off. Nucleic acid, unlike the phosphoproteins, does not split off its phos-
phoric acid when treated by alkali hydrates. And recently nucleic
acid has been found in the sea-urchin's egg 7 where the nuclei are very
small. The author got a substance with some of the properties of nucleic
acid in some quantity from unfertilized eggs of the sea-urchin. It could
not be positively identified, however, as the quantity was too small. In
all these cases, then, it is still uncertain whether the substances described
were really nucleins, and the probability is that they did not contain true
nucleic acid. Further work, however, is necessary on this subject before
a definite statement can be made that nucleic acid is never found in the
cytoplasm. It is certain, however, that most of the phosphoric acid
compounds in the cytoplasm are not nucleic acids.

Are all nucleic acids the same? — The question whether all animal
nuclei contain the same, or different, nucleic acids cannot be answered
definitely, since only two of the animal nucleic acids have been accu-
rately examined, namely that of the sperm of herring and from the
thymus gland of calves. These two acids appear to be identical. They
contain the same bases in the same proportions and they have the same
physical properties. Until the nature of the carbohydrate is discov-
ered it is impossible to say whether they contain the same carbohydrate,
but all indications are that these two nucleic acids are identical. # Since
they come from such widely different sources, it would indicate that
probably the same nucleic acid is found in totally different kinds of cells,



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THE PROTEINS 175

a conclusion of the utmost importance in interpreting the probable role
of nucleic acid in the cell. All other nucleic acids of animal origin,
except guanylic and inosinic acids, have been found to yield the same
splitting products when hydrolyzed, so that they must be closely similar
to thymus nucleic acid, if they are not identical with it.

On the other hand, only two plant nucleic acids have been carefully
examined. These are triticonucleic acid from wheat, and yeast nucleic
acid. These are apparently identical, and they differ from the animal
nucleic acids in having d-ribose, a pentose sugar, in the place of a hexose.
They may also differ in other particulars. The composition of neither
of these acids is exactly known, and particularly the molecular weight
has not been determined. Steudel's analyses indicate that yeast nucleic
acid may be a tri-nucleotide and not a tetra-nucleotide, as Levene thinks.
No one has as yet isolated yeast nucleic acid which on analysis would
yield figures for carbon, phosphorus and nitrogen comparable with a
tetra-nucleotide. But this may be due to the fact that yeast contains a
nucleotidase, and possibly if some of the yeast cells are dead when ana-
lyzed a partial digestion of the nucleic acid may have taken place. Only
fresh, living, active yeast should be used for the preparation of this acid.

Another possibility which complicates the question of the identity
of the nucleic acids is that in the nucleus we may have a polymer of a
tetranucleotide, as Steudel has suggested for the sperm head. He found,
namely, that the viscosity of the solution of the herring sperm heads in
alkali was greater than an equivalent solution of protamine nucleate ; and
he inferred from this a different state of aggregation of the nucleic acid
outside and inside the cell. It is of course possible that some other
factor than that suggested was responsible for the observed result.

The tentative conclusion may with all reserve be drawn from the fore-
going facts, that the nucleic acids of different nuclei of animal tissues
are certainly closely similar if they are not identical ; but that they differ
in their carbohydrate radicles from such plant nucleic acids as have
been examined. It is possible that the hexose component will not be
found to be the same everywhere. Their similarity would clearly indi-
cate that nucleic acids have the same function in all cells. If they inter-
vene actively in ceil metabolism, it must be in connection with some
fundamental cell property such as growth, irritability or respiration
which is common to all cells. It may be, however, that it has only the
function of a supporting structure, or aids in keeping the physical
viscosity of the nucleus what it has to be. In favor of this view it may
be mentioned that it is a fairly stable substance, otherwise it could not
accumulate, and its most probable function would appear to the writer
to be that it serves as a colloidal, gelatinous substratum in the nature
of an organic skeleton to which the specifically active, more labile,



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176 PHYSIOLOGICAL CHEMISTRY

albuminous constituents, possibly of a catalytic nature, may be attached.
Forming a firm union with the acid, these active substances may be thus
confined to, or located in, the nucleus from which they may at times get
free. But nothing positive as to its function can be stated without further
investigation.

It is of interest to recall, in view of the foregoing statement, that all
so-called nuclear stains of a basic nature, with the exception of the mor-
danted stains such as iron hematoxylin, combine with the nucleic acid. .
In thus following the chromatin and chromosomes by means of these
stains, cytologists, if the view stated above of the significance of nucleic
acid is correct, may be following the inert skeletal material of the nucleus,
while the active albuminous material is entirely neglected for the reason
that it does not gel and does not stain with basic dyes. All theories of
inheritance based on the behavior of the nucleic acid of the nucleus, that
is the behavior and number of the chromosomes, must be accepted only
with the greatest reserve, until the function of this substance may be
shown to be something more than a skeletal substance. We have as yet
no chemical evidence that the different chromosomes have different
nucleic acids in them, but such evidence as we have is contrary to this
view. If the chromosomes do differ chemically, as perhaps their indi-
vidual and peculiar shapes and sizes may indicate, it is more probable,
as we shall shortly see, that they differ in their protein or basic rather
than in their acid moieties.

THE BASIC CONSTITUENTS OF THE NUCLEUS.— Nucleic
acid is either a hexa- or tetra-basic acid, probably the former; and it
forms a series of salts. We have now to ask the question with what basic
substances is nucleic acid united in the chromatin t Are the bases organic
or inorganic t

It is probable that some inorganic bases, i.e., calcium, are present;
it is certain that organic bases of a protein nature are always present.
The only nuclei carefully examined in a clean form, free from cytoplasm,
are the sperm heads, and possibly the nuclei of birds' corpuscles. These
always yield some calcium phosphate when dissolved or ashed. It seems
certain that calcium is generally present. MacCallum, from cytological,
microchemical studies, has concluded that nuclei contain no potassium,
since around the outside of the nucleus he generally obtains a deposit of
potassium-cobalto nitrite by his method, but none in the nucleus. But to
his conclusion it may be objected that the place where the precipitate
forms is not necessarily indicative of the location of the soluble salt.
There is, indeed, very little evidence of what inorganic salts or bases we
have in the nucleus itself. This question must be left for further work.
It appears, from some recent work, that iron, contrary to an earlier view,
is not present in all nuclei.



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THE PROTEINS 177

The organic bases which occur in some chromatins are among the
most interesting substances in the cell, whether considered from the
physiological or chemical point of view. Our knowledge of these
bases, the study of which gave Kossel the clew to the constitution
of the proteins, we owe chiefly to Kossel and Miescher and pre-eminently
to the former. These bases are protein in nature and consist either
of basic proteins called protamines or histones, or of other more com-
plex proteins.

The protamines. — If the sperm heads of the salmon, sturgeon, her-
ring and other fishes are extracted with 10 per cent, sulphuric acid, or
hydrochloric acid, there goes into solution about 19 per cent, of the dry,
alcohol- and ether-extracted heads. The nucleic acid remains behind
more or less altered and insoluble. Three extractions of the heads with
10 per cent, sulphuric acid for about half an hour at a time will take out
practically all of the removable base. The substance which goes into
solution as a sulphate is of a protein nature ; when precipitated by alcohol
as the sulphate it is a white, somewhat hygroscopic, amorphous powder,
giving, in the case of the herring, salmon and sturgeon sperm, no Millon,
or xanthoproteic, or tryptophane reaction, but a good biuret reaction.
This substance was named protamine by its discoverer, Miescher, who
obtained it from salmon sperm (Gr. protos, first, amine) . The protamine
from salmon is called salmin.

General properties. The protamines, although individually different,
have the following properties in common : In the free state all are strong
bases, alkaline to litmus, and not precipitated by ammonia. They give
a splendid biuret test, but Millon, xanthoproteic or Adamkiewicz reac-
tions are in many cases negative, but in some protamines positive. They
are digestible by trypsin, but not by pepsin-hydrochloric acid ; they are
readily soluble in water, but not in alcohol, and their sulphates separate
as an oil when the saturated aqueous solution is shaken with ether. They
are not coagulated or changed by heating. They precipitate proteins by
uniting with them in ammoniacal solution, and this is a very delicate
test for them. In this respect they act like metallic bases. Unlike most
proteins, they are precipitated from a neutral solution by neutral solu*
tions of sodium picrate, f errocyanide or tungstate, and they may even
be precipitated in faintly alkaline solutions. The reason for this pecu-
liarity has already been explained. They are such strong bases that their
molecules are electro-positive even in faintly alkaline solutions. On
analysis they consist of carbon, hydrogen, oxygen and nitrogen, but they
contain no sulphur. The elementary analyses of some are as follows:

C H N O Pt CI

Clupein 47.93 7.59 31.68 12.78 — — Free base.

Salmin 22.96 4.32 14.83 6.7 24.73 26.56 Plat, chloride salt

Sturin 24.32 4.49 14.20 8.47 23.10 25.42



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178 PHYSIOLOGICAL CHEMISTRY

The formula for salmin is probably C 80 H 67 N l7 ; that for sturin,
C 80 H 09 N 19 7 . The molecular weight is not yet determined.

The protamines differ from all other proteins in the small number of
different amino-acids they yield on hydrolysis and in the character of
these acids. Kossel found that salmin, one of the simplest, yielded 87
per cent, of its molecule as arginine, and it was this discovery which sug-
gested to him the constitution of the proteins. The composition of the
hydrolytic cleavage products of numerous protamines is given on
page 128.

Does the sperm chromatin consist exclusively of protamine
nucleinate? — The chromatin of the sperm head is supposed to be the
bearer of the hereditary qualities and zoologists have pictured it as com-
posed of individual unite, biophores or determinants, each of which rep-
resents some specific unit-character of the adult. If this hypothesis
were true, we should expect the sperm chromatin to be extremely com-
plex ; more complex indeed than any chromatin in the body, since it is
supposed to represent them all. As a matter of fact, chemical examina-
tion shows this chromatin in the fish sperm to be the simplest found any-
where. The heads of the herring sperm do not contain any tyrosine;
they give no Millon, xanthoproteic or tryptophane test. They contain no
coagulable protein. They have the following composition after extrac-
tion with alcohol and ether :







Average


c


40.99—41.48


41.20


H


5.62— 5.83


5.75


N


20.78—21.44


21.06


P


5.87— 6.33


6.07



Steudel has recently confirmed these figures. Accepting his formula for
the composition of nucleic acid, C 48 H 57 N 15 P 4 3 o, and Kossel's formula for
clupein, or salmin, C 80 H 67 N 17 , there would be required for protamine
nucleate :



Computed for


Found


C7iHiiaN3«Ot & P 4




C 40.97


41.24


H 5.33


5.27


N 20.95


21.09


P 5.80


6.02


O 26.95


20.37



This formula requires 64.8 per cent, nucleic acid and 35.2 per cent,
protamine. He actually isolated 93 per cent, of the calculated amounts
of each of these substances and the deficit was undoubtedly due to the
fact that the methods are not entirely exact. There can be no doubt,
therefore, that the chromatin of herring sperm when fully ripe consists
of a neutral salt of protamine nucleate. Miescher found very similar
relationships in the salmon sperm, the head consisting largely or wholly
of salmin nucleate.



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THE PROTEINS 179

Nature of the union of protamine and nucleic acid. The ease with
which the protamine may be separated from the nucleic acid by acids
or alkalies indicates clearly that the two are in a salt-like union. Prob-
ably the union is between the free amino groups at the end of the chain
of the arginine and the acid radicle of the nucleic acid (Steudel). By
extracting first with alkali these free amino groups of the arginine of
the salmin are decomposed, ammonia being set free and ornithine re-
maining. If, now, the compound is acidified a reunion of the nucleic acid
and protamin does not take place. This is the probable basis of the
Neumann method of preparing nucleic acid. But there can be equally
little doubt that we often have other than salt unions between the pro-
tein and nucleic acid. It is impossible to extract all the protein from
the nuclei of all cells by acid. The union is too firm.

Other basic constituents. Histone. In the sperm of the sea urchin,
Arbacia, the author isolated by acid extraction a basic protein resem-
bling histone in some particulars and protamine in others. About 11 per
cent, by weight of the alcohol and ether extracted, dried whole sperm
was extracted by acid. The arbacin sulphate contained 15.91 per cent,
of nitrogen, whereas protamine sulphate contains about 25.13 per cent.
In this experiment the sperm heads were not separated from the tails.
The substance was not a true histone, for it did not precipitate with
ammonia, except very incompletely. Nucleic acid was also isolated.
Arbacin was strongly basic and gave the Millon test. Only a small pro-
portion of the protein could be extracted by acid from the sperm, indi-
cating that not all of it was in a salt union, or else that the tails made
a very considerable proportion of the whole.

The chromatin of both thymus gland and bird's blood corpuscles con-
tains a basic, simple protein, histone, in a salt union with nucleic acid.
This fact was also discovered by Kossel. These nuclei have been recently
obtained and studied by Ackermann.

The method of isolating the nuclei has already been given (page 162).
The dried nuclei after alcohol and ether extraction contained 3.93 per
cent P; 17.20 per cent. N. If Steudel's formula for nucleic acid is used
in place of the formula employed by Ackermann, it is computed from the
phosphorus that the nuclei contain 43.96 per cent, nucleic acid and
56.M per cent, of histone, if they contain only histone nucleate. From
Steudel's formula nucleic acid contains 15.18 per cent, of N. Hence in
100 grams of the nuclei containing 17.2 grams of nitrogen, 6.67 grams
are in the nucleic acid and 10.53 grams in the histone. Since histone
contains 18.3 per cent. N, the nuclei must contain 57.5 per cent, of histone.
Both nitrogen and phosphorus indicate, therefore, that the nuclei con-
tain 43-44 per cent, of nucleic acid and 56-57 per cent, of histone. Acker-
mann actually extracted by hydrochloric acid (1 per cent.) 63.9 per cent.



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180 PHYSIOLOGICAL CHEMISTRY

(53.9 1) of histone, leaving 46.1 per cent, insoluble nucleic acid instead
of about 44 per cent. Some purine bases undoubtedly went into solution
and the residue contained only 7.79-7.99 per cent, of P and 15 per cent,
of N, so that some histone may have been left unextracted. Although
these figures do not check exactly, the method not being quantitative, it
is clear, nevertheless, that these nuclei consist chiefly, if not entirely,
of histone nucleate, and contain no other protein substance in any quan-
tity. If the molecular weight of nucleic acid is 1,387 and that of histone
about 1,600, which is the simplest formula which can be ascribed to it,
a molecule of chromatin might be simply histone nucleate containing one
molecule of each substance.

It is greatly to be desired that studies similar to these should be made
on other tissues so that we may have a more accurate knowledge of the
composition of the chromatin of as many cells as possible. Only when
this is done will physiological chemistry be able to contribute to the vexed
and vexing question of chromosomal inheritance.

Concerning the nature of the simple protein united with nucleic acid
in other nuclei than these few kinds, nothing is known. Basic proteins
corresponding to histone and protamine have not been isolated from other
cells than those mentioned.

Enzymes in the nucleus. — Many nuclei, and particularly the large
germinal vesicles of starfish eggs when unripe (Asterias vulgaris, etc.)
contain very little of the morphological substance called chromatin. The
greater part of these nuclei consists of a liquid sap which contains protein
matter, if we may conclude from the fine precipitate produced in it by
fixing agents such as mercuric chloride. No one has yet obtained this
nuclear sap for chemical analysis, but there is no question that its admix-
ture with the extra-nuclear cytoplasm produces marked chemical changes
in the latter and greatly stimulates cell respiration. Delage, Loeb and
the author have particularly studied the changes so produced. If unripe
or immature eggs in which the germinal vesicle is intact are placed in
sea-water, some of the eggs rupture the nuclear membrane and the
nuclear sap mixes with the cytoplasm. Some eggs do not rupture the
nucleus spontaneously, but they may be made to do so artificially by
shaking. Eggs in which the nuclear sap has penetrated the cell cyto-
plasm behave very differently from eggs in which the nuclear sap remains
in the nucleus. If rupture of the membrane takes place, the eggs become
very sensitive to oxygen and they will only live about 10-18 hours in
oxygenated sea-water. At the end of that time the protoplasm becomes
opaque and seems filled with a multitude of spherules, the protoplasm
being disintegrated into these spherules. If, however, the nuclear sap
does not penetrate the cell cytoplasm and the nuclear membrane remains



Online LibraryAlbert P. MathewsPhysiological chemistry: a text-book and manual for students → online text (page 20 of 119)