THEIR CHEMICAL PROPERTIES AND
WALTER. JONES, FH.!>.
MONOGRAPHS ON BIOCHEMISTRY
R. H. A. PLIMMER, D.Sc.
F. G. HOPKINS, M.A., M.B., D.Sc., F.R.S.
THE subject of Physiological Chemistry, or Biochemistry, is
enlarging its borders to such an extent at the present time,
that no single text-book upon the subject, without being
cumbrous, can adequately deal with it as a whole, so as to
give both a general and a detailed account of its present
position. It is, moreover, difficult, in the case of the larger
text-books, to keep abreast of so rapidly growing a science
by means of new editions, and such volumes are therefore
issued when much of their contents has become obsolete.
For this reason, an attempt is being made to place this
branch of science in a more accessible position by issuing
a series of monographs upon the various chapters of the
subject, each independent of and yet dependent upon the
others, so that from time to time, as new material and
the demand therefor necessitate, a new edition of each mono-
graph can be issued without re-issuing the whole series. In
this way, both the expenses of publication and the expense
to the purchaser will be diminished, and by a moderate
outlay it will be possible to obtain a full account of any
particular subject as nearly current as possible.
The editors of these monographs have kept two objects
in view : firstly, that each author should be himself working
at the subject with which he deals ; and, secondly, that a
Bibliography^ as complete as possible, should be included,
in order to avoid cross references, which are apt to be
wrongly cited, and in order that each monograph may yield
full and independent information of the work which has been
done upon the subject.
It has been decided as a general scheme that the volumes
first issued shall deal with the pure chemistry of physiological
products and with certain general aspects of the subject.
Subsequent monographs will be devoted to such questions
as the chemistry of special tissues and particular aspects of
metabolism. So the series, if continued, will proceed from
physiological chemistry to what may be now more properly
termed chemical physiology. This will depend upon the
success which the first series achieves, and upon the divisions
of the subject which may be of interest at the time.
R. H. A. P.
F. G. H.
MONOGRAPHS ON BIOCHEMISTRY
THE NATURE OF ENZYME ACTION. By
W. M. BAYLISS, D.Sc., F.R.S.
THE CHEMICAL CONSTITUTION OF THE
PROTEINS. By R. H. A. PLIMMER, D.Sc.
Part I. Analysis.
THE VEGETABLE PROTEINS. By THOMAS B.
THE SIMPLE CARBOHYDRATES AND THE
GLUCOSIDES. By E. FRANKLAND ARMSTRONG,
D.Sc., Ph.D., F.R.S.
ALCOHOLIC FERMENTATION. By A. HARDEN,
Ph.D., D.Sc., F.R.S.
NUCLEIC ACIDS. THEIR CHEMICAL PRO-
PERTIES AND PHYSIOLOGICAL CON-
DUCT. By WALTER JONES, Ph.D.
RESPIRATORY EXCHANGE IN ANIMALS. By
A. KROGH, Ph.D.
LECITHIN AND ALLIED SUBSTANCES. By
H. MACLEAN, M.D., D.Sc.
LONGMANS, GREEN AND CO.,
LONDON, NEW YORK, BOMBAY, CALCUTTA, AND MADRAS.
THEIR CHEMICAL PROPERTIES AND
WALTER JONES, PH.D.
PROFESSOR OF PHYSIOLOGICAL CHEMISTRY IN THE JOHNS HOPKINS MEDICAL SCHOOL
LONGMANS, GREEN AND CO.
39 PATERNOSTER ROW, LONDON
FOURTH AVENUE & 30TH STREET, NEW YORK
BOMBAY, CALCUTTA, AND MADRAS
PREFACE TO THE SECOND EDITION
A COMPARISON of the different editions of a text -book often
brings out interesting historical matters.
When the first edition of this monograph appeared, the
nucleotide structure of plant nucleic acid, though firmly
grounded on the four nucleosides, was nevertheless in the
hypothetical stage ; and this structure was the point of depen-
dence for the analogous nucleotide structure of animal nucleic
acid, so that the two structures must survive or fall together.
The four hypothetical nucleotides have now been prepared
from plant nucleic acid, and analogies dealing with the funda-
mental chemical and physiological properties of the two nucleic
acids have been so frequently found that at the present time
interest in the two structures is directed only to minor points.
A most important development has also occurred on the
physiological side. After it was shown that the purine fer-
mentation is markedly different for different animal species and
that the purine ferments have a successive embryonic develop-
ment, efforts were of course directed to the discovery of evolu-
tionary relations. After thorough examination it became certain
that the organism of the monkey contrasted sharply with that
of man and resembled closely that of the lower animals. This
was a little unexpected, as the organs of the rabbit and guinea-
pig had been found to coincide throughout. However, the
conclusion was accepted without hesitation : in fact every one
was rather pleased, and even took the trouble to emphasize this
notable difference between man and the monkey. But a sub-
sequent examination of apes put the evolutionary matter in a
somewhat different light.
In regard to the most striking factor of purine metabolism
(ability to convert uric acid into the more soluble allantoine)
the lower animals closely resemble one another. Their " uric-
olytic index " is high. But the uricolytic index of man and the
ape is zero : they cannot convert uric acid into allantoine.
Therefore the gap occurs between the ape and the monkey,
not between the ape and man, which constitute a class by
THE nucleic acids constitute what is possibly the best under-
stood field of Physiological Chemistry, yet so far as is known
to the writer no treatise has yet appeared which deals ex-
clusively with this subject. Our information must be acquired
either from widely scattered and often conflicting original
articles which reveal order only by the application of critical
ability, or from incidental chapters of general texts which
appear to have been added more for completeness than for the
information which they contain. Under these conditions the
appearance of a special volume is rather to be expected than
In the present monograph an attempt has been made to
give a comprehensive view of the field as it exists to-day and
at the same time to preserve historical order so far as it
concerns individual priority. At some points this has been
found difficult and at others was made possible only by a trifle
of repetition. It may appear that the method of treatment is
based upon certain rather radical opinions. This is to some
degree true. However, these opinions are not personal, but
can be found stated or clearly implied in all of the more recent
contributions. Their acceptance makes possible a clear ex-
position of the subject and their denial affects only the
arrangement, not the materials, of this monograph.
THE CHEMICAL PROPERTIES OF NUCLEIC ACIDS.
I. INTRODUCTION i
Nucleoprotein, Nuclein, Nucleic Acid . i
II. THE FUNDAMENTAL GROUPS OF NUCLEIC ACIDS 9
Animal and Plant Nucleic Acids . 9
The Final Hydrolytic Products of Nucleic Acids - - 1 1
The Purine Derivatives of Nucleic Acids - - 14
The Pyrimidine Derivatives of Nucleic Acids - 1 7
Syntheses of the Pyrimidine Derivatives - - T 1 9
The Primary Origin of the Pyrimidine Derivatives - 2 1
The Carbohydrate Groups of Nucleic Acids - 2 1
III. PLANT NUCLEIC ACIDS - - 2 3
Yeast Nucleic Acid - - 23
Inosinic Acid - - 23
The Carbohydrate Group of Yeast Nucleic Acid - - - 26
Guanylic Acid - - - 29
The Nucleosides of Yeast Nucleic Acid - 3 1
The Nucleotides of Yeast Nucleic Acid - - - 33
The Partition of Phosphorus in Yeast Nucleic Acid - - 40
The Nucleotide Linkages of Yeast Nucleic Acid - - 42
Triticonucleic Acid - - - - 49
IV. ANIMAL NUCLEIC ACIDS - 50
Thymus Nucleic Acid - 50
The Structure of Thymus Nucleic Acid - 5 1
THE PHYSIOLOGICAL CONDUCT OF NUCLEIC ACIDS.
The Formation of Nucleic Acid in the Body - 56
The Physiological Decomposition of Nucleic Acid - 57
The Metabolism of the Pyrimidine Derivatives - 62
The Formation of Uric Acid from Nucleic Acid - 64
The Formation of Uric Acid from Oxy-purines - - 67
The Formation of Oxy-purines from Amino-purines - - 70
The Synthetical Formation of Uric Acid - - 74
The Physiological Destruction of Uric Acid - 75
The Purine Ferments of Human Organs - -82
The Question of Uncase in the Human Organism - 85
The Distribution of the Purine Ferments - - 87
Nuclease- - 91
The Purine Derivatives of Human Urine - - 98
APPENDIX - - 103
Preparation of Thymus Nucleic Acid - 103
Preparation of Yeast Nucleic Acid - - - T 04
The Analytical Chemistry of the Purine Derivatives - - - 106
Preparation of Guanine and Adenine from Nucleic Acid - - 107
The Analytical Chemistry of the Pyrimidine Derivatives - - 109
Preparation of Thy mine and Cytosine from Thymus Nucleic Acid no
Preparation of Uracil and Cytosine from Yeast Nucleic Acid - in
The Analytical Chemistry of the Nucleosides - -in
Preparation of Guanosine, Adenosine, Uridine, and Cytidine from
Yeast Nucleic Acid - -112
The Analytical Chemistry of the Nucleotides - -115
Preparation of Adenine Nucleotide from Yeast Nucleic Acid - 117
Preparation of Guanine Nucleotide from Yeast Nucleic Acid - 120
Demonstration of the Purine Ferments - - 122
BIBLIOGRAPHY - 129
INDEX - 149
THE CHEMICAL PROPERTIES OF NUCLEIC ACIDS.
Nucleoprotein, Nuclein, Nucleic Acid.
THE early development of nearly every scientific subject is marked by
a set of conditions under which it is extremely difficult or even im-
possible to distinguish the important from the unessential, and unfor-
tunately any misapprehensions which in consequence ar,ise are likely
to be so engrafted upon the nomenclature as to perpetuate themselves
The discovery of nucleic acid happened at a time when the chemistry
of the proteins was little understood and when everything incapable
of being otherwise rationally explained was ascribed to the proteins as
the somewhat mysterious agents of all physiological complexity. The
protein molecule was commonly represented with a sugar group, the
brown pigments of the body were referred to the proteins of the blood,
uric acid was regarded as an intermediate product of protein metabolism,
and it was to be expected that nucleic acid with its high nitrogen
content would be connected with this common source of nitrogen com-
pounds. But the proteins and nucleic acid might easily have been
disentangled had the subject not become confused by the supposed
constitutional ability of the so-called " nucleoproteins " to produce
intravascular clotting. This unfortunate causal association of two
things which have nothing whatever to do with one another gave rise
to a growth of literature that was highly respected in its day and even
at present serves to impress many scientists. " Nuclein," a rather
definite term from the peri of Miescher, became expanded to "nucleo-
protein " by subsequent writers. The nucleoproteins were then sub-
divided for rather trivial reasons, and the components of this subdivision
were confused with one another in a way that suggested chaos when
Kossel rescued "nucleic acid " as the essential factor or " prosthetic
group" of all nucleins and nucleoproteins.
2 NUCLEIC ACIDS
Even at present the terms nuclein and nucleoprotein suggest some-
thing a little disquieting, but all difficulty disappears when the original
sources of these terms are examined in the light of modern discovery.
In the year 1 868 Friedrich Miescher  undertook a chemical
examination of pus cells. Surgical bandages, secured from a neigh-
bouring clinic, were extracted with a dilute solution of sodium sulphate,
and the heavy pus cells thus obtained were easily separated from ad-
herent serum and salt solution by careful decantation. The cells, still
intact, were then submitted to the digestive action of artificial gastric
juice which dissolved the protoplasm, leaving the more resistant nucleus
as an insoluble grey powder, so that cell nuclei free from protoplasm
became available for chemical study. Upon treatment of these insoluble
nuclei with dilute sodium carbonate a solution was obtained in which
acetic acid produced a flocculent precipitate which was found to contain
phosphorus and responded to protein colour tests. This substance, to
which Miescher gave the name nuclein on account of its origin, was the
first known member of what is now a comparatively large class of sub-
stances obtainable from the nuclei of animal and plant cells. Hoppe-
Seyler  prepared one from the nuclei of yeast cells, and Kossel
[i 88 1 ] afterwards prepared another from the red-blood corpuscles of
birds. All of these nucleins are insoluble acids which form soluble
sodium salts. They respond to the protein colour reactions, but differ
from proteins in the phosphorus which they contain and in the resistance
which they offer to the solvent action of artificial gastric juice.
Shortly after the completion of his work with pus cells, Miescher
removed from the laboratory of Hoppe-Seyler in Tubingen to assume
control of the department at Basel where he became intensely inter-
ested in the life of the Rhine salmon in fresh water (see Miescher
). He was able to prove the older suspicion that these animals
never partake of food in their ascent of the Rhine from the sea to the
spawning beds ; for with rare and easily explained exceptions the
alimentary canal was found free from food detritus, and the digestive
fluids were as a rule inactive. As the muscle tissue had greatly de-
creased during the Rhine journey, while the organs of reproduction
had grown enormously, it is necessary to conclude that eggs and sper-
matozoa had been formed from muscle protein.
During the spawning season the spermatic fluid or lachsmilch
can be obtained from these fish in great quantity, and when expressed
from the vas deferens of a living fish, consists of little more than
spermatozoa suspended in a dilute salt solution. The spermatozoa
are composed of head, tail and middle part, it being especially
characteristic that the two latter parts together make up a mass that
is insignificant in comparison with the mass of the head, while the tails
are threads of extreme fineness which dissolve easily in acetic acid,
leaving the heads as insoluble granular masses.
From comparative histological studies of the growing testicle and
also from other considerations we are led rather directly to the con-
clusion that the spermatozoa head is to be regarded as a metamor-
phosed nucleus, so that Miescher was in possession of material in great
quantity admirably adapted to a chemical examination of the cell
nucleus. He found the spermatozoa heads free from protein, and
made up almost exclusively of a single chemical individual a salt of an
organic base rich in nitrogen and of an organic acid containing phos-
phorus (Miescher [1874, i]). The organic base was protamine ; the
acid, nucleic acid. While Miescher had previously isolated " nuclein "
from pus cells, its demonstration lacked the clearness which character-
izes his discovery of protein-free nucleic acid in the spermatozoa heads,
and does not admit of the corollary that nucleic acid is formed in the
body from the decomposition products of protein.
Miescher' s work has been experimentally tested many times, and
has been submitted to the closest critical examination, only to be
found without a flaw (Schmiedeberg : ). He was for-
tunate enough or wise enough to take advantage of the rare opportun-
ity for scientific investigation which was afforded by the Rhine salmon,
and became at a stroke the discoverer of both protamine and nucleic acid.
Miescher came close to another discovery of no less importance
than the isolation of nucleic acid from cell nuclei. Upon warming a
specimen of protamine with nitric acid a yellow spot was formed which
changed to bright red when moistened with alkali. Appreciating the
significance of the reaction, Miescher [1874, 2 ] asked Piccard to make
an examination of salmon sperm for purine bases. Piccard 
made successive extractions of salmon spermatozoa with hydrochloric
acid of increasing strength. The first extract contained only pro-
tamine ; but the final extract, made with boiling acid, produced the
well-known gelatinous purine precipitate with silver nitrate in am-
monia and was found to contain considerable quantities of guanine
and hypoxanthine. Considering the analytical methods at his disposal,
Piccard's results are admirable ; guanine was correct, and adenine, at
that time unknown, was always mistaken for hypoxanthine. But un-
fortunately Piccard added that the composition of salmon sperm as
given by Miescher must be revised to include guanine and hypoxanthine,
which are to be ascribed partly to nuclein and partly to protein. He,
4 NUCLEIC ACIDS
had evidently forgotten that Miescher made his colour reaction with
a substance that was free from protein, and the discovery of the
"alloxuric bases" was therefore left for another.
Kossel observed that purine derivatives are always formed from
nucleins when these substances are submitted to the action of hydro-
lytic agents, and he understood clearly that the bases originated from
the " prosthetic group " (nucleic acid) and not from the protein of the
nuclein. He first found hypoxanthine  and a trace of xanthine
, then guanine : [1883-84], and finally adenine :
[1888, i]. It should be stated that nucleic acids by hydrolysis produce
only the two amitw-purines, guanine and adenine, and that the oxy-
purines formerly observed were always secondary products from these.
As Kossel had not yet discovered adenine it is but natural that he
should have mistaken the substance for hypoxanthine, while the trace
of xanthine which he sometimes found was probably a laboratory
product formed from guanine in the execution of the Neubauer method
 which was employed. Kossel's priority, however, is not in the
least in question, for he himself soon found suitable methods of separat-
ing and identifying the bases (Schindler , Bruhns ).
Kossel's discovery of the "alloxuric bases" was of the utmost im-
1. It gave character to nucleic acid and furnished a definition of
the substance by which it could be distinguished from proteins and
other constituents of the body.
2. It made possible a study of the distribution of nucleic acid in
the body without actually separating the cell nucleus from the proto-
plasm (Kossel [i 88 1] : [1882-83]).
3. It furnished a method of distinguishing true nucleins from
pseudo-nucleins. A substance of the latter class had been prepared
from milk by Lubavin  and another from egg-yolk by Bunge
. These pseudo-nucleins resemble the true nucleins in that they
contain phosphorus and are resistant to the action of pepsin, but they
are not constituents of the cell nucleus nor do they yield purine bases
4. It furnished the refutation (Kossel ) f tne persistent
claims of Liebermann [1888, i]: [1888, 2]: , Pohl , and
Malfatti , that nucleins are merely compounds of protein with
5. It suggested in no uncertain way a chemical connexion between
the cell nucleus and urinary uric acid, and was thus the foundation of
many fruitful investigations in experimental medicine.
But the relation of nuclein to protein remains to be considered.
The presence of a salt, protamine nucleate, in the metamorphosed
nucleus suggests the presence of protein nucleate in the original nuc-
leus ; or that nuclein is simply a salt of protein and nucleic acid. 1 This
assumption cannot be definitely proven, but is supported by a large
amount of evidence, and any other conclusion leads to complication
1. Nucleic acids are polybasic acids and proteins are polyacid
bases, so that a large number of salts of the two substances are to be
expected. One of these salts will have a greater resistance to pepsin
than the others, and will be formed as an end product of the action of
pepsin upon any other salt having a greater proportion of protein in
2. The nucleins are not entirely unaltered by pepsin, they are only
somewhat resistant to its action (Umber ).
3. Artificial nucleins resistant to the action of pepsin are formed
as immediate precipitates when faintly acid solutions of protein and
nucleic acid are brought together (Milroy ).
4. Brief contact of nucleins with dilute caustic soda renders the
protein part precipitable by acetic acid (Altmann ).
5. From solutions of nuclein, picric acid precipitates the protein,
leaving the nucleic acid in solution (Levene ).
6. The presence of protein is apparently without influence upon
the decomposition of nucleic acid by the action of ferments.
7. A solution containing both nucleic acid and protein has an
optical rotation equal to the algebraic sum of the rotations of the two
constituents (Osborne ).
In the year 1889 Altmann  described a method of preparing
protein-free nucleic acids from animal tissues and from yeast, when
the interest which had previously been taken in nuclein began to
decrease. Subsequently, Kossel and Neumann [1894, i] devised a
method of preparing nucleic acid from the thymus gland. Under the
most favourable conditions their procedure gives only passable results
and requires the greatest care in its execution, but it furnished Kossel
and Neumann the material for their wonderful researches on nucleic
acid and made Kossel considerably less interested in nuclein. Finally
Neumann  showed how nucleic acid can easily be prepared
from all of its common sources except yeast (Kowalevsky )
and in such quantity as to place the substance within easy reach of
1 Like the heads offish spermatozoa, the tubercle bacillus contains protamine nucleate
6 NUCLEIC ACIDS
every one. From this time it is difficult to find a serious reference to
"nuclein" in the literature. Wherever the term is used it is em-
ployed as the adjective for nucleic acid. (The "nuclein metabolism"
means the metabolism of nucleic acid.)
Questions relative to nucleoprotein become greatly simplified if
one distinguishes sharply between two classes of these substances
which are called a- and /3-nucleoproteins. The a-nucleoproteins
are prepared from cloudy aqueous gland extracts by precipitation
with acetic acid. The substances thus precipitated can be gotten
into an emulsion with very dilute caustic soda, and by alternate pre-
cipitation with acetic acid and solution in alkali the product can
in a measure be "purified". In this way Wooldridge : 
prepared substances which produced intravascular clotting when in-
jected into animals j 1 but Halliburton and Brodie [1894-95] observed
that this physiological property was lost when the substances were
repeatedly submitted to the process of purification. On the other
hand, Martin  nas shown that snake venom can produce
intra-vascular clotting although it is in no way chemically related
to nucleoprotein. The method of preparing nucleoproteins is so
crude that one is not inclined to attribute their individual pro-
perties to the substances themselves but rather to the impurities
which they contain. 2 Various a-nucleoproteins have been prepared
by Halliburton : : , Gourlay , Halliburton
and Brodie [1894-95], Forrest , Umber , Gamgee and
Jones , Jones and Whipple , and others.
While all of these a-nucleoproteins are of importance only in so
far as they contain nucleic acid, a special interest has attached to the
closely studied nucleoprotein of the thymus. While engaged in a
study of the part played by leucocytes in blood coagulation, Lilien-
feld [1892, i] found that the leucocytes of the thymus contain a
peculiar nucleoprotein which he called leuco-nucleo-histone. By
treatment with acetic acid the substance lost histone and leuco-nuclein
was formed, which in turn could be split into protein and nucleic acid.
These changes were represented thus :
1 Due simply to the thrombase entrapped in them.