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.

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.

Post 500

Linus and Ava Helen Pauling. Angeles National Forest, Thanksgiving Day, 1952.

This is the five-hundredth post that we’ve published on the Pauling Blog, and in this season of thanksgiving we find ourselves in a grateful mood.  Five-hundred posts, surely at least a half-million words and, recently, our 500,000th view.  Great thanks to you, our readers, who continue to seek out and use this resource in steadily increasing measure.

To celebrate this milestone, we are publishing a few excerpts from one of our favorite Pauling manuscripts.  Titled “An Extraordinary Life: An Autobiographical Ramble,” the piece was written by Pauling for presentation to the Institute for the Humanities in Saledo, Texas, April 1989.  The text finds Pauling in an unusually reflective mood, speaking with serenity, at age 88, of a life spent dipping in and out of scientific disciplines in a most remarkable way.

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Young Pauling, ca. 1910s.

[…] I am moderately smart. I estimate that there are 20,000 people in the United States who are smarter than I am, perhaps 15,000 women and 5,000 men. I reached this conclusion because a month after my wife and I got married, we had carried out some intelligence tests, and I discovered she was smarter than I, but we were already married. It was too late for me to do anything about it. Of course, I recognize that there are many physicists who are smarter than I am – theoretical physicists, most of them. There are a lot of smart people who have gone into theoretical physics, so there is a lot of competition there. I console myself with the thought that they may be smarter than I am and deeper thinkers than I am, but I have broader interests than they have. I don’t suppose that there is anybody else in the world who has a good background, knowledge of physics, mathematics, theoretical physics, and who knows a great deal about chemistry – the amount that I know.

When I was eleven years old with no outside inspiration – just library books – I started collecting insects. Not only collecting insects but reading about insects. I was filling my mind with a lot of information about the lepidoptera and diptera and so on. My father, a druggist, died when I was nine. There was another druggist who was a friend of the family to whom I went if I needed some chemicals when I got interested in chemistry, but I wasn’t interested in chemistry yet. I was just interested in insects when I was eleven. I said, “A person who collects insects needs to have a killing bottle.” And I got a Mason jar from my mother. So all I needed now was ten grams of potassium cyanide and perhaps fifty grams of plaster of paris. So Mr. Ziegler, the druggist, gave me ten grams of potassium cyanide and fifty grams of plaster of paris, and I took them home, went out on the back porch, because I knew that potassium cyanide was dangerous, and I dumped the potassium cyanide into the bottle. I mixed the plaster of paris with some water and put it in the bottle on top of it and let it harden. I had my killing bottle. I collected a lot of insects.

Next year I got interested in minerals. I didn’t have very many minerals, at least that I could recognize, only agates. So about all I could do was go around Portland looking for piles of gravel where someone was putting in a house foundation or sidewalk. I’d go through the gravel looking for chunks of agate.

Just think of what the difference is now.  A young fellow gets interested in chemistry and is given a chemical set.   The chemical set doesn’t contain any potassium cyanide. It doesn’t even contain any copper  sulphate  or anything interesting because  they are all  poisonous  substances. Most chemicals are poisonous substances. These young budding chemists don’t have any chance to do anything interesting when they are given a chemical set anymore.   As I look back, I think it is pretty remarkable that Mr. Zieglar, this friend of the family,  would have just turned over one third of an ounce of potassium cyanide to me at age eleven. […]

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Linus and Ava Helen, camping near Palm Springs, 1924.

[…] I  was   very  fortunate  when   I   came  to  the   California   Institute   of Technology.    There was a new experimental technique that had been discovered only eight years before.    This was the determination of the structure of crystals by the x-ray diffraction method.    Roscoe Dickinson,  a  few  years older than I, had been using this technique for three or four years at the California Institute of Technology.    He was the first man to get a Ph.D.  from the California Institute of Technology. He taught me the technique.    I was very much excited about it.    It took only a couple of months for him to teach me how to determine the structure of a rather simple crystal by taking x-ray diffraction photographs of it and then analyzing those photographs.    Perhaps the greatest thing that he taught me was how to assess the reliability of your own conclusions.   He taught me to ask every time I reached some conclusion:

“Have I made some assumption in reaching this conclusion?    And what is the assumption? And what are the chances that this assumption is wrong? How reliable is the conclusion?” I have remembered this ever since and have continued to feel grateful to him ever since. It is possible to delude yourself if you have an original idea into thinking that there are observations that support this idea. Or it is possible when you think that you have developed some idea on the basis of a rational argument that you have made an assumption somewhere that isn’t justified. So this was very important in my development.

I hear people often describing me as a biochemist or as an organic chemist or something else. In fact, I never did like organic chemistry. I liked biochemistry even less. I didn’t have any courses to speak of in organic chemistry and no course at all in biochemistry. No course in any aspect of biology, nothing in medicine. But I have made contributions in the nutritional field and the biochemical field. If I were to go through my some eight hundred scientific papers, and see what fields of science I have made contributions   to,   I  could  say  I  am a x-ray  crystallographer. I am a mineralogist, because the American Mineralogist Society gave me their Roebling Medal which they give every year to an outstanding mineralogist. I am a physical chemist. That was what I called myself originally and what my Ph.D. diploma says. I am a chemical engineer too with a degree and five years of practical experience. I am an analytical chemist. When I was nineteen years old,   I didn’t have enough money to go back to my junior year at Oregon Agricultural College. As a sophomore I had taken the course in Quantitative Chemical Analysis and they gave me a job full time to teach the sophomore  Chemical Analysis. So I am an analytical chemist too. And I am an organic chemist.   I laid the theoretical  foundation for the tetrahedral carbon atom and developed resonance hybrid concept. I explained a lot of things in organic chemistry. I am a biochemist. I am a molecular biologist and sort of originated this field in a sense. I am a geneticist and have made contributions.   I’m an evolutionary scientist. […]

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Pauling in 1989 – an extraordinary life. Photo by Paolo M. Sutter.

[…] In 1937, I was invited to give the prestigious George Fisher Baker Lectures at Cornell University. I went there for one semester. There had been famous chemists who had held this appointment. One requirement was that you write a book. My lectures were on the nature of the chemical bond, and the book came out in 1939, The Nature of the Chemical Bond. It was a bestseller, published by Cornell University Press. After a year the editor of Cornell University Press wrote to me and said, “Your edition of 10,000 copies is just about sold out. Would you prepare a second edition?” And I said, “Well, it hasn’t been a year yet. Nothing much has happened, but there have been some changes in this field. But why should I prepare a second edition of the book?”   He said, “Well, you don’t get any royalties from the book.   It was a condition of your appointment as George Fisher Baker Lecturer in Chemistry that you should write the book and present the manuscript.   There has never been a George Fisher Baker book that has gone into a second edition, but if you write a second edition, Cornell University Press will give you royalties on it.”

Well, that was a really good incentive.    I got busy and added ten pages perhaps and it came out as the second edition in 1940 and ever since then I have collected royalties.   On thinking back on this man, editor of Cornell University Press, he is really a remarkable man in that he should think that it would be unjust to me not to get royalties on that book that had become a scientific bestseller.    He was Amish from Pennsylvania and perhaps this may have something to do with his ethical standards.    It is a good thing that people have ethical standards.

People keep saying to me, “How does it come about that you shifted your field every five or ten years in a remarkable way?” In fact, all that I did was to expand my field of interest. I started out first determining the structure of minerals, and the second job I did was to determine the structure of an intermetallic compound — the first intermetallic compound to have its structure determined. For about ten years I worked on the structure of silicate minerals and of various other inorganic compounds.

So that was one period, but then I got interested in the structure of organic molecules. And there was another technique. We built the first apparatus in the United States to determine the structure of gas molecules by electron diffraction. A friend of mine, Herman Mark in Germany, was the man who built the very first apparatus of this sort. So I began determining interatomic distances, and applying quantum mechanics which I had learned as one of the first people in the field in 1926 when I was in Germany on a Guggenheim Fellowship.   All of this related to the question of the nature of the chemical bond. In the 1930s I formulated several new ideas about chemical bonds.

In 1935 the Rockefeller Foundation had been supporting my work on the crystal structure of the sulphide minerals, and they said to me, “You know, we’re not really interested in the sulphide minerals.    We’re interested in biological substances.”   They had been giving me five thousand dollars a year.   I thought, “What do I know about biological materials?   Not very much.   Hemoglobin, red cells in the blood, molecular weight about 68,000, that has four iron atoms in it.   Iron compounds often are paramagnetic.    So why don’t I apply to the Rockefeller Foundation  and  suggest  that  I  measure  the  magnetic   susceptibility  of hemoglobin and hemoglobin derivatives?”   So I did. And they gave me fifty thousand dollars.    This shows that these fellows in the big foundations can influence  activities  of  scientists.

So we measured  the magnetic susceptibility of blood. Venus blood turned out to be paramagnetic, and arterial blood was diamagnetic,  meaning repelled by a magnet.    Careful measurements  of this sort gave  astonishing  information  about   the  structure  of  the hemoglobin molecule. So then I thought, “Well, what about the rest of the hemoglobin molecule?    There are four iron atoms and 9,996 other atoms.   What are they doing?    So I had better work on the structure of proteins.”  I was giving a talk in 1936 at the Rockefeller Institute for Medical Research about the magnetic properties of hemoglobin.    A man named Karl Landsteiner sent word to me, asking me to come to his laboratory to talk to him.   I did.   He said he was making immunological studies — antibodies, antitoxins.   He wanted to know if I could explain some of his observations.    So I thought about them for four years and finally wrote a paper, and when the second edition of his book came out there was a chapter by me on the molecular structure of antibodies.    I hadn’t changed my course.    I’d just gone on roads that have diverged a  little from the ones I’d been  going  on.

Perutz’s Hemoglobin Breakthroughs and Later Work

Perutz with his hemoglobin molecule, 1959. Image credit: Life Sciences Foundation.

[Part II of our survey of the life of Max Perutz, this time focusing on the years 1941-2002. Published in commemoration of the Perutz centenary, May 2014.]

Knowing that his parents were safe from Nazi persecution and able to return to the United States, circumstances began improving for Max Perutz. The Rockefeller Foundation reactivated his grant, allowing him to support himself while stateside as well as his parents in Cambridge, England. Perutz’s father was also able to find work as a laser operator during the war and afterwards qualified for a pension.

In September 1941, Perutz met Gisela Peiser, who was an accountant at the Society for the Protection of Science and Learning, an organization that assisted Jewish and other academic refugees fleeing from the Nazis. After a quick courtship, they were married the following March and, in December 1944, welcomed their daughter Vivien into the world. That same year, Perutz also found himself back in good stead with the British government and recruited to research ice strength for potential ice stations in the North Atlantic. The research did not work out, so Perutz returned to his work on hemoglobin at Cambridge. The next few years were spent trying to put together a secure source of income for him and his growing family. In the interim, he took out more loans and found a temporary fellowship.

Meanwhile, Perutz’s health continued to suffer. As his chronic gastrointestinal attacks became more unbearable, interfering with his daily activities more and more, Perutz began to seek out help. Most doctors he saw told him it was a psychological problem, but eventually one doctor recognized that the symptoms could be treated by a mixture of atropine and codeine. The remedy helped enough for Perutz to live more or less undisturbed by the problem for several years, though eventually that would change.

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Perutz and John Kendrew, 1962. Image credit: Nobel Foundation.

In 1947, the war now completed, Perutz, along with John Kendrew, was appointed to head the new Research Unit for the Study of the Molecular Structure of Biological Systems (Perutz later shortened the unit’s name to Molecular Biology Research) at the recently established Medical Research Council. Situated at the Cavendish Laboratory in the physics department, the group expanded on Perutz’s earlier application of x-ray crystallography to biological materials. Perutz, in this new administrative role, described his lab management as one where he would “leave people free to do what they wanted…if they were good scientists.”

One of the several student researchers that came through the lab was Francis Crick, who started work in 1949. Perutz had Crick look at the validity of his hemoglobin model, which was the culmination of roughly six years of research. Crick applied his mathematical training to show that the model was “nonsense.” Perutz accepted Crick’s assessment and later reflected that only in England at that time could a student be so critical of their principal investigator. Crick was eventually drawn away from hemoglobin research by James Watson, who came to the lab in 1951 to work under Kendrew on molecular structure, but his impact on the development of Perutz’s hemoglobin structure was long-lived.

Throughout the late 1940s, Perutz also continued his work on glaciers in the Alps and helped found the Glacier Physics Committee in 1947. Though he had trouble recruiting able assistants who could also ski (the first two broke their legs), the work gave Perutz and his family the opportunity to spend summers in the mountains. Perutz’s research led him to conclude that glaciers flowed faster at the surface than at the bottom.

Perutz’s digestive attacks began increasing in intensity in the early 1950s to the point where, in 1954, he was hospitalized for ten days. While there, doctors looked for possible causes but came up empty and could only prescribe bismuth, with little effect. What did help, for reasons Perutz did not understand, was visiting the Alps, and so he arranged for a trip after being released from the hospital. Unfortunately the attacks resumed as soon as he returned to Cambridge, pushing Perutz to his limits – he considered resignation and even contemplated suicide. In desperate straits, he arranged for another trip to the Alps that spring but, once there, continued to get worse and, as an added complication, came down with scurvy.

When he returned, Perutz sought out other doctors who might be able to help, eventually visiting Werner Jacobson, who was also at Cambridge. Jacobson thought Perutz’s symptoms sounded like those of Celiac disease. He suggested that his patient stop eating wheat, or more specifically gluten, which immediately improved Perutz’s condition. Whenever the symptoms appeared again, as they did in the early 1960s, Perutz could trace them back to gluten; he eventually stopped eating any form of bread, since even gluten-free flour contained small amounts of gluten that negatively affected his health.

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Perutz in lecture. Image credit: Nature.

Perutz’s improved physical condition coincided with the final years of his triumphant work on a determination of the structure of hemoglobin. After working out a solution to interpret x-ray diffraction photos of proteins three-dimensionally, Perutz came upon the structure in September 1959, submitting his findings to Nature before heading to the Alps to ski over the winter break. By the time he returned, he was famous.  It was quickly and widely acknowledged that his work comprised a major breakthrough for both chemistry and biology.  As Hugh E. Huxley wrote, in 2002

He was the first person to find out how to determine protein structure by X-ray crystallography, after many years of patient struggle, and he applied the technique to solve the structure of haemoglobin, the oxygen-carrying protein in blood….The results showed that it was possible to see, in the atomic detail necessary to understand mechanisms, the structure of the macromolecules that carry out many of the functions of a living cell. Such knowledge is basic to the revolution that has swept through biology in the past 50 years, and to modern medicine and biotechnology.

By Fall 1962 there were rumors that Perutz would be awarded the Nobel Prize for chemistry. As October arrived, he began receiving calls from the press, but did not quite trust them. As the calls continued, Perutz received a telegram and thought, along with the rest of the lab, that it may be from the Nobel committee. Alas, the message was only from Nature asking how many reprints of his article Perutz wanted. That afternoon however, Perutz received another telegram, the one he had been waiting for. The lab celebrated with a champagne party as Perutz and Kendrew had been awarded the Nobel Prize for Chemistry, and Watson and Crick, along with Maurice Wilkins, would receive the Nobel Prize in Physiology and Medicine.

Perutz continued to work on the hemoglobin structure after his rise to fame, next turning to the question of how the structure changed with the uptake of oxygen. His Nobel lecture described this continued research on the four subunits within hemoglobin that changed their structure as oxygen was taken up; the first description of how proteins changed in structure.

In the years that followed, Perutz focused more on why this change occurred. Aided by automated x-ray diffraction machines and able assistants, Perutz’s lab was able to turn out more measurements than ever before. But the measurements, as Perutz later related, did not make any sense. After one of his research assistants completed his postdoc, Perutz looked closer at his results and realized that the new x-ray instruments had not been calibrated correctly.

In 1967, with all the bugs fixed, Perutz and his team put together the first atomic model of hemoglobin, but Perutz’s questions about why the structure changed still were not answered. By 1970, the lab was able to construct an oxygen-free model, allowing Perutz to compare it with the oxygenated model. As Perutz later described “there came this dramatic moment when between them, the models revealed the whole mechanism.” What he was able to see was how a slight movement of the iron atom triggered a change in the whole molecule. Thus, Perutz felt he was able to explain “all the physiological functions of hemoglobin on the basis of its structure.” The results were published in Nature.

Within the field, objections to Perutz’s explanation were numerous and he spent much of the next two decades refuting criticisms and refining his own explanation. At the same time, his celebrity also rose among scientists as he was increasingly invited to give lectures all over Europe and North America. By 1975 Perutz’s fame outside of scientific circles had grown such that Queen Elizabeth II invited him to visit with her at Buckingham Palace. Afterwards, Perutz expressed his regrets to the Queen’s secretary that “she had made me talk away like an excited little boy about my own doings and that I never asked her anything about hers.” Nonetheless, Perutz did hope that the Queen would enjoy his gift, the autobiography of Charlie Chaplin.

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Max Perutz with his hemoglobin model. Image credit: BBC.

By 1980 Perutz had begun to reach out to broader audiences more intentionally. Shortly after retiring from the chair of the Laboratory of Molecular Biology, Perutz wrote a memoir of his time there. This, in turn, inspired him to compile an account of his experiences during World War II and submit it to the New Yorker. Penned in 1980 but not published until 1985, “Enemy Alien” helped bring Perutz greater levels of fame, as he received more letters after its publication than he did congratulations for his Nobel Prize.

An Italian pharmaceutical company also approached Perutz in 1980 to give a lecture on the social implications of molecular biology. According to a 2001 interview, Perutz told the company that “molecular biology has no social implications,” but that he could talk about “science as a whole.” This spurred him to take more of an interest in broader scientific questions, ultimately leading him to adopt controversial stances combatting criticism of the Green Revolution, DDT use and nuclear power, among other issues in the headlines. It also evolved into an interest in philosophy – Karl Popper’s Open Society and its Enemies proved particularly impactful. By 1989, Perutz expanded his popular lectures into a book of essays, Is Science Necessary? which included writings that he had also done for the New York Review of Books as well as “Enemy Alien.”

While continuing to write for the New York Review of Books up to the end of his life, Perutz also pursued new research on proteins and hemoglobin, taking a particular interest in neurodegenerative diseases like Parkinson’s and Alzheimer’s. In 2001, right before he passed away, Perutz was still at the lab seven to eight hours a day (including lunch and tea), preparing a publication for the Proceedings of the National Academy of Sciences on the common structure of insoluble protein deposits in neurodegenerative diseases. He passed away at the age of 87, unable to reconcile his initial structure with x-ray diffraction photos which showed contradicting features that Perutz concluded arose from three different structures. The results were published in 2002, after Perutz had died, in two separate articles.

Max Perutz (1914-2002)

Max Perutz. Credit: Theresianische Akademie Wien.

[Ed Note: We mark the centenary of Max Perutz’ birth today with the first in a series of posts on his life and his associations with Linus Pauling. Today’s post focuses on his life from 1914-1941.]

Max Ferdinand Perutz was born May 19, 1914 in Vienna, the third child and second son of Hugo and Adele Perutz.  His birth came little more than a month before the assassination of Archduke Franz Ferdinand and the subsequent start of World War I. Vienna was largely untouched by the war, but suffered mightily from the economic depression that followed. The Perutzes, who had accumulated a substantial fortune from family textile concerns, lost their savings to the rapid postwar inflation. Nonetheless, according to biographer Georgina Ferry, the family managed to maintain an income and “within a few years of the war’s end, they were living as well as before.”

In a 2001 interview with Katherine Thompson for the British Library, Perutz said that he remembered little of these early years. He did recall being a “very delicate child,” contracting pneumonia three times before he was six and a very serious fever at age nine. Fortunately, he was able to recover from the fever after his nanny took him to a resort in the Alps for the winter. After World War II, chest x-rays revealed that Perutz had suffered from tuberculosis, the likely cause of his fever.

Perutz’s physical delicacy affected his social life as well; he described himself as a “weakling at school” who had no friends early on since he was sick so often. Because of his condition, Perutz did not excel at most sports. But his many holidays in the Alps led him to develop a lifelong love of rock climbing and skiing. These skills eventually earned Perutz the respect of his peers after he won a prize for the school skiing team.

Perutz attended private primary schools until entering the newly organized Realgymnasium, which brought a shift in focus from classics to modern languages and the sciences. Perutz described his early years of schooling as “eight years of unbearable boredom.” This boredom began to wane as Perutz gravitated toward English literature, an interest enabled by his Anglophile father who saw that he was tutored in English in addition to the more common French. Perutz secretly read Charles Dickens and other British novelists under the bench while at school, later furthering this passion with his first girlfriend, who was from England. Perutz’s parents expected him to take over the family textile business once he was old enough, and were heartened by his developing intellectual prowess.

However, the business route never appealed to Perutz, especially after he was exposed to chemistry by an influential teachers, and at eighteen he began formal pursuit of his interest in chemistry at the University of Vienna. (Protests from his parents were soothed by the help of a friend of Perutz’s older brother, a chemist at Dow.) As with his primary schooling, Perutz was not very impressed by the education that he received at university. He described the curriculum’s lack of mathematical training and decidedly practical emphasis as “chemistry done by heart” because of the reading and memorizing he was forced to do in lieu of actual laboratory work. But ultimately he made it through and, in the process, cultivated a new attraction to physics which he would later fulfill as a graduate student in England.

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Portrait of Perutz drawn by William Lawrence Bragg. Credit: MRC Laboratory of Molecular Biology

From Vienna, Perutz moved on to Cambridge, where he hoped to work with Frederick Gowland Hopkins, the university’s first chair of biochemistry and recipient of the 1929 Nobel Prize in Physiology for his work on the relation between vitamins and growth. Since Perutz showed up without letting anyone know, he did not find out that he could not work with Hopkins until he actually arrived. Chastened, Perutz looked elsewhere and ended up in the Cavendish Laboratory of Physics doing x-ray crystallography. “Without knowing it,” Perutz later recalled, this “was one of the best things I could have done.”  Supported by £500 sent by his father, Perutz settled in and was able to take care of his own finances for the duration of his doctoral studies.  His health continued to suffer though – once in England, he began to experience frequent and painful digestive problems.

The first project that interested Perutz was identifying radioactive deposits dug out from the cliffs in Cornwall. Perutz measured the half-life of the material, but found that it did not correspond to any known elements. Excited that he may have discovered a new element, Perutz shared his findings with Cambridge luminaries Ernest Rutherford and J. D. Bernal, who helped him to determine that the substance was, in fact, radium. Bernal also encouraged Perutz to publish his findings and to present them at a Royal Society soiree. This led to his first publication, “The Iron-Rhodonite from Slag,” which appeared in Mineralogy Magazine in 1937.

At the end of his first year at Cambridge, Perutz spent his summer holiday back in Austria and thought about what he might do for his doctoral dissertation. Felix Haurowitz, then at Charles University in Prague, suggested focusing on hemoglobin, telling Perutz that he could get crystallized hemoglobin from Gilbert Smithson Adair at Cambridge. When he returned and acquired the hemoglobin, Perutz says he “immediately got a lovely x-ray diffraction picture,” which “thrilled” Bernal.

In the midst of his hemoglobin research, Perutz also agreed to assist a man who came to the Cavendish Laboratory looking for researchers to satisfy his own interest in glacier development. Perutz saw this as a perfect opportunity to spend more time skiing in the Alps. He published his work in the Proceedings of the Royal Society in 1939, describing how melting and the movement of water contributed to glacier formation and flow.

In March 1940, Perutz wrapped up his Ph.D., which described the structure of hemoglobin and the x-ray methods used to develop the model. Yet the looming threat and subsequent reality of war overshadowed his findings and began to color components of his world that were much more important than his research.

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Credit: National Portrait Gallery, London.

As World War II approached, the Perutz family, still in Vienna, looked for ways to get out. The Perutzes were ethnic Jews, but Max’s parents were non-observant and Perutz himself had been baptized Catholic. As a young boy, Perutz was very devout, a character trait that he abandoned after his prayers that the Italians not invade Ethiopia were not answered. While his baptism was meant to protect him from anti-Semitism, he claimed that his family “very rarely” experienced discrimination before the Anschluss. Once the Nazis assumed power, the Perutz family quickly left with Max’s brother and sister going to the United States and his parents coming to stay in Cambridge. Hugo and Adele Perutz, used to supporting themselves, lost their businesses and spent all their money leaving Vienna – according to their son, they “were traumatized by suddenly being poor.”

To get them to England, Max both had to prove that he could support them and was also required to pay a thousand pounds, compelling him to sell some of his mother’s jewelry and to borrow funds to cover the rest. Around this time, William Lawrence Bragg, winner of the 1915 Nobel Prize in Physics, came to the Cavendish. Bragg was very excited about Perutz’s work with hemoglobin and helped him to secure a grant with the Rockefeller Foundation in New York. The grant provided £275 per year, enough for Perutz to prove that he could support his parents. But soon the family would come into even more trouble.

In May 1940, just two months after he finished his Ph.D., Perutz was interned by the British government. He was first taken and held in a school at Bury St. Edmunds, east of Cambridge, for one week before being transported to Liverpool. By July, Perutz, along with roughly twelve-hundred others, was shipped across the Atlantic to a camp near Quebec City, Canada, where the residents’ status was upgraded from “internee” to “civilian prisoner of war,” a change that promised access to clothing and army rations. In a 1985 essay for the New Yorker, titled “Enemy Alien,” Perutz wrote,

To have been arrested, interned, and deported as an enemy alien by the English, whom I had regarded as my friends, made me more bitter than to have lost freedom itself. Having first been rejected as a Jew by my native Austria, which I loved, I now found myself rejected as a German by my adopted country.

Perutz’s friends were working on his behalf to have him released, unknown to him since he could receive no communications.

Meanwhile, in Quebec, Perutz tried to make the best of things and organized a “camp university.” Hermann Bondi, a mathematician also from Vienna, taught on vector analysis, while Klaus Fuchs, a student at Bristol who fled Hitler’s persecution for being a communist, taught theoretical physics. For his part, Perutz drew on past research of his own, explaining the atomic structure of crystals to all who might be interested.

The Rockefeller Foundation did not forget about Perutz and arranged a professorship for him at the New School for Social Research in New York City. Hearing rumors that his father had also been interned and worried that he would not be able to obtain a visa to travel once he had been established in the United States, Perutz was eager to go back to England to check on his parents. After several delays and transfers, Perutz arrived back in Cambridge in January 1941, finding his father already released and his friends happy to see him.

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.

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.