On the Formation of Antibodies

By the 1940s, Linus Pauling’s research interests had expanded to include many subjects generally outside the purview of a typical chemist. In particular, immunology was rapidly becoming a fascination of his – one that would come to devour more and more of his time both in and out of the lab. For Pauling, much of the human body could be viewed as a gigantic set of very complicated chemistry problems, and he derived great joy from being able to solve some of these problems. Among his most important immunological discoveries were his elucidation of the role that hemoglobin plays in sickle cell anemia and his theory of antibody formation.  The latter is the topic of today’s post.

In 1940 Pauling published a paper – now his ninth most cited – in the Journal of the American Chemical Society on the subject of antibodies and antigens. This manuscript, titled “A Theory of the Structure and Process of Formation of Antibodies” is fundamentally based around one important assumption that Pauling made about antibody structure, “…that all antibody molecules contain the same polypeptide chains as normal globulin, and differ only in the configuration of the chain; that is, in the way that the chain is coiled in the molecule.”

Antibodies are protein molecules that play an extremely important role in the human body. Their main function is to identify and neutralize foreign objects, called antigens, that have been taken up by the body. Antigens come in many varieties, including high-molecular-weight carbohydrates, lipids, pollen and bacterial cells. It is important to note too that only antigens marked by the body’s systems as “foreign” will set off an antibody response; antigens marked as “self” are tolerated by the body.

Pauling with Dan Campbell, a primary colleague in Pauling's antibody work.

Antigens play an important role in Pauling’s theory, which argues that antigens alone determine the configuration of a specific portion of the antibody molecule. For example, without the presence of an antigen, a normal globulin protein will be synthesized. In the presence of an antigen however, a specific antibody will be produced, a portion of which will be complementary in structure to the antigen that in question. In describing this process, Pauling’s paper first details the four steps that occur in the formation of a normal globulin molecule, which are summarized below.

1. The polypeptide chain is synthesized. The two ends of the chain are free, but the center of the chain is still attached at the site of synthesis.
2. The ends coil into either their most stable, or another very stable, configuration. Hydrogen bonds and other weak forces between amino acids in the polypeptide chain stabilize the two ends.
3. The center of the chain is freed from the site of synthesis.
4. The center coils into its most stable configuration, and the globulin molecule is completed.

Because antibodies are simply modified globulin molecules, the process for their formation is closely related to that of globulin. Summarized below then, are Pauling’s six steps of antibody formation.

1. An antigen is held in place at the site of antibody production and the antibody is synthesized around the antigen molecule.
2. The ends of the newly synthesized antibody coil into a configuration complementary to groups on the antigen and attach to these complementary groups.
3. The center of the chain is freed from the site of synthesis, causing one of two things to happen. If the forces between the ends of the chain are sufficiently strong, both ends will continue to be attached to the antigen, and the antibody will never be completed.
4. If they forces between the ends of the chain and the antigen are weak, one end will dissociate from the antigen.
5. Assuming one end of the chain dissociates from the antigen, the center of the chain coils into its most stable configuration, making a complete antibody.
6. Eventually, the antibody will dissociate from the antigen and float away.

To summarize, Pauling’s theory of antibody formation argues that every antibody has the same configuration in the center of its polypeptide chain, and that the configuration of the ends of the chain are dependent on which antigen is present at the time of the antibody’s synthesis.  In present day, even with our better understanding of antibody synthesis, the core principles of Pauling’s theory – most prominently the idea that each antibody shares a common structure – remain sound.

The entirety of Pauling’s manuscript is available here.  In it, he discusses other topics related to his theory, including the formation of different structures based on antibody to antigen ratios, the characteristics that define a molecule or substance as an antigen, and the compatibility of his theory with experimental results. For more information on Pauling’s immunological work, visit It’s in the Blood: A Documentary History of Linus Pauling, Hemoglobin, and Sickle Cell Anemia.

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.

Invisible Inks

Test screed developed as part of a research program on invisible inks. November 14, 1945.

By 1944 the oxygen meter and propellant projects were running smoothly with only minimal oversight from Pauling.  With more free time available to him, he began looking into new lines of research.  That year, he was contacted by Arthur Lamb, a Harvard professor, regarding a new line of inquiry.  During World War I, Lamb had developed invisible inks for the U.S. government.  He was restarting his work with inks and wanted Pauling’s help.  And so it is that, in September 1944, Linus Pauling became an official investigator in the Office of Scientific Research and Development’s invisible inks project.

The goal for Pauling and his team was to create a series of inks that were truly invisible and could only be developed by a limited number of chemicals. From September to October 1944, Dr. George Wright, William Eberhardt, and Frank Lanni made preliminary examinations of potential methods for developing invisible inks, the specifications of which were not defined in Pauling’s official reports to the OSRD. Once the preliminary tests were complete, Pauling and his team began a wide range of experiments, testing a variety of potential approaches for creating secret inks.

The team began with possible protein-based inks. They applied various proteins including rabbit serum, human saliva, and homogenized milk to standard typing paper. Then, after steaming and ironing the treated page, the team painted it with a mixture of ink, acetic acid and sodium chloride. The combination of acid and ink caused the protein to darken slightly, rendering it legible in well-lit conditions.

The group also tested non-organic inks such as diluted potassium iodide. After drying, the test screed was painted with gold chloride, rinsed, and then treated with a substance referred to only as “the silver physical reagent,” a compound protected by the Office of Censorship.

Page of test screeds developed as part of a research program on invisible inks. 1945.

Pauling and his team needed to find a better way to protect invisible inks from being identified when intercepted by enemy forces. To this end, the team turned its focus toward substances with high immunological specificity; that is, organic substances that reacted with only a limited number of other compounds. The team began with a polysaccharide gum distilled from a bacterium responsible for lobar pneumonia in humans. (Because the gum was largely non-reactive with other chemicals, the paper it was printed on hid it well.) The ink was then masked with an additional coating of a wax-like substance to prevent all but the most immunologically-specific chemicals from developing it. While tedious, the process was effective.

In addition to the use of polysaccharide gum, Pauling and his group examined antibodies and antigens in the hope that they could be used to create inks. In a report to the OSRD, Pauling explained that when a foreign protein (antigen) is introduced to an animal’s bloodstream, the animal produces a highly specific complementary protein (antibody) to neutralize it. When the two proteins combine, they form a stable protein-protein pair.

Initial tests of the solution suggested that the antibody-antigen combination could be highly effective. Unfortunately, as the researchers began practical testing they found it extremely difficult to develop the protein-protein pair without staining or otherwise rendering illegible the paper on which the ink was printed. What’s more, some of the antigens could be developed with non-organic chemicals, greatly reducing their security. Ultimately, the antibody-antigen ink was impractical. Pauling suggested that a few changes might be made to the process, but no record of additional experimentation appears in the Pauling Papers.

Despite having achieved some measure of success with a variety of inks, Pauling suggested that the project might be pushed even further. As he explained in a report,

From the offensive standpoint, it might be considered that the development by the new techniques of substances which are not detectable by the present methods might be useful as a basis for offensive methods.

While Pauling left no traces suggestive of his engaging in this process, it is at least plausible that he and his team did in fact note and retain a number of potential developers for future scientists to test.

In all Pauling and his team created or enhanced around a dozen different ink-developer combinations, ranging from improvements on existing camphor-based Presto pencils to complex processes using albumin, gypsum, and the catalytic reduction of silver. The project was closed with the publication of the “Final Report on Biological SW” dated December 31, 1945.

The Medical Research of Linus Pauling

By Tom Hager

[Ed Note:  In October 2010, Pauling biographer Tom Hager delivered a talk sponsored by the Oregon Health Sciences University which detailed and discussed the various contributions that Linus Pauling made to the medical sciences, including the controversy over his strong interest in orthomolecular medicine.   With the author's permission, excerpts of this talk are being presented on the Pauling Blog over the next three posts.  The full text of Hager's OHSU lecture is available here.  Those with an interest in learning more about Hager's work, including his latest research on food issues and world hunger, are encouraged to visit his blog at http://thomashager.net.]

[Part 1 of 3]

Oil portrait of Linus Pauling, featuring a model of the alpha-helix in the foreground. 1951. Portrait by Leon Tadrick.

By 1939, at the age of 38, Linus Pauling was a full professor and head of the chemistry division at Caltech, as well as the father of four children (three sons, Linus, Jr., Peter, and Crellin; and a daughter, Linda).

He was also beginning to turn his considerable talents toward understanding the complicated molecules inside the human body. He started with proteins.

The Molecules of Life

Determining the structure of proteins at this time was a gigantic problem. Most were difficult to purify, easily degraded, and hard to characterize. Proteins appeared to be not only gigantic, comprising hundreds or thousands of atoms – much too large to solve directly with x-ray crystallography – but also relatively fragile, losing their function (denaturing) after even slight heating or mechanical disturbance. No one at the time was even sure that they were distinct molecules – one popular theory held that proteins formed amorphous colloids, gels that did not lend themselves to molecular study.

Studying them at the molecular level seemed an impossible task with the tools available in the late 1930s. But Pauling took on the challenge. He started with the building blocks of proteins, the amino acids, and directed his growing lab team toward pinning down their precise structures. Then he set himself to figuring out how they formed protein molecules, often building models out of wood, wire, and paper.

He based his approach in part on the ideas of the German biochemist Emil Fischer. Like Fischer, Pauling came to believe that proteins were long molecular chains of amino acids linked end-to-end. Working with Alfred Mirsky in the mid-1930s, Pauling discovered that the denaturing of proteins resulted from breaking weak bonds, called hydrogen bonds, that pinned these chains into specific shapes. Between the early 1930s and early 1950s he made a string of important discoveries about hemoglobin, antibodies (including the most sophisticated work at the time into the structural relationship between antibody and antigen), enzymes, and other proteins.

Foldable paper model of the alpha-helix protein structure published in the Japanese journal Chemical Field, 1954.

In May 1951, he put everything he knew into a celebrated series of seven papers detailing the structures of a number of proteins at the level of individual atoms, including the structure of the single most important basic form of protein, the alpha helix (a hydrogen-bonded helical chain that is a structural component of almost every protein). It was an astounding breakthrough, and it opened the door for an understanding of biology at the molecular level. Within two years, Watson and Crick had used his approach to decipher the structure of DNA.

Biological Specificity

But structure was not everything. Pauling realized that life resulted not from individual molecules, but from the interactions between them. How did organisms make offspring that carried their specific characteristics? How did enzymes recognize and bind precisely to specific substrate molecules? How did antibodies recognize and bind to specific antigens? How did proteins, these flexible, delicate, complex molecules, have the exquisite ability to recognize and interact with target molecules?

It all fell under the heading of biological specificity at the molecular level. Pauling directed much of his attention here during through the 1940s, performing a great deal of careful work on the binding of antigens to antibodies.

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

His findings were surprising. Pauling demonstrated that the precise binding of antigen to antibody was accomplished not by typical chemical means – that is, through covalent or ionic bonds — but solely through shape. Antibodies recognized and bound to antigens because one fit the other, as a glove fits a hand. Their shapes were complementary. When the fit was tight, the surfaces of antibody and antigen came into very close contact, making possible the formation of many weak links that operated at close quarters and were considered relatively unimportant in traditional chemistry — van der Waals’ forces, hydrogen bonds, and so forth. To work, the fit had to be incredibly precise. Even a single atom out of place could significantly affect the binding.

Having demonstrated the importance of complementary structure with antibodies, Pauling extended his idea to other biological systems, including the interaction of enzymes with substrates, odors with olfactory receptors, and to the possibility of complementary structure in genes.

Pauling’s idea that biological specificity was due in great part to complementary “fitting” of large molecules to one another proved to be essential in the development of molecular biology. His research now formed a coherent arc, from his early work on the chemical bond as a determinant of molecular structure, through the structures of large molecules (first inorganic substances, then biomolecules), to the interactions between large biomolecules.

He carried out much of this research during World War II, when he also worked on synthetic plasma substitutes and a fruitless search for ways to produce artificial antibodies.

He had already earned a place among the nation’s leading researchers in the medical applications of chemistry. But his greatest triumph was still to come.

Sickle-Cell Anemia

Toward the end of World War II, Pauling’s reputation was great enough to earn him an invitation to join a national committee that was brainstorming the best structures for postwar medical research. This committee’s work led to the foundation of the National Institutes of Health.

Pauling was the only non-physician asked to join the committee.

At a dinner with other members one night, talk turned to a rare blood disorder called sickle-cell anemia. One of his dinner companions described how red blood cells in the victims were twisted into sickle shapes instead of discs. The distortion appeared to hinder the blood cells’ transport through capillaries, resulting in joint pain, blood clots, and death. The disease primarily affected Africans and African Americans. What caught Pauling’s attention most, however, was one odd fact: Sickled cells appeared most often in venous blood, rather than in the more oxygenated blood found in the arteries.

Pastel drawing of sickled Hemoglobin cells, 1964. Drawing by Roger Hayward.

He thought about this during the next few days. From his previous work with blood, he knew that red cells were little more than bags stuffed with hemoglobin. He had also shown that hemoglobin changed its shape slightly when it was oxygenated. If the red cells were changing shape, perhaps it was because the hemoglobin was altered in some way. What if the hemoglobin molecules in sickle-cell patients were changed in some way that made them clump, stick to one another, as antigens stick to antibodies? Perhaps something had changed that made the hemoglobin molecules complementary in shape. Perhaps adding oxygen reduced the stickiness by changing the molecules’ shape.

He presented his ideas as a research problem to Harvey Itano, a young physician who was then working on his Ph.D. in Pauling’s laboratory. Itano, later joined by postdoctoral fellow John Singer, worked for a year trying to see if sickle-cell hemoglobin was shaped differently from normal hemoglobin. They found no detectable differences in any of the tests they devised. But they kept at it. Finally, in 1949, using an exquisitely sensitive new technique called electrophoresis that separated molecules by their electric charge, they found their answer: Sickle-cell hemoglobin carried more positive charges on its surface.

This was an astounding discovery. A slight change in the electrical charge of a single type of molecule in the body could spell the difference between life and death. Never before had the cause of a disease been traced to a molecule. This discovery – to which Pauling attached the memorable title “molecular disease” – received widespread attention. Itano and Singer’s followup work demonstrated the pattern of inheritance for the disease, firmly wedding molecular medicine to genetics.

Medical Chemistry

It was a great triumph – there was talk of a Nobel Prize in Medicine or Physiology for Pauling – and it led Pauling to make greater efforts in the medical field. He encouraged M.D./Ph.D. candidates, hired physicians to work in his laboratory, and began focusing his own research on medical problems, including developing a new theory of anesthesia.

He was ahead of his time. An example of what the atmosphere was like: Pauling noted that as he went around in the late 1940s seeking funds for a comprehensive marriage of biology and chemistry to attack medical problems, people at funding agencies were telling him that they found the term “medical chemistry” to be “a disturbing description.”

"Mental Deficiency & Brain Chemistry." May 1, 1964.

In the late 1950s, Pauling extended his concept of molecular disease to the brain. After reading about phenylketonuria (PKU) – a condition in which a mental defect can be caused by the body’s inability to metabolize an amino acid, phenylalanine, leading to a buildup of that substance and others in the blood and urine – Pauling theorized that the problem might be caused by a defect in an enzyme needed to break down phenylalanine. PKU, in other words, might be another molecular disease. Now interested in the possibility that there might exist a range of molecular mental defects, Pauling visited a local mental hospital, saw other patients whose diseases seemed hereditary, and decided to seek support for an investigation into the molecular basis of mental disease. The Ford Foundation in 1956 awarded him $450,000 for five years’ work – a vindication of Pauling’s approach and a tribute to his reputation. The grant, however, yielded little in the way of immediate results, with much of the funding going toward testing his (ultimately found to be mistaken) theory of anesthesia.

The long-term results were more significant. Pauling’s immersion in the field, thanks to the Ford grant, led him to read widely in psychiatry and general health, always on the lookout for another molecular disease that might lend itself to new therapy. By the mid-1960s he was coalescing his findings into another overarching theory, this one combining much of what he knew about chemistry and health. He called his new idea “orthomolecular” medicine.