Pauling’s First Hemoglobin Publications: Understanding Oxygen Binding

Pastel drawing of the hemoglobin structure, by Roger Hayward. 1964.

“You know, hemoglobin is a wonderful substance. I like it. It’s a red substance that brings color into the cheeks of girls, and in the course of my hemoglobin investigation I look about a good bit to appreciate it.”

– Linus Pauling, March 30, 1966

Seventy-five years ago, in 1935, Linus Pauling began publishing his research on the protein hemoglobin with a set of papers titled “The oxygen equilibrium of hemoglobin and its structural interpretation” appearing in Science and the Proceedings of the National Academy of Science .

In the fall Pauling extended this work and began collaborating with newly minted Caltech Ph. D. Charles Coryell, on the problem of the binding of oxygen to hemoglobin in the formation of the compound oxyhemoglobin. In April 1936, the duo published a paper specifically devoted to the subject, “The magnetic properties and structure of hemoglobin, oxyhemoglobin, and carbonmonoxyhemoglobin,” an important article which appeared in PNAS.

In order to better understand this early hemoglobin work, it is important to first discuss some of the basics of the hemoglobin molecule. Hemoglobin is a major protein component in the cytoplasm of red blood cells, and is made up of two distinct parts – the heme and the globin. Its primary function is to facilitate gas exchange: it picks up oxygen in the lungs, carries it to the tissues, and returns to the lungs in order to expel the carbon dioxide produced in the tissues.

There are four hemes per hemoglobin molecule, and each is made up of a single iron atom surrounded by a porphyrin ring. Each heme has the ability to bind to a single oxygen dimer, therein giving hemoglobin the capacity to bond with four molecules of O₂. The globin is the main protein component of the molecule. Carbon dioxide, rather than competing with oxygen for a binding site at the heme, instead binds to the globin.

Charles Coryell and Linus Pauling. 1935.

In their 1936 paper, Pauling and Coryell tackled the question of how oxygen binds to hemoglobin by looking at the molecule’s magnetic behavior, using an experiment involving bovine blood and magnets.  In a 1976 interview, Pauling provided this description of their experimental design.

It occurred to me that the same magnetic methods that we had been using to study simple compounds of iron, in order to determine the bond type, could be used to study the hemoglobin molecule. One of my students, Charles Coryell, and I, then got some blood, cattle blood, and put it into an apparatus. It consisted of a balance, which we had fitted out in such a way that a wire was suspended from one arm of the balance through a hole in the base of the cabinet, and held a tube. This tube was placed between the poles of an electromagnet. We filled it with blood, oxygenated blood, and balanced it to measure its weight. Then we passed an electric current through the coils of wire and the apparent weight changed.

From the experimental results, the pair found that oxyhemoglobin contains no unpaired electrons, although free oxygen molecules contain two, and each heme contains four. This was something of a surprise as, quoting from the paper,  “It might well have been expected, in view of the ease with which oxygen is attached to and detached from hemoglobin, that the oxygen molecule in oxyhemoglobin would retain these pair of electrons.”

In spite of this possibly more intuitive expectation, Pauling had earlier theorized that oxygen binds to hemoglobin covalently, a prediction which the experiment confirmed. Indeed, it was found that “the oxygen molecule undergoes a profound change in electronic structure on combination with hemoglobin,” and binds to the iron atom in the heme covalently.

Pastel drawing of Hemoglobin at 100 angstroms, 1964.

This was, however, only one of the striking discoveries that surfaced out of this research.  In a deoxygenated hemoglobin molecule, the bonds between iron and the four porphyrin nitrogen atoms surrounding it are ionic. Nonetheless, upon the binding of oxygen, these bonds become covalent, a rather dramatic change. Pauling and Coryell were keen to point this out:

It is interesting and surprising that the hemoglobin molecule undergoes such an extreme structural change on the addition of oxygen. Such a difference in bond type in very closely related substances has been observed so far only in hemoglobin derivatives.

Clearly something of consequence was being observed.  In their conclusion, the authors noted as much.

It is not yet possible to discuss the significance of these structural differences in detail, but they are without doubt closely related to and in a sense responsible for the characteristic properties of hemoglobin.

Linus Pauling’s work with hemoglobin continued on and off until his death in 1994, and led to a number of important discoveries – most prominent among them the molecular basis of sickle cell anemia. For more information on Linus Pauling’s hemoglobin research, please visit the website It’s in the Blood! A Documentary History of Linus Pauling, Hemoglobin, and Sickle Cell Anemia.

The Theory of the Molecular Evolutionary Clock

Dr. Emile Zuckerkandl, 1986.

“It thus appears possible that there would be no evolution without molecular disease.”
-Linus Pauling. “Molecular Disease, Evolution and Genic Heterogeneity,” 1962.

In the early 1960s, Linus Pauling and Emile Zuckerkandl, a French postdoctoral fellow who had arrived at Caltech in 1959, began researching the characteristics of hemoglobin extracted from a number of different species of animals. Zuckerkandl used a technique called fingerprinting, a process taught to him by a Caltech graduate student named Richard T. Jones, to create patterns of the amino acid sequences in each hemoglobin molecule.

[In her Master of Science thesis (pdf link), Dr. Melinda Gormley described fingerprinting, which was invented by the English chemist Vernon Ingram, as "a two-step process, [that] utilizes paper electrophoresis and paper chromatography. It produces splotches on paper at various locations; each mark corresponds to a peptide (two or more linked amino acids).”]

Once patterns had been prepared for several species, Pauling and Zuckerkandl compared them two at a time, and it was from the results of these comparisons that the theory of the Molecular Evolutionary Clock was developed.

Figures from: "A comparison of animal hemoglobins by tryptic peptide pattern analysis." October 1960. Proc. Natl. Acad. Sci. 46 (October 1960): 1349-1360.

The Molecular Clock differs from other evolutionary theories in that it tracks the evolution of a molecule rather than the evolution of a species. The theory states that, every so often, a mutation occurs in a given hemoglobin molecule. Generally speaking, this mutation is the source of a molecular disease, but will not cause any significant change to any organism other than its host.

Occasionally, however, a mutation will cause a lasting alteration to the molecule, and as the organism with the altered molecule reproduces, the change becomes permanent. More alterations of this nature can then occur on top of the original modification, thus resulting in even more differences in those hemoglobin molecules that have descended from the mutated original.

It is these differences that Pauling and Zuckerkandl were interested in when they compared the fingerprint patterns of different species, and their research led to an important breakthrough. As the duo compared more and more fingerprint patterns in a wider range of combinations, they observed that the number of differences between fingerprints lessened as the two species became more closely related. Pauling later stated that

[Zuckerkandl] found that in the beta chain of the human and the beta chain of the horse, for example, 20 of the 146 amino acids are different; but with human and gorilla, only one is different. It is the same amount of difference, just one amino acid residue, as between ordinary humans and sickle cell anemia patients, who manufacture sickle-cell-anemia hemoglobin.

From there Pauling and Zuckerkandl proposed that the comparative-fingerprinting method could be used to speculate as to how long ago any two species deviated from a common ancestor. Even more specifically, they reached the conclusion that one amino acid would be substituted every eleven to eighteen million years for any given species.

The evolutionary theory of the Molecular Clock was not readily accepted by scientists because it proposed a constant rate of evolution. However, it’s importance has now been noted and more research has been done on Pauling and Zuckerkandl’s original work. See, for instance, the very thorough examination conducted by Dr. Gregory J. Morgan in his 1998 paper “Emile Zuckerkandl, Linus Pauling, and the Molecular Evolutionary Clock, 1959-1965.” [pdf link], as well as Naoyuki Takahata’s “Molecular Clock: An Anti-neo-Darwinian Legacy,” [Genetics, (May 2007) 176: 1-6; not freely available online] which concludes that “a molecular clock is a most remarkable manifestation and a tribute from nature to anyone who studies evolutionary biology.”

For more information on Pauling’s hemoglobin work, please visit the website It’s in the Blood!  A Documentary History of Linus Pauling, Hemoglobin and Sickle Cell Anemia, and for more on Linus Pauling, check out the Pauling Online portal.

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:

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

Flyer for "Abnormal Human Hemoglobin Molecules in Relation to Disease." November 6, 1956.

“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.

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

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

“We 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?”

A sickled red blood cell

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 and the Rockefeller Foundation

Rockefeller Foundation administrator Warren Weaver.

“We are … particularly gratified that the Institute has found it possible to make a substantial contribution which will enable you to direct a larger proportion of our aid to the study of the substances of fundamental biological importance.”
- Warren Weaver to Linus Pauling, December 27, 1934.

It is obvious from much of his scientific work that Linus Pauling possessed a brilliant and uncanny ability to think across and between disciplines. Pauling was also a pragmatic and often business-like researcher who understood the necessity of securing financial support for his projects. The long and fruitful relationship Pauling maintained with the Rockefeller Foundation – and, in particular, a Rockefeller administrator named Warren Weaver – made possible much of Pauling’s most groundbreaking work on hemoglobin and structural chemistry. The full force of this intellectually-fruitful relationship reveals both the importance of interdisciplinarity in scientific work as well as the essential nature of active and timely funding.

Pauling received his first grant from the Rockefeller Foundation in 1932 for a program of research in structural chemistry. Shortly thereafter, in the fall of 1933, Pauling applied for and later received a three-year grant from the Foundation to support his experimental researches.  Pauling’s proposal was bolstered by his recent work in electron and X-ray diffraction, and held great promise of continued theoretical development in the study of the electronic structures of molecules.

In 1934 Pauling received more funding from the Rockefeller Foundation, this time in support of his hemoglobin research. He proposed to study hemoglobin in part because he understood that a great deal of general interest lay in the biomedical application of theoretical chemistry.

It is also clear that Pauling was, at least to a degree, shifting his research focus to match the lines of inquiry that the Foundation was interested in funding. In 1986, Pauling would note

…I’d had one elementary course in organic chemistry and no biochemistry. Didn’t know much about these things. I was getting support from the Rockefeller Foundation. Warren Weaver said to me, “Well it’s alright. We’ve been giving you some money to determine the structure of the sulfide minerals. But the Rockefeller Foundation isn’t really interested in the sulfide minerals. We’re interested in biological molecules and life.” So I said, “Well, I’d like to study the magnetic properties of hemoglobin and see whether the oxygen molecule loses its paramagnetism when it combines with the hemoglobin molecule.” So they said, “Alright, we’ll give you more money.”

And so it was, more or less, that Pauling’s hemoglobin work received Rockefeller support on the order of $70,000 per year circa 1940.

Listen: Pauling discusses the roots of his relationship with the Rockefeller Foundation

Pauling not only sought and gained special assistance from Rockefeller funds, but Rockefeller personnel also contributed to the development of his hemoglobin work throughout the 1930s. Alfred E. Mirsky, a professor in cell biology at the Rockefeller Institute for Medical Research, was one of the first individuals with whom Pauling discussed potential hemoglobin research. Pauling quickly developed a personal friendship with Mirsky and clearly held his colleague in very high regard as a scientist. In a 1944 letter recommending Mirsky for a position at the Carnegie Institution of Washington, Pauling wrote

I do not know any one who is so keenly interested in the development of the field of science involving the applications of chemistry and physics to borderline problems of biology, and especially of genetics, and who has such a penetrating understanding of the work which has been done. I find that every conversation which I have with Dr. Mirsky gives me some valuable idea. He has a masterly ability to coordinate results into a significant whole.

Alfred E. Mirsky

Indeed, over the years Pauling gave a number of lectures at the Rockefeller Institute and continued to benefit from a wide array of academic and personal relationships that began with the Foundation. The Foundation also continued to fund Pauling’s work well into the 1950s, contributing mightily to the “big science” phenomenon that helped define academic research following World War II.

The Rockefeller Foundation was pioneering in its recognition of the importance of supporting interdisciplinary work; in particular, it actively sought to foster research between biology and chemistry. In many ways, Pauling with the prototype scientist that the Foundation was looking to support. Looking back, few can deny the impact that this partnership made on the history of twentieth century science.

For more information on Pauling’s relationship with the Rockefeller Foundation, see the website It’s in the Blood! A Documentary History of Linus Pauling, Hemoglobin, Sickle Cell Anemia. We also strongly recommend the book The Molecular Vision of Life: Caltech, the Rockefeller Foundation, and the Rise of the New Biology (1993), written by the late Dr. Lily Kay.

Linus Pauling, Vitamin C and the AIDS Crisis

Ewan Cameron, Ava Helen and Linus Pauling. Glasgow, Scotland, October 1976.

“Many orthomolecular substances are so free from toxicity that they show beneficial effects over a 10,000-fold range of concentrations. Yet if you take even ten times the amount of aspirin that many patients take, for example, you’d be dead; hundreds of people do die every year from aspirin poisoning. And all of the other major drugs are highly toxic as well.“
- Linus Pauling, December 1986

Today, December 1, 2008, is World AIDS Day. In honor of the fight against AIDS, The Pauling Blog would like to discuss Pauling’s own attempts to find a cure.

Beginning in the early 1930s, stemming from early investigations into the chemical nature of hemoglobin, Linus Pauling became deeply interested in the application of chemistry to medical problems.  Once involved in a long-term study of the substance, he began to recognize the significance of hemoglobin to the health of individuals. In 1949, Pauling coined the term “molecular disease” in reference to a mutation in hemoglobin cells that caused sickle cell anemia.

His interest in medicine did not stop there, however. During World War II, Pauling designed a meter to measure oxygen levels aboard U.S. submarines. He later converted this meter to be used in incubators for premature babies with underdeveloped lungs, saving thousands of lives in the process.

In the early 1970s, Pauling developed an interest in the use of megadoses of vitamins as a means for both preventing and treating disease. He became particularly interested in vitamin C because, even in huge doses, it proved to be non-toxic.  Pauling began recommending its use to prevent colds and other illnesses, eventually suggesting that a high-dosage vitamin C regimen could help cancer patients by fortifying the immune system and potentially destroying carcinogens.

With the onset of the AIDS crisis in the early 1980s, Pauling saw potential for another area in which vitamin C could be put to good use. Though he did not officially endorse vitamin C as a treatment for AIDS until the early 1990s, he commonly noted its possible benefits and lack of side effects.

In 1988, Pauling headed a study on the effects of vitamin C in combating the AIDS virus, measuring the impact that ascorbic acid had on infected T-cells. The results, though not extraordinary, were promising.

In 1990, Pauling and his colleagues published the results of their study in the Proceedings of the National Academy of Sciences, claiming that vitamin C appeared to suppress the growth of the AIDS virus. As was true of Pauling’s previous claims regarding vitamin C and orthomolecular medicine, the studies were at least a source of intrigue to many, though likewise dismissed by a wide cross-section of the medical community.

At the same time that Pauling was embarking on his AIDS research, Ewan Cameron, a researcher at the Linus Pauling Institute of Science and Medicine in Palo Alto, approached Pauling regarding a book he was writing entitled The AIDS Disaster. The book was meant to serve as a comprehensive study of the AIDS virus, describing its history, socio-political context, and related research.

Cameron and Pauling's unpublished manuscript, "The AIDS Disaster." August 1988.

Cameron requested that Pauling serve as co-author, editing the book and providing a chapter on vitamin C and AIDS. Pauling agreed and, in late 1988, the book was completed. Due to a variety of publishing issues, the text never reached bookstore shelves, but several complete versions of the manuscript are held in Cameron’s papers here at Oregon State University.

Until his death in 1994, Pauling continued to emphasize the responsibility of the scientific community to help cure diseases such as AIDS and cancer. He gave frequent lectures on the subject of orthomolecular medicine and continually worked to increase support for medical research.

For additional reading on Ewan Cameron’s AIDS work and research, check out the Cameron Papers finding aid, hosted online by the OSU Libraries Special Collections.  Please also note that a few more of Pauling’s thoughts on the treatment of AIDS with ascorbic acid are linked off of this index page from the Linus Pauling Research Notebooks website.

Pauling’s Methodology: X-ray Crystallography

X-ray apparatus at Linus Pauling's desk, Gates Laboratory, California Institute of Technology. 1925.

“I was very fortunate in having A.A. Noyes suggest to me, or tell me, that I was to work with Roscoe Dickinson on x-ray crystallography, determination of the structure of crystals by x-ray diffraction. This technique gave for the first time detailed information about how atoms are related to other atoms in a crystal and how far apart they are from the other atoms.“
- Linus Pauling, 1988.

As a graduate student, well before Pauling began to research hemoglobin in earnest, he spent a great deal of his time using the technique of X-ray crystallography to determine the crystalline structure of a number of inorganic compounds. Pauling recalled that at that time X-ray crystallography “was a new technique, ten years old when I began. Quite a number of structures had been determined but there was a tremendous field open, a tremendous amount of work that could be done.”

Listen: Pauling discusses the importance of X-ray crystallography to his early structural chemistry research

The young Pauling obviously reveled in the excitement of being able to use a new and powerful technology. “We have a pretty extensive collection of apparatus” he once wrote to William Lawrence Bragg, the senior author of a 1922 textbook that started Pauling on X-ray crystallographic research. Any one of Bragg’s student’s, Pauling remarked, “no matter how physical his training,” need not “be frightened at coming to a chemical laboratory” so well-stocked with mechanical apparatus.

Initially Pauling used the technique of X-ray diffraction to determine the structures of fairly simple inorganic compounds, but later, as his own expertise grew and as he discovered new sources of funding, Pauling oriented this new technology toward complex organic compounds, including hemoglobin.

What was ultimately important to Pauling was not what X-ray crystallography could tell him about the size, structure, or relative placement of atoms within a molecule, but rather, what broader theories that information could then be used to support. His growing allegiance to structural chemistry, his developing ideas about the nature of the chemical bond, and his still nascent interest in biochemical interaction were all fed by his experience of rigorously determining molecular structure through new technological methods.

Pauling’s manuscript notes concerning his early experiments with hemochromogen, for instance, indicate the wide spectrum of experimental results he had to assimilate in order to create a coherent picture of the hemoglobin molecule.

"Outline of Experiments on Hemochromagen," pg. 1. June 25, 1935.

The difficulties presented by the need to combine the information he had obtained from x-diffraction with information from other kinds of experimentation, including solubility and more traditional experimental methods, are readily apparent in Pauling’s notes.  Indeed, the impressive new technology of X-ray crystallography is relegated to just one entry in a list of experimental results.

Ultimately it wasn’t the technology at Pauling’s disposal that helped him become such a successful researcher, but rather his attitude in approaching technology and his ability to use the results it gave him to construct more broadly-applicable and intellectually-powerful theories.

To learn more about Linus Pauling’s use of x-ray crystallography, see the websites Linus Pauling and the Nature of the Chemical Bond: A Documentary History and It’s in the Blood!  A Documentary History of Linus Pauling, Hemoglobin and Sickle Cell Anemia.

Thinking Between Disciplines: Immunological Interests and Beyond

Hand-drawn figures used in "A Theory of the Structure and Process of Formation of Antibodies." July 27, 1940.

“I believe that chemistry will play a very important part in the golden age of biology that is now beginning.”
- Linus Pauling, “Molecular Structure and Biological Specificity,” July 17, 1947.

One of the reasons why Linus Pauling enjoyed such a prolific and diverse scientific career was his ability to combine and draw inspiration from rather disparate interests and research questions.

Indeed, structural chemistry – the discipline with which Pauling is most commonly associated – appealed to Pauling in part because it allowed him to consider the physical causes underlying the chemical nature of certain biological phenomena in concert with known principles of chemical interaction.  In other words, Pauling viewed structural chemistry as the avenue by which he could best utilize the tools not only of chemistry, but of physics and biology as well.

Many of Pauling’s laboratory experiments rested on knowledge and methods borrowed liberally from biology, medicine, chemistry and physics. In a 1946 proposal for a program of fundamental research in biology and medicine at Caltech, Pauling emphasized that the long-established cooperation of the Institute’s divisions of Biology, Chemistry, and Chemical Engineering were resulting in a vigorous and successful “attack” on the “great fundamental problems of biology and medicine.” As he sought to justify the expansion of these interacting programs, Pauling wrote that the “primary features” of their organization were “the presence of a group of men rigorously trained in the exact sciences and interested in attacking…broad problems.”

Of nearly-equal importance was an “unusual spirit of cooperation.” Such ‘unusual cooperation,’ in Pauling’s opinion, could be expected to produce work that was at once “sound but imaginative,” and indebted to “the transfer of ideas among different fields…ranging from quantum mechanics to animal physiology.” Pauling’s ideas on the nature of hemoglobin and sickle cell anemia were two of the ‘sound but imaginative’ ideas that arose out of the broader culture of interdisciplinary laboratory research.

In the 1930s Pauling came under the influence of a prominent immunologist, Karl Landsteiner, who helped to turn his attention and interest towards the mechanism of immunological response. To Pauling, the fundamentals of immune response in the body seemed reminiscent of the folding of hemoglobin in the presence of iron. Both mechanisms underscored the importance of the physical structure of a molecule in influencing its chemical interactions.

Pauling’s work on both the nature of hemoglobin as well as the immunological reaction to antigens and foreign proteins were linked practically, as well as conceptually, to his hemoglobin research. As he came to learn more about immune response, Pauling applied some of this knowledge to increasing the practical value of his work on the development of Oxypolygelatin, a blood substitute created as part of the Pauling’s contributions to the Allied effort during World War II.

An original container of 5% Oxypolygelatin in normal saline. Developed by Linus Pauling as part of his scientific war work research program, mid-1940s.

This project, which was not completed to fruition until 1949, was vexed by certain problems having much to do with the nature of blood in the human body. In a handwritten note from 1945, Pauling suggested that foremost among his concerns vis-a-vis the creation of a suitable blood alternative were both a “lack of toxicity,” and a lack of “antigenicity.”

Pauling’s ideas on the nature of hemoglobin, sickle cell anemia and the blood substitute Oxypolygelatin were all born of his ability to fruitfully-combine the methods of several different disciplines with the expertise of his colleagues and fellow researchers. Even moreso, this remarkable body of work constitutes a clear example of the important place that interdisciplinarity can assume in scientific research.

To learn more about Pauling’s research on hemoglobin, immunology and Oxypolygelatin, please visit the website It’s in the Blood!  A Documentary History of Linus Pauling, Hemoglobin and Sickle Cell Anemia.

Thinking Structurally: The Roots of Pauling’s Hemoglobin Work

Pastel drawing of Hemoglobin at 20 angstroms, 1964. Drawing by Roger Hayward.

“Linus Pauling is one of that select group of individuals whose lives have made a discernible impact on the contemporary world. His contributions to molecular chemistry have been substantial and fully deserving of the recognition that he received in the form of a Nobel Prize in chemistry….Pauling continued to do productive scientific work throughout his lifetime, making a second outstanding contribution in his discovery of the molecular processes involved in sickle-cell anemia. This discovery, if made by anyone who was not already the only person to receive two unshared Nobel Prizes, might well have merited a third prize in medicine.“
- Ted Goertzel. “Linus Pauling: The Scientist as Crusader.” Antioch Review, 38 (1980): 371-382. 1980.

Two of Linus Pauling’s greatest scientific discoveries, his work on the nature of the chemical bond and the discovery of molecular disease, both hinged on his distinctly structural approach to scientific problems.

Having written a doctoral dissertation on the determination of the molecular structure of inorganic compounds in crystalline state, Pauling chose hemoglobin as an object of study in part because he knew that it was hemoglobin’s changing structure that allowed it to carry oxygen to the tissues of the body. While Pauling like to joke that he chose to work on blood because it was easy to obtain, the intellectual challenge of explaining the sigmoid curve of oxygen saturation in hemoglobin profoundly sparked Pauling’s scientific interest.

Notes on hemoglobin, 1935. Note Pauling's caricature of hemoglobin and oxygen sigmoid curve sketch to the upper left, as well as his notes on "magnetic properties" and a possible 3d structure further down the page.

Later, upon learning about the disease sickle-cell anemia, Pauling came to recognize that the potentially molecular and structural basis of the disease could facilitate a deeper investigation into structural studies of the molecule. Hemoglobin, in part because of its association with the bonding and transport of iron atoms, demonstrated extremely changeable magnetic charges and suggested, even from a preliminary acquaintance, the importance of structural changes in chemical function.

By 1934, when Pauling suggested hemoglobin as the organic molecule of choice for his particular research program, he had already laid out a general plan of research that relied heavily on investigations into the structural and electrically-charged nature of organic molecules. In May of 1935, Pauling wrote in his research notebook

At this time I have analyzed the oxygen equilibrium data to make plausible the idea that in hemoglobin the four hemes are arranged at the corners of a square on one side of the globin, being interconnected along the edges of the square, and that in the hemochromogens the hemes are independent of one another; and I have outlined a general program of investigation, consisting mainly of magnetic studies and x-ray studies (anomalous dispersion, radial distribution about iron atoms).

In a way, Pauling had always been thinking structurally about the nature of the hemoglobin molecule, its ability to bind oxygen molecules and, later, its particular pathology in the case of sickle-cell anemia.

In 1937, Pauling delivered an inaugural lecture for the Sigma Xi society of Corvallis, in which he asserted both the importance and the relatively-recent arrival of structural chemistry as a discipline. For Pauling structural chemistry involved “the determination of the structures of molecules – the exact location of the atoms in space relative to one another – and the interpretation of the chemical and physical properties of substances in terms of the structure of their molecules.”

This lecture, entitled “Hemoglobin and Magnetism,” addressed the “new branch of chemistry, modern structural chemistry” through a discussion of some of Pauling’s most recent work on hemoglobin’s magnetic properties.