A Master of Many Fields

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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.


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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.

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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

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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.


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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.


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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

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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.


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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.

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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

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Linus Pauling, 1935

[Part 1 of 6]

Today, Linus Pauling is most commonly known for unraveling the chemical bond, working for peace, and promoting vitamin C. However, this short list barely scratches the surface of Pauling’s work in any number of fields. Beginning today, we will explore a lengthy program of research that Pauling oversaw on the serological properties of simple substances, a title that he appended to fifteen publications authored from 1942 to 1949. Post one in this series will focus primarily on Pauling’s background in biology and the work that led up to his first set of serological publications.

One of Pauling’s first major forays into the world of biology came about through his study of hemoglobin, the molecule responsible for transporting oxygen in the blood. Specifically, in 1934, he launched a study hemoglobin partly as a means to begin a larger inquiry into the structure of proteins.

An investigation of hemoglobin, Pauling quickly decided, would require more than one year to obtain results. Consequently, in November 1934, he applied for a grant from the Rockefeller Foundation to “support researches on the structure of Haemoglobin and other substances of biological importance.”

At the time, the Rockefeller Foundation was keenly interested in funding studies of “the science of life,” and Pauling’s grant request was promptly approved, with the first injection of funds received in July 1935. Although Pauling had originally intended for the grant money to go specifically toward his work on hemoglobin, as he corresponded with his funders he expressed an openness to studying other “interesting biochemical problems,” and indeed this quickly became the case.


A few months later, in 1936, Pauling met Karl Landsteiner, whose ideas would help to shape the course of Pauling’s research for the next several years. Landsteiner was an Austrian biologist and physician best known for discovering the human blood groups. By the time that he met Pauling, he was also actively engaged with topics in immunology.

Over the course of their conversations, Landsteiner passed this interest on to Pauling, who became fascinated by the specificity of antigens (foreign substances that enter into the body) and antibodies (proteins that neutralize antigens and prevent them from causing harm). The human immune system is capable of building thousands of antibodies, each of which reacts with a specific antigen. This specificity is seen in few other physical or chemical phenomena. However, one area in which it is found is crystallization, an area of chemistry with which Pauling was very familiar. This body of knowledge set Pauling down a path to making important contributions to the study of antigen-antibody behavior.

As he sought to learn more, Pauling read Landsteiner’s recently published book, The Specificity of Serological Reactions, finishing it shortly after their initial meeting. The following year, 1937, Pauling and Landsteiner met again and spent several days discussing the most current ideas in immunology. For Pauling, immunology presented two particularly compelling questions: First, what were the forces that enabled the combination of an antibody and its homologous antigen, but no other molecule? Second, how were antibodies produced and how did this means of production allow antibodies and antigens to combine so specifically?


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

In 1939, Pauling decided to shift the bulk of his research focus to the interaction dynamics of antigens and antibodies. As his work moved forward, Pauling came to theorize that the specificity shown by antibodies when combining with antigens depended on how well-matched the shapes of the two molecules were, a theory called molecular complementarity. In other words, antibodies and antigens were able to come together because their shapes complemented one another, like a hand in a glove.

From there, Pauling developed a plan to perform a broad range of experiments that would, he hoped, strengthen this theory and prompt it forward as the accepted explanation for the specificity of serological reactions. To assist in this promising line of inquiry, Pauling hired Dan Campbell, at the time a research fellow at the University of Chicago, to come to Caltech and serve as the Institute’s first faculty member in Immunochemistry. Campbell arrived in January 1940 and remained at Caltech until his death in 1974.

Once relocated to Pasadena, Campbell starting out by working on structural studies of hemoglobin – Pauling’s old research project dating back to 1934. A few months later however, a key shipment of serum antigens arrived from Karl Landsteiner’s laboratory, and both Campbell and Pauling began experimenting on the issue of the day. Initially, the duo encountered only disappointment as they uncovered no results of interest. However, the early setbacks did not stop Pauling. He persevered and, in October, published a landmark article, “A Theory of the Structure and Process of Formation of Antibodies,” which detailed his ideas on molecular complementarity.


In 1941, Pauling began an experimental program on serological reactions focusing on simpler organic compounds whose structure he already knew. In so doing, he also began to add more collaborators. Besides Campbell, the first of these was David Pressman, who earned his doctorate under Pauling and then stayed on at Caltech to support the nascent immunology program until finally leaving in 1947.

In addition to the simple substances work, this trio of researchers also continued other lines of study pertaining to Pauling’s antibodies projects. In early 1942, one of these produced what seemed to be an incredible result: that March, through a press release rather than a conventional journal article, Pauling, Campbell and Pressman announced that they had created artificial antibodies. A wide array of newspapers and magazines picked up the story and interest rapidly grew. However, other scientists could not replicate the trio’s results and skepticism of the group’s claim began to mount. Pauling, however, continued to believe that his team had truly created artificial antibodies, though subsequent efforts found only dead ends.

Undaunted, Pauling continued his experiments on serological reactions in simple substances and, in December 1942, published the first four papers of what would ultimately become a fifteen-paper series. This body of scholarship was the culmination of several years of work conducted by many people including Pauling, his two main collaborators, David Pressman and Dan Campbell, as well as one other non-student colleague. Several graduate students also supported the effort by helping to prepare the necessary compounds and running the experiments; as the publication series ran its course, eight were eventually listed as co-authors. Three graduate students, Carol Ikeda, Miyoshi Ikawa, and David H. Brown, were involved in the first four papers. Beginning next week, we will take a closer look at the details of what this group published.

Pauling, Zuckerkandl and the Molecular Clock

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Dr. Emile Zuckerkandl, 1986.

In 1963, a year after first publishing their ideas on the study of molecules as indicators of evolutionary patterns, Emile Zuckerkandl and Linus Pauling continued to explore what they felt to be a very promising thread of inquiry.

Specifically, the two joined in arguing that the molecular clock method – as they had since termed it – might be used to derive phylogenies (or evolutionary trees) from essentially any form of molecular information. This position was further explicated in “Molecules as Documents of Evolutionary History,” an article published in Problems of Evolutionary and Industrial Biochemistry, a volume compiled in 1964 on the occasion of Soviet biochemist Alexander Oparin’s 70th birthday.

Zuckerkandl and Pauling’s most influential work on this subject was first put forth that same year, in a paper that they presented together at the symposium “Evolving Genes and Proteins,” held at the Rutgers University Institute of Microbiology. The talk, formally published a year later and titled, “Evolutionary Divergence and Convergence at the Level of Informational Macromolecules,” classified molecules that occur in living matter into three groups. Each of these groups was identified according to new terms that the pair had developed that were based on the degree to which specific information contained in an organism was reflected in different molecules. These three categories were:

1.Semantophoretic Molecules (or Semantides), which carry genetic information or a transcript of it. DNA, for example, was considered to be composed of primary semantides.

2. Episemantic Molecules, which are synthesized under control of tertiary semantides. All molecules built by enzymes were considered episemantic.

3. Asemantic Molecules, which are not produced by the organism and do not express (directly or indirectly) any of the information that the organism contains. In their discussion, Zuckerkandl and Pauling were quick to point out that certain asemantic molecules may shift form. Viruses, for example, can change form when integrated into the genome of the host; so too can vitamins when used and modified anabolically.

Semantides were considered most relevant to evolutionary history, but the term never caught on in biology, paleontology, or other allied fields relevant to the study of evolution. Nonetheless, whatever the nomenclature, the “semantides” that Zuckerkandl and Pauling wanted to investigate – DNA, RNA, and polypeptides – proved indeed to be precisely the treasure trove of information on evolutionary history that the duo had hoped would be the case.


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A figure from the French translation of Zuckerkandl and Pauling’s 1964 paper.

Fundamentally, Zuckerkandl and Pauling aimed to elucidate how one might gain information about the evolutionary history of organisms through comparison of homologous polypeptide chains. In examining these substances, the researchers sought specifically to uncover the approximate point in time at which the last common ancestor between two species disappeared. In essence, it is this approach that we speak of when we use the terms “molecular clock” or “evolutionary clock.”

Zuckerkandl and Pauling argued that, by assessing the overall differences between homologous polypeptide chains and comparing individual amino acid residues at homologous molecular sites, biologists and paleontologists would be better equipped to evaluate the minimum number of mutational events that separated two chains.

With this information in hand, researchers would thus be empowered to exhume the details of evolutionary history between species, as inscribed in the base sequences of nucleic acids. This set of data, they believed, would hold even more useful information than would corresponding polypeptide chain amino acid sequences, since not all substitutions in the nucleotides would be expressed by differences in amino acid sequence.


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A table from Zuckerkandl and Pauling’s 1965 Bruges paper.

As their work moved forward, Pauling and Zuckerkandl published another paper, 1965’s “Evolutionary Divergence and Convergence in Proteins.” This publication appeared in Evolving Genes and Proteins, a volume that emerged from a conference that the two had attended in Bruges, Belgium.

By this point, the duo’s idea of the molecular clock, or “chemical paleogenetics,” had elicited opposition from organismal evolutionists and taxonomists, as well as some biochemists. Now referring to their “semantides” simply as informational macromolecules, Zuckerkandl and Pauling used the 1965 meeting to argue strongly against their skeptics. Zuckerkandl chided

Certainly we cannot subscribe to the statement made at this meeting by a renowned biochemist that comparative structural studies of polypeptides can teach us nothing about evolution that we don’t already know.

Pauling likewise added that

Taxonomy tends, ideally, not toward just any type of convenient classification of living forms (in spite of a statement to the contrary made at this meeting).

Directly challenging those present who were attempting to discredit the idea of the molecular clock, the pair insisted that taxonomy tended toward a phyletic classification based on evolutionary history. Since the comparison of the structure of homologous informational macromolecules allowed for the establishment of phylogenetic relationships, the Zuckerkandl-Pauling studies of chemical paleogenetics therefore had earned a place within the study of taxonomy. This, they argued, was true both as a method of reinforcing existing phyletic classifications and also of increasing their accuracy. Specifically, the two claimed that

The evaluation of the amount of differences between two organisms as derived from sequences in structural genes or in their polypeptide translation is likely to lead to quantities different from those obtained on the basis of observations made at any other higher level of biological integration. On the one hand, some differences in the structural genes will not be reflected elsewhere in the organism, and on the other hand some difference noted by the organismal biologist may not be reflected in structural genes.

Indeed, it was these early observations, coupled with additional work conducted by those scientists who took their ideas seriously, that allowed for the development of a successful measure of rates of evolutionary change over time. Without these data, modern paleontologists, physical anthropologists, and geneticists would not be able to accurately determine evolutionary histories. Today, this technique has been systematized and specialized in the field of bioinformatics, which is now foundational to many studies in both biology and medicine.

The taxonomic purpose of the molecular clock, however, was only a byproduct of Zuckerkandl and Pauling’s main ambitions in studying paleogenetics: to better understand the modes of macromolecular transformations retained by evolution; to elucidate the types of changes discernible in information content; and – most importantly for Pauling – to identify the consequences of these changes for a given organism.


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Linus Pauling, 1992.

Despite its considerable potential, the work of Zuckerkandl and Pauling, though conducted at such a critical juncture in a nascent field, was largely forgotten as recently as the early 1990s. In fact, in Allan Wilson and Rebecca Cann’s 1992 article, “The Recent African Genesis of Humans,” it was implied that the concept of the molecular or evolutionary clock was first developed and employed by a Berkeley anthropologist, Vincent Sarich. Sarich had collaborated with Wilson in 1967 to estimate the divergence between humans and apes as occurring between four to five millions years ago.

Pauling was still alive in 1992, and seeing this article he duly wrote to the editor of its publisher, Scientific American, pointing out that that, in fact, he and Zuckerkandl had, in 1962, issued their own estimate of the disappearance of the last common ancestor of gorilla and man. Zuckerkandl and Pauling’s calculations had yielded a divergence at about 7.6 million years before present, which Pauling pointed out was much closer than Sarich’s figure to the more recent estimates of divergence determined by Sibley and Ahlquist in 1984 and 1987. Notably, Pauling and Zuckerkandl’s estimate continues to remain closer to more contemporary notions of 8 to 10 million years.

Today, Emile Zuckerkandl and Linus Pauling are remembered as having first championed the notion of the molecular clock, even if many of the details now deemed as fundamental still needed to be ironed out by an array of scientists who followed. Regardless, as in so many other areas of science, Pauling proved once more to be on the ground floor of a new discipline. This was an academic venture that continued also to serve the younger Zuckerkandl well, as he continued on through a prolific career in science that concluded with his passing in 2013.

Molecules as Documents of Evolutionary History

1986i.155

Linus Pauling and Emile Zuckerkandl, 1986.

[Part 1 of 2]

“Of all natural systems, living matter is the one which, in the face of great transformations, preserves inscribed in its organization the largest amount of its own past history. We may ask the question: Where in the now living systems has the greatest amount of their past history survived, and how it can be extracted?”

-Linus Pauling and Emile Zuckerkandl, 1964

Austria-born Emile Zuckerkandl fled to Paris with his parents to escape the Nazi occupation of his homeland when he was only sixteen. Twenty years Linus Pauling’s junior, this extraordinary scientific mind was nurtured through the study of the biological sciences at the University of Illinois in the United States, and the Paris-Sorbonne University in France. During the course of these studies, Zuckerkandl developed a particularly keen interest in the molecular aspects of physiology and biology, and is now regarded to have been a major contributor to both fields.

Zuckerkandl first met Linus Pauling in 1957, a time period during Pauling himself was spearheading the new study of molecular disease, following his earlier, groundbreaking discovery of the genetic basis of sickle cell anemia. Two years later, Zuckerkandl joined Pauling as a post-doc in the Chemistry and Chemical Engineering Division at the California Institute of Technology. Once arrived, Zuckerkandl was encouraged by Pauling to study the evolution of hemoglobin, a research project that was informed by a basic and crucial assumption that the rate of mutational change in the genome is effectively constant over time.

This assumption was something of a hard sell for biologists at the time, due to the prevailing belief within the discipline that mutations were effectively random. Undaunted, Pauling and Zuckerkandl moved forward with their project and ultimately created a model that, over time, ushered in major changes to the conventional wisdom.


The Pauling-Zuckerkandl project was first revealed to the world in a co-authored paper that appeared in 1962’s Horizons in Biochemistry, a volume of works written and dedicated to the Nobel winning physiologist Albert Szent-Györgyi. The paper, titled “Molecular Disease, Evolution, and Genic Heterogeneity,” is regarded today as a foundational work in its application of molecular and genetic techniques to the study of evolution.

At the time that Pauling and Zuckerkandl wrote this first joint paper, Pauling was, as the title of the work might suggest, very interested in molecular disease; so much so that he was thinking about molecular disease and evolution as occupying two sides of the same coin. Defining life as “a relationship between molecules, not a property of any one molecule,” Pauling believed that disease, insofar as it was molecular in basis, could be defined in exactly the same way. Since both evolution and molecular disease were merely expressions of relationships between molecules, the distinction between the two became blurred for Pauling, who felt that the mechanism of molecular disease represented one element of the mechanism of evolution.

In their paper, Pauling and Zuckerkandl outlined their point of view as follows:

Subjectively, to evolve must most often have amounted to suffering from a disease. And these diseases were of course molecular. …[T]he notion of molecular disease relates exclusively to the inheritance of altered protein and nucleic acid molecules. An abnormal protein causing molecular disease has abnormal enzymatic or other physicochemical properties. Changes in such properties are necessarily linked to changes in structure. The study of molecular diseases leads back to the study of mutations, most of which are known to be detrimental. A bacterium that loses by mutation the ability to synthesize a given enzyme has a molecular disease. The first heterotrophic organisms suffered from a molecular disease, of which they cured themselves by feeding on their fellow creatures. At the limit, life itself is a molecular disease, which it overcomes temporarily by depending on its environment.

These assertions – in particular that “life itself” was a molecular disease – were so strange and seemingly outrageous that many biologists dismissed the paper outright. However, the basic idea that the pair was considering simultaneously sparked the interest of many other scientists who willing to entertain the consequences of this mode of thinking. If Pauling and Zuckerkandl were right, then every vitamin required by human beings stood as a witness bearing testimony to the molecular diseases that our ancestors contracted hundreds of millions of years ago. These “diseases” would manifest negative symptoms only when the curative properties of the nutrients gained from our natural environment through food and drink proved insufficient to dampen their effects.


This line of reasoning provided the impetus for Pauling and Zuckerkandl to begin examining differences between the genetic code as a tool to better understand evolutionary history. Though they did not call it “the molecular clock” at the time, the concept served as the foundation for the genetic analysis of species over long periods of evolutionary history.

Fundamental to Pauling and Zuckerkandl’s argument was the notion that there was no reason to place molecules at specific points “higher” or “lower” on an evolutionary scale. Horse hemoglobin, for example, is not less organized or complex than is human hemoglobin, it is simply different. The same is true for the genetic information contained in the hemoglobin of humans as compared to other primates, such as gorillas.

As Zuckerkandl examined differences of these sorts, he was not surprised to find that the peptide sequences of a gorilla were more similar to a human’s than a horse’s were to either. He and Pauling then began to consider how these sequences must be indicative of the millions of years of separate evolution that had passed since the disappearance of the last common ancestor linking horses and primates.

While compelling in its own right, for Pauling the chief purpose of understanding these differences was to discern crucial information about the human condition and to define the parameters of optimal health. Indeed, Pauling fundamentally believed that an improved understanding of the transitions that the genetic code had undergone would ultimately reveal new and effective treatments for molecular diseases.


1962p.23-table1

Table I from Pauling and Zukerkandl’s 1962 paper.

To demonstrate the efficacy of their methodology, Pauling and Zuckerkandl calculated the number of genetic differences that exist between the alpha and beta chains of hemoglobin peptide sequences in horses, humans, and gorillas, which they then used to determine the time that had elapsed since the erasure of the last common ancestor linking these species. In seeming support of their claims, the authors found that their figures matched pretty closely with data uncovered by paleontologists.

Despite this, the impact of Pauling and Zuckerkandl’s paper dissipated pretty quickly. For other established figures in the field, Pauling and Zukerkandl had failed to prove the essential assumption that evolution should proceed with relative uniformity over time. Lacking a clear reason to accept that genetic change occurs at a constant rate, there was no compelling reason to believe that Pauling and Zuckerkandl’s molecular clock should give an accurate picture of evolutionary history.

Nonetheless, the idea of the molecular clock found a degree of traction among biologists who valued its potential to corroborate and increase the accuracy of existing phylogenetic assignments. And as we’ll discuss in our next post, Pauling and Zuckerkandl continued to explore their ideas, eventually building a body of work that came into far greater favor a few decades later.

Dr. Michael Kenny, Resident Scholar

Dr. Michael Kenny

Dr. Michael Kenny

Dr. Michael Kenny, emeritus professor in the Department of Sociology and Anthropology at Simon Fraser University, recently completed a term as Resident Scholar in the Oregon State University Libraries Special Collections and Archives Research Center. Kenny is the twenty-fourth individual to have conducted work at OSU under the auspices of this program.

Part of Kenny’s scholarly background is in the eugenics movement, and it is this prism that framed his interest in conducting research in the Pauling Papers. Kenny was specifically interested in investigating the changing cultural milieu in which Linus Pauling worked and the ways that this environment may have impacted Pauling’s thinking on issues associated with eugenics.

Kenny was likewise very keen to examine the rhetoric that Pauling used during the years in which the dangers of nuclear fallout were an item of active debate. As it turns out, much of this rhetoric assumed a tone similar to that used by eugenicists contemporary to Pauling. That said, with Pauling and certain of these contemporaries, the use of this rhetoric was not motivated by anything like the ideals that we now commonly associate with the eugenics movement of the early twentieth century.


Rockefeller Foundation administrator Warren Weaver.

Rockefeller Foundation administrator Warren Weaver.

In his research, Kenny leaned in part on a secondary source, Lily Kay’s The Molecular Vision of Life (1993), which examined the development of molecular biology at Caltech during its infancy in the 1930s. Pauling was a central figure in this important chapter of scientific history, having shifted his research program to focus on “the science of life” – specifically, the determination of various protein structures – as funded during the Depression years by the Rockefeller Foundation.

As Kay pointed out in her book, the Rockefeller Foundation harbored a pre-existing interest in eugenics which may have propelled its desire to fund work in the burgeoning field of molecular biology. Rockefeller administrator Warren Weaver, who was Pauling’s main contact with the funding organization, wrote specifically of the Foundation’s interest in exploring “social controls through biological understanding,” and himself considered molecular biology to be the “only way to sure understanding and rationalization of human behavior.”

In his correspondence with Pauling, Weaver likewise suggested that “you are well aware of our interests in the possible biological and medical applications of the research in question.” Queried about the Rockefeller Foundation’s interest in eugenics by Lily Kay in 1987, Pauling replied, “I do not have much to say here,” noting that “my own interest in medical chemistry resulted from my interest in molecular structure.”


James V. Neel

James V. Neel

One major outcome of Pauling’s research on protein structures was his discovery that sickle cell anemia is a molecular disease. This work was conducted in parallel to similar investigations carried out by the human geneticist James V. Neel, a major twentieth century scientist who discovered that sickled cells are the result of a heterozygous mutation that, when it becomes homozygous, leads to sickle cell disease.

For Kenny, James Neel provides a bridge of sorts in the scholarly analysis of Pauling. In addition to his work on sickle cell traits, Neel also was involved in ethnographic research on the indigenous Yanomami population in Brazil. This study was funded by the United States Atomic Energy Commission in the late 1950s and early 1960s, and was motivated by the U.S. government’s desire to more fully understand the consequences that atmospheric radiation might portend for the human gene pool.

The debate over radioactive fallout from nuclear weapons tests during this time was fierce and continually hamstrung by a lack of concrete data. Linus Pauling, of course, was a key figure in the debate, and as Kenny and others have pointed out, he and his opponents used essentially the same data to draw very different conclusions from one another. Indeed, both sides were effectively engaging in the politics of risk assessment in arguing over the likely genetic implications for future generations of radioactive fallout released into the atmosphere by the nuclear testing programs of the era.

Hermann Muller

Hermann Muller

In developing and espousing his strong anti-testing point of view, Pauling was heavily influenced by Hermann Muller, a Nobel Laureate geneticist who is perhaps best known for proving the mutagenic effects of x-rays on fruit flies. According to Kenny, Muller was pretty clearly a eugenicist who spoke often of the need to maintain the purity of the pool of human germ plasm.

For Muller, essentially all mutations caused by radiation were to be viewed as a negative. While he acknowledged that natural selection is indeed the result of mutations that occur over the course of time, Muller believed that an increase in the rate of mutation is very likely to result in negative consequences. In arguing this, Muller pointed out that many mutations are buried and do not emerge until specific reproductive combinations come to pass. As Pauling and James Neel showed in the 1940s, sickle cell anemia is one such situation where this is the case.

Kenny points out that Muller’s ideas are imprinted all over Pauling’s 1958 book, No More War!, and in this book, as well as in his speeches, Pauling frequently used language that drew upon that of Muller and other eugenicists of his time. “I believe that the nations of the world that are carrying out nuclear tests are sacrificing the lives of hundreds of thousands of people now living,” he wrote, “and of hundreds of thousands of unborn children. These sacrifices aren’t necessary.” On other occasions, Pauling more directly echoed Muller, arguing that “we are the custodians of the human race, we have the duty of protecting the pool of human germ plasm against willful damage.”


So given all of this, was Pauling a eugenicist? For Kenny, the answer is no, or at least not “an old fashioned eugenicist in any clear sense.” Rather, Kenny sees Pauling as being one of many transitional figures (fellow Peace laureate Andrei Sakharov is another) working along a historical continuum that exists between the eugenicists of the late 19th and early 20th centuries, and contemporary ideas including genetic counseling and genetic engineering.  One of the more intriguing quotes that Kenny uncovered was Pauling’s statement that

Natural selection is cruel and man has not outgrown it. The problem is not to be solved by increasing mutation rate and thus increasing the number of defective children born, but rather by finding some acceptable replacement for natural selection.

For Kenny, Pauling’s suggestion of a possible replacement for natural selection anticipated contemporary techniques that are now deployed to minimize or negate what would otherwise be devastating hereditary diseases in newborn children. For expectant parents currently opting in favor of genetic counseling, as for Pauling in his day, the goal is to minimize the amount of human suffering in the world, not by proscription or law, but by choice. This ambition, which is global and cosmopolitan in nature – and not dissimilar to contemporary activism concerning global climate change – stands in stark contrast to the racist or nationalist motivations that fueled the eugenics of a different era.

For more on the Resident Scholar Program at the OSU Libraries, see the program’s homepage.