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!


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.

The Martha Chase Effect

Martha Chase and Alfred Hershey, 1953.

Martha Chase and Alfred Hershey, 1953.

The Phenomenon

It pretty well goes without saying that the primary mission of the Oregon State University Libraries Special Collections is to preserve, describe and make available the Ava Helen and Linus Pauling Papers.  Beginning, more or less, with the Pauling centenary in 2001, the main focus of our Pauling-related work has been description and accessibility via the web.  In so doing, we have scanned over one terabyte of data and created, at minimum, tens of thousands of static html pages devoted to the life, work and legacy of Linus Pauling and, to a lesser extent, Ava Helen Pauling.

Knowing this, one might reasonably assume that the top search engine query channeling into the content that we have created would be “Linus Pauling,” or some variant therof.  A reasonable assumption indeed but, as it turns out, quite wrong.  In 2008, as in 2007 and 2006 (a close second in 2005), the top keyword query for those who found our content through search was…”Martha Chase.”

Martha Chase was a geneticist who, in collaboration with Alfred Hershey, made an important contribution to the DNA story as it played out in the early 1950s.  Prior to Chase and Hershey’s work, scientists were mixed on the question as to what, exactly, was the genetic material.  Many researchers, Pauling included, initially felt that the stuff of heredity was contained in proteins.  Others, of course, eventually theorized that DNA was the source of genetic information.  Using an ordinary blender as their primary tool, Hershey and Chase devised a famous experiment which proved conclusively that DNA did, in fact, carry the genetic code.

Diagram of the Hershey-Chase Blender Experiment.  Image by Eric Arnold.

Diagram of the Hershey-Chase Blender Experiment. Image by Eric Arnold.

The breadth of Chase-related content that we have digitized is infinitesimally-small relative to the “reams” devoted to Pauling — this page and this page are pretty much it.  And yet, in the context of search, Martha Chase is the top draw to our resources.  It would seem then, that in the marketplace for information — at least that which is retrieved by search — supply and demand for Martha Chase approach their equilibrium at the two pages devoted to her work on our “Linus Pauling and the Race for DNA” site.

Looking through the web statistics, the phenomenon is remarkably consistent.  Not only has “Martha Chase” been the top search query for our domain over, essentially, the past four years, it was also the top search query for our domain over the final week of 2008.  Indeed, the trend has strengthened to the point where today, those who conduct the simple “Linus Pauling” search in Google will note “martha chase” as a recommended search related to Pauling, though in reality the two had little or no interaction at all.

Learning from the Chase Effect

Looking forward, the Chase Effect has become something that we’re thinking more and more about as we begin to develop new projects for the web.  Our top objective will always be to document Pauling’s impact on any number of fields, but in so doing there likely exists a great deal of opportunity for serving different user groups from what might be called “Chaseian” corners of the web.

To use the old many-fish-in-the-sea analogy, there is a lot of content related to Pauling on the Internet, and though we are the primary contributor to this content, we do compete for pageviews with scads of other extremely diverse projects.  (Take a look at the results for the simple “Linus Pauling” Google search to see how diverse the content providers really are.)  So it’s pretty clear that the Pauling sea is quite large and filled with all manner of creatures.

By comparison, Martha Chase represents a much smaller body of water and, in particular, image searches for her — which is probably where the lion’s share of our successful Chase referrals come from — are dominated by the osulibrary.oregonstate.edu/specialcollections domain.

The idea for future work is to think of the Pauling Papers as a collection of collections in attempting to uncover more Martha Chases.

To an extent we have already, somewhat unwittingly, done this with certain of the Key Participants highlighted on our various documentary history websites.  The Harvey Itano Key Participants page, for example, is the second result returned by Google for “Harvey Itano” searches.  Erwin Chargaff‘s page is seventh,  Arnold Sommerfeld‘s page is eighth and Edward Condon‘s is tenth, to name a few more examples.  In each instance, by developing mini-portals related to specific colleagues important to Pauling’s work, we have created resources that help meet the information demand of a non-Pauling user base.

As we standardize our metadata platforms — upgrading older projects and maintaining the standard for new — and, in the process, increase our capacity to “remix” our digital objects, the idea of enhancing existing mini-portals and creating new ones will emerge as an important consideration for our digitization workflow.  This is something that we’ll be talking a lot more about in the months to come.

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 Theory of Sickle Cell Anemia

It's in the BloodWe owe to Pauling and his collaborators the realization that sickle cell anaemia is an example of an inherited ‘molecular disease’ and that it is due to an alteration in the structure of a large protein molecule, an alteration leading to a protein which is by all criteria still a haemoglobin.
– Vernon M. Ingram, 1957.

Of the four Documentary History websites that the OSU Libraries Special Collections has produced, “It’s in the Blood!  A Documentary History of Linus Pauling, Hemoglobin and Sickle Cell Anemia” is, in certain respects, the most unique.

For one, “the blood site” — its usual in-house appellation — is the only of our Documentary Histories not to have been written by Pauling biographer Tom Hager.  On the contrary, the idea for the blood site arose out of a history of science master’s thesis that Melinda Gormley — then a graduate student and now a professor at OSU — developed from research done in the Ava Helen and Linus Pauling Papers. As Dr. Gormley documented in this article (PDF, see pp. 8-9) it took the better part of two years to repurpose the text of her dissertation into a format suitable for the web.

Gormley’s thesis topic was relatively broad — “The Varieties of Linus Pauling’s Work on Hemoglobin and Sickle Cell Anemia,” (PDF, 1.8 MB) — and, as a result, the swath of content covered in the website is similarly wide.  The website begins its narrative in 1930, ends it in 1994, and along the way discusses Pauling’s contributions to areas ranging from immunology to Scientific War Work to evolutionary theory to orthomolecular psychiatry.  All of these topics will be addressed in future posts on this blog.

The heart of the blood site, however, is Pauling’s research on sickle cell anemia. Sickle cell anemia is a terrible disease that predominantly effects inhabitants of sub-Saharan Africa or those who can trace their lineage to that region.  The disease is a painful one, characterized by drastically-malformed red blood cells, and manifesting itself in a host of health maladies and, often, shortened lifespans.

Many folks who are semi-acquainted with the Pauling legacy know that he was, in some way, important to the modern understanding of sickle cell anemia.  But how? Well, Linus Pauling was the first individual to correctly theorize that sickle cell anemia is a disease that locates its source to the molecular level — in the process Pauling likewise became the first individual to postulate the concept of a molecular disease.

What then, exactly, was Pauling’s theory of sickle cell anemia?  That is the question that we aim to explore in this post.

Linus Pauling probably wasn’t a true freak-of-nature genius in the manner of an Einstein or a Mozart.  On the contrary, the likely secret of his profound success as a scientist was at least threefold in nature: 1) he possessed a relentless work ethic; 2) he was a very clear and concise thinker who conceptualized his ideas well and understood the efficiencies inherent to leading teams of researchers as opposed to going it alone; 3) and most importantly, he was deeply interested in, and capable of concretely understanding, radically-disparate areas of scientific study.  All three of these traits reveal themselves in the sickle cell anemia story.

Pauling first encountered the problem of sickle cell anemia rather by accident.  At a dinner in 1945, Pauling sat in the audience of an informal presentation by physician Dr. William Castle, wherein it was noted that the shape of red blood cells in sickle cell patients varied depending on whether the blood was venous or arterial —  normal in arterial blood, sickled in venous blood.  Clearly this suggested that the oxygen content in sickle cell blood played a major role in its molecular architecture. By his own recollection, “within two seconds,” Pauling concluded that the oxygen piece of the equation suggested that hemoglobin must be involved in the sickling mechanism — a conclusion that he could reach because of his keen understanding of the structural chemistry of hemoglobin.

In 1960, Pauling provided this description of his initial thoughts on how malformed hemoglobin could lead to sickled red blood cells.

…immediately I thought, “could it be possible that this disease, which seems to be a disease of the red cell because the red cells in the patients are twisted out of shape, could really be a disease of the hemoglobin molecule?” Nobody had ever suggested that there could be molecular diseases before, but this idea popped into my head. I thought, “could it be that these patients can manufacture a special kind of hemoglobin such that the molecules are sticky and clamp on to one another to form long rods, which then line up side by side to form a long needle-like crystal, which as it grows inside of the red cell becomes longer than the diameter of the cell and thus twists the red cell out of shape?”

From here, Pauling delegated many of the details necessary to verifying his thinking on the sickle cell problem to a team of Caltech graduate students led by Harvey Itano.  (This was common practice for Pauling, and helps explain how he was able to generate over 1,100 published papers in ninety-three years of living)  Using a variety of methods including electrophoresis, the Itano team, in the words of a 1950 Caltech press release

found a difference – slight but still unmistakable – between normal hemoglobin and that of a sickle-cell anemia patient.  Sickle-cell hemoglobin proved to have a greater positive electrical charge, under the proper chemical conditions, than did the hemoglobin from a normal person.  Such a difference in electrical properties can only mean a difference in molecular architecture, in the way in which the hemoglobin molecules are constructed.

In other words, Pauling was right: sickle cell anemia was a molecular disease and malformed hemoglobin was the cause.

In 1956, an English chemist named Vernon Ingram, using a new technique called fingerprinting, (Pauling provides a rather technical description of the method here) proved conclusively that sickle cell anemia was an inherited disease as well.  Moreover, sickle cell anemia was found to be caused by an astonishingly small change at the molecular level.  Physicist John Hopfield described it this way

On the surface of the ten-thousand atom molecule, there is a slight change. A small group of a few atoms on the edge of the molecule is replaced by another small group of atoms. That’s all that happens – an exchange of a few atoms. Yet it’s enough to make people very ill. The effect of the change is to create a sticky point between an abnormal molecule and its neighbor, causing molecules to pile up on each other.

Just as Linus Pauling predicted, after dinner, in 1945.

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.