Linus Pauling’s legacy can be judged partly by his foresight in the selection of research projects and collaborators. With a view to the future of evolutionary biology, he insisted that his postdoctoral fellow, Emile Zuckerkandl, examine the evolution of hemoglobin. This decision heralded one of the beginnings of contemporary molecular evolution, a field Zuckerkandl and Pauling helped to delimit as the study of the history of “one-dimensional” molecules, protein, RNA, and DNA. Over the last forty years, molecular evolution research has grown and flourished. However, one cannot understand Pauling’s fruitful choice of this research agenda apart from considerations of the larger goals of Pauling’s life. Exploring the evolution of molecules, Pauling hoped, would strengthen the argument against nuclear testing and increase our
understanding of molecular disease.
Introduction
I
n the early 1960s, Emile Zuckerkandl and Linus Pauling proposed the simple but controversial idea of a “molecular evolutionary clock.” The molecular clock hypothesis, as it came to be known, proposed that the rate of evolution in a given protein molecule is approximately constant over time. More specifically, it proposed that the time elapsed since the last common ancestor of two proteins would be roughly proportional to the number of amino acid differences between their sequences. The molecular clock, therefore, would not be a metronomic clock—that is, its “ticks and tocks” would not be uniform—but would instead be a clock based upon random mutation events. In practice, a molecular clock would allow biologists to date the branching points of evolutionary trees.The molecular clock hypothesis, while rarely cited among Pauling’s most important discoveries, has proven to be very influential. The UC Berkeley biologist Alan Wilson claimed that the molecular clock is the most significant result of research in molecular evolution. In his book Patterns of Evolution, Roger Lewin describes the molecular clock as “one of the simplest and most powerful concepts in the field of evolution.” Francis Crick, co-discoverer of the structure of DNA, called the molecular clock a very important idea that turned out to be much truer than most thought when it was proposed.
Zuckerkandl and Pauling’s Comparative Hemoglobin Research Pauling’s research in molecular biology had its roots in the hemoglobin investigations he first undertook in the 1930s when he and Charles Coryell, a postdoctoral fellow, both motivated by purely chemical questions, examined the magnetic properties of hemoglobin. Hemoglobin research was conducted under Pauling’s supervision for the next two decades. In 1949, using Tiselius moving band electrophoresis, Harvey Itano and Linus Pauling, with help from S. J. Singer and I. C. Wells, showed that molecules of sickle-cell hemoglobin moved differently than normal molecules of hemoglobin in an electric field. Accordingly, they coined the term “molecular disease” to describe sickle-cell anemia. (It was later shown that sickle-cell hemoglobin had one amino acid residue different than normal hemoglobin.)
Pauling began to think about evolution at the molecular level in a focused way during the 1ate 1950s. This interest had both scientific and political dimensions.
Pauling was aware of early evolutionary work with hemoglobin through reading Karl Landsteiner’s landmark book The Specificity of Serological Reactions in 1936. In the second chapter of his book, Landsteiner discusses work that uses chemical differences to measure differences between species.
For example, he considers the discovery that the shapes and angles of hemoglobin crystals are characteristic for each species, and the differences between cr ystals are more pronounced between species that are more distantly related to one another. In the fall of 1937, while Pauling was the George Fisher Baker Lecturer in Chemistry at Cornell University, Landsteiner and he spoke at length about serology. The meeting had a lasting effect on Pauling, affecting the trajectory of his subsequent research into both hemoglobin and immunology.
In the latter half of his life, Pauling became more interested in humanistic issues. His wife Ava Helen, who was perhaps more liberal and politically aware than he, influenced his political and ethical development. His ethical framework was made explicit—Pauling believed that the correct moral code should be based upon the minimization of human suffering. Accordingly, this ethical principle provided a motivational force that influenced two other elements in his life that led to his evolutionary work with hemoglobin—his Portrait of Linus Pauling,
1958
spirited protest against the testing of nuclear weapons, and his interest in disease, especially molecular diseases such as sickle-cell anemia.
Between 1950 and 1963 Pauling became increasingly involved in the debate over the genetic effects of radioactive fallout. This in turn led him to think more deeply about mutation, molecular disease, and evolution, and provided motivation to conduct research on molecular evolution with Zuckerkandl. By 1955, Pauling was quoting the views of prominent geneticists such as Herman Muller, Kurt Stern, and Alfred Sturtevant in his ongoing debate with William Libby over the dangers of natural radiation and man-made radiation. In 1958 Pauling debated Edward Teller, the Hungarian physicist known as “the father of the H-bomb,” on KQED, an educational television channel based in San Francisco. Teller brought up the question of genetic damage, arguing that very small amounts of radioactivity might be helpful rather than harmful. Countering this type of argument required that Pauling discuss evolution through mutation and natural selection.
After Life magazine refused to publish a reply to a defamatory pro-testing article by Teller and Latter, Pauling began writing his book No More War! In this popular book, Pauling included a six-page section called
“Mutation and Evolution.” To write his book and participate in the fallout debate, Pauling read, discussed, and became quite proficient in genetics and evolutionary theory. However, he needed a collaborator to pursue these questions further.
Emile Zuckerkandl was born into a prominent Viennese family in 1922.
During the Second World War the Zuckerkandls fled Austria, first to Paris, and then to Algiers. After the war and a year’s biological study at the Sorbonne in Paris, Zuckerkandl undertook graduate studies in physiology at the University of Illinois under the direction of C. Ladd Prosser. On completing his master’s degree, Zuckerkandl returned to France, completed a doctoral degree at the Sorbonne, and secured a job at a marine laboratory in Roscoff, Brittany.
His early work on the molting cycle of crabs developed into an interest in the roles of copper oxidases and hemocyanin in the molting cycle.
Although the position at the marine laboratory was pleasant, secure, and allowed Zuckerkandl to meet internationally respected biologists such as Ernst Mayr dur ing the summers, Zuckerkandl and his wife, Jane, considered returning to America to escape the isolation of the remaining three seasons. Taking the advice of Professor Alfred Stern and others, Zuckerkandl wrote to Linus Pauling, who was planning a trip to France, and arranged a meeting with him in Paris in the summer of 1957. In a hotel in Paris, Zuckerkandl proposed a research project on hemocyanin and copper oxidases. Pauling was receptive. The famous chemist was impressed by the young researcher and recommended him for a post-doctoral fellowship in chemistry under his direction.
In September 1959, Emile and Jane Zuckerkandl arrived at Caltech.
Zuckerkandl recounted his first meeting with Pauling: “He said, you know
this subject of yours on hemocyanin and copper oxidases, I think the results are going to be difficult to interpret and I think you would do better to work on a protein about which more is known, … why don’t you work on hemoglobin?” Pauling suggested that Zuckerkandl analyze the hemoglobin of var ious pr imates using the newly invented electrophoretic-chromatographic technique of “finger-printing,” recently popularized by Cambridge hemoglobin researcher Vernon Ingram. This technique combined two “one-dimensional” techniques of paper chromatography and paper electrophoresis to for m unique two-dimensional patterns of hemoglobin cleaved into pieces. Using the technique on the hemoglobin of various species, Pauling hoped they could draw evolutionary conclusions. Pauling arranged for Zuckerkandl to work with his graduate student Richard T. Jones in Professor Walter Schroeder’s laboratory, since at that time Pauling did not have a laboratory of his own.
For the first two or three months of Zuckerkandl’s appointment, Jones taught Zuckerkandl how to finger-print proteins. After Zuckerkandl had perfected the technique, he widened the number of species in the analysis from primates to include cow, pig, shark, bony fish, lungfish and Echiurid
“worm.” Using this technique, Zuckerkandl, Pauling, and Jones drew qualitative conclusions from their comparative study, which they completed in the summer of 1960. Their study showed that the gorilla, chimpanzee, and human patterns were almost identical in appearance. The further the evolutionary distance from the primates, the more different was the hemoglobin “finger-print.” While the qualitative differences were clear, measur ing quantitative differences would require a more detailed description of the amino-acid sequences.
At this time, three rival laboratories were working on the complete amino acid sequences of two components of human hemoglobin (the alpha and beta chains): Walter Schroeder’s laboratory at Caltech; Gerhard Braunitzer’s laboratory at the Max Planck Institute in Munich; and Lyman Craig’s laboratory at the Rockefeller Institute in New York City. Max Delbrück, returning to Caltech from a visit to Braunitzer’s laboratory in Germany in the spring of 1960, brought back the sequence of the thirty terminal residues of the human beta chain, and Zuckerkandl was able to compare it with the preliminary results of Schroeder’s group. Through this comparison, Zuckerkandl correctly inferred that the alpha and beta chains are homologous, that is, they have a common ancestor and arose as distinct chains through a duplication event. Once Schroeder returned from sabbatical in Denmark, he and Zuckerkandl discussed whether or not the similarity in sequence was evidence for common ancestry. Unfortunately, Schroeder disagreed with Zuckerkandl’s inference and they did not publish the discovery. However, the idea that the hemoglobin chains were homologous legitimated further evolutionary analysis of the different chains within a single species as well as chains from different species.
In early 1961, Zuckerkandl began working with Schroeder in determining the amino acid composition of gorilla hemoglobin using an automatic amino-acid sequencer. The results of the compositional analysis, published in Nature, showed that the alpha chains of gorilla and human hemoglobin probably differed by only two residues and the beta chains by one. (It was later found that the gorilla and human alpha chain actually differed by only one residue.) Zuckerkandl and Pauling used these quantitative results in their next paper to calculate the time of divergence between gorilla and human using the evolutionary molecular clock.
In late November 1960, Pauling accepted an invitation to submit a paper to be published in a volume dedicated to Albert Szent-Györgyi, the Nobel Prize-winning discoverer of vitamin C. On June 22, 1961, Pauling wrote to inform Dr. Bernard Pullman, an editor of the volume, that he would write on “The Molecular Basis for Disease.” Zuckerkandl recounts how Pauling came down from his office to Schroeder’s lab, a floor below, to ask him to collaborate on the paper: “I said, I would with pleasure, and he said, ‘you know it is for Szent-Györgyi, so we should say something outrageous!’ ” This set the tone for much of the future collaboration between Pauling and Zuckerkandl—Pauling would be invited to submit to a Festschrift volume without peer review, the article would be written by Zuckerkandl, and together they would publish the pioneering paper on molecular evolution. After Zuckerkandl traveled to Seattle and Berkeley to check some final details with geneticists there, the historic paper was finally completed and sent to the publishers on November 1, 1961. The Szent-Györgyi paper was written by Zuckerkandl, and many people did find parts of it outrageous, especially those traditional biolog ists and anthropologists who disputed the whole idea of the molecular clock.
The most novel feature of the historic article, titled, “Molecular Disease, Evolution, and Genic Heterogeneity,” is the first application of the then-unnamed molecular evolutionary clock. The idea of using the number of amino acid substitutions to make temporal divergence estimates evolved as Zuckerkandl wrote the paper. In the article, Zuckerkandl and Pauling explicitly assume the homology of the globin genes: “in the course of time the hemoglobin chain genes duplicate, . . . the descendants of the duplicate genes ‘mutate away’ from each other, and the duplicates eventually become distributed through translocations over different parts of the genome.” In a somewhat cautious manner, the authors then compared horse and human alpha chains to calibrate the clock:
It is possible to evaluate very roughly and tentatively the time that has elapsed since any of the hemoglobin chains present in a given species ...
diverged from a common chain ancestor. ... From paleontological evidence it may be estimated that the common ancestor of man and horse lived ...
between 100 and 160 million years ago. ... the presence of eighteen differences between human and horse alpha chains would indicate that
each chain had nine evolutionary effective mutations in 100 to 160 millions [sic] of years. This yields a figure of 11 to 18 million years per amino acid substitution in a chain of about 150 amino acids, with a medium [sic] figure of 14.5 million years.
Using the figure of 14.5 million years per amino acid substitution per 150 residue polypeptide, Zuckerkandl and Pauling calculated the time of derivation from the common chain ancestor of the gorilla alpha and human alpha and gorilla beta and human beta chains, as 14.5 and 7.3 million years respectively. They note that the inferred divergence of gorilla and human of 11 million years, although a little lower than was thought, was consistent with the range estimated by paleontologists.
Pauling wove the idea of the molecular clock into numerous lectures he gave over the next five years. Often he would combine a lecture on molecular evolution with a lecture on science and peace. Pauling believed that, through the detailed determination of amino acid sequences of hemoglobin and other molecules, we would obtain much information about the course of the evolutionary process.
In September 1964, the Institute of Microbiology at Rutgers University held the seminal symposium, “Evolving Genes and Proteins,” organized by Vernon Bryson and Henry Vogel. In many ways, this conference marks the beginning of the modern field of molecular evolution. Many eminent biologists were present to hear Zuckerkandl deliver a collaborative paper,
“Evolutionary Divergence and Convergence in Proteins,” that some consider to be most influential of Pauling’s later career. This lengthy piece, written by Zuckerkandl, finally named the molecular evolutionary clock and derived the mathematical function that characterizes it.
Interestingly their derivation did not mention any selective processes.
However, the remainder of the lengthy text gives ample evidence to suggest that natural selection leads to different probabilities of substitution at each site, and is consistent with Zuckerkandl’s claim that natural selection is perfectly compatible with the clock. The 1965 article represented the pinnacle and culmination of the previous five fruitful years of collaboration between Linus Pauling and Emile Zuckerkandl.
Early Reaction to the Molecular Evolutionary Clock The biological and anthropological communities were at first unreceptive to the idea that evolution at the molecular level might proceed at a constant rate. One of the first confrontations between the champions of the new molecular approach and the heirs of the organismal orthodoxy occurred at a milestone conference entitled “Classification and Human Evolution” at Burg Wartenstein, Austr ia, in the summer of 1962.
Zuckerkandl presented a paper whose title introduced the new controversial term “molecular anthropology.” A “restricted committee”
meeting consisting of B. Campbell, T. Dobzhansky, M. Goodman, G. A.
Harrison, H. P. Klinger, E. Mayr, G. G. Simpson, and Zuckerkandl
considered the utility of the molecular approach for anthropology and the study of evolution. Only Morris Goodman, who had used immunological properties of proteins to reconstruct phylogenies, shared Zuckerkandl’s optimism about the utility of the molecular approach. Simpson and Mayr were skeptical of the clock hypothesis and the study of molecular evolution in general. Concerning the use of amino acid sequences as discontinuous characters, Simpson argued that they had no important advantage over morphological characters. For example, Simpson pointed out that the clock ignores variation in the rates of evolution and would be highly inaccurate when applied to short lapses of time. Furthermore, they were skeptical of the use of single characters, as they took a molecule to be, to accurately measure evolutionary rates and similarity between species.
“Seemingly contradictory evidence (e.g., that of the hemoglobins as reported by Zuckerkandl in this book) indicates merely that in certain characters Homo and its allies [e.g., gorilla] retain ancestral resemblances and that these are not the characters involved in their radical divergence
…, “ Simpson wrote in 1963. Two years later he reiterated his well-received comments even more strongly:
Zuckerkandl has shown that “From the point of view of hemoglobin structure, it appears that gorilla is just an abnormal human, or man an abnormal gorilla, and the two species form actually one continuous population.” From any other point of view other than that properly specified, that is, of course, nonsense. What the comparison seems really to indicate is that in this case, at least, hemoglobin is a bad choice and has nothing to tell us about affinities, or indeed tells us a lie. … Of course, as Zuckerkandl points out, we should not use just one kind of molecule but many, preferably proteins. However, if one can be misleading, so can many!
In a 1963 piece, Ernst Mayr echoed Simpson’s criticism, “Man’s shift into the niche of the bipedal, tool-making, speech-using hominid necessitated a drastic reconstruction of his mor phology, but this morphology did not, in turn, require a revamping of his biochemical system.” He wrote: “Different characters and character complexes thus diverged at different rates.” Interestingly, Mayr and Simpson appear to be little concerned with defending any strong dependencies between evolution at the molecular and organismal levels.
A 1964 meeting at Rutgers proved to be a battle-ground between the molecularly and organismally inclined biologists, but nonetheless was marked by an unusually fruitful exchange among biochemists, molecular biologists, evolutionists, geneticists, taxonomists, exobiologists, and microbiologists. Zuckerkandl wrote to Pauling informing him that a long paper was needed, given the mostly negative reaction of people to what he now called “chemical paleogenetics.” But the fast pace of advances in molecular biology, coupled with a growing appreciation for the value of the field, was helping to turn the tide.
Conclusion
The 1960s saw the development of the new field of molecular evolution.
The series of largely theoretical papers that arose from the collaboration between Zuckerkandl and Pauling were founding documents of the new discipline. In these early years, there was no disciplinary journal. Instead, Zuckerkandl and Pauling published in Festschrift volumes. As an eminent scientist, Pauling was often invited to submit papers to these volumes. He then co-opted Zuckerkandl’s co-authorship and they published papers
The series of largely theoretical papers that arose from the collaboration between Zuckerkandl and Pauling were founding documents of the new discipline. In these early years, there was no disciplinary journal. Instead, Zuckerkandl and Pauling published in Festschrift volumes. As an eminent scientist, Pauling was often invited to submit papers to these volumes. He then co-opted Zuckerkandl’s co-authorship and they published papers