The Tantalizing Prospect of Artificial Antibodies

Linus Pauling and Dan Campbell, 1943.

[Part 2 of 3]

By late 1939, Linus Pauling had thrown himself wholeheartedly into the study of antibodies, specifically how they work and how they are made. He’d already developed a few memorable and unique hypotheses, though by 1940 they were still yet to be proven correct or otherwise.

In January 1940, a researcher named Dan Campbell arrived at Caltech with the intent of working on problems in immunochemistry. He and Pauling began collaborating, and ended up co-authoring several papers together. Later in 1940, after a first, erroneous paper, Pauling published another in which he claimed:

all antibody molecules contain the same polypeptide chains as normal globulin, and differ from normal globulin only in the configuration of the chain; that is, in the way that the chain is coiled in the molecule.

In other words, shape was what determined the effect of antibodies. Pauling acknowledged the potential for flaws lying within his hypothesis, but adopted it because of his inability to otherwise “formulate a reasonable mechanism whereby the order of amino-acids residues would be determined by the antigen.”

Nobody knew about the genetic basis of amino acids at the time. As a result, while Pauling’s hypothesis on how antibodies worked eventually turned out to be correct, his coupled hypothesis on how they were formed was still wrong. Regardless, Pauling’s theory “ruled the roost amongst immunochemists” for almost 20 years. It wasn’t even until 1949 that people began to seriously question Pauling’s model, and when that finally happened, it was because the model failed to account for the development of immunity to antibodies.

In July a colleague at Caltech, Max Delbrück – a German researcher who had arrived in the US on a study abroad trip and never went home – showed Pauling a paper written by Pascual Jordan, another German scientist. Jordan claimed that identical molecules were attracted to each other, and opposite molecules repelled from one another, because of quantum mechanical resonance. Pauling declared the idea “baloney,” and decided to write a paper debunking Jordan’s theory. He asked Delbrück to co-author it, which he did with hesitation, as the young scientist was nervous about signing his name to a paper attacking the theories of the famous and well-respected Jordan. The duo finished the paper and published it in Science, but unfortunately for them, the work went largely unnoticed.

Since the start of the Battle of Britain in 1940, Pauling had been doing some side research on explosives, propellants, and other “war work” in anticipation of United States involvement in the war raging across Europe and the Asia. In 1941, U.S. engagement finally came about with the attack on Pearl Harbor. Pauling and his laboratory at Caltech switched their main priority to war work, as did just about every other research laboratory in the country. As part of this realignment of efforts, Pauling received a grant to begin a research project with the goal of creating gelatin-based blood plasma artificial antibodies, to be used for military medical purposes.

Pauling hypothesized that if a generic protein, such as beef globulin, was very carefully denatured, then placed in the presence of an antigen and revitalized, it would grow and mold itself to the antigen. If this worked, medical technicians would someday be able to create antibodies “made to order.” The potential import of the idea was clear: “the end result of a million years of evolution” would become available “by the quart.” Successful implementation would represent a massive breakthrough in medical technology, and Pauling was convinced that he was just the man to do it. He picked Dan Campbell and David Pressman to be his primary assistants and the group got to work.

The process was slow and complicated; it involved a mixture of 1-2% beef globulin, dissolved in 0.9% sodium chloride, then very slowly heated to 57˚F. Once at that temperature, alkali would be carefully added to bring the mixture to a pH of 11, before very delicately and slowly returning the mixture to pH 0. To further complicate issues, the process produced a large amount of nitrogen, which had to be removed, but the process of removing the nitrogen created carbon monoxide and other “undesirable substances.” The Pauling group tested their antibodies on rabbits, mice, and guinea pigs predominantly, as their labs lacked the space for more extensive animal research.

In March 1942, instead of submitting his ideas to a peer-reviewed scholarly journal, Pauling issued a press release to Science News, claiming that his lab had developed artificial antibodies.

Text of Pauling's artificial antibodies press release, March 1942.

Text of Pauling’s artificial antibodies press release, March 1942.

The scientific community was vexed by this highly unusual action. Numerous scientists questioned the strength of the data, saying that the reports released by Pauling and Campbell were vague and weak. Pauling asserted that while the data they had gathered was not definitive, it was strong enough to make the statement that artificial antibodies had been produced. Pauling said that his artificial antibodies, though weak, were still real, and that he was going to continue improving on them. Specifically, he claimed that the antibodies had been used to prevent mice from getting pneumonia.

Pauling’s reputation helped to coax certain colleagues in the direction of his arguments. And after some skillful debating, the Rockefeller Foundation offered $31,000 to continue the research, while the federal Office of Science Research and Development offered $20,000.

Despite the skepticism of his peers, Pauling was enthusiastic and eager to continue research on this line of work. He had the funding and the desire, and he was convinced it was only a matter of time before he could start mass-producing artificial antibodies. However, he was about to run into some issues outside of his laboratories.


Salvador Luria, 1912-1991

Salvador Luria, ca. 1970s. Image courtesy of the Massachusetts Institute of Technology Museum.

The microbial geneticist Salvador Edward Luria would have celebrated his centenary birthday this month, and it is to him that we turn our attentions today.

Luria was born in Turin, Italy, on August 13, 1912, the second son of David and Esther Luria. As a boy, his attitude toward school was lukewarm – he received his best grades in math and literature courses, but never fully developing a passion for learning. He eventually decided to study medicine due to the influence of his parents, graduating from the University of Turin Medical School in 1935. It was during medical school, however, that he became interested in the ways in which modern physics could be used to solve problems in biology and genetics.

Upon graduating, Luria decided to combine his love for biology and physics by studying radiology. He finished physics and radiology courses at the University of Rome, where he learned about recent theories regarding genes as a molecule and also about bacteriophages, which are viruses that infect bacteria. In 1938 Luria moved to Paris, where he became a Research Fellow at the Institute of Radium. In order to escape Nazi persecution, Luria immigrated to New York in 1940 (later becoming a U.S. citizen in 1947), where he received a Rockefeller Foundation fellowship and worked as a Research Assistant in Surgical Bacteriology at the College of Physicians and Surgeons at Columbia University.

Max Delbrück, 1949. Image courtesy of the Archives, California Institute of Technology.

During the annual meeting of the American Physical Society in December 1940, Luria met Max Delbrück, who introduced him to the bacteriophage experiments that they would work on together later that summer, including an investigation into the mechanisms by which phage multiply within bacteria. The duo discovered that different phage strains interfere with each other when attacking bacteria.

In 1943 Luria moved yet again to Indiana University where he thrived, forming close friendships and training numerous graduate students.  It was at Indiana that conducted one of his most important studies, showing that bacteria mutated spontaneously into phage-resistant forms. Later, again in collaboration with Delbrück, Luria developed the “fluctuation test” for calculating bacterial mutation rates. This work provided statistical evidence for the existence of genes in bacteria, which established microbes as suitable subjects for genetics research. The work was also a source of great recognition for Luria within the biological community.

Throughout his career, Luria was not only a scientist, but also an outspoken policy advocate, and as a result he quickly gained the attention of Linus Pauling. On May 15, 1957, Pauling wrote to Luria for the first time, asking if he would be willing to sign his name on the “Appeal by American Scientists” – the beginnings of what would become Pauling’s famous United Nations Bomb Test Petition. Pauling sent a cover letter to Luria along with a copy of the appeal, which urged scientists to support an international agreement to stop the testing of nuclear bombs in the atmosphere. Pauling wrote in the document,

Each added amount of radiation causes damage to the health of human beings all over the world and causes damage to the pool of human germ plasm such as to lead to an increase in the number of seriously defective children that will be born in future generations.

Luria supported Pauling in this effort, and so began a series of exchanges over many years in which the two scientists requested one another’s support for various political, social and environmental causes.

One such letter was penned on March 25, 1965, when Luria wrote to Pauling regarding a Vietnam War protest that a group in Boston had recently sponsored. In it, he noted his exasperation with the contemporary political climate – one which reminded him all too much of his early life in Europe.

We now feel that the time for polite questioning is past and that something more drastic and dramatic is needed. Also, most of us feel the need to bear witness publicly of our personal refusal to acquiesce in a policy that is immoral and criminal.  The situation in my mind has the same nightmare quality I felt in Germany in the ‘thirties and in France during the Algerian war. In both situations not enough intellectuals were able or willing to stand up and be counted.

Luria thought that the public’s conscience could possibly be stirred if a group of National Academy of Science members resigned from the organization while issuing a public announcement stating that the action was being taken in protest of the war in southeast Asia. He asked Pauling if this would be wise and Pauling responded by stating that he would agree to resign from the NAS if nine other members also agreed.

Pauling to Luria, March 29, 1965. pg. 1.

pg. 2.

Over time, Luria continued down his career path, first by moving to the University of Illinois and then to the Massachusetts Institute of Technology, where he served as an adviser for both the reorganization of its biology department and for the development of its microbiology department. His collaborations with Delbrück likewise continued over the course of what would become a long and successful career.

In 1969 the duo received, alongside Alfred Hershey, the 1969 Nobel Prize in Physiology or Medicine for “discoveries concerning virus replication and genetics and the importance of contributions to the biological and medical sciences.” True to his convictions, Luria donated part of his prize money to the peace movement, helping to organize the Vietnam War Moratorium in Boston. Meanwhile, the three scientists, Luria, Delbrück and Hershey, became known as the “Phage Group,” an informal assembly who worked with seven strains of bacteriophage, comparing their findings and results.

Luria continued to be engaged in the humanities as an activist well into the 1970s, during which time he participated in debates over genetic engineering, among other issues. In 1972, he also set up and directed a new center for cancer research at MIT, expanding the molecular and cellular biology programs there. All the while he wrote prolifically, a body of work which included a popular science book, Life: The Unfinished Experiment. The book was a huge success and he continued to publish essays and opinion articles on scientific and political issues until his death from a heart attack in Lexington, Massachusetts, on February 6, 1991.

The Triple Helix

We have seen Paulings paper on Nucleic Acid. Have you? It contains several very bad mistakes. In addition, we suspect he has chosen the wrong type of model.

-James Watson, letter to Max Delbrück, February 20, 1953.

We were very interested to see that a model of the Pauling-Corey “triple helix” structure of DNA has been built by Farooq Hussain.  As Hussain notes on his website, the model was constructed based on drawings published by Linus Pauling and Robert Corey in their paper detailing the incorrect structure.

The proposed triple helix structure of DNA. Model by Farooq Hussain.

Hussain’s representation of the triple helix is striking; especially so when compared with Watson and Crick’s far more elegant and intuitive double helix, surely the most famous molecule in history.

Double helix model, courtesy of P. Shing Ho.

Indeed, Watson was well within his right to feel confident in his February letter to Delbrück.  As he noted before closing, “Today I am very optimistic since I believe I have a very pretty model, which is so pretty I am surprised no one has thought of it before.”

For more on the triple helix, see our write-up on the subject, published in April 2009.

The Watson and Crick Structure of DNA

Francis Crick and James Watson, walking along the the Backs, Cambridge, England. 1953.

Today, our series on models of DNA is concluded with a discussion of the correct structure determined by James Watson and Francis Crick. Although they made an unlikely pair, the two men succeeded where one of the era’s leading scientists – Linus Pauling – failed, and in the process they unraveled the secrets of what may be the most important molecule in human history.

In the fall of 1951, James Watson was studying microbial metabolism and nucleic acid biochemistry as a postdoctoral fellow in Europe. It didn’t take long for him to tire of these subjects and to begin looking for more inspiring research. He became interested in DNA upon seeing some x-ray photos developed by Maurice Wilkins. He then tried to talk his way into Wilkins’ lab at King’s College, but was denied and ended up studying protein x-ray diffraction in the Cavendish Laboratory at Cambridge University. Here he was assigned space in an office to be shared with an older graduate student named Francis Crick, a crystallographer. At the time, Crick was studying under Max Perutz, and was also becoming bored with his research. Watson and Crick hit it off immediately and before long, Watson’s interest in DNA had worn off on Crick. Although neither of them were experts in structural chemistry, they decided to attempt to solve the structure of DNA. As Watson put it, their planned method of attack would be to “imitate Linus Pauling and beat him at his own game.”

The pair’s first attempt at the structure in the fall of 1951 was very quick, and also unsuccessful. Interestingly, however, it was quite similar to Linus Pauling and Robert Corey‘s own attempt about a year later. Watson and Crick came up with a three stranded helix, with the base rings located on the outside of the molecule and the phosphate groups found on the inside. This left them with the problem of fitting so many negatively charged phosphates into the core without the molecule blowing itself apart. In order to solve this problem, they turned to Pauling’s own The Nature of the Chemical Bond. They were looking for positive ions that would fit into the core of DNA, therefore canceling the negative charge. They found magnesium and calcium to be possibilities, but there was no significant evidence that these ions were in DNA. However, there was no evidence against it either, so they ran with the idea.

Watson and Crick assumed – as would Pauling in his later attempt – that the finer details would fall into place. Overjoyed at solving DNA so quickly, they invited Wilkins and his assistant, Rosalind Franklin, to have a look at their structure. Expecting praise, they were undoubtedly surprised when Franklin verbally destroyed their work. She told them that any positive ions found in the core would be surrounded by water, which would render them neutral and unable to cancel out the negative phosphate charges. She also noted that DNA soaks up a large amount of water, which indicates that the phosphate groups are on the outside of the molecule. All in all, Franklin had no positive feedback for Watson and Crick.  And she was, at it turned out, correct. After the visit, Watson and Crick attempted to persuade Wilkins and Franklin to collaborate with them on another attempt at the structure of DNA, but their offer was declined.

Diagram of the double-helix structure of DNA. August 1968.

When Sir William Lawrence Bragg, the head of the Cavendish laboratory, heard about Watson and Crick’s failure, he quickly sent them back to other projects. Almost a year passed with Watson and Crick accomplishing no significant work on DNA. Although they weren’t building models, DNA was still at the front of their minds and they were gathering information at every opportunity. In the fall of 1952, Peter Pauling, the second eldest of Linus and Ava Helen Pauling’s four children, arrived at Cambridge to work as a graduate student. Jerry Donohue, another colleague of Pauling’s from Caltech, also arrived at the same time and was assigned to share an office with Watson and Crick. As a result, Peter also fell in with the group. Therefore, as the quest for DNA progressed, Linus Pauling was provided with a general idea of Watson and Crick’s work with DNA through contact with Peter. However, the opposite also proved true.

When Pauling and Corey submitted their manuscript on the structure of DNA in the last few days of 1952, Peter passed on to Watson and Crick the news that his father had solved DNA. Although the two men were crestfallen by this information, they decided to soldier on with their own program of research, figuring that if they published something at the same time Pauling that did, they might at least be able to share some of the credit.

Around this time, the pair added an important piece of information that they had learned from Erwin Chargaff, a biochemist. He had told them that the four different base rings in DNA appeared to be found in pairs. That is, one base ring is found in the same relative amounts as another. This first correlation constitutes one pair, and the remaining two bases make up the other pair. Interestingly enough, Chargaff had also told Pauling this same thing in 1947. However, Pauling had found him to be annoying and, as a result, disregarded his tip. Chargaff’s information did, however, prove to be crucial for Watson and Crick, who were slowly piecing together the basics of the DNA structure.

When Watson and Crick finally received Pauling’s manuscript via Peter in early-February 1953, they were surprised – not to mention elated – to see a structure very similar to their own first attempt. Bragg, a long time competitor of Pauling’s, was so pleased to see Pauling’s unsatisfactory work that he allowed Watson and Crick to return to DNA full time. The pair wasted no time, and had soon spread the news about Pauling’s model to all of Cambridge. Watson even told Wilkins about the manuscript, and was rewarded with the permission to view Franklin’s most recent DNA x-ray patterns. These beautifully-clear photos immediately confirmed Watson’s suspicion that DNA was a helix, adding yet another piece of important information.

Based on all of the information that they had gathered, Watson and Crick began rapidly building models. One model, which Watson called “a very pretty model,” contained the wrong structures for two base rings. Fortunately, Donohue, who was an excellent structural chemist, set them right. After his correction, Watson and Crick noticed that hydrogen bonds would form naturally between the base pairs. This explained Chargaff’s findings, and also showed the potential for replication of the molecule. The rest of the model came together quickly, and Watson and Crick began to write up their structure.

Eventually, Linus Pauling began to catch wind of the recent work that Watson and Crick had been doing with DNA. His first actual glimpse of their work came in March 1953 when Watson sent a letter to Max Delbrück, a colleague of Pauling’s, that included a brief description and rough sketches of the structure. Although Watson had asked Delbrück not to show the letter to Pauling, Delbrück could not resist. Pauling marveled at the simplicity and functionality of the structure, but still retained confidence in his own structure. Only a few days later, Pauling received an advance copy of the Watson and Crick manuscript, but he was still not convinced they had solved DNA. In April, Pauling finally traveled to England, and only after seeing the model in person and comparing it to Franklin’s DNA photographs was he certain that Watson and Crick had solved the structure of DNA.

On April 25, 1953, Watson and Crick’s article, “A Structure for DNA” was published in Nature. James Watson, Francis Crick, and Maurice Wilkins would go on to share the Nobel Prize in Physiology or Medicine for 1962 “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material.” Unfortunately, Rosalind Franklin died of cancer at age 37 and, for many years, was given only minor credit for her considerable contributions related to the discovery of the DNA structure.

For more information on Watson and Crick and DNA, please visit the website Linus Pauling and the Race for DNA: A Documentary History. For more information on Linus Pauling and his research, visit the Linus Pauling Online Portal.