Dr. Pauling’s Prediction of a Mutation in Beta-Globin Which Causes Sickle Cell Anemia and How This Prediction Impacted My Research

[Guest post written by John Leavitt, Ph.D., Nerac, Inc., Tolland, CT.]

Linus Pauling lecturing on sickle cell anemia, Kyoto, Japan. 1955

In September 2010, the company BlueBird Bio announced that it had cured a patient with the hemoglobinopathy, beta-thalessemia, by correcting the genetic defect in beta-globin that this patient inherited from his parents. This came 61 years after Linus Pauling and his associate, Harvey Itano, explained the cause of hemoglobinopathies such as sickle cell anemia. Beta-thalessemia like sickle cell anemia is caused by an inherited mutation in the beta-globin gene, just a different mutation. In the case of thalessemia, the defective beta-globin gene product disappears, whereas the defective beta-globin in sickle cell anemia remains stable to wreak havoc on the body. BlueBird Bio accomplished this first cure of a hemoglobinopathy by removing the blood-forming hematopoietic stem cells from the patient, engineering his cells ex vivo with a correct beta-globin gene, and then putting the cells back into the patient. The stem cell transplant sustained itself and produced red blood cells which functioned normally in the circulatory system. For the first time in this patient’s 18 year-old life, he did not have to have a monthly blood transfusion.

In late September 1981, when I gave a seminar at the Pauling Institute of Science and Medicine in Palo Alto, CA, I noticed that Dr. Pauling was smiling during the talk. He was aware of the discovery of the muscle isoform of actin by his friend Albert Szent-Györgyi and knew about the structure and function of actins (the subject of my talk). After reading Dr. Pauling’s 1949 paper on the molecular nature of the sickle cell trait, I understood that he was seeing during my talk the very same experiments in my discovery of a mutant human beta-actin that he and Harvey Itano had performed, which led to the prediction of a mutation in the hemoglobin protein that caused sickle cell anemia. His paper was the very first to describe the molecular genetic basis of a human disease. By 1981 there was plenty of conceptual evidence to suggest how I could look for mutations in proteins using electrophoretic separation of complex mixtures of cellular proteins. In 1949 though, Dr. Pauling was way ahead of his time. In his and Itano’s case, the plan was well thought out based upon years of characterization of oxygen bonding to the heme of the globin molecule. By contrast, I was very lucky to find a mutation in the most abundant structural protein of the cell, cytoskeletal actin in a human fibrosarcoma cell.

Harvey Itano.

It was probably evident in 1949 that hemoglobin amounts to about 95 percent of the total protein of a mature red blood cell; so these cells were essentially bags of hemoglobin molecules – globin polypeptides with attached heme moieties with an iron atom that bound oxygen. The heme-bound iron carries oxygen through the arterial system to cells for respiration. After delivery of oxygen to tissues, these red blood cells (RBCs) return carbon dioxide to the lungs through the venous system for expiration. In sickle cell anemia, after RBCs deliver oxygen throughout the body, the RBCs take on a sickled shape, clog the venous system and lyse, causing a wide variety of systemic problems. Pauling and Itano predicted that this change in RBC architecture was a direct consequence of “two to four” charged amino acid changes in the globin complex, which consists of two beta-globin subunits and two alpha-globin subunits (this was not known then). Because of the science that came after their discovery, we know now that the genetic mutation in the beta-globin moiety is a single amino acid exchange of glutamic acid to valine resulting from a single nucleotide transition (A to T transition) in codon 6 of the beta-globin gene encoding the 147 amino acid polypeptide. Thus two positive charges were added to the hemoglobin molecule by this mutation. Pauling and Itano concluded that these charge alterations caused RBC sickling.

Important discoveries can be quite simple. The figure below is the key experiment carried out by Pauling and Itano, an electrophoretic separation of hemoglobin based upon its isoelectric point (net charge). Because of the mutation in codon 6 present in both inherited beta-globin alleles, the hemoglobin complex migrated to the right of the normal hemoglobin by approximately “two to four” positive charges (panel B compared with panel A). At pH 6.9 the normal hemoglobin was shown to have an isoelectric point of 6.87, migrating as a negative ion, whereas the mutated hemoglobin had an isoelectric point of 7.09 migrating as a positive ion. We know now that this electrophoretic change in the hemoglobin complex described by Pauling and Itano is due to the loss of a single negative charge in a glutamic acid residue (replaced with an uncharged valine residue) near the N-terminus of the two beta-globin moieties of the hemoglobin molecule. Today, the fact remains that this is the only mutation in hemoglobin that causes sickle cell anemia, although other beta-globin mutations cause other hemoglobinopathies like beta-thalessemia. Panel C shows the electrophoretic behavior of hemoglobins in a heterozygous carrier of the disease-causing mutation (Panel D is a control mixture of the globins in panels A and B). Much more insight about these phenomena is discussed in the Pauling and Itano paper but the charge alteration in hemoglobins is the basic observation.

Pauling experiment

(click to enlarge)

Fast-forward to 1976. I decided to look for evidence of charge-altering mutations in a protein profile of about 1,000 visible proteins (polypeptides) comparing normal and neoplastic cells by looking for Pauling and Itano’s evidence of mutations, e.g. minor charge alterations in proteins in the protein profile. A technique had just been developed by Patrick O’Farrell which permitted high-resolution separation of virtually all major protein gene products of the cell.

An elegant study was performed by Greg Milman at the University of California at Berkeley who demonstrated that one could predict the occurrence of mutations in the relatively minor protein, the enzyme hypoxanthine phosphoribosyltransferase (HPRT), in HeLa cells by the positional changes in the HPRT polypeptide in high-resolution two-dimensional polyacrylamide gels within complex profile of proteins separated both by their charge (isoelectric point) and their molecular weight. When I saw Milman’s result I decided to use this technique to compare normal and neoplastic human cells to see if I could identify charge alterations similar to those demonstrated by Pauling and Itano in hemoglobin and by Milman in HPRT.

I labeled the proteins of normal and tumor-forming human fibroblasts with S35-methionine and separated them using O’Farrell’s two-dimensional technique (isoelectric point separation is a tube gel followed by molecular weight sieving in an SDS slab gel). Then I fixed the proteins in the two-dimensional slab gel and stained these proteins with Coomassie blue dye.

mutant actin further annotated

With the dye you could only see the most abundant proteins and I was surprised to see this pattern of actins in the tumor-forming fibroblasts shown above. This image is actually the image of the radioactive protein pattern in the region of actins (pI 5.3 to pI 5.1, molecular weight Mr about 42,000) developed after a very short autoradiographic exposure to X-ray film (a digital image). Normally you only see one beta-actin spot barely separated from the gamma-actin spot. Gamma actin is a second isoform of actin encoded by a separate gene which differs by only four amino acids from beta-actin. Normally there is about twice as much beta-actin at isoelectric point 5.2 as gamma actin and both actins together amount to 5-10 percent of the total cellular protein. But half of the normal beta-actin was missing and a new more negative spot at isoelectric point 5.3 appeared. I was able to show that this was a new form of beta-actin by tryptic peptide separation and other criteria. The observation that the new variant migrated slower in the second dimension as a larger protein was later attributed to a frictional effect in the gel sieve due to an altered conformation caused by the amino acid change.

These observations and other differences in protein expression between the normal and tumor-forming fibroblasts were published in the Journal of Biological Chemistry in February 1980. A second paper was published a month later demonstrating that a T-cell leukemia also had a beta-actin anomaly which suggested loss of a beta-actin allele. It is now well established that reorganization of the actin cytoskeleton occurs when cells become cancerous, although mutations in the structural gene may be less common. These alterations can also be caused by changes in actin-binding proteins.

Later in the year, with my colleagues at the Max-Planck in Goettingen Germany, Klaus Weber and Joel Vandekerckhove, I published the sequences of the normal human beta- and gamma-actins and the mutant beta-actin in Cell. The normal and mutated sequence of human beta-actin is shown in the figure below.

The simple electrophoretic difference between the mutant and normal beta-actin was a single amino acid exchange of a neutral glycine for a negatively charged aspartic acid at amino acid residue 244 in the 374 amino acid polypeptide chain, an observation similar to Pauling and Itano’s hypothesis 32 years earlier. An amino acid exchange at this residue in the actin polypeptide chain had never been observed in any eukaryote. Two years later I cloned the mutant and wildtype human beta-actin genes at the Pauling Institute and formally proved the existence of the mutation at the level of the gene. This mutation was caused by a single nucleotide change in the gene. Several years later my colleagues and I demonstrated that acquisition of this simple mutation contributed to the tumorigenic phenotype of the cells in which it arose.

actin sequence with arrow

The sequence of human beta-actin and its amino acid 244 mutation (the most highly conserved protein in eukaryotes).

Ed Note: This week marks the sixth anniversary of the creation of the Pauling Blog.  Birthed to help promote the unveiling of a postage stamp, the blog, 461 posts later, has developed into a resource of consequence with an audience that is steadily growing.  For those who might be interested in how the project operates, please see this post that we ran one year ago.

As always, we thank you for your continued readership.  We plan to keep researching and writing, so please keep coming back!


An Era of Discovery in Protein Structure

Linus and Ava Helen Pauling, Oxford, 1948.

[The Paulings in England: Part 4 of 5]

Though metals were consuming a good portion of his time during his fellowship at Oxford, Linus Pauling’s other projects never strayed far from his thoughts.  High on the list were the mysteries of proteins, whose structures and functions were slowly starting to be unraveled.

Pauling’s interest in proteins was spurred in the mid-1930s when the Rockefeller Foundation began to look most favorably upon the chemistry of life when deciding where their grant money would go. Early on, Pauling set out to tackle hemoglobin and though his affair with the molecule lasted for the remainder of life, Pauling certainly didn’t limit himself to the study of just one protein.

At a time when most were looking at proteins from the top down, trying to sort out the complicated data produced by an x-ray diffraction photograph of an entire protein, Pauling was working from the bottom up, in the process determining the structures of individual amino acids – the building blocks of proteins.

A specific protein that kept coming back into view over the years was keratin. In the 1930s, the English scientist William Astbury had studied the structure of wool, which along with hair, horn, and fingernail is made up primarily of this enigmatic protein, keratin. Astbury proposed that the structure was akin to a flat, kinked ribbon, but Pauling disagreed. “I knew that what Astbury had said wasn’t right,” Pauling recalled, “because our studies of simple molecules had given us enough knowledge about bond lengths and bond angles and hydrogen-bond formation to show that what he said wasn’t right. But I didn’t know what was right.” Pauling attempted to construct a model at the time, but could not match his structure to the measurements dictated by Astbury’s blurry x-ray diffraction images. Pauling wrote the project off as a failure and continued pursuing other interests.

In 1945 Pauling found himself seated next to Harvard medical Professor William B. Castle on a railroad journey from Denver to Chicago. Castle was a physician working on the nature of sickle cell anemia and the conversation that he shared with Pauling planted a seed in Pauling’s mind about the cause of this debilitating disease.

In the bodies of those suffering from sickle cell anemia, red blood cells assume a sickled shape when they are in the deoxygenated venous system but retain their normal flattened disk shape in the oxygen-rich arterial system. Noting this, Pauling suggested that perhaps the source of the problem could be a defect in the oxygen-carrying protein itself: hemoglobin.

Amidst his travels in Europe, Pauling continued to act on this idea as maestro from afar, directing the scientists in his Caltech laboratory to continue searching for differences in the hemoglobin of normal and sickled cells. In the meantime, he sought out and communicated new ideas gleaned from meetings such as the Barcroft Memorial Conference on Hemoglobin, held at Cambridge in June 1948. Pauling’s research team, in particular Harvey Itano and S. Jonathan Singer, were able to show experimentally that his hunch had been right, and less than a year after his return to Pasadena a paper was published that established sickle cell anemia as the first illness to be revealed as a truly molecular disease.

Linus and Peter Pauling at the model Bourton-on-the-water, England. 1948.

While in England, Pauling had occasion to interact closely with a number of scientific greats.  Among these were his close friend Dorothy Crowfoot Hodgkin, who is credited as a pioneer in the development of protein crystallography and was the winner of the 1964 Nobel Prize for Chemistry.  Likewise, Pauling conversed with Max Perutz, a protege of Sir William Lawrence Bragg‘s at the Cavendish Laboratory at Cambridge, who would go on to discover the structure of hemoglobin and receive the Nobel Prize for Chemistry in 1962.  While fruitful in many respects, these interactions served to increase Pauling’s feelings of urgency as concerned the race to determine the structure of proteins.

Bragg shared the 1915 Nobel Prize in Physics with his father for their early development of X-ray crystallography, and though there existed a long-standing scientific rivalry between Pauling’s and Bragg’s laboratories, it wasn’t until Pauling saw, with his own eyes, the work that was being done that he admitted he was “beginning to feel a bit uncomfortable about the English competition.” As he wrote to his colleague Edward Hughes back at Caltech

It has been a good experience for me to look over the x-ray laboratory at Cambridge. They have about five times as great an outfit as ours, that is, with facilities for taking nearly 30 x-ray pictures at the same time. I think that we should expand our x-ray lab without delay.

This realization prompted Pauling to get researchers in his lab started on work with insulin – an arduous and complicated process that required sample purification and crystallization prior to x-ray investigation. In relaying research findings from English scientists working on insulin to his partners back in Pasadena, Pauling intimated that

It is clear that there is already considerable progress made on the job of a complete structure determination of insulin. However, there is still a very great deal of work that remains to be done, and I do not think that it is assured that the British school will finish the job. I believe that this is the problem that we should begin to work on, with as much vigor as possible, under our insulin project.

Little did Pauling know that, while laying in bed, using little more than a piece of paper, a pen and a slide rule, he would soon make a major breakthrough in protein chemistry on his own.

The Medical Research of Linus Pauling

By Tom Hager

[Ed Note:  In October 2010, Pauling biographer Tom Hager delivered a talk sponsored by the Oregon Health Sciences University which detailed and discussed the various contributions that Linus Pauling made to the medical sciences, including the controversy over his strong interest in orthomolecular medicine.   With the author’s permission, excerpts of this talk are being presented on the Pauling Blog over the next three posts.  The full text of Hager’s OHSU lecture is available here.  Those with an interest in learning more about Hager’s work, including his latest research on food issues and world hunger, are encouraged to visit his blog at http://thomashager.net.]

[Part 1 of 3]

Oil portrait of Linus Pauling, featuring a model of the alpha-helix in the foreground. 1951. Portrait by Leon Tadrick.

By 1939, at the age of 38, Linus Pauling was a full professor and head of the chemistry division at Caltech, as well as the father of four children (three sons, Linus, Jr., Peter, and Crellin; and a daughter, Linda).

He was also beginning to turn his considerable talents toward understanding the complicated molecules inside the human body. He started with proteins.

The Molecules of Life

Determining the structure of proteins at this time was a gigantic problem. Most were difficult to purify, easily degraded, and hard to characterize. Proteins appeared to be not only gigantic, comprising hundreds or thousands of atoms – much too large to solve directly with x-ray crystallography – but also relatively fragile, losing their function (denaturing) after even slight heating or mechanical disturbance. No one at the time was even sure that they were distinct molecules – one popular theory held that proteins formed amorphous colloids, gels that did not lend themselves to molecular study.

Studying them at the molecular level seemed an impossible task with the tools available in the late 1930s. But Pauling took on the challenge. He started with the building blocks of proteins, the amino acids, and directed his growing lab team toward pinning down their precise structures. Then he set himself to figuring out how they formed protein molecules, often building models out of wood, wire, and paper.

He based his approach in part on the ideas of the German biochemist Emil Fischer. Like Fischer, Pauling came to believe that proteins were long molecular chains of amino acids linked end-to-end. Working with Alfred Mirsky in the mid-1930s, Pauling discovered that the denaturing of proteins resulted from breaking weak bonds, called hydrogen bonds, that pinned these chains into specific shapes. Between the early 1930s and early 1950s he made a string of important discoveries about hemoglobin, antibodies (including the most sophisticated work at the time into the structural relationship between antibody and antigen), enzymes, and other proteins.

Foldable paper model of the alpha-helix protein structure published in the Japanese journal Chemical Field, 1954.

In May 1951, he put everything he knew into a celebrated series of seven papers detailing the structures of a number of proteins at the level of individual atoms, including the structure of the single most important basic form of protein, the alpha helix (a hydrogen-bonded helical chain that is a structural component of almost every protein). It was an astounding breakthrough, and it opened the door for an understanding of biology at the molecular level. Within two years, Watson and Crick had used his approach to decipher the structure of DNA.

Biological Specificity

But structure was not everything. Pauling realized that life resulted not from individual molecules, but from the interactions between them. How did organisms make offspring that carried their specific characteristics? How did enzymes recognize and bind precisely to specific substrate molecules? How did antibodies recognize and bind to specific antigens? How did proteins, these flexible, delicate, complex molecules, have the exquisite ability to recognize and interact with target molecules?

It all fell under the heading of biological specificity at the molecular level. Pauling directed much of his attention here during through the 1940s, performing a great deal of careful work on the binding of antigens to antibodies.

Drawings of antibodies and antigens made by Linus Pauling in the 1940s.

His findings were surprising. Pauling demonstrated that the precise binding of antigen to antibody was accomplished not by typical chemical means – that is, through covalent or ionic bonds — but solely through shape. Antibodies recognized and bound to antigens because one fit the other, as a glove fits a hand. Their shapes were complementary. When the fit was tight, the surfaces of antibody and antigen came into very close contact, making possible the formation of many weak links that operated at close quarters and were considered relatively unimportant in traditional chemistry — van der Waals’ forces, hydrogen bonds, and so forth. To work, the fit had to be incredibly precise. Even a single atom out of place could significantly affect the binding.

Having demonstrated the importance of complementary structure with antibodies, Pauling extended his idea to other biological systems, including the interaction of enzymes with substrates, odors with olfactory receptors, and to the possibility of complementary structure in genes.

Pauling’s idea that biological specificity was due in great part to complementary “fitting” of large molecules to one another proved to be essential in the development of molecular biology. His research now formed a coherent arc, from his early work on the chemical bond as a determinant of molecular structure, through the structures of large molecules (first inorganic substances, then biomolecules), to the interactions between large biomolecules.

He carried out much of this research during World War II, when he also worked on synthetic plasma substitutes and a fruitless search for ways to produce artificial antibodies.

He had already earned a place among the nation’s leading researchers in the medical applications of chemistry. But his greatest triumph was still to come.

Sickle-Cell Anemia

Toward the end of World War II, Pauling’s reputation was great enough to earn him an invitation to join a national committee that was brainstorming the best structures for postwar medical research. This committee’s work led to the foundation of the National Institutes of Health.

Pauling was the only non-physician asked to join the committee.

At a dinner with other members one night, talk turned to a rare blood disorder called sickle-cell anemia. One of his dinner companions described how red blood cells in the victims were twisted into sickle shapes instead of discs. The distortion appeared to hinder the blood cells’ transport through capillaries, resulting in joint pain, blood clots, and death. The disease primarily affected Africans and African Americans. What caught Pauling’s attention most, however, was one odd fact: Sickled cells appeared most often in venous blood, rather than in the more oxygenated blood found in the arteries.

Pastel drawing of sickled Hemoglobin cells, 1964. Drawing by Roger Hayward.

He thought about this during the next few days. From his previous work with blood, he knew that red cells were little more than bags stuffed with hemoglobin. He had also shown that hemoglobin changed its shape slightly when it was oxygenated. If the red cells were changing shape, perhaps it was because the hemoglobin was altered in some way. What if the hemoglobin molecules in sickle-cell patients were changed in some way that made them clump, stick to one another, as antigens stick to antibodies? Perhaps something had changed that made the hemoglobin molecules complementary in shape. Perhaps adding oxygen reduced the stickiness by changing the molecules’ shape.

He presented his ideas as a research problem to Harvey Itano, a young physician who was then working on his Ph.D. in Pauling’s laboratory. Itano, later joined by postdoctoral fellow John Singer, worked for a year trying to see if sickle-cell hemoglobin was shaped differently from normal hemoglobin. They found no detectable differences in any of the tests they devised. But they kept at it. Finally, in 1949, using an exquisitely sensitive new technique called electrophoresis that separated molecules by their electric charge, they found their answer: Sickle-cell hemoglobin carried more positive charges on its surface.

This was an astounding discovery. A slight change in the electrical charge of a single type of molecule in the body could spell the difference between life and death. Never before had the cause of a disease been traced to a molecule. This discovery – to which Pauling attached the memorable title “molecular disease” – received widespread attention. Itano and Singer’s followup work demonstrated the pattern of inheritance for the disease, firmly wedding molecular medicine to genetics.

Medical Chemistry

It was a great triumph – there was talk of a Nobel Prize in Medicine or Physiology for Pauling – and it led Pauling to make greater efforts in the medical field. He encouraged M.D./Ph.D. candidates, hired physicians to work in his laboratory, and began focusing his own research on medical problems, including developing a new theory of anesthesia.

He was ahead of his time. An example of what the atmosphere was like: Pauling noted that as he went around in the late 1940s seeking funds for a comprehensive marriage of biology and chemistry to attack medical problems, people at funding agencies were telling him that they found the term “medical chemistry” to be “a disturbing description.”

In the late 1950s, Pauling extended his concept of molecular disease to the brain. After reading about phenylketonuria (PKU) – a condition in which a mental defect can be caused by the body’s inability to metabolize an amino acid, phenylalanine, leading to a buildup of that substance and others in the blood and urine – Pauling theorized that the problem might be caused by a defect in an enzyme needed to break down phenylalanine. PKU, in other words, might be another molecular disease. Now interested in the possibility that there might exist a range of molecular mental defects, Pauling visited a local mental hospital, saw other patients whose diseases seemed hereditary, and decided to seek support for an investigation into the molecular basis of mental disease. The Ford Foundation in 1956 awarded him $450,000 for five years’ work – a vindication of Pauling’s approach and a tribute to his reputation. The grant, however, yielded little in the way of immediate results, with much of the funding going toward testing his (ultimately found to be mistaken) theory of anesthesia.

The long-term results were more significant. Pauling’s immersion in the field, thanks to the Ford grant, led him to read widely in psychiatry and general health, always on the lookout for another molecular disease that might lend itself to new therapy. By the mid-1960s he was coalescing his findings into another overarching theory, this one combining much of what he knew about chemistry and health. He called his new idea “orthomolecular” medicine.

Remembering Harvey Itano

Portrait of Harvey Itano, 1954. Image courtesy of the Caltech Institute Archives.

“The discovery by Dr. Itano of the abnormal human hemoglobins has thrown much light on the problem of the nature of the hereditary hemolytic anemias, and has changed these diseases from the status of poorly understood and poorly characterized diseases into that of well understood and well characterized diseases.”

-Linus Pauling, 1955.

We were saddened to learn of the death of Harvey A. Itano, emeritus professor of pathology at the University of California, San Diego.  Dr. Itano passed away on May 8, 2010 at the age of 89.

Best known professionally for his work on sickle cell anemia, Itano’s early personal history makes for fascinating reading.  According to this excellent obituary issued by UCSD

Itano was born in Sacramento, CA on November 3, 1920, the oldest of four children of Masao and Sumako Itano, originally of Okayama-ken, Japan.  A star student at UC Berkeley, he graduated in 1942 with highest honors in chemistry.  He was unable to attend his own graduation ceremony, because he and his family were confined to internment camps established after the bombing of Pearl Harbor for the detention of Japanese and Japanese-Americans living in the western US.  In recognition of his outstanding achievements as a student, having earned the highest academic record in his class, then-UC President Robert Gordon Sproul personally awarded him the University Medal during his internment.

[…] He was released from the camp on July 4, 1942, the first of the Nisei (second generation Japanese-Americans) to be released to attend colleges and universities.  He attended the St. Louis School of Medicine, where he earned his MD in 1945 before continuing his studies at California Institute of Technology, earning a PhD in Chemistry and Physics in 1950.

It was at Caltech that Itano came into contact with Linus Pauling, his major professor during his doctoral studies and research colleague for the duration of a four year post-doctoral stint in Pasadena.  Over the course of this time period, Itano, Pauling and their collaborators made a series of significant contributions to the field of molecular biology.

Most prominent among these contributions was a 1949 paper published in Science, titled “Sickle Cell Anemia, A Molecular Disease.”  Authored by Pauling, Itano, S. Jonathan Singer and Ibert C. Wells, the paper presented experimental evidence in support of Pauling’s theory that sickle cell anemia could be traced to significant abnormalities in the hemoglobin molecules of those suffering from the disease.  The paper was quickly recognized to be the first solid proof of the existence of a “molecular disease.”

In his book Force of Nature, Pauling biographer Thomas Hager comments on the importance of this discovery.

People had theorized in broad terms about the molecular basis of disease before, but no one had ever demonstrated it the way Pauling’s group did….By pinpointing the source of a disease in the alteration of a specific molecule and firmly linking it to genetics, Pauling’s group created a landmark in the history of both medicine and molecular biology.

Itano spent much of his long career furthering the breakthroughs signaled in the 1949 paper.  Among other achievements, he developed a “rapid diagnostic test” for sickle cell anemia which would quickly indicate whether or not a given blood sample would sickle.  With S. J. Singer, Itano also described the condition of sicklemia, an intermediate and less severe stage of sickle cell anemia in which a patient’s blood contains a mix of normal hemoglobin and sickled hemoglobin cells.

Harvey Itano and Linus Pauling. 1980s.

Linus Pauling held Itano in high regard, both as a scientist and as a person.  In a lengthy award nomination that Pauling composed for Itano in 1955, Pauling describes the specifics of Itano’s contribution to the team’s molecular disease breakthrough while noting his “great natural ability and thoroughly sound training in chemistry and related sciences as well as in medicine.”  Of the man, Pauling wrote

His success must also be attributed in part to his excellent personality.  He is quiet and pleasant in manner, and is well liked by all of his associates.  During his eight years at the California Institute of Technology he made many friends, and he was uniformly successful in effective collaboration with a number of co-workers.  He is original, clearheaded, keen, and critical in his scientific work.

Itano maintained a keen interest in his rich genealogical background, and those who wish to learn more about his story are encouraged to visit the Itano family history website.  A great deal more about Itano’s role in the sickle cell anemia and molecular disease story is likewise available at It’s in the Blood!  A Documentary History of Linus Pauling, Hemoglobin and Sickle Cell Anemia.

Mastering Genetics: Pauling and Eugenics

Illustration from Medical World News article,

Illustration from Medical World News article, “Sickle Cell Anemia” December 3, 1971.

“I have suggested that the time might come in the future when information about heterozygosity in such serious genes as the sickle cell anemia gene would be tattooed on the forehead of the carriers, so that young men and women would at once be warned not to fall in love with each other.”
-Linus Pauling, August 15, 1966

After declaring sickle-cell anemia to be a “molecular disease” in the late 1940s, Pauling spent more than a decade describing the cause of the disease and the significance of its unique origins to his fellow academics. Unfortunately, though interested, his colleagues seemed more concerned with the concept of a molecular disease than its real world application in genetics and medicine. Beginning in 1958, Pauling became a vocal advocate of genetic counseling, focusing especially on sickle-cell anemia among the African American population. His efforts went largely unnoticed by both researchers and the general public alike.

Frustrated with his unsuccessful endorsement of genetic counseling, Pauling chose to take his ideas a step further. In 1962, Pauling began a public campaign in support of negative eugenics – the restriction of human breeding and childbirth as a means of minimizing the sharing of hereditary diseases. He advocated genetic testing as a requirement for obtaining a marriage license. Perhaps even more controversial, Pauling recommended placing legal restrictions on marriage and childbirth between carriers of hereditary diseases.

Listen: Pauling on Marriage Tests and the Disclosure of Genotype Information

Pauling recognized the difficulty of controlling and monitoring a program of this magnitude. Without being able to easily identify carriers of various diseases, the public could not effectively choose sexual partners, thus lessening the potential effectiveness of employed eugenics. As a solution, in the late 1960s, Pauling began suggesting a means of visibly marking disease carriers – a tattoo on the forehead, clearly marking the individual as the carrier of a specific disease. Not surprisingly, this suggestion engendered a great deal of criticism. He was compared with the likes of Hitler by his critics who drew parallels between the proposed tattoo and the yellow star worn by Eastern European Jews during the reign of the Nazi party.

In reflecting upon Pauling’s stance, it is important to note that he was not interested in positive eugenics – the manipulation of genetic combinations as a means of developing a superior human. Rather, he intended only to minimize human suffering and found the idea of building a “super race” highly undesirable. Pauling was also a critic of the concept of genetic purity. He was concerned with purifying the human gene pool of harmful diseases, but he was not motivated by the desire to manipulate intelligence, appearance, strength, etc.

Pauling insisted that his ideas, though extreme, were meant to decrease human suffering rather than to segregate and belittle. Though Pauling faced many critics, he did have supporters as well. Nobel laureate Sir Peter Medawar agreed with Pauling, famously stating,

It is humbug to say that such a policy violates an elementary right of human beings. No one has conferred upon human beings the right knowingly to bring maimed or biochemically crippled children in the world.

During the 1960s, Pauling’s critics began discussing the effect that negative eugenics could have on evolution. Roderic Gorney, a psychiatrist, argued that over a long enough period of time, eugenics could redirect and even supersede the process of natural selection.

For example, consider the effect of negative eugenics in relation to sickle-cell anemia. An individual with sickle-cell anemia has two sickle-cell alleles. Typically, sufferers of sickle-cell anemia are plagued by a host of related health problems, often leading to an early death. Some individuals, however, possess only one sickle-cell allele. These individuals exhibit some sickling of the blood cells, but are otherwise able to live normal, healthy lives. Because sickle-cell anemia is a hereditary disease, it is passed on in Mendelian fashion. As a result, a person with a single sickle-cell allele, when paired with a healthy individual, has a 25% chance of giving birth to a child with one sickle-cell allele. When paired with another single-trait individual, there exists a 50% chance that a child will have one sickle-cell trait, and a 25% chance that the child will be afflicted with full sickle-cell anemia.

Gorney argued that sickle-cell anemia, if left alone, would eventually be removed from the human gene pool. He explained that, because individuals suffering from sickle-cell anemia rarely live to procreate, few instances of sickle-cell anemia are added to the collective gene pool. Similarly, a single-allele individual has a statistical opportunity to produce children with sickle-cell anemia when paired with another carrier. These offspring will die at a young age, further reducing the number of carriers present in the next generation. As a result, over a period of time, the number of sickle-cell carriers would decrease to nothing.

Negative eugenics, however, allows sickle-cell carriers to identify other carriers and instead mate with healthy individuals, producing more children with a single sickle-cell allele. If this process were to continue indefinitely, more and more humans would be heterozygous for sickle-cell anemia, rendering it virtually impossible for natural selection to remove the disease from the human gene pool. This argument could, in fact, be applied to any similar hereditary disease.

“Bad Genes and Marriage,” New York Post, October 21, 1968.

Pauling acknowledged Gorney’s concerns but countered that, without eugenics, preventative medicine would have a much more damaging effect. Pauling felt that modern medicine (antibiotics, chemotherapy, prescription drugs, etc.) helped prolong the lifespan of sick or diseased individuals, sometimes allowing them to procreate and pass along hereditary diseases. As such, modern medicine was effectively undoing natural selection, leaving negative eugenics as the best hope for maintaining a balanced, healthy population.

In the early 1970s, Pauling began to run into trouble. His main focus throughout his eugenics campaign was the elimination of sickle-cell anemia, a disease that had originated in Africa where it became common among the native population because of its ability to prevent malaria. When slave traders brought African captives to North America, sickle-cell anemia was introduced to the United States. Due to racial segregation and the social mores that developed in the U.S. over the intervening 300 years, very few individuals outside of the African American population were afflicted with sickle-cell disease. For these reasons, Pauling advocated blood testing among the African American population. As the Civil Rights movement gained momentum, Pauling’s suggestions were seen as racist, and even as an attempt to cast African Americans as genetically inferior and meriting legal restrictions on their rights to marriage and procreation.

Frustrated and embarrassed by the criticism that he was receiving, Pauling fell silent on the topic of eugenics. In the past, when faced with heavy opposition, Pauling had always held his ground. But this episode was different. By the end of 1972, Pauling had given up his negative eugenics campaign and turned to other means of improving the human condition.

For more information on Pauling and his work with genetics, visit “It’s in the Blood! A Documentary History of Linus Pauling, Hemoglobin and Sickle Cell Anemia” or Linus Pauling Online.

Mutations and Malaria: Pauling’s Adventure in Genetics

Pastel drawing of Hemoglobin at 100 angstroms, 1964.

During the 1940s, Pauling had established sickle-cell anemia as a molecular disease, a pioneering concept that synthesized biology and chemistry in a revolutionary manner. Other interests had pulled him away from this important work, however, for the better part of a decade.

Then, in the early 1960s, he was introduced to research suggesting that rates of malaria infection in areas with a high rate of sickle-cell anemia were greatly reduced. On top of this existing research, Pauling also came across a reference to a particularly interesting African legend regarding the origin of malaria resistance. Intrigued, he decided to dig a little deeper and, before long, he had dedicated a small portion of his lab to the problem.

Early in his research, Pauling found that the protozoan parasites responsible for malaria were not able to penetrate and replicate in sickled blood cells — e.g, cells containing deformed hemoglobin. Even more interesting, Pauling discovered that individuals with only one sickle-cell allele did not suffer from the effects of sickle-cell anemia but were still highly resistant to the malaria disease.

By examining these findings, Pauling developed a set of basic rules explaining the sickle-cell and malaria interactions. They are as follows:

1. Individuals with only normal hemoglobin do not possess the deformed hemoglobin molecules present in individuals possessing either one or two sickle-cell alleles. As a result, these individuals are not resistant to malaria.
2. Those with the homozygous recessive sickle-cell trait suffer from sickled blood cells, resulting in a variety of health complications including stroke, ulcers, bacterial bone infection, kidney failure, and heart problems. Victims of the dominant form of sickle-cell anemia have a significantly shorter lifespan than the average human, often dying in infancy. Nevertheless, these individuals are not afflicted by the malaria disease.
3. Other individuals are heterozygous for the sickle-cell trait, meaning that they experience some sickling of the blood cells, but enough of their blood cells appear normal that they are able to survive without experiencing the health difficulties associated with sickle-cell anemia. Like those with the full sickle-cell anemia disease, these individuals enjoy significant resistance to the malarial disease.

Pauling stated that the human populations inhabiting malarial zones in Central Africa were becoming predominantly comprised of heterozygotes. He explained that an individual homozygous recessive for the sickle-cell trait would probably die before reaching sexual maturity, therefore not producing any children with the sickle-cell disease. Those without the sickle-cell trait would be vulnerable to malaria. In malarial regions, this group would have a high mortality rate, many of them dying before reproducing. The third group, those with only one sickle-cell allele, does not suffer from the effects of full sickle-anemia and are immune to malaria. As a result, these individuals are best suited to malarial regions and are able to procreate, giving birth to more heterozygotes who can, in turn, continue the genetic trend.

The sickle-cell trait is a hereditary disease, passed from parent to child in the Mendelian fashion. Each parent provides the child with one of the two alleles which will determine whether the child will have normal or sickled blood. Two individuals with sickle-cell anemia will invariably produce children with sickle-cell anemia. A pair in which one parent has sickle-cell anemia and the other is a carrier (meaning they have one trait rather than two) will have a 50% chance of producing a child with sickle-cell anemia and a 50% chance of producing a child with only one sickle-cell allele. A couple in which both parents carry only one sickle-cell allele will have a 25% chance of producing a child with sickle-cell anemia, a 25% chance of producing a child without the sickle-cell trait, and a 50% chance of producing a child with only one sickle-cell allele.

The following series of Punnett squares demonstrates the transfer of alleles in the case of sickle-cell anemia:

Sickle-Cell Anemia Punnett Square

Based on this thinking, Pauling argued that only the people with one sickle-cell allele would live to have children, approximately 50% of which would be born with one sickle-cell allele. He argued that this trend could continue indefinitely, probably until a mutation eliminated the sickle-cell disease entirely, leaving all peoples in malarial zones homozygous for an anti-malarial gene.

Listen: Pauling on the effect of sickle cell disease on the spread of malaria

With his theory firmly in place, Pauling turned his attention to sickle-cell anemia in non-malarial zones. Pauling was primarily concerned with the presence of sickle-cell anemia in the African American population of the southeastern United States. Because malaria is not endemic to the southern U.S., Pauling feared that a positive mutation was unlikely to occur, and the sickle cell mutation was not being removed from the gene pool as quickly as new, harmful mutations were occurring. As a result, the number of individuals suffering from sickle-cell anemia could only continue to increase.

In order to counteract this trend, Pauling spoke out in support of eugenics as a means of controlling and gradually diminishing the presence of sickle-cell anemia in the United States.

In the 1960s and 1970s, Pauling made headlines by giving talks on the subject. He was introducing the concept of beneficial mutations to a public not necessarily comfortable with certain implications of the phenomena. The humanitarian components of his efforts earned him praise from various medical groups, though his advocacy of eugenics created some concern among politicians, religious conservatives, and secular ethicists alike.

For more information on Pauling’s work with sickle-cell anemia and malaria, visit It’s in the Blood or take a look at the OSU Special Collections homepage.

The Importance of the Concept of Molecular Disease

The idea of Dr. Linus Pauling that an abnormal hemoglobin molecule might be responsible for the sickling process initiated the study of the hemoglobin molecule in hereditary anemias.
– Harvey Itano. “Clinical States Associated with Alterations of the Hemoglobin Molecule.” Archives of Internal Medicine, 96: 287-97, 295. 1955.

During his lengthy career, Linus Pauling maintained a long-running interest in the relationships between chemistry and the human body. In the mid-1930’s, he began to work extensively with the hemoglobin molecule. As we’ve seen in previous posts, this research would eventually lead to many important discoveries and his coining of the term “molecular disease.”

Sickle cell anemia was the first disease to be classified as a molecular disease. As was mentioned in this post, Pauling first learned of the disease in the spring of 1945 when Dr. William B. Castle, a physician and Professor of Medicine at Harvard University, described it at a meeting of the Medical Research Association. As Dr. Castle listed off the characteristics of the disease, Pauling, through the prism of his deep knowledge of the structural chemistry of hemoglobin, developed an almost-immediate formulation of sickle cell anemia as a disease of the hemoglobin molecule, rather than of the entire blood cell.

Listen: William Castle recounts his first meetings with Linus Pauling…

Listen: …and Pauling responds in kind

A few months later, Pauling would pass this idea on to Harvey Itano, who was completing his doctorate in chemistry at Caltech. Itano conducted a series of initial experiments on the hemoglobin molecule, all of which failed. After months of tedious investigation, however, Itano, Dr. S. J. Singer and Dr. Ibert C. Wells – both of them newly-minted Ph.D.’s – were able to use the techniques of electrophoresis to identify a significant distinction. The paper “Sickle Cell Anemia, a Molecular Disease” was then published in the fall of 1949 and the concept of molecular disease was instantly established.

Listen: Pauling describes the Itano, Singer and Wells electrophoresis experiments

Although Pauling wasn’t the first to think about diseases in terms of molecular aberrations, no one prior to the Pauling-Itano group had concretely demonstrated their existence. After their initial success, Singer and Itano continued to expand on the original research, eventually discovering a less-severe case of sickle cell anemia called sicklemia. The duo also described the manner in which sickle cell anemia is inherited. As such, not only did Pauling and his colleagues identify the exact source of the disease, they also provided a link to genetics and confirmed Pauling’s view that analysis on a molecular level can provide valuable information. Later, Itano would discover more abnormal hemoglobin molecules, and the molecular analysis of diseases would continue.

Since Pauling’s coining of the term “molecular disease,” many other diseases have been similarly categorized: Hemophilia, Thalassemia, Alzheimer’s Disease and Muscular Dystrophy to name a few. (Though it could also be argued that every heritable disease can be classified as a molecular disease because these diseases require a modified genetic component that can be passed from parent to child.)

Thalassemia, for example, is also a disease of the hemoglobin molecule. However, while sickle cell anemia is caused by the production of abnormal hemoglobin, Thalassemia, conversely, involves the abnormal production of hemoglobin. More specifically, in cases of Thalassemia, the rate of production of a specific globin chain is decreased, which then causes the formation of abnormal hemoglobin molecules.

Pauling’s conceptualization of sickle cell anemia as a disease of the hemoglobin molecule jump-started years of research pertaining to abnormal hemoglobins and opened many new doors in the study of inherited diseases. Although he wasn’t directly involved in the discovery of the abnormal hemoglobin molecules, Pauling’s development of the concept of molecular disease was achievement enough to significantly raise his stature in the medical community (at least for a while) and further cement his status as a scientist of world-historical importance.

For more information on molecular disease and other related topics, please visit the website “It’s in the Blood! A Documentary History of Linus Pauling, Hemoglobin, and Sickle Cell Anemia.”