Sickle Cell Research to the Present and the Future

Three-dimensional rendering of sickle cell anemia blood cells. Credit: National Institutes of Health.

By Dr. Marcus Calkins, Part 3 of 3

Forty years after Linus Pauling and his lab demonstrated the molecular basis for Sickle cell disease and James Watson speculated that upregulation of fetal hemoglobin may protect from the disease, methods to control fetal hemoglobin specifically in red blood cells began to be developed. The molecular biology revolution of the late 20^th century had produced extensive knowledge about the molecular systems that drive fetal hemoglobin production, but harnessing that intricate knowledge has taken another thirty years.

Hematopoietic Stem Cells (1990s)

Since the advent of radioactivity research, it has been well-established that red blood cells have a short lifespan of only about 115 days and are continually produced from precursors in the bone marrow. In order to replace defective blood cells in an individual, a protocol for whole-body irradiation and allogenic bone marrow transplant was pioneered by a group of doctors in Seattle in the 1970s, as a treatment for cancer patients.

However, the ability to specifically isolate and identify hematopoietic stem cells from patients was only developed in the 1990s. At that time, nuclear dye exclusion and flow cytometry characteristics were used to isolate the stem cells, but since then, a variety of cell surface markers have been identified, and protocols to isolate, expand and differentiate hematopoietic stem cells have become standardized. In addition, scientists have learned to modify the hematopoietic stem cells at a genetic level, creating the possibility that stem cells may be extracted, genetically modified ex vivo, and then used to reconstitute the bone marrow of patients with blood diseases.

For patients with Sickle cell disease, it may therefore be possible to extract hematopoietic stem cells and inactivate the BCL11A gene, which normally suppresses fetal hemoglobin. The red blood cell progeny of these altered stem cells would then produce fetal hemoglobin that could mask the effects of the disease-causing mutation in the β-globin gene. Afterward, the modified stem cells could be transplanted back into the same patients from which they were isolated, providing the person with a continual supply of red blood cells that expresses fetal hemoglobin and are resistant to sickling.

Gene Therapy and Genome Editors (2000s-2010s)

The final component of a therapy for Sickle cell disease has recently been realized, as it is now feasible to efficiently inactivate BCL11A in isolated hematopoietic stem cells. In the last two decades, several systems of modifying the genome (gene editors) have been developed. Although the first editors to be produced may still find clinical use, CRISPR has quickly overtaken previous technologies to become the most widely applied and well-known gene editing platform.

In the late 1990s, researchers invented two key methods of using proteins to make targeted edits to the genome. These early gene editor proteins are called TALENs and Zinc fingers, both of which are being tested in clinical trials today. Each of these editors is able to target highly specific DNA sequences and make an incision in the DNA helix at a predictable site. Once the DNA strand is incised, error-prone DNA repair processes are activated to fix the incision, often resulting in random base insertions, deletions and changes. In this way, the genetic code is disrupted in some cells, and these random disruptions often serve to inactivate the targeted gene. If those cells with an inactivated gene can be identified and expanded, whole populations of cells with the genetic alteration can be established.

The comparative difficulty of using TALENs and Zinc finger proteins instead of CRISPR is that targeting a particular site in the genome often requires a major technical effort. Since the proteins themselves target the DNA sequence of interest, each target sequence must have its own specialized editor. The introduction of CRISPR/Cas9 in the early 2010s allowed researchers to target various DNA sequences much more easily. This system uses a short guide RNA molecule for DNA targeting, so the same protein can be used to cut any genomic site. Since generating these guide RNAs is a relatively simple procedure, the amount of effort required to design and execute genome edits is greatly reduced.

Theoretically, TALENs, Zinc fingers and CRISPR could all be used to inactivate BCL11A in hematopoietic stem cells. However, design of an appropriate TALEN or Zinc finger might require relatively large investments of money and time.  On the other hand, CRISPR promises to be a more cost-effective and faster approach to editing the genome. In academic studies, CRISPR is already widely used and far more common than the other editing technologies for making genetic modifications to laboratory model organisms. However, with human patients, safety and efficacy greatly outweigh effort and cost. Time will tell which gene-editing platform proves to be most cost-effective, efficient and safest for clinical use.

A New Clinical Reality (2020s)

With these new tools at hand, Watson’s dream of increasing fetal hemoglobin in Sickle cell disease patients is finally within sight. At least two major collaborations to perform ex vivo gene therapy for Sickle cell disease have been initiated since 2018. Both use gene editors to inactivate the BCL11A gene and promote fetal hemoglobin expression in red blood cells.

One collaboration between Bioverativ and Sangamo is testing a protocol for gene editing with a Zinc finger. The estimated completion date for this trial is 2022. Another collaboration that has received a great deal of attention, and was recently published in the New England Journal of Medicine, involves CRISPR Therapeutics and Vertex Pharmaceuticals. This trial is among the first to attempt CRISPR in a clinical setting, and the results are highly anticipated by the research and medical communities, not only for their impact on Sickle cell disease, but also as a bellwether for the use of CRISPR in medical practice. So far the results of the trial are encouraging. As of December 2020, the first two patients to receive therapy were reportedly doing well and were free from symptoms more than one year after receiving the treatment. While this news is exciting, there is still much work to be done before the technique can be applied to a wider population.

It has taken many years and many twists, but the visions of the 1950s are finally beginning to be realized, bringing us to the cusp of an exciting new dawn in medicine. The slow march toward a cure for Sickle cell disease clearly demonstrates that through patience and continued investment in scientific discovery, we can continue to achieve the dreams of our predecessors and plant new seeds for future generations to reap the harvest.  

Sickle Cell Research in the Wake of Pauling and Watson

Harvey Itano, 1954. Image credit: Caltech Archives.

By Dr. Marcus Calkins, Part 2 of 3

The molecular defect in Sickle cell disease was demonstrated by Linus Pauling’s lab in 1949, and one year prior, James Watson had proposed that increasing the level of fetal hemoglobin may provide a means by which the disease could be cured. These two publications provided a direction for what may soon become a real-life cure for the disease. However, many practical questions needed to be answered before a cure could be realized, or even imagined in a realistic sense. The first of these questions had to do with the composition of hemoglobin protein.     

Defining the structure of hemoglobin (1950s-1960s)

The project in the Pauling lab was led by a formidable graduate student of Japanese descent named Harvey Itano. It is noteworthy that despite his birth in Sacramento and his honorific as valedictorian of the University of California, Berkeley class of 1942, Itano was interned at Tule Lake during World War II. He was only released from detention to attend medical school at Washington University in St. Louis. Upon graduating with his M.D., he came to Pauling’s lab to pursue a Ph.D. His widely celebrated doctoral thesis was built on the Sickle cell disease project. After finishing this training in biochemistry and publishing his high-impact paper with Pauling, Itano opened his own lab in which he continued working on hemoglobin.

By applying many of the same methods pioneered in Pauling’s lab, Itano’s small group became central players in identifying aberrant forms of hemoglobin that lead to disease. Because of this careful and detailed work, Itano and other scientists began to suspect that the protein subunits that make up hemoglobin may be derived from multiple, highly similar genes. At the time, this idea represented a major paradigm shift, as it was previously assumed that each enzyme should correspond to a single gene.

This idea also brought up an evolutionary question of how such similar genes might come to exist. In a memorial biography, Itano’s friend and colleague R.F. Doolittle summarized Itano’s forward thinking on the genetic and evolutionary basis of enzyme constituents as follows:

In his thorough review of all the data, Harvey noted that in sheep and cattle, however, there were two kinds of end group (valine and methionine), and concluded that there had to be two each of two kinds of polypeptide in human adult hemoglobins. He went on to discuss how gene duplications could account for all the various hemoglobin chains, including myoglobin and foetal hemoglobin. These were thoughts well ahead of their time.

These major scientific contributions and others led to Itano’s election to the National Academy of Sciences, U.S.A. He was the first of many Japanese Americans to achieve that honor.

The exact change in hemoglobin protein responsible for causing Sickle cell disease was identified at the amino acid level ten years after Itano’s paper with Pauling, when V.M. Ingram used protease digestion to show that the disease came from a glutamine-to-valine mutation in the β-globin subunit of the hemoglobin protein complex. Although the molecular structure of DNA was accurately modeled by Watson and Crick in 1954, revealing tantalizing clues as to how genetic information can be encoded and passed from cell to cell and parent to child, the precise genetic mutation underlying Sickle cell disease would remain unknown for a few more decades, until the molecular biology revolution.

Molecular Switch from Fetal to Adult Hemoglobin (1970s-1990s)

In order to institute the cure dreamed up by Watson, much needed to be learned about hemoglobin composition, genetics, and control in red blood cells. This basic knowledge was largely generated through new technologies developed in the latter part of the 20^th century. The cell and molecular biology revolution began in the mid-1970s and brought with it new techniques for manipulating DNA and other molecules. These advances allowed scientists to define genes and genetic structures, and to begin to understand how gene expression is regulated.

With regard to hemoglobin, it was discovered that two sets of genes (five on Chromosome 11 and three on Chromosome 16) encode the subunit proteins for the hemoglobin molecular complex. Each set contributes one subunit to the complex, and the subunits that are present in the blood change, depending on the developmental stage of the individual. In essence, a switch occurs just after the time of birth, wherein expression of fetal hemoglobin is turned off and adult hemoglobin is turned on. The mechanics of this switch, including the DNA sequences and proteins that make it work, were elucidated during this fruitful period of biological research.

Once the mechanics of the fetal hemoglobin to adult hemoglobin molecular switch were known, it became apparent that one gene, BCL11A, is a dominant factor in shutting down fetal hemoglobin production. If this gene can be silenced or disrupted, fetal hemoglobin expression will continue. Furthermore, there is strong evidence from molecular, cellular and clinical studies that continued expression of fetal hemoglobin will at least partially prevent the harmful accumulation of mutant hemoglobin aggregates and thereby prevent Sickle cell disease.

However, turning off the BCL11A gene is not an easy task. The molecular system evolved over millions of years to function robustly under almost any environmental condition or situation. Like a train hurtling down a track, such developmental programs are extremely hard to redirect, and if they are completely derailed, the individual may experience catastrophic effects. Thus, precise control of only the BCL11A gene, precisely in red blood cells, is necessary to realize the therapy first imagined by Watson. This level of control is possible to achieve in cells outside the body, but inside the body, cells are resistant to change, and our ability to target only one cell type at a time remains relatively primitive. Thus, a cure for Sickle cell disease still needed a method for regulating BCL11A specifically in red blood cells.

The Slow March Toward a Cure for Sickle Cell Disease

Pastel drawing of sickled hemoglobin cells by Roger Hayward, 1964

By Dr. Marcus Calkins

[Ed Note: This is the first of three posts examining the history of sickle cell treatment up to present day. It is authored by Marcus J. Calkins, Ph.D., “a proud OSU alumnus” (Chemical Engineering, B.S., 1999), who now works as a scientific communications service provider and educator in Taipei, Taiwan. In submitting this piece, Calkins emphasized that he has “taken inspiration from Linus Pauling’s research activities, teaching methods and moral character for many years.”]

In 2020, Jennifer Doudna and Emmanuelle Charpentier shared a Nobel Prize for their discovery and development of the CRISPR gene editor. One of the first clinical applications for CRISPR promises to be an ex vivo gene therapy for Sickle cell anemia. If it works, this medical technology will be a major breakthrough in biomedicine, representing the culmination of more than a century of research on Sickle cell disease that encompasses a wide range of topics.

Despite the lifetimes of work that have led to our current exciting position on the precipice of a cure for Sickle cell disease, the basic molecular features of the disease were defined seven decades ago by another Nobel Prize winner, Linus Pauling. The intervening 70 years of work have been required for scientists to learn how we might apply the foundational knowledge to actual patients in a real-life clinical setting. While the pace of progress may seem agonizingly slow to those outside biomedical research, the ground that has been covered is immense, and entire fields of biomedicine needed to be built and optimized before a truly feasible treatment technology could be invented.

Sickle Cell Disease (1910)

Sickle cell disease was first described over the period from 1910 to about 1924. During this time, a series of case reports detailed approximately 80 people of African descent, who had an odd morphology of red blood cells resembling a crescent or a sickle. In many cases, this sickle-like morphology was associated with a devastating condition involving severe anemia and early death. Furthermore, scientists learned that the red blood cell sickling could be exacerbated by depriving the blood of oxygen, either by adding carbon dioxide to cells in a dish or restricting blood flow in the patient. These clinical observations laid the foundation for basic scientists to postulate that the condition was related to hemoglobin, the protein that carries oxygen in red blood cells.

The first person to make this suggestion was Pauling. At some time in 1945, he was chatting with a colleague on the train from Denver to Chicago, when he learned about the difference in sickling between oxygenated and deoxygenated blood. According to the account of his colleague, Pauling was also informed that the sickled red blood cells show birefringence when viewed under a polarizing microscope, which would suggest an alignment of molecules within the cells.

However, by Pauling’s account of the conversation, his immediate guess that Sickle cell disease is caused by a defect in the hemoglobin protein complex was based entirely on the difference in the sickling properties of oxygenated and deoxygenated blood. Notably, Pauling later stated that the idea of Sickle cell disease being singularly caused by the hemoglobin molecule came to him in “two seconds,” but gathering evidence and refinement of the idea took at least three years.

In his public talks, Pauling often emphasized the fact that in the first years of the study, his students performed many experiments but could not identify any obvious biochemical differences between the hemoglobin molecules of patients and control individuals. From his repeated emphasis of this fact, one might speculate that the translation of a two-second idea to a three- or four-year demonstration would have been frustrating for such a quick-minded individual, though Pauling never said as much. Alternatively, he may have simply been emphasizing the challenges and slow, steady nature of rigorous scientific pursuit.

The Molecular Defect and a Potential Cure (1949)

In 1949, prior to the double helix model of DNA and before stem cells were described, the Pauling lab published a paper titled “Sickle Cell Anemia, a Molecular Disease.” In this work, Pauling and his students definitively showed that a slightly abnormal form of hemoglobin is found exclusively in patients with the cell sickling phenotype. Using a 30-foot-long Tiselius apparatus that they had constructed for electrophoresis, a small two-electron difference could be detected in the overall charge of hemoglobin molecules from Sickle cell patients and unaffected individuals. Meanwhile, carriers of the disease had a mixture of the two hemoglobin isoforms.

Importantly, Pauling’s group found that the defect in hemoglobin is not related with its ability to bind oxygen. Instead, it was later shown that the slight change in molecular charge affects the way hemoglobin proteins interact with each other, as would be predicted from the birefringence observation. This aberrant interaction causes the formation of long molecular scaffolds that change the shape of the red blood cell and lead to its dysfunction.

With this publication, Sickle cell disease became the first disorder to be associated with a single molecule. It was also the first with a known genetic basis. In his publication of the same year, J.V. Neel showed that Sickle cell disease follows an autosomal recessive inheritance pattern, meaning that each parent must contribute one copy of the mutated gene for a child to develop the disease. The cell sickling phenotype can occur to some degree in people who only carry one mutant allele, but only those with two copies experience the pernicious effects of the disease. This information, combined with Pauling’s study, established the essential basis for our understanding of Sickle cell disease and serves as a model for many other genetic diseases.

Surprisingly, James Watson (prior to his famed work on the structure of DNA) contributed a prescient idea to Sickle cell disease treatment, when he speculated that cells could be protected by expression of another form of hemoglobin, fetal hemoglobin. Watson made this prediction in 1948, just one year before Pauling’s powerhouse publication. His suspicion was an extension of reports that red blood cell sickling did not happen in the blood of infants who would later develop the condition as children and adults.

The stage was thus set for a Sickle cell disease cure. After the theoretical basis was determined, onlookers might have expected a cure for the disease to be found within a few years. However, extension of the ideas of Pauling and Watson has required incredible efforts by myriad scientists over the course of the next seven decades to create a potential new clinical reality.

Finding Resources for Basic Science and Medical Research

Linus Pauling, 1949

[Exploring Linus Pauling’s popular writings on the shape of post-war science, part 4 of 5.]

“Our job ahead.” Chemical and Engineering News, January 1949

The onset of 1949 brought with it the beginning of Linus Pauling’s one-year term as president of the American Chemical Society, and Pauling’s article “Our job ahead” outlined the message that he wished to convey to the society. In it, Pauling specifically addressed the financial concerns being faced by the ACS as well as the scientific community at large.

The society’s problems centered on the need to manage operating costs and member remunerations in the midst of rising costs of living. More broadly though, Pauling saw for the society a responsibility to try to improve financial conditions for science as a whole. Pauling argued that the destruction of war, in tandem with the massive consumption of natural resources required by the war effort, had resulted in increasing levels of poverty throughout the world. Pauling encouraged the ACS to do its part to combat the problem by supporting and participating in global interdisciplinary scientific cooperation.

Pauling also pushed for ACS support of basic research, believing that work of this sort was most likely to lead to significant breakthroughs. Doing so would be made all the more effective by the creation of a National Science Foundation, which would issue and administer unrestricted grants on behalf of the federal government. It was Pauling’s ultimate vision that the majority of research dollars be provided by the federal government, with supplementary funding being made available by state governments, permanent endowments, private foundations, and industry.

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“Chemistry and the world of today.” Chemical and Engineering News, September 1949.

The themes put forth by Pauling in his initial message to the ACS – particularly the need for a National Science Foundation – were continued in his presidential address, delivered in fall 1949.

Pauling opened his talk with a broad question, “What can I say under the title ‘Chemistry and the World of Today?'” His answer was “that I can say anything, discuss any feature of modern life, because every aspect of the world today – even politics and international relations – is affected by chemistry.”

Pauling’s all-roads-lead-to-chemistry perspective informed his strong support of a potential National Science Foundation and his firm belief in the value of basic research. He lamented the ongoing struggle for funding faced by so many of his colleagues, and pressed the notion that even applied science was dependent on advances in basic science. Moreover, Pauling suggested that applied science often received the credit for ideas that had initially been discovered or cultivated by basic researchers.

Above all, Pauling believed that, in the post-war era, “…a nation’s strength will lie largely in the quality of its science and scientists.” That noted, Pauling emphasized that government funding for scientific research should not be funneled toward military channels. To this end, it was the responsibility of the ACS, as an organization representing American chemists, to make its voice heard in the fight for the creation of a National Science Foundation.

During Pauling’s presidential year, the concept of the NSF had been put forth in political circles but had not yet been acted upon. Looking forward to that day (which would, in fact, come the next year) Pauling put forth an ideal scenario where the NSF would fund $250 million a year in research, while science-dependent industries would fund an additional $75 million. Of this latter contribution, Pauling believed that private funding ought to be considered as a form of insurance rather than charity, since it was certain to fuel the scientific discoveries necessary to drive industrial development.

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“Structural chemistry in relation to biology and medicine.” Second Bicentennial Science Lecture of the City College Chemistry Alumni Association, New York, December 7, 1949. Baskerville Chemical Journal, February 1950. 

At the end of 1949, Pauling gave another high profile public lecture, this time to the City College Chemistry Association in New York. In this talk, he focused on the relationships between structural chemistry, biochemistry, and molecular medicine.

Pauling began by citing the role that chemistry had played in catalyzing immense achievement in medicine over the preceding half-century, referencing in particular the discovery and refinement of chemotherapeutic agents including antibiotics. That said, Pauling was quick to point out that scientists still had a very poor understanding of the principles and structural attributes underlying chemotherapeutic functions. It was Pauling’s belief that “…if a detailed understanding of the molecular basis of chemotherapeutic activity were to be obtained, the advance of medicine would be greatly accelerated,” and that structural chemistry was fast approaching a point where it could produce this understanding. Once done, Pauling suggested that the decade or two that followed would surely offer significant advancements in the scientific understanding of medicine and the development of new pharmaceuticals.

Pauling then identified a collection of major areas where he thought biomedical research should be focused. The first involved developing a detailed molecular structure of 1) chemotherapeutic substances (i.e., antibiotics and other medications), 2) the organisms against which they are directed (bacteria, viruses, etc.), and 3) the human organism which they are meant to protect. A second major program of work should delve into the nature of the forces involved in the intermolecular interactions between the above substances and organisms.

Pauling pointed out that the last quarter century had seen great progress in the first goal – the science of organic chemistry had been developed and the structures of many organic compounds had been confirmed. But progress elsewhere, though promising, had not come about so quickly. For questions related to the physiology of disease-causing organisms and of the human body itself, advancements were fated to be slow simply due to the immensity of the task. In addition, structural chemistry was a fairly new field and, although it was growing quickly, the stock of previous discoveries upon which one might expand was finite (research thus far had mainly focused on the structures of amino acids and peptides).

The latter goal, an understanding of the intermolecular interactions between chemotherapeutic substances and the organisms they are meant to treat or defeat, had seen the least progress of all. It was a complicated task for sure, and though he had very little data in hand, Pauling offered a back of the envelope theory about what might be going on, speculating that

…some drugs operate by undergoing a chemical reaction with a constituent of the living organism, and that others operate by the formation of complexes involving only forces that are usually called intermolecular forces.

Regardless, Pauling felt that work in these areas would prove integral to the conduct of future medical research, and he put forth his own work on sickle cell anemia as an example of how other investigations might unfold. Specifically, Pauling and his team had discovered that the hemoglobin present in the red blood cells of afflicted individuals differed structurally from normal hemoglobin. Were other investigators able to develop a similar molecular understanding of a given disease, producing new treatments would be that much easier, since chemotherapeutic agents could be tailored to fit a particular molecular architecture. Work of this sort would

…represent the first time that a chemotherapeutic agent had been developed purely through the application of logical scientific argument, without the significant interference of the element of chance.

Similar to his call for an NSF, Pauling encouraged the creation of an institute for medical chemistry that would train a new generation of students to apply chemistry to medical problems. Doing so, in Pauling’s view, ought to be prioritized due to its potential significance to the health and happiness of all people.

Dr. Edna Suárez-Díaz, Resident Scholar

Dr. Edna Suárez-Díaz delivering her Resident Scholar lecture at the Valley Library, Oregon State University.

Dr. Edna Suárez-Díaz is the most recent individual to complete a term as resident scholar in the Oregon State University Libraries Special Collections and Archives Research Center. A professor in the Department of Evolutionary Biology at the Universidad Nacional Autónoma de México in Mexico City, Suárez-Díaz is an accomplished scholar of molecular evolution and molecular disease who serves on the editorial boards of Osiris and Perspectives on Science, among other publications.

Dr. Suárez-Díaz’ current research focus is the geopolitics of disease with a particular interest in approaches taken toward blood diseases in the twentieth century. This project brought her to Corvallis to study components of the Ava Helen and Linus Pauling Papers, focusing on Linus Pauling’s work on sickle cell anemia.

Pauling is, of course, well known for his discovery that sickle cell anemia traces its origin to the molecular level, a concept first published in 1949 with Harvey Itano, S. J. Singer and Ibert Wells. As Suárez-Díaz noted in her resident scholar lecture, the group’s finding that the basis of a complex physiological disease could emerge from a simple change in a single molecule made a profound impact on the history of biomedicine. Indeed, it is not an overstatement to suggest that the concept of a molecular disease led to massive shifts in post-war research and public policy.

Importantly, these shifts were accompanied by technological breakthroughs that enabled many other laboratories to explore new ideas related to molecular disease. In particular, the modernization of gel electrophoresis techniques served to democratize research in a way that had previously not been possible. When the Pauling group was conducting their initial experiments, electrophoresis was a tool that lay within the grasp of only a handful of well-funded laboratories. As the equipment and methodology required to do this work became less expensive, practitioners around the world began to enter the field and the impact was profound.

Suárez-Díaz is particularly interested in blood diseases and malaria in developing countries, noting that these afflictions were to the “Third World” what cancer was for industrialized societies and the middle classes. It is important to note as well that there exists a strong connection between blood diseases (like sickle cell anemia) and malaria, as the mutations that gave rise to blood diseases also provide a certain degree of immunity to malaria. As such, the geographic distribution of blood diseases correlates closely with malaria epidemiology, a phenomenon that has had consequences for public health campaigns over time, including decisions to use DDT on a massive scale in attempting to eradicate the mosquitoes that carry the disease.

Harvey Itano and Linus Pauling, ca. 1980s.

It is also interesting to chart Linus Pauling’s role – or lack thereof – in the further development of molecular disease as a field of study. Though he and three colleagues essentially created a new discipline with their 1949 paper, Pauling gradually became marginalized within the community, in part because he devoted so much of his time to political activism during the 1950s. His departure from Caltech in 1963 further distanced his scientific activities from what had, by then, become a truly international body of work.

As such, while undeniably important, Pauling’s contributions to the field might now be seen as one of many important nodes in a transnational network of scientists and practices. Moving forward, Suárez-Díaz’ work will continue to explore this transnational network, touching upon several other key issues including G6PD deficiency and the genetic consequences of atomic fallout.

Now in its eleventh year, the Resident Scholar Program at OSU Libraries has provided research support for more than two dozen visitors traveling from locations across the United States as well as international scholars from Germany, Brazil and, now, Mexico. New applications are generally accepted between January and April. To learn more, please see the Resident Scholar Program homepage.

Pauling at UCSD: Season of Tumult

 

[Part 2 of 3 in a series exploring Linus Pauling’s years on faculty at the University of California, San Diego.]

As his program on orthomolecular psychiatry began to take off, Pauling’s work as an activist moved forward with as much zeal as ever. Despite criticism that his association with the Center for the Study of Democratic Institutions (CSDI) and his protests against the Vietnam War made no sense in the context of his scientific career, Pauling had stopped viewing his interests as an activist and his scientific research as being separate branches of a single life.

Pauling happened to be at the University of Massachusetts a mere five days after Martin Luther King Jr. was assassinated. Invited to deliver a series of lectures as the university’s first Distinguished Professor, Pauling fashioned his remarks around the topic of the human aspect of scientific discoveries. Reflecting on the tumult of the previous week, Pauling told his audience that it was not enough to mourn the fallen civil rights leader. Rather, individuals of good conscience were obligated to carry King’s legacy forward by continuing the work that he began.

In keeping with this theme over the course of his lectures, Pauling emphasized the scientist’s responsibility to ensure that discoveries be used for the good of all humanity and society, rather than in support of war and human suffering. Scientific inquiry should also emphasize solutions to current issues, he felt, pointing to the lack of equality in access to medical care in the United States as one such issue. Pauling saw his work in orthomolecular medicine as potentially solving this problem: vitamins were fairly inexpensive, more accessible, and could, he believed, significantly improve one’s mental and physical well-being.

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Notes used by Pauling for his talk, “The Scientific Revolution,” delivered as a component of the lecture series, “The Revolutionary Age, the Challenge to Man,” March 3, 1968.

Pauling made similar connections to his work on sickle cell anemia.

Though he was no longer involved in the daily operations of the CSDI, he continued to participate in a public lecture series that the center sponsored throughout his time in San Diego. In one contribution to a series titled “The Revolutionary Age: The Challenge to Man,” Pauling put forth a potential solution to sickle cell disease. As science had succeeded in identifying the gene mutation responsible for the disease, Pauling believed that forms of social control could be used to prevent carriers of the mutation from marrying and procreating. Over time, Pauling reasoned, the mutation would eventually be phased out.

Pauling specifically called for the drafting of laws that would require genetic testing before marriage. Should tests of this sort reveal that two heterozygotes (individuals carrying one normal chromosome and one mutation) intended to marry, their application for a license would be denied. Pauling put forth similar ideas about restricting the number of children that a couple could have if one parent was shown to be a carrier for sickle cell trait.

In proposing these ideas, Pauling aimed to ensure that his discovery of the molecular basis of sickle cell disease was used to decrease human suffering. Likewise, he felt that whatever hardships the laws that he proposed might cause in the short run, the future benefits accrued from the gradual elimination of the disease would justify the legislation.

Partly because he called this approach “negative eugenics,” Pauling came into harsh criticism for his point of view; indeed, his ideas on this topic remain controversial today. In a number of the lectures that he delivered around the time of his CSDI talk, however, Pauling took pains to clarify that his perspective was not aligned with the broader field of eugenics, a body of thought to which he was opposed. On the contrary, Pauling’s focus was purely genetic and his specific motivation was borne out of a desire to eliminate harmful genetic conditions.

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Bruno Zimm. Credit: University of California, San Diego

At the end of February 1968, Pauling turned 67 year old, and the University of California regents used his age as a mechanism to hold up discussions about his obtaining a permanent appointment in San Diego. Sixty-seven, the board argued, was the typical retiring age within the UC system. Moreover, the UC regents were empowered to veto any age-related retirement exceptions and, given his radical political views, Pauling was unlikely to receive any support at all from the group, much less an exception.

One of the stated reasons why the regents harbored concerns about Pauling’s politics was his increasingly strident rhetoric. Pauling frequently commended student strikes and demonstrations, and although he emphasized nonviolence as the most effective means to foster social change, he encouraged students to recognize that authorities may incite violence through tactics of their own. In these cases, he felt that retaliation was justified, even necessary.

Pauling also believed that the regents and their trustees wielded too much power; for him they were part of a system that largely inhibited social progress and took power away from students. For their part, the regents saw Pauling in a similar light: a dangerously powerful radical who was constraining the university’s capacity to grow.

Realizing that, in all likelihood, Pauling was soon to be forced out, his UCSD colleagues Fred Wall and Bruno Zimm began searching for a way to shift the governing authority for his reappointment to the university president, Charles Hitch, with whom Pauling had maintained a positive relationship. After months of negotiations, Zimm succeeded in winning for Pauling a second year-long appointment.

Pauling expressed gratitude to Zimm for his efforts, but the slim possibility of a permanent position at UCSD had emerged as a source of lingering dismay. Looking for a longer term academic home, Pauling began considering other universities that might also provide better support for his research.

Over time, Ava Helen had also found herself frustrated with UCSD and La Jolla in general. In particular, she disliked their rental house and missed their previous home in Santa Barbara, where she had been able to tend a beautiful garden. As 1968 moved forward, the couple began spending more and more time at Deer Flat Ranch, with Ava Helen hinting that she would like to make the ranch their permanent home in the coming years.

A Master of Many Fields

Linus and Ava Helen Pauling, Oxford, 1948.

[The serological properties of simple substances – part 4 of 6]

By the Spring of 1946, having published no fewer than twelve articles – over a little more than three years – on the serological properties of simple substances, Linus Pauling’s busy life began to get in the way of continued advancement of his research program. Perhaps chief among competing interests was a separate fifteen-year joint research program, funded by a $300,000 grant, that Pauling and George W. Beadle, the head of Biology at Caltech, were in the midst of setting up.

Pauling had also returned to studies of sickle cell anemia with the arrival of Dr. Harvey Itano in the fall of 1946. He was likewise engaged with new inquiries in inorganic chemistry that reached a crescendo with a famous article, “Atomic Radii and Interatomic Distances in Metals,” published in March 1947. From there, the dawn of 1948 saw Pauling moving to England, where he served as George Eastman Professor at Oxford University. Not long after, he received the Presidential Award for Merit for work done during World War II. Clearly there was much going on in Pauling’s world.

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

Nonetheless, consequential progress continued to be made in the serological program with the thirteenth paper – an important one – coming into print in April 1948, while Pauling was still in England. This article, written by Pauling along with David Pressman and John Bryden, marked a continuation of the precipitation experiments that had been carried out in the previous two papers, but this time with a different antiserum and antigen substitute. The Paper XIII experiments determined that antibodies are rigid and cannot change shape to bond to a different antigen.

Significantly, these data also confirmed that structural complementarity was responsible for the reaction’s specificity, affirming Pauling’s early notions of a “hand in glove” fit. Furthermore, the paper’s findings established that the principal forces involved in the complementary bonds were Van der Waals interactions – very weak bonds induced by sheer proximity. In short, the experiments verified the importance of intermolecular interaction in the specificity of serological reactions, a significant breakthrough.

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With Pauling now having returned stateside, the year 1949 saw the publication of the final two serological articles, one released in January and another during the summer. Paper XIV, written by Pauling and Arthur Pardee, was fashioned as a response of sorts to disagreements that had been expressed by other scientists concerning Pauling’s interpretations of his experimental results.

The paper specifically focused on experiments utilizing simple antigens and purified antibodies, rather than the antisera that Pauling had been using. These trials found that, although the behavior of simple antigens was different when matched with purified antibodies rather than antisera, “…the earlier work, carried out with serum, is presumably reliable.” In making this statement, Pauling and Pardee cited the non-specific combination of dye molecules along with other components of the serum for past results that had varied slightly.

Illustration of the antibody-antigen framework, 1948.

The last article in the serological properties series, Paper XV, appeared in the Journal of the American Chemical Society in August 1949; Pauling and Pressman were its authors. The article detailed the results of experiments using an antiserum with two or more positive charges. This experimental set-up, Pauling hoped, would allow him to determine the difference in combining power between antibodies containing only one negative charge as well as those containing two negative charges. The duo discovered that the antibody would only combine strongly with antigens that contained two negatively charged groups in specific positions. From this, Pauling concluded that the attraction between the negative charges of the antigen and the positive charges of the antibody are very strong.

After completing the fifteenth paper, Pauling largely left immunology behind in favor of the work that he and Itano were doing on sickle cell anemia. In 1950 and 1951, Pauling and several collaborators also published multiple articles delineating protein structures. In addition, it was during this time that Pauling began to really ramp up his peace work, delivering more and more lectures on the topic as the years went by.

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The fifteen articles that comprise Pauling’s serological properties series were published over a span of seven years. During that period, Pauling worked with twelve collaborators, several of whom were graduate students. By the conclusion the project, hundreds of experiments, using dozens of compounds, had been run.

Particularly given the fact that he lacked any sort of formal background in immunology, the massive impact that Pauling made on the field is truly impressive. By the time that he moved on to other topics, Pauling’s work had served to raise the level of immunological knowledge by orders of magnitude. He is credited now with having discerned a relatively complete understanding of both antibody structure as well as the reaction mechanics underlying the interplay between antigens and antibodies. He also applied the vast collection of data that he had compiled to develop a theory of antibody formation. Of this, biographer Tom Hager wrote

For fifteen years…until a new, more powerful theory of antibody formation was put forward, Pauling’s idea led the field. His antibody work again expanded his growing reputation as a master of many fields.

Pauling himself believed that this work had solved “the general problem of the nature of specific biological forces” and that this understanding would “permit a more effective attack on the many problems of biology and medicine.”

Indeed, Pauling’s work with antibodies was influential even outside of the field of immunology. In 1990, journalist Nancy Touchette declared, “In his 1946 paper [“Molecular Architecture and Biological Reactions”], Pauling prophesied about the future of biology and medicine and why understanding the nature of complementarity is so important to the future of the field.” Five years later, at a Pauling symposium held at Oregon State University just a few months after Pauling’s death, molecular biologist Francis Crick stated flatly that Pauling “was one of the founders of molecular biology.” Once again, Linus Pauling had revolutionized a scientific field while following his curiosity and intuition.

A Period of Rapid Advancement in Pauling’s Immunological Work

Dan Campbell and Linus Pauling in a Caltech laboratory, 1943.

[Part 3 of 6 in a series investigating Pauling’s work on the serological properties of simple substances.]

In April 1943, only four months after releasing his first four papers on the serological properties of simple substances, Linus Pauling was ready to publish more. His fifth paper in the series reported out on the results of hapten inhibition experiments that his lab had conducted using two different antibodies. In the experiments, “measurements were made of the inhibitory effect of each of twenty-six haptens on one antigen-antibody reaction, and interpreted to give values of the bond-strength constant of the haptens with the antibody.”

The results of the experiments, with particular attention paid to the twenty-six hapten molecules, were then discussed in the context of their possible molecular structure. In this discussion, Pauling pointed out that some of the polyhaptenic molecules did not produce participates, a detail that was explained as having been caused by steric hindrance, or the inability for a reaction to take place due to molecular structure.

David Pressman was again a co-author of the paper, as were two graduate students, John T. Maynard and Allan L. Grossberg. Grossberg would stay with Pauling’s lab until 1946 – two years after completing his war-time master’s degree – and was involved with three more papers from the series. He later went on to work with Pressman at the Roswell Park Memorial Institute and eventually became associate chief of cancer research there.

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Pauling’s immunological work was quickly producing exciting new results, momentum that was recognized by The Rockefeller Foundation, which awarded Pauling another grant in June 1943. Pauling also began delivering lectures on his serological research, notably including the Julius Stieglitz Memorial Lecture in January 1944.

Articles six, seven, and eight of the serological series were each published a few months apart from one another, beginning in March 1944. Pauling co-authored these papers with previous collaborators Pressman, Campbell and Grossberg, and also with Stanley Swingle, a research fellow and instructor who had earned his Ph.D. at Caltech in 1942.

Paper VI put forth more evidence for the Marrack-Heidelberger framework theory, for which Pauling had first announced his support in Paper I. The experiments specified in Paper VI made use of fifty different substances possessing either one, two, or three haptenic groups. The results of these trials indicated that a substance containing two different haptenic groups would only form a precipitate when antisera binding to both of those two groups were present. Of this finding the article states, “this provides proof of the effective bivalence of the dihaptenic precipitating antigen, and thus furnishes further evidence for the framework theory of antigen-antibody precipitation.”

In the seventh paper, published in May 1944, Pauling returned to the simple theory for calculating the inhibition of precipitation that he had developed in Paper II, published at the end of 1942. In his discussion, Pauling reported that his laboratory’s experiments found general qualitative agreement with the theory, but the numbers tended to be off. In seeking a more reliable equation, Pauling worked to improve the theory, accounting now for the fact that a single antiserum can contain slightly different antibody molecules with assorted combining powers.

This new and improved theory, and the equation that accompanied it, agreed with experimental results much better than had the original proposal. Indeed, by accounting for variations in the antibodies, Pauling and his colleagues had succeeded in developing a “quantitative theory of the inhibition by haptens,” which would prove important to much of the work that was to come.

Paper VIII, “The Reactions of Antiserum Homologous to the p-Azobenzoic Acid Group,” appeared in October 1944 and shared the results of experiments done with a new type of antibody. Previously, experiments had been conducted with antisera homologous to two different acid groups. However, in these new investigations, the Caltech researchers used antisera homologous to another type of acid group. In doing so, Pauling and his colleagues were attempting to gauge optimum acidity levels for serological reactions; to identify the types of antigens that most readily cause precipitation; to likewise identify haptens that inhibit precipitation; and to measure the strength of their inhibiting power.

Despite Pauling’s extensive involvement in studying reactions of antibodies and antigens, he still had time for other research interests. In February 1945, Pauling and Campbell announced that they had created a usable substitute for blood plasma, the result of three years of work supported by military contracts. Shortly thereafter, Pauling learned a few key details about sickle cell anemia while meeting with the other members of the Medical Advisory Committee. He immediately thought that hemoglobin was involved and went on to experimentally prove that the disease located its source on the molecular level; a first in the history of science.

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Arthur Pardee, 1980

June, July, and September of 1945 each saw the publication of another serological article: Papers IX, X, and XI respectively. The final two of this set featured the addition of a pair of new collaborators. John Bryden, a co-author for Paper X, completed his master’s degree around the time that the article was published, and Arthur Pardee was in the middle of his doctoral program when he worked on Paper XI. Pardee also worked on the experiments described in Paper XIV, although the article was published after he had completed his Ph.D. and returned to Berkeley. Pardee later went on to enjoy a hugely successful career as the Chief of the Division of Cell Growth and Regulation of the Dana Farber Cancer Institute at Harvard Medical School.

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Karl Landsteiner

Papers IX and X shared the results of still more inhibition experiments. The experiments reported on in Paper IX largely confirmed Karl Landsteiner’s discovery on the combining of antiserum and antigen, or antiserum and hapten. Landsteiner had found that less bonding occurred between antibody and antigen or antibody and hapten if the substituent groups on the binding molecule were different from the antigen that created the antibody. The Pauling group confirmed this theory and, in addition, described the forces that affect hapten inhibition. Pauling believed that it had to do with intermolecular forces “including electronic van der Waals attraction…the formation of hydrogen bonds, and steric hindrance,” a supposition that would play a crucial role in later papers in which Pauling explained the incredible specificity that governs the behavior of these molecules.

Paper X studied the effect of molecular asymmetry on serological reactions. In this series of experiments, Pauling and two collaborators, David Pressman and John Bryden, had prepared an antiserum with an optically inactive immunizing antigen; e.g., a molecule that does not rotate plane polarized light. However, even though the immunizing antigen was not optically active, the antibodies in the serum combined more strongly with one configuration over an optically active hapten, which does rotate light, than in the other configuration. Pauling and his colleagues hypothesized that this was due to the presence of optically active amino acid residues in the antibody molecules.

Paper XI, published in September 1945, discussed reactions of antisera with various antigen substitutes. In this instance, the Pasadena group measured the precipitate formed by these reactions to gauge the inhibiting power of the haptens. They then correlated hapten-inhibiting power to molecular structure, suggesting that if a substance mixed with antisera more readily, then the structure of the molecule might be smaller. They ultimately discovered that if a hapten structure matched an immunizing azoprotein structure, the haptenic group exhibited a strong inhibitory effect.

In February 1946, Pauling and co-authors Pressman, Grossberg, and Leland Pence published the twelfth serological article. This was Grossberg’s fourth and final contribution; ultimately, he served as co-author on more of the series than did any other collaborator, save David Pressman and Dan Campbell. New to the series was Leland Pence, an assistant professor of organic chemistry at Reed College who had been collaborating with Pauling since 1942.

Prior to Paper XII, all previous experiments carried out by the lab had used negatively charged or neutral compounds. Paper XII presented the results of experiments that used a positively charged antibody. Pauling and his collaborators found that, even when using positively charged antibodies, hapten inhibition occurred the same way, with the same factors, as was the case with a negative or neutral compound. That said, one important difference that was observed was the ideal acidity for maximizing precipitates; when using a positively charged antibody, the pH required for the optimum amount of precipitate was much lower.

Analyzing Precipitation Reactions Between Simple Substances

Linus Pauling, 1942

[Part 2 of 6 in a series investigating Pauling’s work on the serological properties of simple substances.]

The first four papers published by Linus Pauling and his Caltech colleagues on the serological properties of simple substances described general aspects of the precipitation reactions that occur between antibodies and antigens. This work was spurred by a fundamental conundrum: Pauling and many others knew that antibodies and antigens would react to form solid precipitates. However, because the chemical structures of these precipitates were, at the time, so difficult to determine, scientists had been unable to decipher crucial details about the antibodies and the antigens that combined to form them.

Pauling’s solution to this problem was to investigate the products of a reaction that utilized, in part, a chemical compound whose structure he already knew. The constituents of these products were a simple organic compound consisting of carbon, oxygen, and hydrogen, combined with one or more haptenic groups – small molecules that spur the formation of antibodies when coupled with a larger molecule. Employing this methodology would, Pauling felt, allow him to better approximate the make-up of the antibody, because the experiment now involved only one unknown structure.

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In order to run the experiments, Pauling set up a standard protocol for preparing the compounds that he needed. Each experiment required three types of compounds: simple antigens used in the precipitation reactions; immunizing antigens used to create antibodies; and antisera, which are liquids containing antibodies formed through the coagulation of blood. Pauling used this method for all of his serological reaction experiments.

Pauling and his collaborators obtained the antisera by injecting rabbits (some of them housed in Pauling’s yard and cared for by his children) with immunizing antigens. The rabbits then produced antibodies to combine with and neutralize the immunizing antigens. Once the last injection was carried out, the scientists drew blood from the rabbits, allowed it to clot, and collected the antiserum.

The reactants for Pauling’s experiments – immunizing antigens and simple antigens – were either purchased or prepared by Pauling and his collaborators, typically the graduate students.

For each precipitation test, equal portions of antiserum and a saline solution containing a simple antigen were mixed together. Typically, four to six different concentrations of antigen were used. The mixtures stood at room temperature for one hour, then were refrigerated overnight. The next day, a centrifuge was used to separate out the precipitates, which were then washed with saline solution and analyzed. Pauling’s method of analysis involved measurements of nitrogen, arsenic, carbon, and hydrogen. From there, the amount of a given antibody in the precipitate was determined using the nitrogen measurements.

The initial set of experiments used twenty-seven different compounds as the antigen, each containing between one and four haptenic groups. All of the polyhaptenic substances – those that had more than one haptenic group per molecule – formed precipitates, but none of the monohaptenic substances did. This finding supported the framework theory, devised by the British chemist John Marrack in 1934, that postulated that multivalent antibody molecules could combine with polyhaptenic molecules to form large aggregates, which would become precipitates. On the other hand, Marrack suggested, if multivalent antibody molecules combined with monohaptenic molecules, only small complexes would form and these would not precipitate.

Pauling summarized this work in a set of four papers that were published in the December 1942 issue of the Journal of the American Chemical Society.

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John Richardson Marrack

Pauling’s first article, “Precipitation Reactions between Antibodies and Substances Containing Two or More Haptenic Groups,” served primarily to provide support for Marrack’s framework theory. Eight years before, Marrack had stated that antibodies were multivalent; in other words, they can bond to more than one antigen molecule. In order for them to bind in this way, the molecules must be properly oriented such that the binding sites fit together. This causes the formation of a lattice-like structure which grows until it is too large to stay in solution and precipitates out.

As noted above, Pauling’s experiments found that “simple antigens containing two or more haptenic groups per molecule were found to give precipitates with the antisera, whereas the seven monohaptenic substances failed to precipitate,” a discovery that confirmed the validity of the Marrack-Heidelberger framework, or lattice theory.

The second paper in this installment was titled “The effects of changed conditions and of added haptens on precipitation reactions of polyhaptenic simple substances.” The alterations to conditions that were tested by Pauling included allowing the mixture to rest longer, changing its temperature, and altering its pH. Having confirmed his own belief, in Paper I, that antibodies are multivalent, Pauling used Paper II to first note his assumption – and provide evidence for – bivalence.

In addition, Pauling used this paper to publish an equation that could be employed to find the amount of a precipitated compound in a given solution based on solubility, equilibrium constant, and total amount of hapten. Notably, the equation led Pauling to deduce “that in each case the maximum amount of precipitate is produced by an amount of antigen approximately equal to the amount of antibody,” an idea that unfolded more fully in the following paper.

The equation published by Pauling in Paper II.

Paper III, “The composition of precipitates of antibodies and polyhaptenic simple substances; the valence of antibodies,” further explores the supposition of bivalence through an examination of the ratio of antibody to antigen in precipitates.

While the bulk of Pauling’s experiments focused on dihaptenic antigens, some used trihaptenic antigens, and others used tetrahaptenic antigens. Through careful analyses of the different precipitates that resulted, Pauling was able to determine that the ratio of antibody to antigen in any given precipitate was approximately 1:1.

This finding suggested that most antigens could have only two antibody molecules attach to them, even if they possessed more than two haptenic groups, since the antibody molecules were relatively large and interfered with one another’s attachment. Pauling also used the one-to-one ratio to conclude that most antibody molecules possess two binding sites. The major development of this paper – the near one-to-one ratio – was “taken to indicate bivalence of most of the antibody molecules.”

The last paper of the first installment, Paper IV, reported the results of initial experiments on the inhibition of precipitation in the presence of hapten. Pauling and his colleagues had tested precipitate inhibition in three basic ways: by altering temperature, by augmenting the amounts of hapten present in their mixtures, and by isolating the effects of twenty-four specific haptens. These experiments found that adding haptens to a mixture of antibodies and antigens inhibited the precipitation of the antibody-antigen complex.

Furthermore, Pauling concluded that the structure of the haptens correlated with their inhibition power and detailed the relative values of each hapten’s bond strength. He then used the hapten inhibition data from these experiments to update his earlier equation for finding the amount of antibody precipitated.

Next week, we’ll examine eight more papers that Pauling published on the topic over the next three years and explore the ways in which this body of research evolved and expanded during that time.

The Serological Properties of Simple Substances

Linus Pauling, 1935

[Part 1 of 6]

Today, Linus Pauling is most commonly known for unraveling the chemical bond, working for peace, and promoting vitamin C. However, this short list barely scratches the surface of Pauling’s work in any number of fields. Beginning today, we will explore a lengthy program of research that Pauling oversaw on the serological properties of simple substances, a title that he appended to fifteen publications authored from 1942 to 1949. Post one in this series will focus primarily on Pauling’s background in biology and the work that led up to his first set of serological publications.

One of Pauling’s first major forays into the world of biology came about through his study of hemoglobin, the molecule responsible for transporting oxygen in the blood. Specifically, in 1934, he launched a study hemoglobin partly as a means to begin a larger inquiry into the structure of proteins.

An investigation of hemoglobin, Pauling quickly decided, would require more than one year to obtain results. Consequently, in November 1934, he applied for a grant from the Rockefeller Foundation to “support researches on the structure of Haemoglobin and other substances of biological importance.”

At the time, the Rockefeller Foundation was keenly interested in funding studies of “the science of life,” and Pauling’s grant request was promptly approved, with the first injection of funds received in July 1935. Although Pauling had originally intended for the grant money to go specifically toward his work on hemoglobin, as he corresponded with his funders he expressed an openness to studying other “interesting biochemical problems,” and indeed this quickly became the case.

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A few months later, in 1936, Pauling met Karl Landsteiner, whose ideas would help to shape the course of Pauling’s research for the next several years. Landsteiner was an Austrian biologist and physician best known for discovering the human blood groups. By the time that he met Pauling, he was also actively engaged with topics in immunology.

Over the course of their conversations, Landsteiner passed this interest on to Pauling, who became fascinated by the specificity of antigens (foreign substances that enter into the body) and antibodies (proteins that neutralize antigens and prevent them from causing harm). The human immune system is capable of building thousands of antibodies, each of which reacts with a specific antigen. This specificity is seen in few other physical or chemical phenomena. However, one area in which it is found is crystallization, an area of chemistry with which Pauling was very familiar. This body of knowledge set Pauling down a path to making important contributions to the study of antigen-antibody behavior.

As he sought to learn more, Pauling read Landsteiner’s recently published book, The Specificity of Serological Reactions, finishing it shortly after their initial meeting. The following year, 1937, Pauling and Landsteiner met again and spent several days discussing the most current ideas in immunology. For Pauling, immunology presented two particularly compelling questions: First, what were the forces that enabled the combination of an antibody and its homologous antigen, but no other molecule? Second, how were antibodies produced and how did this means of production allow antibodies and antigens to combine so specifically?

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Dan Campbell and Linus Pauling in a Caltech laboratory, 1943.

In 1939, Pauling decided to shift the bulk of his research focus to the interaction dynamics of antigens and antibodies. As his work moved forward, Pauling came to theorize that the specificity shown by antibodies when combining with antigens depended on how well-matched the shapes of the two molecules were, a theory called molecular complementarity. In other words, antibodies and antigens were able to come together because their shapes complemented one another, like a hand in a glove.

From there, Pauling developed a plan to perform a broad range of experiments that would, he hoped, strengthen this theory and prompt it forward as the accepted explanation for the specificity of serological reactions. To assist in this promising line of inquiry, Pauling hired Dan Campbell, at the time a research fellow at the University of Chicago, to come to Caltech and serve as the Institute’s first faculty member in Immunochemistry. Campbell arrived in January 1940 and remained at Caltech until his death in 1974.

Once relocated to Pasadena, Campbell starting out by working on structural studies of hemoglobin – Pauling’s old research project dating back to 1934. A few months later however, a key shipment of serum antigens arrived from Karl Landsteiner’s laboratory, and both Campbell and Pauling began experimenting on the issue of the day. Initially, the duo encountered only disappointment as they uncovered no results of interest. However, the early setbacks did not stop Pauling. He persevered and, in October, published a landmark article, “A Theory of the Structure and Process of Formation of Antibodies,” which detailed his ideas on molecular complementarity.

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In 1941, Pauling began an experimental program on serological reactions focusing on simpler organic compounds whose structure he already knew. In so doing, he also began to add more collaborators. Besides Campbell, the first of these was David Pressman, who earned his doctorate under Pauling and then stayed on at Caltech to support the nascent immunology program until finally leaving in 1947.

In addition to the simple substances work, this trio of researchers also continued other lines of study pertaining to Pauling’s antibodies projects. In early 1942, one of these produced what seemed to be an incredible result: that March, through a press release rather than a conventional journal article, Pauling, Campbell and Pressman announced that they had created artificial antibodies. A wide array of newspapers and magazines picked up the story and interest rapidly grew. However, other scientists could not replicate the trio’s results and skepticism of the group’s claim began to mount. Pauling, however, continued to believe that his team had truly created artificial antibodies, though subsequent efforts found only dead ends.

Undaunted, Pauling continued his experiments on serological reactions in simple substances and, in December 1942, published the first four papers of what would ultimately become a fifteen-paper series. This body of scholarship was the culmination of several years of work conducted by many people including Pauling, his two main collaborators, David Pressman and Dan Campbell, as well as one other non-student colleague. Several graduate students also supported the effort by helping to prepare the necessary compounds and running the experiments; as the publication series ran its course, eight were eventually listed as co-authors. Three graduate students, Carol Ikeda, Miyoshi Ikawa, and David H. Brown, were involved in the first four papers. Beginning next week, we will take a closer look at the details of what this group published.