The Serological Properties of Simple Substances

1935i.1

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.


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.


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.

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The Arrival of Dan Campbell at Caltech

Dan Campbell, ca. 1940s.

Dan Campbell, ca. 1940s.

[Part 1 of 2]

As a scientist, Linus Pauling is remembered by many for combining his expertise in chemistry with other fields. Often times Pauling would start off thinking about a problem from a chemical perspective and end up learning about a field entirely new to him, like cellular biology or medicine. Though this sort of cross-disciplinary work is more commonplace today (partly because of the example that Pauling provided), in the 1930s it was fairly rare for scientists to combine different fields of study. This given, pioneers of the cross-disciplinary approach often found it difficult to identify like-minded researchers with whom to collaborate. Fortunately for Pauling, a man with a very wide network, other researchers often found him.

After delivering a talk about hemoglobin in 1936, Pauling was pleasantly surprised to be consulted by Austrian medical researcher Karl Landsteiner. For many years, Landsteiner had been trying to understand how antibodies in the immune system work, and he believed that Pauling’s knowledge of medicine and chemistry could help him in his investigations. An antibody is a disease-fighting macromolecule that targets and rids the body of unwanted foreign substances, such as viruses and incompatible blood types. Landsteiner wanted to know how antibodies can target specific foreign substances with such precision. This encounter drew Pauling’s attention to the field of immunology, which would eventually become an important part of his research and would remain so for many years to come.

Pauling’s communications with Landsteiner spurred an interest in looking into the chemistry of antibodies and their substrates, antigens. At the time, however, most of Pauling’s focus was necessarily occupied with finishing up his previous program of grant-funded research on protein structures. Furthermore, Pauling was not an immunologist and the demands on his time were such that he could do little more than keep immunology in the back of his mind.

It wasn’t until 1939 that Landsteiner once again brought Pauling’s full attention back to antigens when he used Pauling’s theory of protein structure in a discussion about antibodies. Reading Landsteiner’s article sparked several ideas for Pauling which quickly led to his drafting a rudimentary theory of antibody chemistry. Six months later he found the perfect opportunity to test some these ideas.


Image extracted from a glass plate display, “Pictures of Antibodies,” prepared for the First International Poliomyelitis Conference, New York, 1948. The caption accompanying this image reads: “…[An] antibody-antigen framework which may precipitate from a solution or be taken up by phagocytic cells.”


In January 1940, immunologist Dan Campbell first visited Caltech on a fellowship. Campbell was an Ohio native who had been trained at Wabash College in Indiana and George Washington University in St. Louis, before receiving a doctoral degree from the University of Chicago, where he was subsequently hired as an assistant professor. During his tenure at Chicago, Pauling invited Campbell to spend a fellowship period at Caltech.  Campbell was only scantly familiar with the Institute, but was aware of the reputation of its chemistry department and accepted Pauling’s offer largely on this basis.

Due to his unfamiliarity with the institution, by the time of his arrival in Pasadena Campbell had still not yet identified a research project on which to collaborate. Pauling advised Campbell to consider different researchers before making his final decision on where and with whom he might work. In the end, after asking around, Campbell chose to collaborate with Pauling on his theory of immunology.

This was a fortuitous decision, for several reasons.  First, in addition to immunology, Campbell had a background in biophysics and chemistry, which made him a perfect candidate to test and develop Pauling’s antigen theory. More importantly, as Campbell began his initial investigations, it became apparent that Pauling’s ideas were flawed and that Pauling’s knowledge of chemistry alone would not be sufficient to make further progress in immunological research.


Campbell and Pauling, 1943.

Pauling had alleged that antibodies were similar to denatured proteins; that is, a protein that has lost its secondary and tertiary structures and has unfolded into an amino acid chain. Pauling’s theory anticipated that antibodies were an unfinished protein that required specific antigens in order to fold into the proper secondary and tertiary structures.

According to this model, antibodies would only form hydrogen bonds and thus would coil around chemically complementary antigens. As such, the theory explained how antibodies are able to bind unambiguously to their complementary molecules. However, Campbell’s results did not support all of Pauling’s ideas. Though his research showed that antibodies were in fact proteins, their physical structure before and after binding to antigens remained unclear.

Pauling’s lack of evidence for his theory of antibody structure and composition limited him to publishing only a single theoretical paper in which he explained his ideas about antibodies. In July 1940 the Journal of the American Chemical Society featured Pauling’s “A Theory of the Structure and Process of Formation of Antibodies.” The article received much attention and, despite the lack of evidence, was widely acclaimed, though it failed to provide a definitive explanation for antibody structure.

After the publication of the piece, Campbell once again tested Pauling’s theory, and this time his results were much more confusing, to say the least. Initially, it appeared that Campbell had succeeded in creating artificial antibodies by simply denaturing beef globulins (a protein found in blood) and later allowing them to refold around an antigen.

Word of these results greatly excited Pauling, who began to envision the mass production of antibodies using Campbell’s method. Reality turned out to be not so simple; when students and postdoctoral fellows tried to replicate Campbell’s experiment, they were unable to obtain the same results. Looking back now, it seems most likely that Campbell’s research assistants had misinterpreted the results of his experiment.

Pauling knew that he would need more time with Campbell to refine his theory, but that could only happen if Campbell’s position at Caltech was secured. In 1942 Pauling arranged for the Institute to offer Campbell an assistant professorship, which he accepted. By 1950 Campbell had become a full professor.

Combining immunology and chemistry proved to be a commendable approach for tackling many health concerns of the time. Likewise, Campbell’s presence was crucial to the development of Caltech’s immunochemistry department, which over a span of five years grew from a single office (Campbell’s) to a space occupying most of the third floor of Caltech’s Church Laboratory. Students and professors alike flocked to the growing department to discuss questions and engage in research on immunology, using chemistry as the basis of their approach. From the outset, both Pauling and Campbell benefited from one another’s expertise while colleagues at Caltech, and their partnership would continue to yield fruit for many years.

Post 500

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

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.


Young Pauling, ca. 1910s.

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. […]


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

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. […]


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

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.

The End of Artificial Antibodies

Illustration of bivalent antibodies attaching to complementary antigen molecules. Image extracted from a glass plate display, “Pictures of Antibodies,” prepared for the First International Poliomyelitis Conference, New York. The caption accompanying this image reads: “In vitro or in vivo bivalent antibodies may become attached to complementary portions of antigen molcules.”

[Part 3 of 3]

Though highly controversial, Linus Pauling’s claim that he had created artificial antibodies gave him a boost in funding. Many of his backers realized that if Pauling was correct he had just revolutionized modern medicine and they were just as eager as he was for his project to succeed. Since he now had more money, Pauling hoped he could expand his staff, though the war greatly prohibited this effort. Pauling lamented:

Unfortunately the amount of war work which is being done now here is so great that the usual seminars and informal discussions of science have decreased somewhat in number, but still a good bit of work in pure science is being carried on.

To help remedy this situation, Pauling corresponded frequently with William C. Boyd of Boston University’s School of Medicine, trying to persuade him to transfer to Caltech over the summer. Boyd refused, stating that he had too many other medical defense projects, though suggesting that “perhaps it will not be too late when the war is over, unless it goes on for 10 years or more, as some pessimistic writers predict.” Pauling also failed to hire two other well-known researchers, Henry F. Treffers, and A.M. Pappenheimer. Both declined because he was only able to offer a one year position. Indeed, from 1942-1943, Pauling actively tried to find staff for his lab, offering a $3,500 one-year position and draft deference. Despite this, most of the competent researchers he wanted were otherwise employed doing war work. He was able to hire Leland H. Pence in December of 1942, and in 1945 Frank Johnson visited from Princeton and worked at the lab in Caltech a bit. These individuals were, however, the exceptions, and Pauling was generally unsuccessful with his offers of employment.

Pauling was also facing other staff issues that were relics of the era. Among his group were two employees who were born and raised in the United States, but whose families were Japanese, Carol Ikeda and Miyoshi Ikawa. All too cognizant of the forthcoming policy of internment, Pauling began corresponding with Michael Heidelberg of Columbia University, hoping that he could temporarily trade employees as a method of getting Ikeda, at least, to a less hostile location. The plan did not work out, and Heidelberger ended one of his letters to Pauling bemoaning the fact that “…unfortunately a lot of wholly patriotic people are going to suffer.” Pauling was eventually able to get both Ikeda and Ikawa into graduate programs, though doing so took a substantial amount of work.


Dan Campbell and Linus Pauling, 1943.

Even though William Boyd had refused a job, he and Pauling continued to correspond frequently. Boyd was critical of Pauling’s theories on antibodies, warning his colleague that “preconceived notions evidently play a big role in the field [of immunochemistry.]” Boyd told Pauling to be careful with his research and his declarations, as he had often made arguments that he felt infallible only to have his colleagues inform him that they were unconvinced.

In August 1942, five months after issuing his controversial press release, Pauling finally published his research on artificial antibodies in an article titled “The Manufacture of Antibodies in vitro,” which appeared in the Journal of Experimental Medicine. As with the press release, Pauling’s paper was somewhat lacking in detail and many scientists found it hard to replicate his experiments. Those who did, such as Pauling’s valued colleague Karl Landsteiner, were unable to obtain the same results that Pauling had reported. Despite this, Pauling remained convinced that his research was valid and worth pursuing. However, Pauling’s collaborator Dan Campbell seemed to be the only one who could successfully produce the antibodies, and even those were weak and ineffective.

In early 1943 the Rockefeller Foundation assigned Frank Blair Hanson to assume some of the work that Warren Weaver had been conducting, and right away it was clear that Hanson was notably “less entranced than Weaver with Pauling’s work.” Despite the fact that he agreed to continue funding Pauling’s artificial antibody research, he was skeptical of its worth and began polling immunologists across the country to that end. They did not respond favorably – even Landsteiner believed that there was a less than 50% chance that Pauling had actually created artificial antibodies. As a result of these lackluster opinions, Hanson cut Pauling’s funding by half.

Pauling proceeded nonetheless, his enthusiasm still strong. His next move was to submit a patent application, “Process of Producing Antibodies.” The response that he received was not what he wanted to hear:

The claims are again rejected for lack of utility as no evidence has been presented to show that the antibodies alleged to be produced by the claimed have any utility at all… The claims are rejected as being too broad, functional, and indefinite…the claims are rejected for lack of invention…all the claims are rejected.

Pauling was disheartened by this response but still confident that he could salvage the project. However, shortly afterward he received a letter from the Rockefeller Foundation informing him that they did not approve of researchers using their funds to apply for medical patents. Because of the letter, and the general ineffectiveness of the research, Pauling opted not to pursue his patent application any further.

Illustration of the antibody-antigen framework. The caption accompanying this image reads: “…[An] antibody-antigen framework which may precipitate from a solution or be taken up by phagocytic cells.”

As 1943 progressed, Pauling continued to piece together a better understanding of how antibodies adhere to antigens. Later that year, he and Campbell published a paper in Physiological Review which further elaborated on their idea that shape was the primary determinant of antibody functions. They wrote that while many of their colleagues at other institutions felt that antibody formation adhered to a “lattice theory,” they did not, because their research showed that the structures created by antibody/antigen precipitation were not regular enough. Instead, they coined the phrase “framework theory” to describe their idea.

By the winter of 1943-1944, Pauling had at last concluded that the artificial antibody research was going nowhere. This was a difficult admission for Pauling because, despite the fact that progress in the research was still slow, he was convinced that he could make it work if given more time. Unfortunately for him, the war effort demanded quicker results and actively prohibited greater focus on antibodies. In the end, he decided to abandon the artificial antibodies research. Pauling never retracted his support for the work, though many years later Dan Campbell admitted that a laboratory technician had “shaded” the results to fit what he thought Pauling wanted to see.


The failure of the artificial antibodies project allowed Pauling to move on to more productive lines of research. He continued to build his ideas on how exactly antibodies function, and by 1945 he was able to prove that shape was indeed what caused antibodies to adhere to antigens. In Pauling’s description, the antibodies would fit to the antigens like a glove, at which point they adhered, not due to orthodox chemical bonds, but because of another weak, poorly understood force.

The force that causes antibodies to bind with antigens is called the van der Waal’s force. It is a very weak, almost imperceptible subatomic bond between two molecules existing in extremely close proximity. Historically, due to their weakness, van der Waal’s forces had been ignored as viable components of biochemical reactions. However, Pauling was able to show that the extremely tight fit between antibodies and antigens exposed a large surface area across which the van der Waal’s force could become a factor. The fit had to be precise, as even one or two atoms being out of place would effectively break the hold that the van der Waal’s force put into place. This concept, known variously as molecular complementarity or biological specificity, cast a great deal of light on a central mystery of molecular biology. With it, Pauling was additionally able to confirm his earlier hypothesis that antibodies are bivalent.

As World War II drew to a close, Pauling shifted his focus away from antibodies and back towards a more general study of the shape, creation, and function of proteins. Pauling’s focus on proteins was long lived, stretching at least twenty-five years from 1933-1958. His foray into antibodies was notably shorter, a nine-year interlude from 1936-1945. Yet in this time, he had managed to dramatically impact immunochemistry with his discovery that antibodies are bivalent and his insights into how they work. These accomplishments are made even more impressive when considering that Pauling was neither an immunochemist nor an immunobiologist by trade. As was so often the case during his life, he threw himself into the challenge with characteristic enthusiasm, and managed to make major contributions to the field.

Thinking about the Creation of Antibodies

Drawings of the interaction between an antibody and azoprotein by Linus Pauling. 1940s.

[Part 1 of 3]

During a period of about a decade, beginning in 1936, my principal research effort was an attack on the problem of the nature of life, which was, I think, successful, in that the experimental studies carried out by my students and me provided very strong evidence that the astonishing specificity characteristics of living organisms…is the result of a special interaction between molecules…

-Linus Pauling, 1991

As one would expect, the Great Depression made it extremely difficult to acquire funding for scientific research. Luckily for Linus Pauling, he had on his side the patronage of the Rockefeller Foundation, and a close friendship with Warren Weaver, the head of the foundation’s Physical Sciences Division. Weaver wanted Pauling to get more involved in biological research, specifically protein research. Pauling was hesitant, but Weaver controlled the funding and convinced Pauling to move forward.

Alfred Mirsky

By 1933 Pauling was heavily involved in research on proteins, specifically their shape and function. In 1935 he began working with Alfred Mirsky, a Rockefeller Foundation scientist, with whom Pauling concluded that all proteins are structured as chains, and that the shape of a given protein determines its function and behavior.

A large component of Pauling and Mirsky’s research was on protein denaturation, effectively the breakdown or death of proteins. It was known that modest heating, mild acids, milk alkalis, or agitation, such as beating eggs with a fork, all serve to denature a protein. However, Mirsky discovered that proteins that were slowly denatured at lower temperatures could be resuscitated and the process reversed.

Karl Landsteiner

In the Spring of 1936, Pauling began another collaboration, this time with Karl Landsteiner, an Austrian scientist who won a Nobel prize for discovering and developing the field of blood typing. Landsteiner invented the ABO system, and uncovered methods for making blood transfusions safe. In this research Landsteiner observed that, in instances where the wrong blood type is used in a transfusion, antibodies attacked the transfused blood. Pauling was intrigued by Landsteiner’s work, and began reading about antibodies; he was interested and puzzled by what he found. While the scientific community knew that antibodies worked, how exactly they worked and how exactly they were formed were still unknown.

At the time, there were four main schools of thought regarding the creation of antibodies: the Antigen-Incorporation theory, the Side-Chain theory, the Instruction theory, and the Selection theory.

The Antigen-Incorporation theory, originally proposed by Hans Buchner in 1893, proposed that antibodies were actually the byproduct of antigens “splintering” in the human body and becoming incorporated into it. Despite the fact that this theory had been largely disproven at the time, it was proposed again by E. Hertzfeld and R. Klinger in 1918, by W.H. Manwaring in 1926, by Locke, Main, and Hirsch also in 1926, and finally once more by Gustave Ramon in 1930.

The Side-Chain theory was posited by the famous Paul Ehrlich in 1897, who argued that the body’s immunological reaction to antigens was “only a repetition of the processes of normal metabolism.” Ehrlich thought that cells would digest certain antigens in the same way that they digested nutrients. After repeated assimilations, or too large of an assimilation, the cells would overcompensate and release antibodies. His theory included a number of issues that the scientific community could not solve at the time, and it took over sixty years for the model to be improved upon.

The Instruction theory states that the body uses antigens as a template, then manufactures antibodies to specifically combat the antigen that the antibody is based off of. Pauling eventually belonged to this school of thought, as did Landsteiner, Michael Heidelberger, Felix Haurowitz, and Jerome Alexander. This group was far from unified however; the only point on which adherents to this school agreed was that antigens acted as templates. How antibodies worked, and how they were produced, was still a highly contentious question.

The final theory was the Selection theory, which was in concept almost identical to Ehrlich’s Side-Chain theory, except that its explanations were based on more modern mechanisms. Instead of general metabolic processes, quantum mechanical forces were proposed to be the cause of the attraction between antigens and antibodies. This school of thought became more popular near the end of World War II and in the post-war era.

Pauling described Antibodies as “fantastically precise little weapons,” and found it fascinating that they could identify and attack invading molecules that were different from safe molecules by only a few atoms. Antibodies are made of pure protein, are remarkably similar to one another, are relatively enormous, and also attack vastly different types of molecules.

Pauling and Landsteiner were especially vexed by how antibodies could target varied molecules so precisely when they were so similar. Pauling proceeded to read Landsteiner’s book on antibodies, and began to wonder if shape affected antibodies as much as it affected regular proteins. Landsteiner had arrived at a similar conclusion, and in 1939 published a note in Science suggesting that shape was what determined the effect of antibodies.

Diagram included in Pauling’s article, “A theory of the structure and process of formation of antibodies.” 1940.

Pauling expanded upon this idea, and in 1940 published a paper in which he hypothesized that antibodies were built as chains of non-specific proteins which collided with antigens, then compressed and shaped themselves around the antigen, “like wet clay pressed against a coin.” The paper created quite a stir, and generated a lot of support for the notion of using chemistry to solve biological questions. Unfortunately for Pauling, it later turned out that his hypothesis was deeply flawed.

Another argument developed in the 1940 paper was that antibodies are bivalent – that is, they have two sites which can bind to antigens. In addition to being bivalent, Pauling hypothesized that each of the “arms” of an antibody could latch onto different kinds of antigens. While Pauling was incorrect on the latter part – antibodies can only grab onto one type of antigen – he was correct that they are bivalent.

Pauling had gotten off to a strong and noticeable start in the field of immunology. Whether correct or incorrect, he was making progress towards a greater understanding of how the body protects itself. As the clouds of war began to reach across the Atlantic and Pacific towards the United States, Pauling’s new and growing knowledge was going to be put to the test.

Pauling110

Linus Pauling. Lecturing at the Concepts of Chemical Bonding Seminar, Oslo University, Oslo, Norway. 1982.

Today marks the 110th anniversary of Linus Pauling’s birth, which occurred in Portland, Oregon on February 28, 1901. As has become tradition on the Pauling Blog, we are celebrating this occasion by looking back at Pauling’s life in increments of twenty-five years.

1911

At the tender age of ten, young Linus was already at a crossroads in his life. First and foremost, his father Herman had died of a perforated ulcer the previous summer, thus throwing the Pauling family into something akin to chaos. Herman was a pharmacist and businessman of middling success, and his death was a source of major financial concern for his widow Isabelle and their three children, Linus, Pauline (age 9) and Lucile (age 7). From this point on, Linus’s childhood was certainly informed, if not dominated, by the continual need to contribute to the household income. His mother’s only asset of consequence was the family home, which she boarded out on a regular basis in an attempt to make ends meet. But as time passed and Belle’s own health faded, her only son was frequently called upon to assist with the family finances, leading Linus to assume any number of odd jobs, from delivery boy to film projectionist to grocery clerk.

Young Linus, ca. 1910s.

It was at this same time that the boy’s interest in science was beginning to flower. The previous year Herman had written a letter to the Portland Oregonian newspaper indicating that his son was a “great reader” keenly interested in ancient history and the natural sciences. In 1911 Pauling’s scientific impulses continued to flourish in the form of an insect collection that he maintained and classified using books checked out from the Portland library. Not long after, as with many scientists of his generation, Linus would develop an interest in minerals and begin compiling a personal collection of classified stones that he found.

1936

By the age of thirty-five, Pauling had already established himself as among the world’s pre-eminent structural chemists and was well on his way to making a major impact in the biological sciences. In 1936 Pauling met Karl Landsteiner of the Rockefeller Institute, a Nobel laureate researcher best known at the time for having determined the existence of different blood types in human beings. In their initial meeting, Pauling and Landsteiner discussed Landsteiner’s program of research in immunology, a conversation that would lead to a fruitful collaboration between the two scientists. Importantly, his interactions with Landsteiner would lead Pauling to think about and publish important work on the specificity of serological reactions, in particular the relationship between antibodies and antigens in the human body.

Linus Pauling, 1936.

The year also bore witness to a major change at the California Institute of Technology: in June, Arthur Amos Noyes died. Noyes had served as chairman of the Caltech Chemistry Division for some twenty-seven years and was among the best known chemists of his era. His death ushered a power vacuum within the academic administration at Caltech, by then an emerging force in scientific research. Three of Pauling’s colleagues cautiously recommended to Caltech president Robert Millikan that Pauling be installed as interim chair of the department. Millikan agreed and offered the position to Pauling, but was met with refusal. At the time of the proposal,  Pauling was the object of some degree of criticism within the ranks at Caltech – certain of his peers felt him to be overly ambitious and even reckless in his pursuit of scientific advance – and the suggestion that Pauling assume division leadership was hardly unanimous. Millikan’s terms likewise did not meet with Pauling’s approval; in essence he felt that he would be burdened with more responsibility but would not gain in authority. The impasse would not last long however, as Pauling would eventually accept a new offer in April 1937 and begin a twenty-one year tenure as division chief.

1961

A busy year started off with a bang when the sixty-year-old Pauling was chosen alongside a cache of other U.S. scientists as “Men of the Year” by Time magazine. By this period in Pauling’s life his peace activism was a topic of international conversation and early in the year Linus and Ava Helen followed up their famous 1958 United Nations Bomb Test Petition with a second “Appeal to Stop the Spread of Nuclear Weapons,” issued in the wake of nuclear tests carried out by France. As a follow-up, the Paulings organized and attended a May conference held in Oslo Norway, at which the attendees (35 physical and biological scientists and 25 social scientists from around the world) issued the “Oslo Statement,” decrying nuclear proliferation and the continuation of nuclear tests.

Group photo of participants in the Oslo Conference, 1961.

While Pauling’s attentions during this period were increasingly drawn to his peace work, he did make time for innovative scientific research. Of particular note was his theory of anesthesia, published in July in the journal Science. Pauling’s idea was that anesthetic agents formed hydrate “cages” with properties similar to ice crystals. Owing to the nature of their molecular structure, these cages would impede electrical impulses in the brain, thus leading to unconsciousness. In a review article published one year later, the pharmacologist Chauncey Leake described the theory as “spectacular,” though for reasons that are still unclear it failed to gain traction with the larger scientific community.

1986

By age eighty-five, Pauling’s interests centered largely upon his continuing fascination with vitamin C. Having already published monographs focusing upon ascorbic acid’s capacity to ward of the common cold and the flu, Pauling was ready to put his thinking together into a general audience book that would discuss the path to happier and healthier lives. The result was How to Live Longer and Feel Better, a modest critical and commercial success that helped bolster the reputation and the finances of the struggling Linus Pauling Institute of Science and Medicine.

Pauling at 85.

Many of the recommendations that Pauling made in How to Live Longer… were fairly typical of most health promotion books: a sensible diet, regular exercise and no smoking. The major exception to this moderate approach was the famed author’s stance on vitamin supplementation. In biographer Thomas Hager‘s words

Pauling was now advising between 6 and 18 grams of vitamin C per day, plus 400-16,000 IU of vitamin E (40-160 times the RDA), 25,000 IU of vitamin A (five times the RDA), and one or two ‘super B’ tablets for the B vitamins, along with a basic mineral supplement.

This staunch belief in the value of megavitamins would stay with Pauling until his death eight years later, in August 1994.

Pauling’s Methodology: Electrophoresis

Diagram of a Tiselius electrophoresis apparatus.

Diagram of a Tiselius electrophoresis apparatus.

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

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

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

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

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

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

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

Listen:  Pauling discusses the evolution of electrophoresis work at Caltech

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

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

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

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

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