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.

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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.

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.”

Pauling’s Methodology: Electrophoresis

Diagram of a Tiselius electrophoresis apparatus.

Diagram of a Tiselius electrophoresis apparatus.

[Electrophoresis image extracted from the published version of Arne Tiselius’ Nobel lecture, December 13, 1948.  A digitized version of this lecture is available here courtesy of the Nobel Museum.]

The item of $7,500 for apparatus, supplies, animals would permit us to use the large number of animals required for some of our projected researches, and should permit also the construction of a Tiselius apparatus for the electrophoretic separation of antibody fractions by the suggested method of combination with charged haptens, and for other investigations.
– Linus Pauling, budget request letter to Warren Weaver. January 2, 1941.

Though, by the late 1930s, X-ray crystallography had become important to Linus Pauling’s research on the structure of complex organic proteins, the newly developed technique of electrophoresis eventually became the technology that defined his work on sickle cell anemia.  Indeed, Pauling was one of the first in a generation of scientists to effectively use the technique of electrophoresis to explain a biological phenomenon.

Lying at the core of Pauling’s interest in sickle cell disease was this question: What really made normal hemoglobin and the hemoglobin from someone suffering from sickle cell anemia different? Though Pauling and his fellow researchers theorized that the answer lay in differences between the structures of the hemoglobin molecules themselves, and also figured that magnetic properties somehow played a role, they had yet to find or develop a method suitable for testing their ideas.

As it turned out, Pauling and his colleagues had to do both: they found and they developed.

The Pauling group seized upon the new technique of electrophoresis but manipulated it considerably to fit their own research agenda. Pauling attributed the idea of using electrophoresis in the first place to one of his graduate students, Harvey Itano. Later Pauling and Itano sought advice, assistance and collaboration with others who were also using the technique, including Karl Landsteiner and Arne Tiselius, both accomplished researchers and close colleagues of Pauling’s. After the construction at Caltech of an electrophoretic machine, Stanley Swingle, a general chemistry instructor at the Institute, developed a number of mechanical improvements while Harvey Itano and Seymour Jonathon Singer conducted research using the apparatus.

After much trial and error, electrophoresis emerged as one of the more important experimental methods used to determine the difference in electrical charge between normal hemoglobin and sickle cell hemoglobin.

Listen:  Pauling discusses the evolution of electrophoresis work at Caltech

The results of Pauling’s electrophoretic experiments, reported in his group’s groundbreaking 1949 paper, “Sickle Cell Anemia, a Molecular Disease,” promoted the argument that sickle cell anemia was not only a pathology resultant of differential protein folding patterns, but that it was also inherited in a simple Mendelian pattern. In other words, sickle cell anemia was both ‘molecular’ and ‘genetic,’ and by seeing it as such, Pauling suggested certain therapies that directly addressed both the structural and the genetic components of the disease.

Even as late as the 1960s Pauling was still looking for ways to use electrophoresis in his research. He mentions, in a handwritten note, that of the ‘likely developments’ in biology, control of molecular and genetic diseases could possibly be obtained through the “electrophoresis of sperm.”

(Though the idea may sound strange today, Pauling was an advocate for the controversial notion of positive eugenics — that is the planned and controlled production of healthy offspring, primarily through genetic counseling. We’ll talk more about this component of Pauling’s thinking in a later post.)

In more ways than one, electrophoresis was a new technology that required the coordinated effort of a number of trained individuals. Though it took several years to fine-tune both the method and the instruments, the results were well worth the wait.

To learn more about Linus Pauling’s use of electrophoresis, please visit the website It’s in the Blood!  A Documentary History of Linus Pauling, Hemoglobin and Sickle Cell Anemia.