Abnormality of Hemoglobin Molecules
in Hereditary Hemolytic Anemias

ABNORMALITY OF HEMOGLOBIN MOLECULES IN
    HEREDITARY HEMOLYTIC ANEMIAS*
LINUS PAULING
Profeuor of Chemistry, California Institrdte of Technology,
       Pasadena, California
T WENTY years ago, after having worked for a decade on the
determination of the structure of relatively simple inorganic
and organic molecules, I became interested in hemoglobin. This
interest arose from the consideration of the structural origin of the
sigmoid oxygen equilibrium curve.l It was soon extended to include
the denaturation of hemoglobin and other proteins' and the mag-
netic properties of hemoglobin and its derivatives.3-11 The study of
magnetic properties has been especially fruitful in providing in-
formation about the nature of the bonds formed by the iron atoms
in hemoglobin with the neighboring atoms of the porphyrin ring
system, the globin, and attached molecules such as the oxygen
mo1ecule.3J*-1*
The discovery of the abnormal hemoglobins was the result of
the consideration of hypothetical molecular mechanisms of the
disease. In the spring of 1945 I, together with eight men from
medical schools of the country, was serving as a member of the
Medical Advisory Committee which assisted in the preparation of
the Bush Report. I6 One evening Dr. William B. Castle, Professor
of Medicine in Harvard University, mentioned to the other mem-
bers of the Committee the disease sickle-cell anemia, with which
he had had some experience. He told about the discovery of the
disease by Dr. J. B. Herrick, in 1910,1 and described the character-
istic change in shape of the red corpusdes and the effect of oxygen
in preventing the sickling and of carbon dioxide in accelerating it.
I suggested that the action of carbon dioxide was to accelerate the
dissociation of oxygen from oxyhemoglobin, through the Bohr-
HasselbaIch effect (it had in fact been clearly stated by Hahn and
Gillespie1B in 1927 that sickling occurs only when the partial pres-
* Lecture delivued April 29, 1954.
                     216

ABNORMALITY OF HEMOGLOBIN MOLECULES IN ANEMIA   217
sure of oxygen is small), and I pointed out that the relation of
sickling to the presence of oxygen clearly indicated that the hemo-
globin molecules in the red cell are involved in the phenomenon
of sickling, and that the difference between sickle-cell-anemia red
CO~JUS&S and normal red corpuscles could be explained by postu-
lating that the former contain an abnormal kind of hemoglobin,
which when deoxygenated has the power of combining with itself
into long rigid rods, which then twist the red cell out of shape.
The opportunity to test this idea arose when Dr. Harvey A. Itano
came to the California Institute of Technology, in the fall of 1946.
He had been a student of Professor Edward A. Doisy, of St. Louis
University SchooI of Medicine, where Dr. Itano had received his
M.D. degree in 1945. Dr. Doisy suggested that he work with me,
and the opportunity for doing so arose in the course of his year as
an intern, when he was awarded an American Chemical Society
Predoctoral Fellowship in Chemistry, for the three years 1946 to
1949. In a letter to Dr. Itano I suggested that he investigate the
hemoglobin from the red cells of sickle-cell-anemia patients, in
order to see whether it was different from normal adult human
hemoglobin. On his arrival in Pasadena in September, 1946, he
began this investigation. He verified the published report+* that
carbonmonoxyhemoglobin, like oxyhemoglobin, prevents sickling
of the red cells, and found that some other hemoglobin derivatives,
including alkyl isocyanide-ferrohemoglobin, ferrihemoglobin,
ferrihemoglobin azide, and ferrihemoglobin cyanide similarly pre-
vent sickling. He developed a rapid diagnostic test for sickle-cell
anemia and sickle-cell trait, based on the use of a chemical reducing
agent.lg Most of the properties of the hemoglobin from the blood
of sickle-cell-anemia patients were found to be the same, to within
the error of determination, as those of hemoglobin from normal
individuals, but it was finally clearly shown, by careful measure-
ment of electrophoretic mobility, that the blood of the patients
contains nearly 100 per cent of an abnormal hemoglobin, differing
from normal adult human hemoglobin, and that the blood of the
parents of patients contains an approximately half-and-half mixture
of the abnormal hemoglobin and normal adult human hemo-
globin. This electrophoretic work was carried out with the col-
laboration of Dr. S. J. Singer and Dr. Ibert C. Wells.

218                LINUS PAULING
THE INHERITANCE OF SICKLE-CELL ANEMIA
The electrophoretic patterns reported in the first publicatiorP
are shown in Pig. 1. They were made by electrophoresis for 20
hours at a potential gradient of 4.73 volts per centimeter of solu-
tions of carbonmonoxyhemoglobins in phosphate buffer of 0.1
ionic strength and pH 6.90. The peaks ti and b, representing
normal hemoglobin and hemoglobin from the red cells of patients
with sickle-cell anemia, are single peaks, corresponding in each
(a) Normal          (cl Sickle - call trait
      (b) Sickle -cell anemia   (d) Mixlure of (a) and (b)
FIG. 1. Longsworth scanning diagrams of carbonmonoxyhemoglobin in
phosphate buffer of 0.1 ionic strength and pH 6.90, taken after 20 hours
electtophoresis at a potential gradient of 4.73 volts pu cc&meter.
case to an electrophoretically homogeneous material. The electro-
phoretic mobilities are different for the two hemoglobins; in fact,
at this pH the mokxules of normal hemoglobin move toward the
anode, showing that they have a negative electric charge, and those
of sickle-cell-anemia hemoglobin move toward the cathode, show-
ing that they have a positive charge. The isoelectric points in phos-
phate buffer of ionic strength 0.1 were found to be 6.87 (in pH
units) for normal adult human carbonmonoxyhemoglobin and 7.09
for sickle-cell-anemia hemogiobin. The difference between these
values is nearly the same as that between the observed values 6.68
for normal ferrohemoglobin and 6.91 for sickle-cell-anemia ferro-
hemoglobin.

ABNORMALITY OF HEMOGLOBIN MOLECULES IN ANEMIA  219
The electrophoretic diagram of a solution containing a mixture
of normal carbonmonoxyhemoglobin and sickle-cell-anemia car-
bonmonoxyhemoglobin, in equal amounts, is shown as d in Fig. 1,
and that of hemoglobin from the red cells of a parent of a patient
is shown as c. The blood from which this hemoglobin was obtained
showed the characteristic properties of sickle-cell trait (sicklemia) ;
the cells could be made to sickle, on removal of oxygen, but less
readily than the cells of a sickle-cell-anemia patient. It is seen that
the sicklemic hemoglobin is a mixhue of two hemoglobins, pre-
sumed to be normal adult human hemoglobin and sickle-cell-
anemia hemoglobin, with the normal hemoglobin present in an
amount somewhat greater than 50 per cent.
The indication of a genetic basis for the sickling of erythrocytes
had been recognized by Emmel" in 1917; and Taliaferro and
Huck,** at a time when the distinction between sicklemia and
sickle-cell anemia was not cleady understood, suggested that a
single dominant gene was involved. The inheritance of sickle-cell
disease was then clarified by Neel, who in 1947" had suggested
"that there is present in the colored population a certain factor
which, when heteroygous, may have no discernible effect, but
usually results in sickling, and, when homozygous, tends to result
in sickle cell anemia." In 1949 he reported" that every one of 42
tested parents of children with sickle-cell anemia was found to be
sicklemic, their blood containing red cells which could be made to
sickle, though less readily than that of the sickle-cell-anemia pa-
tients. He concluded that sickle-cell anemia is the result of the
homozygous condition of the sickle-cell gene, and sicklemia the
result of the heterozygous condition. Beet'" arrived at the same
conclusion independently and almost simultaneously. The electro-
phoretic patterns shown in Fig. 1 had permitted this inference
to be drawn before Neel's paper and Beet's paper were published.
Moreover, the gene responsible for the sickling process could be
identified with an alternative pair of alleles of which neither one is
recessive or dominant, one allele being responsible for a part of the
process of manufacture of normal adult human hemoglobin, and
the other for the manufacture of sickle-cell-anemia hemoglobin.
The fact that all the red cells of a sicklemic individual can be made
to sickle by removal of oxygen shows that the cells are not of two

2.20                LINUS PAULING
classes, one containing normal hemoglobin and the other abnormal
hemoglobin, but that each cell contains a mixture of the two kinds
of hemoglobin. The presence of a larger amount of normal than
of abnormal hemoglobin in the blood of sicklemic individuals
indicates that the process of manufacture of the abnormal hemo-
globin is somewhat less efficient than that of normal hemoglobin.
It was suggested in the first paper on sickle-cell-anemia hemo-
globiP that the two genes in the heterozygous individual might
compete for a common substrate in the synthesis of two different
enzymes essential to the production of the two different hemo-
globins, or that competition for a common substrate might occur
at a later stage in the series of reactions leading to the synthesis of
the two hemoglobins themselves.
An investigation of the amount of abnormal hemoglobin in the
blood of sicklemic individuals was carried out by Wells and
Itan0,2B who found, using the electrophoretic method, that the
amount of sickle-cell-anemia hemoglobin varied from 24 per cent
to 45 per cent in 42 individuals with sicklemia. Neel, Wells, and
Itano*' reported a study of 32 sicklemic individuals who were mem-
bers of 7 Negro families, comprising 74 individuals altogether.
The amounts of abnormal hemoglobin, ranging from 22.3 per
cent to 45.2 per cent, showed significant differences between
family means. A postulate to explain the apparent inheritance of
a factor determining the amount of abnormal hemoglobin in
sicklemic blood was made by Itano.28 He suggested that the differ-
ences can be attributed to differences in the rate of synthesis of
normal hemoglobin, and that the evidence requires that there be
at least three rate-determining modifications of the mechanism of
synthesis of normal hemoglobin.
Additional contributions to the problem of the genetics of nor-
mal and abnormal hemoglobins have been made by Nee12B-a1 and
other workers.aP.38
   THE PROPERTIES OF SICKLE-CELL-ANEMIA HEKJGLOBIN
         AND THE MECHANISM OF SICKLING
Sickle-cell-anemia hemoglobin is closely similar to normal adult
human hemoglobin in most of its properties.zo The two proteins
have approximately the same sedimentation and diffusion constants,

ABNORMALITY OF HEMOGLOBIN MOLECULES IN ANEMIA   221
and hence nearly the same molecular weights. The acid-base titra-
tion curves of both hemoglobins in the neighborhood of neutrality
are linear, a change of 1 pH unit of the solution being associated
with the change in charge of the hemoglobin of about 13 electronic
charges per molecule. The normal molecule has about three more
negative charges than the abnormal molecule in this region. In the
search for the structural basis for this difference samples of
porphyrin dimethyl esters were prepared from the two hemo-
globins, and the samples were shown by their x-ray powder
photographs and by identity of their melting points and mixed
melting points to be identical. The difference in structure was
hence attributed to a difference in the globins.
An investigation by Schroeder, Kay, and We&P of the adno
acid composition of normal adult human hemoglobin (from nor-
mal Negro individuals) and sickle-cell-anemia hemoglobin gave
results indicating that the hemoglobins do not differ with respect
to their content of basic and acidic amino acids; the investigators
concluded that sickle-cell-anemia hemoglobin probably contains
slightly less leucine and more serine than normal hemoglobin, and
possibly less valine and more threonine. These amino acids do not
contribute directly to the net charge of the proteins, but they might
affect the folding or coiling of the polypeptide chains in such a
way as to change the acid or basic constants of other groups.
Havinga'" investigated the phosphorus content, optical rotation,
ease of separation of hemes and globin, and number of terminal
amino acid residues of normal adult human hemoglobin and
sickle-ceil-anemia hemoglobin, and found no significant differences
between the two proteins. Globins carefully prepared from the two
hemoglobins were investigated electrophoretically by Havinga and
Itanos6 and fouad to have the same dtierence in electrophoretic
mobility as the hemoglobins themselves. On denaturation by treat-
ment with 4 N guanidinium chloride for 1 hour at 4O C. and
removal of the guanidinium chloride by dialysis, the globins were
found to have increased markedly in heterogeneity, and to have
essentially the same electrophoretic properties. These results in-
dicate that the normal and abnormal hemoglobin molecules might
be composed of the same polypeptide chains, folded, however, in
different ways, and that on denaturation with guanidinium ion the

222                 LINUS PAULING
resulting denatured proteins have the same complex of conQua-
tions. The interesting possibility exists that the gene responsible. for
the sickle-cell abnormality is one that determines the nature of the
folding of polypeptide chains, rather than their composition.
It was pointed out by Sherman"' in 1940 that sickled red ceils are
observed under the polarizing microscope to be birefringent,
whereas normal cells are optically isotropic. Ponders8 suggested,
on the basis of this observation, that in sickled cells the hemoglobin
molecules assume an orderly or paracrystalline arrangement, which
is responsible for the sickling. A detailed mechanism of the
sickling process was suggested in the first paper on sickle-cell-
anemia hemoglobin,2o as follows: "We can picture the mechanism
of the sickling process in the following way. It is likely that it is
the globins rather than the hemes of the two hemoglobins that are
different. Let us propose that there is a surface region on the globin
of the sickle-cell-anemia hemoglobin molecule which is absent in
the normal molecule and which has a configuration complementary
to a diflerent region of the surface of the hemoglobin molecule.
This situation would be somewhat analogous to that which very
probably exists in antigen-antibody reactions.3n The fact that
sickling occurs only' when the partial pressures of oxygen and
carbon monoxide are low suggests that one of these sites is very
near to the iron atom of one or more of the hemes, and that when
the iron atom is combined with either one of these gases, the com-
plementariness of the two structures is considerably diminished.
Under the appropriate conditions, then, the sickle-cell-anemia
hemoglobin molecules might be capable of interacting with one
another at these sites suf5ciently to cause at least a partial align-
ment of the molecules within the cell, resulting in the erythrocyte's
becoming birefringent, and the cell membrane's being distorted
to accommodate the now relatively rigid structure within its con-
fines. The addition of oxygen or carbon monoxide to the cell might
reverse these effects by disrupting some of the weak bonds between
the hemoglobin molecules in favor of the bonds formed between
gas molecules and iron atoms of the hemes."
A more detailed discussion of the effect of oxygen was made
possible by the results of an investigation of the combination of
hemoglobin with alkyl isocyanides.40 It was found that ethyl iso-

ABNORMALITY OF HEMOGLOBIN MOLECULES IN ANEMIA  223
cyanide, isopropyl isocyanide, and tertiary butyl &cyanide differ
greatly in their combining powers with hemoglobin, although they
have essentially the same combining power with heme; and this
fact was interpreted as showing that the Four hemes in the hemo-
globin molecule are buried within the globin: "Out postulate
provides an obvious explanation of the action of oxygen in pre-
venting the sidding of sickle-cell-anemia erythrocytes. We have
visualized the sickling processZo as one in which complementary
sites on adjacent hemoglobin molecules combine. It was suggested
that erythrocytes containing oxyhemoglobin or carbonmonoxy-
hemoglobin do not sickle because of steric hindrance of the at-
tached oxygen or carbon monoxide molecule. This steric-hindrance
effect might be distortion of the complementary sites through
forcing apart of layers of protein, as is suggested by the isocyanide
experiments."
Substantiation of this picture was soon obtained through micro-
scopic investigations. Rebuck, Sturrock, and Monaghan," sub-
stantiating the work of Sherman,37 observed that in the earIy stages
of sickling the intracellular hemoglobin forms anisotropic aggre-
gates, suggestive of incipient crystallization. Per& and Mitchi-
son,4z at the suggestion of Dr. C. A. Stetson of the Rockefeller
Institute, compared the dichroism of sickled cells and hemoglobin
crystals, and additional studies of the same sort were reported by
Perutz, Liquori, and Eirich.`s These investigators found that the
dichroism of the sickled cells corresponds to an orientation of the
hemoglobin molecules such that the normal to the plane of the
heme groups is perpendicular to the long axis of the crystal needles
and of the sickled cells. (This statement is based on the paper of
Perutz, Liquoti, and Eirich, +43 there is some conflict with the earlier
paper.4a) They also pointed OU~`~,*~ that the solubility of sickle-
cell-anemia hemoglobin is much smaller than that of normal
hemoglobin or of either normal oxyhemoglobin or sickle-cell-
anemia oxyhemoglobin. A detailed study of the solubilities of
mixtures of sickle-cell-anemia hemoglobin and other hemoglobins
has been made by Itano," who has shown that a solubility meas-
urement provides a simple way of determining roughly the amount
of sickle-cell-anemia hemoglobin present in a mixture of hemo-
globins. A most significant investigation was then reported by

LINUS PAULING
FIG. 2. Hemoglobin formed in stroma-free solutions of deoxygenated sickle-
cell-anemia hemoglobin. Phase photomicrography, X 375. From John W.
Harris, Pror. Ser. Exptl. Sol. and Med. 75, 197 (1950).

ABNORMALITY OF HEMOGLOBIN MOLECULES IN ANEMIA   225
FIG. 3. Sickled erythrocytes in oxygen-depleted whole blood from a patient
with sickle-cell anemia, demonstrating the similarities in shape to tactoids
formed in stroma-free solutions of their deoxygenated hemoglobin. Phase
photomicrography, X 373. From John W. Harris, Proc. Sot. &pti. Biol. and
Med. 75, 197 (19~0).

226                LINUS PhULlNG
Harris.46 He showed that a stroma-free solution of sickle-cell-
anemia hemoglobin with concentration 15.2 or 23.5 g. per loo ml.
on deoxygenation forms birefringent spindle-shaped bodies vary-
ing in length from 1 to 15 I(. He identified these bodies as tactoids
(liquid crystals) of the nematic type. His photomicrograph is
shown as Fig. 2, which may be compared with Fig. 3, a similar
photomicrograph (375 x magnification) of sickled erythrocytes.
It hence seems probable that sickle-cell anemia can be described
as a molecular disease, resulting from the difference in molecular
structure of sickle-cell-anemia hemoglobin and normal adult
human hemoglobin. The properties of the abnormal hemoglobin
are such that when deoxygenated the molecules combine with one
another to form long molecular strings, which, through inter-
molecular attraction, aggregate into tactoids. These tactoids have
enough mechanical strength to distort the red cell, changing the
viscosity of the blood, and causing the clinical and pathological
manifestations of the disease.
.+?`he close approximation in structure of sickle-cell-anemia hemo-
globin to normal adult human hemoglobin is strikingly shown by
the antigenic properties, investigated by Goodman and Campbell.4o
They found that, whereas large differences in serological specificity
are shown by human fetal hemoglobin and human adult hemo-
globin, suggesting that only a few antigenic groups are shared in
common by these two hemoglobins, only a small difference in
antigenic specificity could be demonstrated between sickle-cell-
anemia hemoglobin and normal adult human hemoglobin. Anti-
serums obtained by injection of rabbits with these two forms of
adult human hemoglobin showed no differences in antigenic
specificity. The injection of chickens with these two forms of adult
hemoglobin produced antiserums with a significant, though small,
difference in properties, indicating that the two hemoglobins have
a predominance of antigenic groups in common, but that a small
number are different.
The results of an investigation by Ingbar and Kass" of the num-
ber of titratable sulfhydryl groups (two groups per molecule in
normal hemoglobin, and three in sickle-cell-anemia hemoglobin)
may provide an additional clue as to the difference in structure of
the molecules.

ABNORMALITY OF HEMOGLOBIN MOLECULES IN ANEMIA   227
OTHER ABNORMAL HEMOGLOBINS, FETAL HEMOGLOBIN,
       SYNERGY WITH TYALASSEMIA
During the five years since the discovery of sickle-cell-anemia
hemoglobin three other abnormal varieties of adult human hemo-
globin (named hemoglobin C, D, and E, respectively; A represents
normal adult human hemoglobin, and S sickle-cell-anemia hemo-
globin) have been discovered. These abnormal hemoglobins,
either alone or in synergistic interaction with sickle-cell-anemia
hemoglobin or with thalassemia, have been observed in association
with five diseases, which, together with a sixth disease resulting
from the simultaneous existence in the individual of sicklemia and
thalassemia minor, had not previously been recognized as clinical
entities. Another interesting complication in the constitution of the
blood has also been recognized, the continued manufacture of fetal
hemoglobin by anemic individuals.
Hemoglobin C was discovered by Itano and Neel'= and has been
further investigated by Neel,40-"1 Spaet et al.,=' Ranney et al.,5s*s~
and Smith and Conley. 53 The difference in electrophoretic mobility
between hemoglobin C and normai adult human hemoglobin is
nearly twice as great as the difference between sickle-cell-anemia
hemoglobin and normal hemoglobin: the electric charge of hemo-
globin C differs by about 5 electronic charges from that of A.
Hemoglobin C was first found to be present together with S in
the blood of some anemia patients. These patients may be described
as carrying one allele for C and one for S: they are heterozygous in
hemoglobin C and also in hemoglobin S, having inherited one of
the two abnormalities from each parent. On investigation, the red
cells of one parent were found to contain a mixture of A and C,
and of the other parent to contain a mixture of A and S. Dilution
of S with C does not inhibit the sickling tendency so much as
dilution of S with A, and in consequence the individuals of type
SC are anemic, their disease being called sickle-cell : hemoglobin-C
disease. The heterozygous condition in C does not lead to a
pathologic state. Sickle-cell: hemoglobin-C disease can be readily
dzerentiated from sickle-cell anemia on clinical grounds.4B-5S The
characteristic differences in electrophoretic behavior of A, S, and C
are shown in the paper electrophoresis patterns of Fig. 4, repro-

128                LINUS PAULING
duced from the work of Smith and Conley.SJ Under the conditions
of this investigation, which was carried out in Verona1 buffer at
pH 8.6 and ionic strength 0.06, all three hemoglobins are nega-
tively charged, with A having the largest negative charge and C the
smallest charge.
FIG. 4. Paper electrophoresis patterns of hemoglobins. Verona1 buffer, 0.06
ionic strength, pH 8.6; Whatman paper No. 3, 6 X 6 inches, 15 mils, 380
volts. The migration begins at the dotted line; hemoglobin A is the fastest
moving, hemoglobin S has intermediate mobility, and hemoglobin C migrates
most slowly. From Ernest W. Smith and T. Lockard Conley, Bull. Johar
Hopkins Hosp. 93, 94 (1953).
Using paper electrophoresis, Smith and Conley*" made an in-
vestigation of the hemoglobins of 500 white persons and 500
Negroes. In the 500 white persons they found no evidence of the
presence of any hemoglobin differing electrophoretically from nor-
mal adult human hemoglobin. Hemoglobin S was found to occur
in 8.4 per cent of the 500 Negroes surveyed, and hemoglobin C
in 2 per cent. Seven patients with sickle-cell : hemoglobin-C disease
were found among the 500 Negroes.
If the incidence of the heterozygous state AC is 2 per cent, the

ABNORMALITY OF HEMOGLOBIN MOLECULES IN ANEMIA  229
Occurrence of the C allele is 0.01, and it can be predicted that
homozygous hemoglobin-C disease should occur among Negroes
with frequency O.Ol', or one in 10,000. It was reported by Spaet
and co-worker+ and Ranney and co-workers53 that patients with
a mild hemolytic anemia have been found with only hemoglobin C
in their red cells, and it is probable that they are homozygous in
hemoglobin C, and that their disease is homozygous hemoglobiti-C
disease.
An interesting complication in the hemoglobin pattern has been
resolved through the discovery that anemic individuals may con-
tinue to manufacture fetal hemoglobin long after they have passed
the fetaf stage of development. Wells and Itano, in the course of
FIG. 5. Electrophorctic pattern of the hemoglobin of a patient with sickle.
cell:hemoglobin-C disease, showing three hemoglobins, C, S, and F, from left
to right.
their investigation of the ratio of S to A in sicklemic individuals,*"
discovered that there is present in the blood of patients with an
abnormally mild form of sickle-cell anemia a small amount, 5 per
cent to 20 per cent of the total, of a protein other than S. They
identified this protein as A, on the basis of its electrophoretic
pattern, but they pointed out that the patients could be assumed,
from the facts that both parents were sicklemic and that hemo-
globin S predominated, to be homozygous in S, and that the
presence of A was anomalous. Itano and Neelds reported that one
patient with sickle-cell : hemoglobin-C disease gave a hemoglobin
electrophoretic pattern showing S and C in large amounts (39 per
cent and 48 per cent, respectively) and an additional hemoglobin,
13 per cent, with the electrophoretic mobility, at the pH used
(pH 6.50), of A. The electrophoretic pattern of this patient is
given in Fig. 5. It was pointed out by Singer, Chernoff, and Singer56
that there is present in the blood of many patients with sickle-cell

230                LINUS PAULING
                    .>
anemia and other hematologic disorders a hemoglobin fraction
that is resistant to alkali denaturation, and that the properties of
this fraction permit it to be identified with fetal hemoglobin, F;
in particular, these authors suggested that the hemoglobin reported
by Wells and Itano to be present in small amounts in the blood of
some sickle-cell-anemia patients is hemoglobin F, and not A. At
about the same time Liquoris reported that he had found that as
much as 50 per cent of the hemoglobin in the blood of patients
with thalassemia is fetal hemoglobin, and Rich** found that two
patients with thalassemia major had in their red cells no hemo-
globin other than F. It is now recognized that anemic patients may
continue to manufacture significant amounts of F throughout their
lives, and that the presence of F ameliorates their disease. Hemo-
globin F is identified, in relation to A, only with difficulty by elec-
trophoretic methods, but it is easily identified by its resistance to
alkali denaturation and by its characteristic ultraviolet absorption
spectrum. 68-81 Goodman and Campbell" verified the identification
of the minor hemoglobin in patients with sickle-cell anemia by
studying its antigenic properties, which were found to be either
identical with or closely similar to those of fetal hemogiobin.
Thalassemia (Cooley's anemia, Mediterranean anemia) is a
hereditary anemia that results from a gene-controlled interference
with the process of synthesis of adult human hemoglobin. It was
first clearly recognized by Cooley and Lee.ej The disease is largely
confined to persons derived from the northern shores of the
Mediterranean. The thalassemia allele in the homozygous form
produces a very serious anemia, thalassemia major, which usually
terminates fatally in childhood. In heterozygous form the allele
produces a mild anemia, called thaIassemia minor. In some parts of
Italy, notably the region around Ferrara, the incidence of thalas-
semia minor is about 10 per cent, corresponding to a probability
of the allele of 5 per cent8"
In 1953 Powell, Rodarte, and Nee166 reported one case of a
disease due to the combination of thalassemia minor and sicklemia.
The resulting sickle-cell : thalassemia disease is a more serious dis-
ease than thalassemia minor. Other patients with the disease have
been described by Sturgeon, Itano, `and Valentinea' and by NeeI;
ltano and Lawr&ce.R" `A case of thalassemia:hemo@obin-C'disease

ABNORhUL1l-Y OF HEMOGLOBIN MOLECULES IN ANEMIA  :w
is mentioned in a footnote at the end of a paper by Kaplan, Zuelzer,
and NeeLsl
The third abnormal hemoglobin to be discovered, hemogIobin
D, was found by ItanoB* in five members of a single family. Two
of the family were patients with a disease resembling sickle-cell
anemia but somewhat milder than the normal form of this disease.
The electrophoretic patterns of the blood of these patients were
closely similar to those of hemoglobin S. The hemoglobin from the
red cells of the three other individuals gave an electrophoretic
pattern like that obtained from sicklemics. It was found, however,
by measurement of the volubility of the hemoglobins that an ab-
normal hemoglobin is present, with electrophoretic properties like
that of S, and solubility like that of A. This hemoglobin, hemo-
globin D, seems not to interfere with the process of sickling so
much as does A, so that the state of double heterotygosity SD lea&
to a moderately serious anemia: The blood of etih of the two
patients with sickle-cell:hemoglobin-D disease was found to con-
tain a small amount (6 per cent to 12 per cent) of F.
Two individuals containing the fourth abnormal hemoglobin, E,
have been discovered by Itano, Bergren, and Sturgeon. One of them
is an anemic patient with thalassemia: hemoglobin-E disease.`O The
mother of the patient has thalassemia minor, and no hemoglobin
other than A in her red cells.To The red cells of the patient contain
41 per cent F and 59 per cent E. The father of the patient is of
type AE, with 72 per cent A and 28 per cent E (no F) ; he is a
carrier of E (personal communication from Itano, Bergren, and
Sturgeon). The patient has inherited thalassemia minor from the
mother and the allele for E from the father. The thalassemia allele
seems to have completely suppressed the manufacture of A.
Hemoglobin E shows a striking change in electrophoretic mo-
bility with change in pH. At pH 6.5, in cacodylate buffer of ionic
strength 0.1, its mobility is greater than that of A and slightly less
than that of S, and at pH 8.8, in 0.01 F disodium hydrogen phos-
phate, its mobility is nearly identical with that of C. The absorptidn
spectrum, solubility, and lability to alkali denahxation of E are
similar to those of A.
A summary of the hereditary hemolytic anemias related to ab-
normal hemoglobins is presented in Fig. 6. There are five forms of

232                LINUS PAULING
adult human hemoglobin: A, S, C, D, and E. Assuming that these
five correspond to five alleles occupying the same locus in a
chromosome, there are fifteen possible combinations, the five
homotygous states AA, SS, CC, DD, and EE, and the ten het-
erozygous states AS, AC, AD, AE, SC, SD, SE, CD, CE, and DE.
Of these fifteen states nine have been found to occur. In addition
to sickle-cell anemia, six other types of hereditary anemia involving
an abnormality of hemoglobin have been recognited.`l
A m Th'>SS>SD>AS +ThsSC>CC
FIG. 6. A chart representing possible combinations of the alleles A, S, C, D,
and E. The horizontal row at the bottom represents simultaneous occurrence
with thalassemia minor. The amount of fetal hemoglobin usually present is
also indicated: F means a few per cent, F means 10 per cent or more. Ob-
served conditions are shown within heavy borders. At the top the seriousness
of different kinds of anemia is indicated.
The seriousness of these diseases (not including AC + Th and
E + Th, for which the number of patients is too small to permit an
estimate) is indicated at the top of Fig. 6. There is some variability
in the seriousness of the diseases, in part as the result of the extent
to which compensation of abnormal hemoglobins is achieved
through the manufacture of fetal hemoglobin. The mildest anemias
are SC and CC. Not all SC and CC individuals are anemic. Al-
though they probably have a greater than normal rate of hemolysis,
they may be able to compensate completely with a greater than
normal rate of production of red blood cells.52~`2
The conditions CD, DD, SE, CE, DE, EE, and DA + Th have
not yet been observed; it may be found that they are associated
with anemia.

~BNOKMALITY OF HEMOGLOBIN MOLECULES IN ANEMIA  233
Except for the fetal-hemoglobin fraction, the hemoglobin of
patients with thalassemia major has properties reported to be the
same as those of normal adult human hemoglobin,58 and it is USU-
ally considered that thalassemia is not associated with the produc-
tion of an abnormal hemoglobin. There are some facts, however,
that indicate that thalassemia hemoglobin is a fifth abnormal kind
of adult human hemoglobin, with properties so closely similar to
those of normal adult human hemoglobin that the differences have
escaped detection. It has been observed that thalassemia minor in-
volves a greater interference with the synthesis of the normal
hemoglobin than of abnormal hemoglobin. For example, whereas
in sicklemic individuals the ratio A/S is always greater than 1, this
ratio is much less than 1 in thalassemia: sickle-cell disease. Two
patients reported by Neel, Itano, and Lawrences8 were found to
have the ratios A:S:F equal to 20:61:19 and 11:84:5, respec-
tively. The hemoglobin of the patient with thaiassemia: hemo-
globin-E disease contains no detectable amount of normal adult
hemoglobin, but only E and F. The thalassemia allele interferes
with the manufacture of normal adult hemoglobin, and seems not
to interfere seriously with the manufacture of the abnormal hemo-
globins. The simplest explanation of this fact is that the thalassemia
allele occupies the same locus in the chromosome as the alleles for
the other abnormal hemoglobins, and is itself responsible for an
abnormal globin in which the abnormalih/ is of such a nature as to
interfere with the step of inclusion of the hemes in the molecule.
If this postulate is correct it should be possible to show a difference  .
between thalassemia hemoglobin and normal adult human hemo-
globin.
Under this circumstance there would be six alleles occupying the
same locus, A, S, C, D, E, and Th (more than six if there is more
than one thalassemia allele). The six states at the bottom of the
triangle in Fig. 6 should then be written ATh, STh, CTh, DTh,
ETh, and ThTh. Of the possible twenty-one combinations of the
six alleles, fourteen would be ascribed to known individuals.
The possibility that the thalassemia gene is allelomorphic with the
sickle-cell gene has been discussed by Neel."" He pointed out that
both of the children of a patient with thalassemia:sickle-cell dis-
ease and married to a normal woman exhibited thalassemia minor,

234               LINUS PAULING
and mentioned that if either of these children had been normal
(without either thahassemia minor or sickIemia) the hypothesis of
aIlelomorphism would have to be abandoned. Silvestroni and
Bianco, who a number of years ago described the disease resulting
from simultaneous inheritance of thalassemia minor and sick-
lemia,78 have also discussed the genetic aspects of thalassemia and
sickling, and have concluded that the two are not allelomorphic.T4
    SICKLE-CELL-ANEMIA HEMOGLOBIN AND M,+LARM
A number of interesting questions about the origin and heredity
of the abnormal hemoglobins remain to be answered. It has, for
example, been pointed out by LehmannTs that sickle-cell anemia
seems to be a far less serious disease in Africa than in America.
A possible explanation may be that the patients in Africa for some
reason manufacture a larger amount of fetal hemoglobin than those
.h-r America; the question can presumably be answered by a
thorough investigation of the hemoglobins of Africans, which has
not yet been carried out.
The question of the continued high incidence of the sickle-cell
allele, despite its continued loss because of the lethal character of
the homozygous condition, has been raised by Neel,O" who has
suggested three alternative explanations: (1) continued produc-
tion of the allele through mutation; (2) the existence of an ab-
normal genetic mechanism that favors the heterozygous condition,
AS, over the normaI condition, AA; (3) a positive selection of the
heterozygote, perhaps through increased fertility. The first ex-
planation has to be rejected because the rate of mutation that would
be required is far greater than any that has ever been observed for
any organism. There now exists evidence indicating that the third
alternative provides the correct explanation, and that malaria is
involved. It was first suggested by Brainls that the presence of S
in the red cells might give protection against malaria parasites, and
thus confer an advantage to the sicklemic individual that would
balance the disadvantage of the lethal homozygosity. Lehmann"
wrote that "The lethal tendency of a gene potentially causing
sickle cell anemia may thus be counteracted by its conferring a re-
sistance to malaria similar to that found in early infancy." A test
of the hypothesis was carried out recently by Allison,`@ who in-

ABNORMALITY OF HEMOGLOBIN MOLECULES IN ANEMIA  235
fected 1S healthy adult Africans with the sickle-cell trait and 15
similar healthy adult Africans without the sickle-cell trait with
P. fdcipawm, by subinoculation with 15 ml. of blood containing
a large number of trophotoites or by being bitten by heavily in-
fected anopheles mosquitoes, in which the presence of sporozoites
was continued by dissection of the mosquito. The infection was
established in 14 out of the 15 Africans without the sickle-cell
trait, and in only 2 of the 15 with this trait. It was concluded by
Allison that the abnormal erythrocytes of individuals with the
sickle-cell trait are less easily parasitized by P, fdcip~wn than are
normal erythrocytes, and that accordingly those who are het-
crozygous for the S allele have a selective advantage over aormal
individuals in regions where malaria is hyperendemic. It is, of
course, not unreasonable that the abnormal hemoglobin may be
less effective than normal hemoglobin in nourishing the parasites.
The question of the origin of sickle-cell anemia has been in-
vestigated especially by Lehmann, who has studied the incidence of
the ability of red cells to sickle in several parts of the world.Te-s* In
Africa the incidence of the sickle-cell allele is highest in the north
snd east, diminishing somewhat toward the west and south, and
being virtually zero in South Africa itself. In India the majot
groups of the population, classed as Dravidians, show no sickling,
and among the pre-Dravidians living in the hills of Southern
India the Veddoids alone have a high incidence of sickling. A
high incidence of sickling was found also in a Veddoid community
of Southern Arabia, although sickling is absent among the Semitic
Arabs. Some sicklemic individuals are found in Italy, and in Greece
there are a few communities, each of a few hundred individuals,
with a high incidence of sickling. Lehmann concIudeP that it is
not unlikely that the sickle-cell gene originated, presumably
through a mutation, in a Veddoid community in Southern Arabia,
and that it spread from this point to India, the Mediterranean
region, and especially to Africa.
             MOLECULAR DISEASES
Sickle-cell anemia has been described as a mole&u disease.*0 It
may be that all diseases can be described as molecular diseases,
inasmuch as the human body and the vectors of disease are al! com-

236                LINUS PAULING
posed of molecules. For example, carbon monoxide poisoning is
the result of combination of molecules of carbon monoxide with
molecules of hemoglobin. Erythroblastosis fetalis involves the in-
teraction of the molecules or haptenic groups of an Rh antigen on
red cells with molecules of the homologous antibody. An inborn
error of metabolism such as alcaptonuria results from the failure
of the body to manufacture molecules of the enzyme that, if
present, would catalyze the oxidation of homogentisic acid in the
body. Any hereditary disease may be said to be a molecular dis-
ease, if the genes are described as molecules, in that it involves an
abnormality of a gene,
There is, however, one sense of the expression molecular dis-
ease that permits it to be applied to sickle-cell anemia and the other
diseases associated with the abnormal hemoglobins. These diseases
have been shown to result from the manufacture by the patient of
abnormal molecules. in the place of normal molecules which are
manufactured by normal individuals, and the abnormal molecules
have been characterized. I think that it is not unlikely that many
diseases will in the course of time be found to be molecular dis-
eases in this sense. The discovery of the abnormal molecules re-
sponsible for other molecular diseases may be far more ditlicult
than the discovery of the abnormal hemoglobins. Hemoglobin is
unique among the proteins of the human body because of its
presence in very large amount, about I per cent by weight of the
body, and because of the ease with which it can be obtained from
the individual and characterized. Other substances in general are
present in far smaller amounts. I have estimated that there are of
the order of 100,000 different kinds of proteins in the human
body. If the number of substances making up the human body is
about 100,000, the average amount of each substance present
(aside from water) is about 100 mg., and some important sub-
stances may be present in far smaller amounts. The isolation and
identification of abnormal forms of molecules of these substances
might be extremely difficult. Because of the insight into the nature
of disease that would be provided by the discovery of additional
molecular diseases, however, it seems important to prosecute
investigations along this Sine with much vigor.

ABNORMALITY OF HEMOGLOBIN MOLECULES IN ANEMIA  237
ACKNOWLEDGMENTS
It is a pleasure for me to acknowledge the financial support of
the work on hemoglobin received from The Rockefeller Founda-
tion during the entire score of years that the work has been carried
on in our laboratories, and from the Public Health Service of the
National Institutes of Health (Research Grant 257, on Investiga-
tions of the Chemistry of Blood) during the period 1946 to 1953.
I am glad also to have an opportunity to express my thanks to my
co-workers, Alfred E. Mirsky, Charles D. Coryell, Fred Stitt,
Donald S. Taylor, Richard W. Dodson, Charles D. Russell, S. J.
Singer, Ibert C. Wells, Robert B. Corey, Ray D. Owen, W. A.
tiroeder, Lois M. Kay, Alexander Rich, E. Havinga, D. H. Camp-
bell, Morris Goodman, Robert C. C. St. George, William R.
Bergren, Phillip Sturgeon, and especially Harvey A. Itano, who
bas been the discoverer or co-discoverer of each gf the four known
abnormal forms of adult human hemoglobin.
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