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.

A Theory of the Denaturation of Proteins

Alfred E. Mirsky, 1960s

In 1935, as a result of being prompted toward the biological sciences in order to keep his Rockefeller Foundation funding, Linus Pauling began his research on proteins. Hemoglobin, the oxygen-binding agent in blood, was his first target; but as he became more aware of the complex nature and diversity of proteins, he began contemplating broader topics related to the subject – one of which was the theory of protein denaturation.

In the spring of 1935, Pauling traveled to the Rockefeller Institute in New York City, where he met Dr. Alfred Mirsky. Mirsky was a Rockefeller scientist who had previously conducted denaturation research, and because of his new interest in the subject, Pauling arranged for Mirsky to spend fifteen months working with him at Caltech. Although initially hesitant, Mirsky eventually agreed, and the pair began collaborating in the summer of 1935.

In July 1936, the duo’s paper, titled “On the Structure of Native, Denatured, and Coagulated Proteins” was published in the Proceedings of the National Academy of Sciences.  In this paper, the authors loosely describe protein denaturation as “the loss of certain highly specific properties by the native protein,” and provide examples of the types of changes that have been experimentally observed.

In so doing, Pauling and Mirsky point out that while many proteins in their native form have been crystallized, no denatured protein exist in this state. Likewise, in proteins that act as enzymes, denaturation causes a disappearance of the enzymatic activity.  And one fact that was of particular interest to Pauling was that the process of denaturation is occasionally reversible.

Early Pauling notes on the characteristics of protein denaturation, ca. 1935

As researchers are now aware, any given protein has a certain structure – or rather, four different structural levels – that needs to be maintained in order for the molecule to function correctly. Although this crucial bit of information was still unknown at the time of Pauling and Mirsky’s research, the authors essentially touch on this exact detail in their 1936 paper:

Our conception of a native protein molecule (showing specific properties) is the following. The molecule consists of one polypeptide chain [the amino acid sequence] which continues without interruption throughout the molecule (or in certain cases, of two or more such chains); this chain is folded into a uniquely defined configuration, in which it is held by hydrogen bonds…

The collaborators further posited that, as a result of this “structure equals function” characteristic of proteins, denaturation is “characterized by the absence of a uniquely defined configuration” and can be accomplished in a number of different ways, including heating, subjection to ultraviolet light, or an attack by certain reagents.

In presenting their theory of denaturation, Pauling and Mirsky associated both the heating of the protein and its treatment with certain reagents, as leading to the disruption or complete rupturing of hydrogen bonds.  From there they pointed out that ultraviolet light is not able to break a sufficient quantity of hydrogen bonds, and therefore must affect the molecule differently – an impact which they predicted to be an attack on the main polypeptide chain. Consequently, they suggested that denaturation caused by ultraviolet light was irreversible, while methods that disrupt the more easily re-formed hydrogen bonds would be reversible.

Although Pauling and Mirsky weren’t correct in every aspect of their denaturation theory (ultraviolet light does not disturb the polypeptide chain, and denaturation involves more than just the disruption of hydrogen bonds), it provided a strong start for further work.  The Pauling-Mirsky theory also touched on many details of the structure of proteins in their native forms, a field of inquiry that would not be completely elucidated for many years to come.

For more information on Linus Pauling, please visit the Linus Pauling Online portal. For more information on Alfred Mirsky, visit his key participants page within the It’s in the Blood! A Documentary History of Linus Pauling, Hemoglobin, and Sickle Cell Anemia site.

Pauling’s Battle with Glomerulonephritis

Pauling family portrait taken in 1941. Back of photograph is annotated, “1941. Daddy very ill.”

[Part 1 of 5]

On March 7, 1941, Linus Pauling stood before distinguished colleagues prepared to deliver an address in response to his receipt of the prestigious William H. Nichols Gold Medal, presented by the New York chapter of the American Chemical Society.

Before Pauling began his recitation, he spoke candidly to his audience. He thanked the award committee for his selection and expressed gratitude that the acceptance of this award had provided him with an opportunity to reconnect with old friends.

On this rare occasion, however, it was apparent to all in attendance that Pauling’s physical health was suffering. His face was bloated and he reportedly lacked the enthusiasm that he was so well known to exude. Addressing the observations of many of his peers, Pauling joked, “Several of [my old friends] said to me tonight that I appeared to be getting fat. This is not so.”

Just that morning, Pauling had awoken to find his face so bloated that his eyes were nearly swollen shut. His tongue felt enlarged and his voice was flat. Over the previous few weeks, Pauling had been experiencing noticeable swelling, weight gain, and chronic fatigue but he could not identify the cause of his ailments.

With his audience, Pauling half-heartedly pondered over the cause of his puffed-up appearance. He compared the experience to childhood memories of unfortunate encounters with poison oak.

Yesterday I must have bumped into something similar…while I was wondering what the responsible protein could have been, I decided that it was a visitation – that I was being punished for thinking wicked thoughts.

The following evening Linus and Ava Helen had dinner at Alfred Mirsky‘s residence. Pauling was examined by another guest at the dinner party, Dr. Alfred E. Cohen, a cardio specialist from the Rockefeller Medical Institute. After ruling out problems with Pauling’s heart, Dr. Cohen remained perplexed by Pauling’s condition. Nothing appeared to be wrong with the forty-year-old man other than his extreme edema. Concerned by the severity of the swelling however, Dr. Cohen recommended that Pauling come into his office the following day for a more thorough examination and lab work-up.

Adhering to the physician’s recommendation, the Paulings met Dr. Cohen in his office at the Rockefeller Medical Institute the next day. After a battery of lab tests, Pauling was diagnosed with Bright’s disease – a potentially fatal renal disease that results in the degradation of the kidneys. At the time, little was known about Bright’s disease and the majority of the medical community considered it to be a terminal condition.

After receiving this diagnosis, Pauling was fortunately referred to a leading specialist in renal diseases, Dr. Thomas Addis, head of the Clinic for Renal Disease at Stanford. Dr. Addis was a pioneer in the field of nephrology and his treatment plan, at the time, was new and revolutionary. Had Pauling not been referred to Dr. Addis’ care, the treatment he would have received elsewhere would almost surely have killed him.

Under the guidance of Dr. Addis, Pauling’s condition was effectively treated by alternative means – a low-protein, low-sodium diet – rather than the polysaccharide infusions that would have reduced his edema but done little to improve his health.  By May, Pauling reported improvements in his overall well-being and by August, the edema had completely disappeared.

Since Pauling’s time of diagnosis, Bright’s disease has been reclassified and redefined. Now it is believed that Pauling was affected by what is currently termed acute glomerulonephritis.

Acute glomerulonephritis is characterized by inflammation of the kidneys due to an immunological response. Damage to the small clusters of capillaries within the kidney, known as glomeruli, results in what can most simply be described as a “leaky kidney.” When the glomeruli are damaged, proteins leak from the bloodstream into the urine through the damaged portions of the kidney. Thus glomerulonephritis consequentially leads to excessive protein loss. Glomerulonephritis profoundly effects the body’s ability to function, because the nephritic kidneys are unable to properly filter the blood.

In his 1941 speech, Pauling had wondered aloud about a protein that was responsible for his swollen condition. The culprit protein can now perhaps be identified as albumin. As proteins leak from the bloodstream into the urine, blood proteins, called albumin, exit the bloodstream. These proteins are known to be essential in the regulation of blood osmotic pressure. Without sufficient albumin in the bloodstream, the body becomes incapable of efficiently extracting excess fluid from the body cavity. This excess fluid then remains trapped in the body and ultimately results in excessive swelling – such as the bloating that Pauling experienced in 1941.

Although the albumin did not cause Pauling’s condition, the loss of this blood protein due to the nephritis appears to have resulted in the symptoms that he was experiencing at his award ceremony. Therefore, contrary to his original speculation, it was the absence, rather than the presence, of a protein that caused his extreme fluid retention.

Over the next series of posts, we’ll explore the details of Pauling’s battle with this frightening disease, and learn more about the people and methods who saved Linus Pauling’s life.