Molecular Disease and Evolution

by
Linus Pauling

(Rudolf Virchow Lecture, 5 November 1962)

The universe is made up of aatter and radiant energy. The human body is
made up of molecules ~ molecules of all sorts; Little molecules, such ac the
water molecule, consisting of only three atoms - a very important molecule,
which 1s present in larger mumbers than any cther in the human body; larger
molecules, of medium size, such as those that constitute the vitamins: and
many very large golecules, protein molecules, polysaccharide molecules, nu~
cleic acid molecules.

I believe that it is likely that a huuan being mamifactures 50,000 or
LOU ,000 different kinds of protein molecules. A representative protein uole-
cule, such as hemoglobin, is built of about 10,000 atoms. It has a well de-
fined structure; for most of the protein molecules not a single atom is out
of place.

The protein molecules of different kinds are manufactured by gensa, which
are themselves molecules of deoxyribonucleic acid. Bach one of use inherits
half of his complement of genes, approximately 50,000 from his father, and the
other half, approximately 50,000, from his mother. It is these molecules,
100,000 molecules of DNA, that make the Guman being what he is, that confer
his characters upon hin.

These are the most important molecules in the world. The pool of human
gere plasm is a precious heritage of the human race.

A few years ago it was diacovered that some diseases are molecular diseases,
diseases of protein molecules. A gene, a volecule of deoxyribonucleic acid,
may be damaged by cosmic radiation or some other mutagenic agent in such « way
that a few atoms are out of place. This gene then duplicates itself in its new,
mitated, form. Moreover, when it serves its other function, the function other
than self-duplication, it determines the nature of a protein molecule, which it
has the responaibllity of manufacturing. A miteted gene produces an altered
protein molecule, with a few atoms different from the corresponding normal pro-
tein molecule.

Molecular disease is closely connected with evolution. The appearance of
the concept of good and evil that was interpreted by Man as his painful expulsion
from Paradise probably wes a molecular disease that turned out to be evolution.

Among the molecular diseases there are many that involve enzymes. For ex-
auple, the disease phenylketonuria, which ia responsible for 1% of the institu-
tionalized mentally defective individuals in the United States, is a simple mole-
cular disease that is reesonabiy well understood. One person in elbhty has an
~2D~

abnormal gene that is called the gene for phenylketonuria. A normal person has
two genes that manufacture, independently of one another, ean enzyme is the liver
that catalyzes the oxidation of phenylalanine to tyrosine. This is a mechanism
for converting part of the phenylalanine in our food, which is present in excess
over our need, into another amino acid, tyrosine, which is then used in various
ways in the human body. One person in eighty has one normal gene, which mam-
factures this enzyme, and one abnormal gene (the gene for phenylketonuria), which
does not manufacture the enzyme, or which manufactures an abnormal enzyme mole-
cule that is lacking in enzyme activity.

These people, the carriers of a single gene for phenylketonuria, manufactwre
only 50% as mach of the enzyme as normal individuals; but this 50% is enough to
take care of the phenylalanine that they ingest. They are called phenyl ketonuric

heterogygotes. They are not damaged significantly by carrying the gene in single
dose.

However, when two of these heterozygotes marry one another there occurs the
great lottery, the greatest of all lotteries in the world, in which the prospec-
tive child, the fertilized ovum, carries out the selection of one or the other of
the pair of genes that the father has and of one or the other of the pair of genes
that the mother has. On the average, a quarter of the children inherit the de-
fective gene from the father and also the defective gene from the mother. They
have the defective gene in double dose, and they manufacture none of the enzyme
that catalyses the oxidation of the phenylalanine to tyrosine, When such a homo-
zygote eats his food, containing ordinary protein, the phenylalanine builds up
in his blood stream and cerebrospinal fluid to concentrations es great as fifty
times that in normal individuals. This high concentration of phenylalenine and
of other substances made from it interferes with the growth and function of the
brain in such a way as to cause him to be mentally defective y perhaps with an
I.q. a8 low as 20. In addition, the phenylketonuria genes in double dose cause
him to have severe eczema and other somatic difficulties,

It has been recognized in recent years that it is possible to treat this dis-
ease, phenylketomiria. A diagnosis of the disease may be made at an age a8 early
as one month, and the infant then may be fed a diet of protein hydrolysate fron
which most of the phenylalanine has been removed. Children treated in this way
seem to develop in an essentially normal manner.

Many molecular diseases that have arisen in the course of evolution have
been controlled in a somewhat similar manner. Human beings require many vitanins.
Pellagra is an example of a vitamin deficiency disease - a molecular disease that
originated through a mutation, perhaps millions of years ago, and was then cured
by the heterotrophic process of eating other organiams that manufacture the vita-
min. Scurvy and other avitaminoses are also diseases of thia sort. It is not
customary for us to admit that we have these diseases, because we treat them as
@ matter of habit by eating what is called a proper diet.

Organisms such as the red bread mould are able to manufacture not only all
~3-

of the vitamins, but aleo all of the axino acids. At some tise in our evolu-
tionary history we suffered mitations that resulted in the loss of oux power
to mamifacture the verious enzymes involved in these syntheses. Each of thase
mutations produced in qur predecessors a disease ~ one disease for each vita-
min that we now require, and one disease for each of the nine amino acids that
ate esgential for man. Most of us keep these diseases under control by inges-
ting the proper food.

I have been especially interested in the hemoglobinepathies, which are the
diseases, Including sickle-cell anemia, to which the name molecular disease vas
first applied. I remeber very well the tinue, sane fifteen years ago, when
three of ny students - Dr. Harvey Itens, ie. 3. 7. Singer, and Dr. I, ¢. Wells,
carried out the crucial saperiment that showed that sickle-cell anemia. ia a
disease of the hemoglobin molecule. i had made thie prediction three years
earlier, and Dr. Itano had worked for three years, toward the end with Dr.
Singer and Or. Wells, to test it.

Patients with aickle-cell anamia are anemic because their red cells tend
to twist out of shape. These deformed celis are then recognised by the spleen
a6 abnormal, and are destrayed so rapidly as to make 1t impossible for the
patient to manufacture new erythrocytes fast enough to prevent anemia from de~
veloping. Moreover, the deformed cells are sticky; they clamp on te one another
and clog up the capillaries in such a way as to interfere with the flow of blood
ami thus to cause different organs of the body to be damaged by anoxia. This
disease, involving deformmtion of the red cell, might seem to be a classic dis-
ease of cella, as deacribed by Rudolf Virchow. However, the fact that the cells
sickle only in the venous circulation and regain their normal shape in the arter-
jal circulation seemed to me, in 1945, to provide very strong indication that
the disease is in fact a disease of the hemoglobin molecule, which ia preaent aa
hemoglobin in the venous bloed and as a different unlecule, oxyhennglobin, in arter-
dai blood.

We all know that protein aalecules tend to be aticky ~ it is hard for a pro-
tein molecule to keep fram being sticky. If a solution of protein molec Les,
mamifactured by some living organiam and selected by the evolutionary proveas
of trial and error so ag not to be sticky, but to remain in solution inetead of
forming an insoluable coagulum, is disturbed a bit by warming, even to as low 4
temperature as 60°C, go that the moleculea become slightly unfolded (denatured),
than the characteristic property of stickiness makes iteelf evident; the dena~
tured protein molecules clamp onto one another, to form an insoluble coagulun
of denatured protein. Tt need not surprise us that, although the noraal hemo--
globin molecules, selected. by the evolutionary process, are able to remain aep-
arated from one another even in the concentrated solution (30% protein) that is
inside the red cell, « change in structure reeulting from a gene uutation may
Cause the altered henoglabin molecule to have a sticky region on its surface,
euch ae to make it tend to clamp onte another one, which would clamp onte a third
ong, & fourth one, and so on, to form a long rod of these molecules. These rods
would then line up side by side, attracted by the Van der Waals forces of attrac-
tion, to form a sort of needle-like crystal that would grow longer and Longer
oh.

until, es 1t became longer than the diameter of the red cell, it would twist
the red celi cut of shape, and would deform the red cell membrane, making it
sticky and causing the red cells to get tangled up with one another in the
Capillaries and causing the spleen to destroy these red cells, and thus pro-
duce the manifestation of the disease. We accordingly have a molecular explana~
tion of the manifestations of the disease, based upon the hypothesis that is a
disease of the hemoglobin molecule, a molecular disease in which the abnormal
molecule is manufactured by a mitated gene. We can alec understand that the
molecules of exihemoglobin, molecules of hemoglobin to which molecules of oxygen
molecules to interfer with the Van der Waals forces of attraction and thus to
prevent the sickling of the red cella in the arterial circulation.

The incidence of the gene for phenylketonuria ie small enough to permit it
to be explained as the result of a study state determined by the rate at which
new genes for phenylketonuria are produced by mutation and the rate at which the
phenylketonuria genes are removed from the pool of human germ plasu by the death
without propeny of the phenylketonuria bhomoaygotes. But the incidence of the
gene for sickle~sell heaoglobin is much too great to be explained in this way.

It was recognised that the sickle-cell gene mst carry some advantageous charac~
ter, to compensate the disadvantage of death of the sickle-cell homosygotes with-
out propeny. The suggestion was made by Dr. Russell Brain that the hetervunygotes,
carrying one sickle-cell gene, are protected against malaria ~ he had noticed that
there is a higher incidence of sickling in villages in Africa where malaria is
enienic than in other villages, where malaria is not endemic. Dr. Anthony Alli-
son, of Oxford, then carried out an experiment that provided good evidence that
the sickle-cell heterosygotes are protected against aalignant subtertian malaria
(Plesmodium falctparus). We can accordingly understand why the sickle-cell gene
spread in the Africian population. A heterozygote, carrying a sickle-cell gene
newly formed through sutation, was protected against malaria. Half of his children
inherited the sickle-cell gene, and, because of their protection against malaria,
they helped in rapidly spresding the game through the population. Finally, the
incidence of the gene approached the steady-state value. In marriages between
heterozygotes, who would then make up a large fraction of the population, one
quarter of the children would inherit two normal genes for hemoglobin, and would,
in large part, die of malaria; one quarter would inherit two sickle-cell genes,
~§-

and would die of sickle-cell anemia; but one half would be heterozygotes, Like
their parents, and would be protected against malaria and would not have the
disease sickle-cell anemia. This process gives a yield of only fifty percent
in children, but only recently hae the yield of fifty percent been thought to
be unsatlefactary.

The next step in the process should be a mutation that would mamfacture a
kind of hemoglobin auch that in the homozygous state it would provide protection
against malaria and would not produce a disease such as sickle-cell anenta. This
newly mtated gene could spread rapidly through the population, provided that the
double heterozygotes, in the new gene and in the sickle-cell gene, were aldo
protected against salaria and did not have « serious disease. It seems not un-
likely that another knowa form of abnormal hemoglobin, hemoglobin C, represents
a step in this direction.

Gince the discovery of sickle-cell anemia hemoglobin 14 years ago some scores
of other abnormal human hemoglobins have been discovered. These abnormal hemo-
globing are associated with many different diseases.

The nature of the difference between sickle-cell-anemia hemoglobin (hemoglo-
bin 8) and normal adult bumen hemoglobin (hemoglobin A) has now been discovered,
through the efforts principally of Vernon M. Ingram and his collaborators. In-
mediately after the discovery of hemoglobin 5S, Dr. Walter A. Schroeder and his
associates in the California Institute of Technology made an amino-ecld analysia
of hemoglobin 3 and hemoglobin A. They were able to report that the amino-acid
composition of the two hemoglobins {a closely similar, with no amino-acid repre-
sented by a difference of more than two residues, Ingram then developed a new
and powerful way of investigating the structure of hemoglobin molecules, which
he called the fingerprint method - it 1s also called the peptide-pattern method.
The sample of hemoglobin is split into peptides by the proteolytic action of an
enzyme, such as trypsin. About twenty-six peptides, containing on the average
about twelve amino-acid residues each, are obtained in the mixture produced in
this way. The mixture is then separated into the constitutent peptides by a two~
dimensional process carried out on a sheet of fliter paper. The separation on
the basis of mobility in an electric field is carried out along the horizontal
axis of the sheet of filter paper, and then separation by the chromatographic
method, involving a flowing solvent, is carried out in the vertical dtrection.
a

In thia way Ingram was able to show that hemoglobin 5 differs fran hemoglobin
A only in the replacement of a single amino-acid residue in one-half of the
hemoglobin molecule vy the residue of angther amino acid.

Schroeder and his associates in Pasadena found that hemoglobin molecules
usually consist of four polypeptide chaias, two of one kind and two of anather
Kind. The normal adilt human hemoglobin molecule contains two alpha cheins,
which have the sequence val-leu-ser-pro-ala... (total sl residues), measured
from the free-amino end, and two beta chains, which have the sequence val-his-
leu-~thr-pro-glu-... (total 146 residues). Ingram and Schroeder found that the
alpha chains of hemoglobin & are the same as those of hemoglobin A, but the
beta chains are different: the beta chain of hemoglobin 3 has valine in the
sixth position, in place of glutamate; the other 145 residues are the sane.

The amino-acids sequence has been determined for many other abnormal hemo--
élobins. For every one of these studied so far, the mutation involves only a
single amino-acid residue. Thus, for hewoglobin C there ie lysine in the sixth
reéiiue of the beta chain, in place of the glutamate of hemoglobin A or the
Valine of heuoglobin 3. For hemoglobin § there ts lysine in the twenty-sixth
position, in place of glutamate.

Other abnormal hemoglobins involve an abnormality in the alpie. chain,
rather than the beta chain. An interesting exauple is hemoglobin Mo ton”
the alpha chain of normal adult hemoglobin the $&th residue is pAistidine. tate
residue of histidine ig know to be close to the tron atom of the name group.
Histidine usually carries a positive charge, because of the attachumt of ¢
proton to the imidazole ring. In nemoglobin Meoston the alpha chain has tyrosine
in the 58th position, in place of histidine. Because the tyrosine residue does
not carry a positive charge, we may expect that it would be easter for the iron
atom of the heme group to assume an extra positive charge, leading to a ferrt
heme group, combeining tripesitive tron, in place of the normal ferro heme, con-
taining bipositive iron. The presence of tripositive iron in heumglobin con-
verte it into ferrihenoglocin (also called methemoglobin); and the carriers of
the gene for hemoglobin 4, do in fact have a disease, a foru of mrbhenc-
gloolinenenta.

Accordingly in this disease, as in sickle-cell anemia, the know difference
in smino-acid sequence of the abnormal hemoglobin provides a reasonable explana-
~T-

tion of the manifestations of the disease produced by the molecular abnormality.

Some interesting conclusions about the process of evolution have been
reached on the basis of the comparison of amino-acid sequence of hesoglobin
molecules of animals of different species, carried out especially by my colla-
berator Dr. Eaile Zuckerkandl. It has been found that the peptide pattern of
hemoglobins of animals of different species can be correlated reasonably well
with the generally accepted ideas about evolutionary relationships between the
apecies. For exeuple, the peptide patterns of gorilla hemoglobin and chimpan-
see hemoglobin are almost identical with these of human hemoglobin. The peptide
pattern of Rhesus monkey hemoglobin is somewhat different from that of iunan
hemoglobin. Still greeter differences frou human hemoglobin are shown by the
patterns of cow hemoglobin, horse hemoglobin, pig hemoglobin, and the hesoglobings
of other mammals. The differences are greater still for fish hemoglobin and
woru hemoglobin.

A detailed study of horse hemoglobin nas shown that the alpha chains differ
from those of human hemogiobin by about 15 amino-acid substitutions, as do also
the beta chains of the two hemoglobins. If we accept 130,000,000 years as the
time that has passed aince the evolutionary lines of horse and human separated,
as estinated by paleontologists, we conclude that each chain has on the average
suffered an evolutionarily effective mutation every 14.5 million years. We may
then use this value to discuss other evolutionary epochs.

The goriille alphs chain and the human alpha chain differ in two residues,
and the gorilla beta chain and human beta chain differ in one; the average, 1.5,
indicates that about 11 uiliton years has gone by since the derivation of theae
chains from their comson chain ancestor ~ that is, that the evolutionary Lines
leading to the present-day gorillas and present-day human beings separated from
one another about 11 million years ago. The estimates made by paleontologists
for this epoch range from 10 million to 35 million years.

Another interesting question ig that of the biechemical 4ifferences between
adult tuman beings and human fetuses. The numan fetus mamifactures a special
kind of hemogichin, called hemoglobin F. In hemoglobin F the beta chains are
abaormal. These abnormal beta chains, which are called gamsa chains, differ
from adult human beta in 36 of the 146 amino-acid residues. Accordingly we cal-
~g~

culate, assuming that there has been a constant rate of evolutionarily effec-
tive mitation, that the gauna chains and the beta chains separated fron ane
another about 260 million years ago; that is, at the begiming of the Carboal-
Lferous period.

This epoch was, of course, long before human beings had cose inte exis-
tence. Other mawuale also have fetal elebine differing from the adult hemo~
g@icbin for the species, and we might conclude that the fetal forma of different
macmels separated from the adult foras some 260 aillion years ago, and in 4
sense coastitute a group of species different trom the adult group. With ree-
pect to hemoglobin, a human fetus reseubles a fetal horse more closely than a
human adult.

I believe that it will be possible, through the detailed determination of
auino-acid sequences of hemoglobin molecules and of other mlecules, to obtain
mch information about the course of the evolutionary process, and to illuminate
the question of the origin of species.

Moreover, I believe that the continued atudy of the molecular structure of
the human body and the nature of molecular disease will provide information that
will contribute to the control of disease and will significantly diminish the
amount of huuan suffering. Molecular biology and molecular medicine are new
fields of science that can be greatly developed for the benefit of mankind.

 
References

L. Pauling, H. A. Itano, 3. J. Ginger, and I. C. Wells, “Sickle Cell Aneala,
& Molecular Disease," Science 110, 543 (1949).

L. Pauling, "Abnormality of Hemoglobin Molecules in Hereditary Hemolytic
Anenies", The Harvey Lectures. XOL, 216, Academic Presa Inc., 1955.

. Zuckerkandl and L. Pauling, vee Disease, Evolution, and Gente Hetero-
geneity," Bord: « 189, Academic Press, Inc., 1962.

 

¥. M. Ingram, "fhe Hemoglobins in Genetics and Evolution,” Colusbia Univer-
nity Press, 1963.