David Pressman

pressman-1937

David Pressman, 1937

[Part 6 of 6 in our series exploring Linus Pauling’s work on the serological properties of simple substances, and the colleagues who assisted him in this work.]

After a meeting with Karl Landsteiner in 1936, Linus Pauling began serious investigations into the link between antibodies and antigens, compiling notes for what would eventually become his serological series, a collection of fifteen papers published during the 1940s. Landsteiner had specifically piqued Pauling’s curiosity on the question of the human body’s specificity mechanism – e.g., how could the body produce antibodies tailored to lock onto and fight specific antigens?

Pauling ultimately surmised that the answer lie in the shape of the molecules, and in the type and number of bonding sites. He described this as a “lock and key” mechanism, otherwise termed as molecular complementarity. Throughout this project, which made a significant impact on the modern study of immunology, Pauling enlisted the help of many undergraduate, graduate, and doctoral students, including a promising young scholar named David Pressman.


David Pressman was born in Detroit, Michigan in 1916. He attended Caltech as an undergraduate, studying under Pauling and completing his degree in 1937. He stayed in Pasadena for his doctorate, earning it in 1940. During this time, he became a part of Pauling’s quest to unravel the structure of proteins, and was particularly involved with the antibody and antigen work.

By this point, Pauling and his colleague Dan Campbell felt confident enough in what they had learned about antibody specificity to attempt creating artificial antibodies. Pauling was enthusiastic about the practical application that such an endeavor might promise for physicians. Warren Weaver, Pauling’s primary contact at the Rockefeller Foundation, which was funding the work, cautioned Pauling against becoming overconfident, but still granted him enough money to hire Pressman full-time. Thus began Pressman’s career in immunology.

At Pauling’s request, Pressman stayed on at Caltech as a post-doc, and during this time the two became friends. In 1943, after failing to prove that they could synthesize antibodies, Pauling’s research team changed their focus from understanding the structural components of antibodies and antigens, to looking for the binding mechanism that allowed antibodies to attach to specific antigens through Van der Waals bonds. One outcome of this was their development of the theory of complementarity, a “lock and key” model in which molecules fit together because of the high levels of specificity that they show for one another.

Pressman authored three papers with Pauling during this phase, including a very important one titled “The Nature of the Forces between Antigen and Antibody and of the Precipitation Reaction,” published in Physiological Reviews. In this paper, the researchers discussed the historical significance of immunology within the context of structural chemistry. Speaking of the tradition in which they worked, Pauling and his colleagues wrote that “two of the most important advances in the attack on the problem of the nature of immunological reactions were the discovery that the specific precipitate contains both antigen and antibody, and the discovery that antibodies, which give antisera their characteristic properties, are proteins.”  In this paper, they also theorized that the immune system depends on structural and chemical forces to function.


pressman-1960s

Pressman (at right) in the lab, ca. early 1960s.

In 1947, Pressman decided to pursue an interest in cancer research and moved on to the Sloan Kettering Institute in New York City to investigate the use of radioactive tracers as they pertained to cancer treatment.  The West Coast was never far from his thoughts however, and he often wrote back to friends comparing the two regions and asking for information about life in Pasadena. Of his new arrangements he observed, “The mechanics of living take a much greater part of the time in New York, so that I do not have as much time to do as much as I would like to or could do in Pasadena.”

Pressman’s first few years at Sloan-Kettering were difficult, not only because of the nature of the research that he was conducting – a continuation of the research that he started with Pauling – but because he was frequently forced to move both his lab and his residence, a source of continuous disruption for himself and his family. Sloan Kettering had just been established in the early 1940s and wasn’t formally dedicated until the year after Pressman moved there. Though it eventually became one of the nation’s leading biomedical research institutions, Pressman’s early experiences there coincided with institutional growing pains.

Eventually, as the environment at Sloan-Kettering became more stable, Pressman settled in to his position and provided Pauling with regular updates on his progress. The two often traded manuscripts back and forth, and each solicited technical advice from one another on their specific endeavors, which gradually grew further afield as time moved forward. At Kettering, Pressman continued to study antibody specificity and explored the potential use of radioactive antibodies for tumor localization to develop immunotoxins. In 1954, he left New York City for the Roswell Park Institution in Buffalo, remaining there until his death.


pressman-19610227

60th birthday greetings sent to Pauling by David and Reinie Pressman, February 1961.

Pauling and Pressman remained in frequent contact for many years, focusing their voluminous correspondence primarily on work that Pressman continued to do as an outgrowth of their time together in Pasadena.  In July 1961, Pressman wrote that he and a colleague, Oliver Roholt, had potentially made a breakthrough with regard to the sequencing of the polypeptide chain associated with the region of specific binding sites in antibodies. He sent his manuscript, “Isolation of Peptides from an Antibody Site,” to Pauling for review prior to submission to Proceedings of the National Academy of Science. Pauling felt that the manuscript had been put together too quickly and challenged Pressman to do better. He annotated the manuscript with numerous suggestions, most of which Pressman adopted. Less than a week later, Pressman sent the manuscript back to Pauling with the corrections and Pauling transmitted it in to PNAS, where it was received favorably.

The late 1960s were a period of great activity and advancement for Pressman. In 1965, he received the Schoellkopf Medal, a prestigious award granted by the Western New York section of the American Chemical Society. In 1967, he became assistant director at Roswell and, in 1968, he published a book, The Structural Basis of Antibody Specificity. By all outside indications, Pressman’s life was going well.


In 1977 however, tragedy struck when Jeff Pressman, David and Reinie Pressman’s son, committed suicide at the age of 33. Jeff was an up-and-coming professor of political science at MIT, where he was well-liked by faculty and students. Up until a few months before his death, Jeff had seemed happy, both with his career and his life at home. In a letter to Pauling, Pressman described Jeff’s descent into depression as sudden, severe, and uncharacteristic. He also documented the events leading up to his son’s suicide, conveying that he and his wife had become increasingly convinced that the responsibility for the tragedy lay at the feet of a rheumatologist to whom Jeff had been seeking assistance for back pain.

Believing Jeff’s back pain to be primarily muscular in cause, the rheumatologist had prescribed Indocin in January 1977. According to multiple sources that Pressman later consulted, Indocin was a mood-changer, so much so that other patients had reported sudden depressive symptoms and, in severe cases, committed suicide a few months after starting the medication. To complicate matters, the rheumatologist had increased Jeff’s dose to a level that few patients could tolerate well, and had done so more rapidly than was advisable. When Jeff began complaining of insomnia, the rheumatologist prescribed two additional medications, both of which had the potential to worsen his depression. Jeff finally stopped taking Indocin, but the effects lingered. Jeff’s wife, Katherine, reported that Jeff had felt increasingly hopeless about his depression, even though he continued to work at MIT up until his death.


pressman-plaque

David Pressman’s former secretary, Cheryl Zuber, posing with a plaque mounted in Pressman’s honor at the Cancer Cell Center, Roswell Memorial Institute, 1981.

In the wake of Jeff Pressman’s death, his colleagues at MIT published a collection of political essays dedicated in his honor. The dedication specifically called out Jeff’s commitment to his students and his impact as a teacher. In it, his colleagues wrote, “He cared deeply about public affairs and immersed himself in them because he genuinely felt that government at its best could improve peoples’ lives.”

Nonetheless, the loss took its toll and, for David Pressman, the only source of solace that he could identify was a return to work. In 1978, his focus in the laboratory was on localizing radio-iodinated antitumor antibodies. He later wrote to Pauling about chronic shoulder pain that he was experiencing, as he was aware of Pauling’s vitamin research and was in search of an alternative to the shoulder replacement surgery that had been recommended by his physician. Pauling put forth an argument for a megadose of vitamins, but Pressman was eventually diagnosed with osteoarthritis. By the end of the year, he was slowing down, both in his work and in his correspondence.

Two years later, in June 1980, Pauling received the news that David Pressman had jumped from the roof of Roswell Park Memorial Institute. In a letter to Pauling informing him of her husband’s death, Reinie Pressman cast about for answers. She wrote at length about the health problems that he had been experiencing, including partial hearing loss, prostate trouble, and chronic problems associated with the osteoarthritis in his right shoulder. She also confided that “You were a significant part of Dave’s happier past.” Pauling replied in kind, stating

I was very fond of David. Also, I owe much to him, because of the vigor and effectiveness with which he tackled scientific problems during the eight years that he worked with me. Much of the success of our program in immunochemistry was due to his contribution.

A Master of Many Fields

1948i.39

Linus and Ava Helen Pauling, Oxford, 1948.

[The serological properties of simple substances – part 4 of 6]

By the Spring of 1946, having published no fewer than twelve articles – over a little more than three years – on the serological properties of simple substances, Linus Pauling’s busy life began to get in the way of continued advancement of his research program. Perhaps chief among competing interests was a separate fifteen-year joint research program, funded by a $300,000 grant, that Pauling and George W. Beadle, the head of Biology at Caltech, were in the midst of setting up.

Pauling had also returned to studies of sickle cell anemia with the arrival of Dr. Harvey Itano in the fall of 1946. He was likewise engaged with new inquiries in inorganic chemistry that reached a crescendo with a famous article, “Atomic Radii and Interatomic Distances in Metals,” published in March 1947. From there, the dawn of 1948 saw Pauling moving to England, where he served as George Eastman Professor at Oxford University. Not long after, he received the Presidential Award for Merit for work done during World War II. Clearly there was much going on in Pauling’s world.


sci7.00

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

Nonetheless, consequential progress continued to be made in the serological program with the thirteenth paper – an important one – coming into print in April 1948, while Pauling was still in England. This article, written by Pauling along with David Pressman and John Bryden, marked a continuation of the precipitation experiments that had been carried out in the previous two papers, but this time with a different antiserum and antigen substitute. The Paper XIII experiments determined that antibodies are rigid and cannot change shape to bond to a different antigen.

Significantly, these data also confirmed that structural complementarity was responsible for the reaction’s specificity, affirming Pauling’s early notions of a “hand in glove” fit. Furthermore, the paper’s findings established that the principal forces involved in the complementary bonds were Van der Waals interactions – very weak bonds induced by sheer proximity. In short, the experiments verified the importance of intermolecular interaction in the specificity of serological reactions, a significant breakthrough.


With Pauling now having returned stateside, the year 1949 saw the publication of the final two serological articles, one released in January and another during the summer. Paper XIV, written by Pauling and Arthur Pardee, was fashioned as a response of sorts to disagreements that had been expressed by other scientists concerning Pauling’s interpretations of his experimental results.

The paper specifically focused on experiments utilizing simple antigens and purified antibodies, rather than the antisera that Pauling had been using. These trials found that, although the behavior of simple antigens was different when matched with purified antibodies rather than antisera, “…the earlier work, carried out with serum, is presumably reliable.” In making this statement, Pauling and Pardee cited the non-specific combination of dye molecules along with other components of the serum for past results that had varied slightly.

sci7.002.1-antibodies-right-600w

Illustration of the antibody-antigen framework, 1948.

The last article in the serological properties series, Paper XV, appeared in the Journal of the American Chemical Society in August 1949; Pauling and Pressman were its authors. The article detailed the results of experiments using an antiserum with two or more positive charges. This experimental set-up, Pauling hoped, would allow him to determine the difference in combining power between antibodies containing only one negative charge as well as those containing two negative charges. The duo discovered that the antibody would only combine strongly with antigens that contained two negatively charged groups in specific positions. From this, Pauling concluded that the attraction between the negative charges of the antigen and the positive charges of the antibody are very strong.

After completing the fifteenth paper, Pauling largely left immunology behind in favor of the work that he and Itano were doing on sickle cell anemia. In 1950 and 1951, Pauling and several collaborators also published multiple articles delineating protein structures. In addition, it was during this time that Pauling began to really ramp up his peace work, delivering more and more lectures on the topic as the years went by.


The fifteen articles that comprise Pauling’s serological properties series were published over a span of seven years. During that period, Pauling worked with twelve collaborators, several of whom were graduate students. By the conclusion the project, hundreds of experiments, using dozens of compounds, had been run.

Particularly given the fact that he lacked any sort of formal background in immunology, the massive impact that Pauling made on the field is truly impressive. By the time that he moved on to other topics, Pauling’s work had served to raise the level of immunological knowledge by orders of magnitude. He is credited now with having discerned a relatively complete understanding of both antibody structure as well as the reaction mechanics underlying the interplay between antigens and antibodies. He also applied the vast collection of data that he had compiled to develop a theory of antibody formation. Of this, biographer Tom Hager wrote

For fifteen years…until a new, more powerful theory of antibody formation was put forward, Pauling’s idea led the field. His antibody work again expanded his growing reputation as a master of many fields.

Pauling himself believed that this work had solved “the general problem of the nature of specific biological forces” and that this understanding would “permit a more effective attack on the many problems of biology and medicine.”

Indeed, Pauling’s work with antibodies was influential even outside of the field of immunology. In 1990, journalist Nancy Touchette declared, “In his 1946 paper [“Molecular Architecture and Biological Reactions”], Pauling prophesied about the future of biology and medicine and why understanding the nature of complementarity is so important to the future of the field.” Five years later, at a Pauling symposium held at Oregon State University just a few months after Pauling’s death, molecular biologist Francis Crick stated flatly that Pauling “was one of the founders of molecular biology.” Once again, Linus Pauling had revolutionized a scientific field while following his curiosity and intuition.

A Period of Rapid Advancement in Pauling’s Immunological Work

1943i11-600w

Dan Campbell and Linus Pauling in a Caltech laboratory, 1943.

[Part 3 of 6 in a series investigating Pauling’s work on the serological properties of simple substances.]

In April 1943, only four months after releasing his first four papers on the serological properties of simple substances, Linus Pauling was ready to publish more. His fifth paper in the series reported out on the results of hapten inhibition experiments that his lab had conducted using two different antibodies. In the experiments, “measurements were made of the inhibitory effect of each of twenty-six haptens on one antigen-antibody reaction, and interpreted to give values of the bond-strength constant of the haptens with the antibody.”

The results of the experiments, with particular attention paid to the twenty-six hapten molecules, were then discussed in the context of their possible molecular structure. In this discussion, Pauling pointed out that some of the polyhaptenic molecules did not produce participates, a detail that was explained as having been caused by steric hindrance, or the inability for a reaction to take place due to molecular structure.

David Pressman was again a co-author of the paper, as were two graduate students, John T. Maynard and Allan L. Grossberg. Grossberg would stay with Pauling’s lab until 1946 – two years after completing his war-time master’s degree – and was involved with three more papers from the series. He later went on to work with Pressman at the Roswell Park Memorial Institute and eventually became associate chief of cancer research there.


Pauling’s immunological work was quickly producing exciting new results, momentum that was recognized by The Rockefeller Foundation, which awarded Pauling another grant in June 1943. Pauling also began delivering lectures on his serological research, notably including the Julius Stieglitz Memorial Lecture in January 1944.

Articles six, seven, and eight of the serological series were each published a few months apart from one another, beginning in March 1944. Pauling co-authored these papers with previous collaborators Pressman, Campbell and Grossberg, and also with Stanley Swingle, a research fellow and instructor who had earned his Ph.D. at Caltech in 1942.

Paper VI put forth more evidence for the Marrack-Heidelberger framework theory, for which Pauling had first announced his support in Paper I. The experiments specified in Paper VI made use of fifty different substances possessing either one, two, or three haptenic groups. The results of these trials indicated that a substance containing two different haptenic groups would only form a precipitate when antisera binding to both of those two groups were present. Of this finding the article states, “this provides proof of the effective bivalence of the dihaptenic precipitating antigen, and thus furnishes further evidence for the framework theory of antigen-antibody precipitation.”

In the seventh paper, published in May 1944, Pauling returned to the simple theory for calculating the inhibition of precipitation that he had developed in Paper II, published at the end of 1942. In his discussion, Pauling reported that his laboratory’s experiments found general qualitative agreement with the theory, but the numbers tended to be off. In seeking a more reliable equation, Pauling worked to improve the theory, accounting now for the fact that a single antiserum can contain slightly different antibody molecules with assorted combining powers.

This new and improved theory, and the equation that accompanied it, agreed with experimental results much better than had the original proposal. Indeed, by accounting for variations in the antibodies, Pauling and his colleagues had succeeded in developing a “quantitative theory of the inhibition by haptens,” which would prove important to much of the work that was to come.

Paper VIII, “The Reactions of Antiserum Homologous to the p-Azobenzoic Acid Group,” appeared in October 1944 and shared the results of experiments done with a new type of antibody. Previously, experiments had been conducted with antisera homologous to two different acid groups. However, in these new investigations, the Caltech researchers used antisera homologous to another type of acid group. In doing so, Pauling and his colleagues were attempting to gauge optimum acidity levels for serological reactions; to identify the types of antigens that most readily cause precipitation; to likewise identify haptens that inhibit precipitation; and to measure the strength of their inhibiting power.

Despite Pauling’s extensive involvement in studying reactions of antibodies and antigens, he still had time for other research interests. In February 1945, Pauling and Campbell announced that they had created a usable substitute for blood plasma, the result of three years of work supported by military contracts. Shortly thereafter, Pauling learned a few key details about sickle cell anemia while meeting with the other members of the Medical Advisory Committee. He immediately thought that hemoglobin was involved and went on to experimentally prove that the disease located its source on the molecular level; a first in the history of science.


1980Pardee

Arthur Pardee, 1980

June, July, and September of 1945 each saw the publication of another serological article: Papers IX, X, and XI respectively. The final two of this set featured the addition of a pair of new collaborators. John Bryden, a co-author for Paper X, completed his master’s degree around the time that the article was published, and Arthur Pardee was in the middle of his doctoral program when he worked on Paper XI. Pardee also worked on the experiments described in Paper XIV, although the article was published after he had completed his Ph.D. and returned to Berkeley. Pardee later went on to enjoy a hugely successful career as the Chief of the Division of Cell Growth and Regulation of the Dana Farber Cancer Institute at Harvard Medical School.


landsteiner

Karl Landsteiner

Papers IX and X shared the results of still more inhibition experiments. The experiments reported on in Paper IX largely confirmed Karl Landsteiner’s discovery on the combining of antiserum and antigen, or antiserum and hapten. Landsteiner had found that less bonding occurred between antibody and antigen or antibody and hapten if the substituent groups on the binding molecule were different from the antigen that created the antibody. The Pauling group confirmed this theory and, in addition, described the forces that affect hapten inhibition. Pauling believed that it had to do with intermolecular forces “including electronic van der Waals attraction…the formation of hydrogen bonds, and steric hindrance,” a supposition that would play a crucial role in later papers in which Pauling explained the incredible specificity that governs the behavior of these molecules.

Paper X studied the effect of molecular asymmetry on serological reactions. In this series of experiments, Pauling and two collaborators, David Pressman and John Bryden, had prepared an antiserum with an optically inactive immunizing antigen; e.g., a molecule that does not rotate plane polarized light. However, even though the immunizing antigen was not optically active, the antibodies in the serum combined more strongly with one configuration over an optically active hapten, which does rotate light, than in the other configuration. Pauling and his colleagues hypothesized that this was due to the presence of optically active amino acid residues in the antibody molecules.

Paper XI, published in September 1945, discussed reactions of antisera with various antigen substitutes. In this instance, the Pasadena group measured the precipitate formed by these reactions to gauge the inhibiting power of the haptens. They then correlated hapten-inhibiting power to molecular structure, suggesting that if a substance mixed with antisera more readily, then the structure of the molecule might be smaller. They ultimately discovered that if a hapten structure matched an immunizing azoprotein structure, the haptenic group exhibited a strong inhibitory effect.

In February 1946, Pauling and co-authors Pressman, Grossberg, and Leland Pence published the twelfth serological article. This was Grossberg’s fourth and final contribution; ultimately, he served as co-author on more of the series than did any other collaborator, save David Pressman and Dan Campbell. New to the series was Leland Pence, an assistant professor of organic chemistry at Reed College who had been collaborating with Pauling since 1942.

Prior to Paper XII, all previous experiments carried out by the lab had used negatively charged or neutral compounds. Paper XII presented the results of experiments that used a positively charged antibody. Pauling and his collaborators found that, even when using positively charged antibodies, hapten inhibition occurred the same way, with the same factors, as was the case with a negative or neutral compound. That said, one important difference that was observed was the ideal acidity for maximizing precipitates; when using a positively charged antibody, the pH required for the optimum amount of precipitate was much lower.

Analyzing Precipitation Reactions Between Simple Substances

1942i.2

Linus Pauling, 1942

[Part 2 of 6 in a series investigating Pauling’s work on the serological properties of simple substances.]

The first four papers published by Linus Pauling and his Caltech colleagues on the serological properties of simple substances described general aspects of the precipitation reactions that occur between antibodies and antigens. This work was spurred by a fundamental conundrum: Pauling and many others knew that antibodies and antigens would react to form solid precipitates. However, because the chemical structures of these precipitates were, at the time, so difficult to determine, scientists had been unable to decipher crucial details about the antibodies and the antigens that combined to form them.

Pauling’s solution to this problem was to investigate the products of a reaction that utilized, in part, a chemical compound whose structure he already knew. The constituents of these products were a simple organic compound consisting of carbon, oxygen, and hydrogen, combined with one or more haptenic groups – small molecules that spur the formation of antibodies when coupled with a larger molecule. Employing this methodology would, Pauling felt, allow him to better approximate the make-up of the antibody, because the experiment now involved only one unknown structure.


In order to run the experiments, Pauling set up a standard protocol for preparing the compounds that he needed. Each experiment required three types of compounds: simple antigens used in the precipitation reactions; immunizing antigens used to create antibodies; and antisera, which are liquids containing antibodies formed through the coagulation of blood. Pauling used this method for all of his serological reaction experiments.

Pauling and his collaborators obtained the antisera by injecting rabbits (some of them housed in Pauling’s yard and cared for by his children) with immunizing antigens. The rabbits then produced antibodies to combine with and neutralize the immunizing antigens. Once the last injection was carried out, the scientists drew blood from the rabbits, allowed it to clot, and collected the antiserum.

The reactants for Pauling’s experiments – immunizing antigens and simple antigens – were either purchased or prepared by Pauling and his collaborators, typically the graduate students.

For each precipitation test, equal portions of antiserum and a saline solution containing a simple antigen were mixed together. Typically, four to six different concentrations of antigen were used. The mixtures stood at room temperature for one hour, then were refrigerated overnight. The next day, a centrifuge was used to separate out the precipitates, which were then washed with saline solution and analyzed. Pauling’s method of analysis involved measurements of nitrogen, arsenic, carbon, and hydrogen. From there, the amount of a given antibody in the precipitate was determined using the nitrogen measurements.

The initial set of experiments used twenty-seven different compounds as the antigen, each containing between one and four haptenic groups. All of the polyhaptenic substances – those that had more than one haptenic group per molecule – formed precipitates, but none of the monohaptenic substances did. This finding supported the framework theory, devised by the British chemist John Marrack in 1934, that postulated that multivalent antibody molecules could combine with polyhaptenic molecules to form large aggregates, which would become precipitates. On the other hand, Marrack suggested, if multivalent antibody molecules combined with monohaptenic molecules, only small complexes would form and these would not precipitate.

Pauling summarized this work in a set of four papers that were published in the December 1942 issue of the Journal of the American Chemical Society.


marrack

John Richardson Marrack

Pauling’s first article, “Precipitation Reactions between Antibodies and Substances Containing Two or More Haptenic Groups,” served primarily to provide support for Marrack’s framework theory. Eight years before, Marrack had stated that antibodies were multivalent; in other words, they can bond to more than one antigen molecule. In order for them to bind in this way, the molecules must be properly oriented such that the binding sites fit together. This causes the formation of a lattice-like structure which grows until it is too large to stay in solution and precipitates out.

As noted above, Pauling’s experiments found that “simple antigens containing two or more haptenic groups per molecule were found to give precipitates with the antisera, whereas the seven monohaptenic substances failed to precipitate,” a discovery that confirmed the validity of the Marrack-Heidelberger framework, or lattice theory.

The second paper in this installment was titled “The effects of changed conditions and of added haptens on precipitation reactions of polyhaptenic simple substances.” The alterations to conditions that were tested by Pauling included allowing the mixture to rest longer, changing its temperature, and altering its pH. Having confirmed his own belief, in Paper I, that antibodies are multivalent, Pauling used Paper II to first note his assumption – and provide evidence for – bivalence.

In addition, Pauling used this paper to publish an equation that could be employed to find the amount of a precipitated compound in a given solution based on solubility, equilibrium constant, and total amount of hapten. Notably, the equation led Pauling to deduce “that in each case the maximum amount of precipitate is produced by an amount of antigen approximately equal to the amount of antibody,” an idea that unfolded more fully in the following paper.

1942-equation

The equation published by Pauling in Paper II.

Paper III, “The composition of precipitates of antibodies and polyhaptenic simple substances; the valence of antibodies,” further explores the supposition of bivalence through an examination of the ratio of antibody to antigen in precipitates.

While the bulk of Pauling’s experiments focused on dihaptenic antigens, some used trihaptenic antigens, and others used tetrahaptenic antigens. Through careful analyses of the different precipitates that resulted, Pauling was able to determine that the ratio of antibody to antigen in any given precipitate was approximately 1:1.

This finding suggested that most antigens could have only two antibody molecules attach to them, even if they possessed more than two haptenic groups, since the antibody molecules were relatively large and interfered with one another’s attachment. Pauling also used the one-to-one ratio to conclude that most antibody molecules possess two binding sites. The major development of this paper – the near one-to-one ratio – was “taken to indicate bivalence of most of the antibody molecules.”

The last paper of the first installment, Paper IV, reported the results of initial experiments on the inhibition of precipitation in the presence of hapten. Pauling and his colleagues had tested precipitate inhibition in three basic ways: by altering temperature, by augmenting the amounts of hapten present in their mixtures, and by isolating the effects of twenty-four specific haptens. These experiments found that adding haptens to a mixture of antibodies and antigens inhibited the precipitation of the antibody-antigen complex.

Furthermore, Pauling concluded that the structure of the haptens correlated with their inhibition power and detailed the relative values of each hapten’s bond strength. He then used the hapten inhibition data from these experiments to update his earlier equation for finding the amount of antibody precipitated.

Next week, we’ll examine eight more papers that Pauling published on the topic over the next three years and explore the ways in which this body of research evolved and expanded during that time.

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?


1943i11-600w

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.

John Kendrew (1917-1997)

Kendrew, John

John Kendrew building a model of myoglobin. Credit: MRC Laboratory of Molecular Biology.

[Ed Note: Today we remember Sir John Kendrew, who would have turned one-hundred years old on March 24th.]

The Cavendish Laboratory at Cambridge University was an exciting place to be in the 1950s. While James Watson and Francis Crick worked themselves into a frenzy in their race with Linus Pauling to discover the structure of DNA, lab-mate John Kendrew worked quietly alongside another future Nobel laureate, Max Perutz, as they too competed with Pauling in another arena: the molecular structure of various proteins.

For Kendrew however, this pursuit was not considered to be a competition against Pauling. Rather, he felt his corner of the laboratory to be working in tandem with researchers at Caltech in their joint pursuit of a common goal. For Kendrew, whoever got there first was beside the point. Indeed, when Perutz and Kendrew received the Nobel Prize for Chemistry – one year prior to Pauling’s receipt of his Peace Nobel – Kendrew credited Pauling as having been a source of inspiration and direction for his work on the atomic structure of myoglobin.


kendrewperutz_nobelportrait-220x220

John Kendrew and Max Perutz, 1962.

Sixteen years Pauling’s junior, John Cowdery Kendrew was born in Oxford, England on March 24, 1917. He received an appointment for study at Cambridge in 1939 and was working on reaction kinetics before the outbreak of World War II called him away to support the Allied effort.

By the time that he had reached the rank of Wing Commander in the Air Ministry Research Establishment, Kendrew had developed relationships with several important scientific contacts. Perhaps chief among these colleagues was the crystallographer J.D. Bernal, who also influenced Pauling’s protein work in the late 1930s. Bernal encouraged Kendrew to contact Max Perutz at the Cavendish Laboratory once his military service was completed. After receiving similar advice from Pauling, Kendrew began working with Perutz in 1945. His early research at the lab was conducted in support of his Ph. D. thesis – an x-ray diffraction study of hemoglobin in fetal and adult sheep.

In the late 1940s, Kendrew and Perutz established the Cavendish MRC Unit for the Study of the Molecular Structure of Biological Systems, and together they attacked the chemical structure of proteins using X-ray crystallography, with a particular interest in whale myoglobin. Although the research excited Kendrew, he was sometimes perplexed by the cross-disciplinary nature of what he was trying to accomplish. In a later interview with the Journal of Chemical Education, he remembered, “one of the problems was the lack of professional label. By profession, I was a chemist working on a biological problem in a physics lab.”

Nonetheless, Kendrew and Perutz were avidly pursuing the structure of keratin when the Pauling family visited the Cavendish in 1948. Pauling himself had done some preliminary work on the protein about ten years earlier, but after failing to build a satisfactory chain, he had abandoned the effort and moved on to other structures. Seeing the steady progress that Kendrew and Perutz were making reignited his own interest in the structure. Not long after, while lying in bed with a severe sinus infection, he worked on a rough sketch of a keratin model, which eventually inspired his signature proteins breakthrough: the alpha-helix.

Shortly after Pauling published his landmark 1951 paper, “The Structure of Proteins: Two Hydrogen-Bonded Helical Configurations of the Polypeptide Chain,” in which he introduced the alpha and gamma helixes, Pauling invited Kendrew to visit Pasadena and lecture at Caltech. Kendrew, impressed and eager to discuss Pauling’s findings, made preparations to stop in southern California as part of an already scheduled trip to San Francisco and Seattle. The visit proved thought-provoking for both scientists, and Kendrew returned to the Cavendish brimming with fresh ideas.


1954i.57-peter-600w

Peter Pauling, 1954.

In their early exchange of correspondence, Pauling’s communications (as was typical) were usually formal and brief. On the contrary, Kendrew’s enthusiasm about both his and Pauling’s work is spelled out in long, detailed paragraphs. In due time, Pauling’s writing broadened not only in length, but in a personal dimension as well.  Importantly, between a letter dated October 8, 1956 and another written on November 22, 1957, Pauling switched from referring to his correspondent as “Dr. Kendrew” to “John,” and Kendrew responded in kind.

Without doubt, one catalyst for this shift was Kendrew’s mentorship and guidance of Linus’ second-oldest son, Peter Pauling, a budding crystallographer who was pursuing his doctorate at the Cavendish. Despite his promise and pedigree, once Peter had settled in, many scientists at Cambridge had begun to express concern about his level of commitment to and interest in his work.

Amidst a flurry of letters from Peter’s Cambridge professors that ranged from outright condemnation of his behavior to genuine concern for his future, a 1953 letter from Kendrew comes across as amiable but firm. In it, he expresses serious doubts about Peter’s ability to attain a Ph.D. unless he undergoes “a considerable revolution during the summer.” The message also urges the elder Pauling to alter other travel plans and come to England to address the matter in person. Ultimately, Pauling declined to do so and, fortunately, Peter initiated the revolution for which Kendrew had expressed hope. A year later, Kendrew penned another letter in which he assured Pauling that he had observed in Peter’s work both a genuine interest and a more stringent ethic.

Kendrew was not merely a fair-weather supporter of Peter’s endeavors. When Peter ran into serious personal trouble at Cambridge in 1955, Kendrew proved invaluably resourceful. Most notably, he helped Peter transfer his fellowship and remaining doctoral research to the Royal Institution of London, where former Cavendish chief Sir Lawrence Bragg was now directing the Davy-Faraday research lab.  Kendrew and Bragg later assisted Peter in moving yet again – this time to University College, London – when he could not complete his dissertation in the requisite amount of time allotted by the Royal Institution.

In a number of letters, Pauling repeatedly expressed his gratitude to Kendrew for so carefully tending to Peter’s well-being and educational progress, choppy though it was. These circumstances only served to cement a friendship between the two; one that developed alongside the great professional respect with which they had always extended to one another.


1953i.19-kendrew-900w

Kendrew posing at a proteins conference held at Caltech, 1953.

On the other hand, Caltech and the Cavendish regularly found themselves to be in professional competition with one other, and this did lead to occasional friction between friends. In one instance, Kendrew sought out Pauling’s assistance with a rather complicated labor shortage that had partly been caused by Pauling himself. Shortly after Peter’s departure from Cambridge and Bragg’s resignation from his leadership post in the Cavendish, Kendrew wrote to Pasadena, asking for assistance. The gravity of the moment was especially amplified for Kendrew, who was presumably a tad annoyed by Pauling’s having convinced a mutual colleague, Howard Dintzis, to leave the Cavendish for Caltech the previous year. In his letter, Kendrew made a request:

I am writing to ask whether you would be good enough to let me know if you hear of any good man who would like to come to work on the myoglobin project in the near future. As you may have heard from Howard Dintzis, owing to a continuation of unforeseen circumstances I shall be totally without collaborators from January onward.

Pauling replied kindly, but did not include any recommendations.


In 1957, Kendrew succeeded in delineating the atomic structure of myoglobin. Two years later, Max Perutz successfully mapped the structure of hemoglobin. When Lawrence Bragg approached Pauling with the idea of nominating Kendrew for the Nobel Prize in Chemistry, Pauling suggested that the award be split three ways between Kendrew, Perutz, and Robert Corey, a colleague of Pauling’s at Caltech. Bragg disagreed and instead nominated the British chemist Dorothy Crowfoot Hodgkin, a pioneer in X-ray crystallography. Ultimately, Pauling’s final nomination of Kendrew and Perutz in 1962 included Hodgkin as well. As it turned out, Kendrew and Perutz split that year’s prize, and Hodgkin took the 1964 award for herself.


The remainder of Kendrew’s career was spent working less directly on scientific research and more intently on public policy. Like Pauling, Kendrew believed that scientists bore an obligation beyond scientific research and discovery. As he expressed in a 1974 interview

[Scientists] have special knowledge, and their most important responsibility is communication; because it is bad enough to try and foresee the effects of some scientific or technological advance given all the facts, but without them it is impossible…it is all the more important for scientists to communicate and make what they are doing understood at the government level and publicly through the media.

Jojn Kendrew Award gallery, EMBL ATC 11.2016

Wall of Honor at the European Molecular Biology Laboratory.

In the same year that he gave that interview, Kendrew helped to establish the European Molecular Biology Laboratory in Heidelberg, where he acted as director until his retirement in 1981. The lab has since created the John Kendrew Award to recognize and honor outstanding contributions made by the laboratory’s alumni.

Peter Pauling at Cambridge, 1953-1954

1954i.036-peter

Peter Pauling, 1954.

[The life of Peter Pauling, part 5 of 9]

In the first months of 1953, with his office mates scrambling to determine the molecular structure of DNA before his own father could beat them to it, Peter Pauling was mostly concerned with the English weather. He had been at Cambridge University since the fall of 1952 when he began his PhD program in physics at the university’s Cavendish Laboratory, and in that time he judged that he had seen a mere two full days of sun and was now officially fed up.

His father, by contrast, was mostly concerned with finishing his most recent edition of The Nature of the Chemical Bond, for which he had often solicited Peter as a source to provide example problems and solutions prior to his departure for England. As he was now beginning his graduate research, however, Peter was too busy to provide much assistance for this edition.

Instead, he was mostly occupying himself with a muscle camera developed by Hugh E. Huxley, a molecular biologist studying the physiology of muscle with Max Perutz’ Medical Research Council (MRC) Unit of Molecular Biology at Cambridge. Taking pictures of fibrous and globular proteins – beginning with insulin and tropomyosin – Peter applied the Cochran-Crick theory, with the goal of determining the helical structure of these protein molecules. This inquiry was, in principle, made possible by Linus Pauling’s work from less than a decade prior.

Since 1947, when the MRC unit was founded by Sir Lawrence Bragg, John Kendrew and Max Perutz had endeavored to use x-ray crystallography to determine the molecular structure of hemoglobin in sheep. By the time that Peter arrived at Cambridge, however, hemoglobin had proven to be an untenable object of study, and Kendrew’s focus had shifted to myoglobin. Whereas hemoglobin is found mostly in the blood, myoglobin is generally found only in muscle tissue. Both are proteins that carry oxygen to cells. Problematically, myoglobin is one fourth the size of hemoglobin, and too small for the era’s techniques of x-ray analysis.

To solve this issue, sperm whale myoglobin was used in hopes that the molecular details of the larger, oxygen-rich proteins of a diving mammal would be more observable with the tools then available. “Stranded whales are the property of the Queen,” Peter explained to his father as he discussed this work, “but we have an agreement with her to get a piece of meat if one comes ashore.” Nonetheless, though availed of samples from beached whales in the United Kingdom and from countries as far afield as Peru, Kendrew could not render the x-ray diffraction patterns with complete certainty.


 

myoglobin

Sperm whale myoglobin image created by John Kendrew.

In 1953, Perutz realized that by comparing the diffraction patterns of natural whale myoglobin crystals to crystals soaked in heavy metal solutions – a procedure called multiple isomorphous replacement – the positions of the atoms in myoglobin could be more accurately determined. Accordingly, Peter was tasked with making countless measurements in support of this effort.

Peter wrote to his father often over the next two years as he struggled to complete this project, which was the focus of his PhD. In particular, Peter asked for advice on how one might best get heavy metal atoms onto myoglobin, detailing his attempts to use everything from saltwater to telluric acid, which was used to produce salts rich in metallic contents, such as the element Tellurium.

Indeed, Peter’s work proceeded slowly, not least of all because of his knack for keeping things entertaining. Shortly after Watson and Crick’s discovery of DNA, for example, he fabricated a letter of invitation from his father, Linus Pauling, to Francis Crick, requesting Crick’s presence at an upcoming conference on proteins at Caltech. “Professor Corey and I want you to speak as much as possible during the meeting,” the impostor Pauling said to Crick in the fake letter, even urging him to consider lecturing at Caltech as a visiting professor. Linus Pauling had appeared to sign the letter himself, his signature skillfully forged. The letter proved so convincing that Crick actually replied, accepting the invitation to speak at the conference.

Before long, it became apparent that the entire communication was, in fact, a practical joke. Lawrence Bragg, the director of the Cavendish Laboratory, where Crick himself worked, was scheduled to speak at the proteins conference in the same time slot that the fake letter had proposed for Crick. Were it not for this, the deception might have gone even farther, since upon seeing his son’s forgery Linus himself was almost convinced that he had written the letter and had simply forgotten about it amidst the relentless pace of his schedule.

Ever a stickler for the details, however, Pauling noticed a grammatical error in the document that he would never have made. From there, he deduced the letter as having been authored by his mischievous son. For this transgression, Linus subtracted a five-pound fine from the $125.00 check that he sent to Peter each month.