Sickle Cell Research in the Wake of Pauling and Watson

Harvey Itano, 1954. Image credit: Caltech Archives.

By Dr. Marcus Calkins, Part 2 of 3

The molecular defect in Sickle cell disease was demonstrated by Linus Pauling’s lab in 1949, and one year prior, James Watson had proposed that increasing the level of fetal hemoglobin may provide a means by which the disease could be cured. These two publications provided a direction for what may soon become a real-life cure for the disease. However, many practical questions needed to be answered before a cure could be realized, or even imagined in a realistic sense. The first of these questions had to do with the composition of hemoglobin protein.     

Defining the structure of hemoglobin (1950s-1960s)

The project in the Pauling lab was led by a formidable graduate student of Japanese descent named Harvey Itano. It is noteworthy that despite his birth in Sacramento and his honorific as valedictorian of the University of California, Berkeley class of 1942, Itano was interned at Tule Lake during World War II. He was only released from detention to attend medical school at Washington University in St. Louis. Upon graduating with his M.D., he came to Pauling’s lab to pursue a Ph.D. His widely celebrated doctoral thesis was built on the Sickle cell disease project. After finishing this training in biochemistry and publishing his high-impact paper with Pauling, Itano opened his own lab in which he continued working on hemoglobin.

By applying many of the same methods pioneered in Pauling’s lab, Itano’s small group became central players in identifying aberrant forms of hemoglobin that lead to disease. Because of this careful and detailed work, Itano and other scientists began to suspect that the protein subunits that make up hemoglobin may be derived from multiple, highly similar genes. At the time, this idea represented a major paradigm shift, as it was previously assumed that each enzyme should correspond to a single gene.

This idea also brought up an evolutionary question of how such similar genes might come to exist. In a memorial biography, Itano’s friend and colleague R.F. Doolittle summarized Itano’s forward thinking on the genetic and evolutionary basis of enzyme constituents as follows:

In his thorough review of all the data, Harvey noted that in sheep and cattle, however, there were two kinds of end group (valine and methionine), and concluded that there had to be two each of two kinds of polypeptide in human adult hemoglobins. He went on to discuss how gene duplications could account for all the various hemoglobin chains, including myoglobin and foetal hemoglobin. These were thoughts well ahead of their time.

These major scientific contributions and others led to Itano’s election to the National Academy of Sciences, U.S.A. He was the first of many Japanese Americans to achieve that honor.

The exact change in hemoglobin protein responsible for causing Sickle cell disease was identified at the amino acid level ten years after Itano’s paper with Pauling, when V.M. Ingram used protease digestion to show that the disease came from a glutamine-to-valine mutation in the β-globin subunit of the hemoglobin protein complex. Although the molecular structure of DNA was accurately modeled by Watson and Crick in 1954, revealing tantalizing clues as to how genetic information can be encoded and passed from cell to cell and parent to child, the precise genetic mutation underlying Sickle cell disease would remain unknown for a few more decades, until the molecular biology revolution.

Molecular Switch from Fetal to Adult Hemoglobin (1970s-1990s)

In order to institute the cure dreamed up by Watson, much needed to be learned about hemoglobin composition, genetics, and control in red blood cells. This basic knowledge was largely generated through new technologies developed in the latter part of the 20th century. The cell and molecular biology revolution began in the mid-1970s and brought with it new techniques for manipulating DNA and other molecules. These advances allowed scientists to define genes and genetic structures, and to begin to understand how gene expression is regulated.

With regard to hemoglobin, it was discovered that two sets of genes (five on Chromosome 11 and three on Chromosome 16) encode the subunit proteins for the hemoglobin molecular complex. Each set contributes one subunit to the complex, and the subunits that are present in the blood change, depending on the developmental stage of the individual. In essence, a switch occurs just after the time of birth, wherein expression of fetal hemoglobin is turned off and adult hemoglobin is turned on. The mechanics of this switch, including the DNA sequences and proteins that make it work, were elucidated during this fruitful period of biological research.

Once the mechanics of the fetal hemoglobin to adult hemoglobin molecular switch were known, it became apparent that one gene, BCL11A, is a dominant factor in shutting down fetal hemoglobin production. If this gene can be silenced or disrupted, fetal hemoglobin expression will continue. Furthermore, there is strong evidence from molecular, cellular and clinical studies that continued expression of fetal hemoglobin will at least partially prevent the harmful accumulation of mutant hemoglobin aggregates and thereby prevent Sickle cell disease.

However, turning off the BCL11A gene is not an easy task. The molecular system evolved over millions of years to function robustly under almost any environmental condition or situation. Like a train hurtling down a track, such developmental programs are extremely hard to redirect, and if they are completely derailed, the individual may experience catastrophic effects. Thus, precise control of only the BCL11A gene, precisely in red blood cells, is necessary to realize the therapy first imagined by Watson. This level of control is possible to achieve in cells outside the body, but inside the body, cells are resistant to change, and our ability to target only one cell type at a time remains relatively primitive. Thus, a cure for Sickle cell disease still needed a method for regulating BCL11A specifically in red blood cells.

The Theory of the Molecular Evolutionary Clock

Dr. Emile Zuckerkandl, 1986.

Dr. Emile Zuckerkandl, 1986.

It thus appears possible that there would be no evolution without molecular disease.”
-Linus Pauling. “Molecular Disease, Evolution and Genic Heterogeneity,” 1962.

In the early 1960s, Linus Pauling and Emile Zuckerkandl, a French postdoctoral fellow who had arrived at Caltech in 1959, began researching the characteristics of hemoglobin extracted from a number of different species of animals. Zuckerkandl used a technique called fingerprinting, a process taught to him by a Caltech graduate student named Richard T. Jones, to create patterns of the amino acid sequences in each hemoglobin molecule.

[In her Master of Science thesis (pdf link), Dr. Melinda Gormley described fingerprinting, which was invented by the English chemist Vernon Ingram, as “a two-step process, [that] utilizes paper electrophoresis and paper chromatography. It produces splotches on paper at various locations; each mark corresponds to a peptide (two or more linked amino acids).”]

Once patterns had been prepared for several species, Pauling and Zuckerkandl compared them two at a time, and it was from the results of these comparisons that the theory of the Molecular Evolutionary Clock was developed.

The Molecular Clock differs from other evolutionary theories in that it tracks the evolution of a molecule rather than the evolution of a species. The theory states that, every so often, a mutation occurs in a given hemoglobin molecule. Generally speaking, this mutation is the source of a molecular disease, but will not cause any significant change to any organism other than its host.

Occasionally, however, a mutation will cause a lasting alteration to the molecule, and as the organism with the altered molecule reproduces, the change becomes permanent. More alterations of this nature can then occur on top of the original modification, thus resulting in even more differences in those hemoglobin molecules that have descended from the mutated original.

It is these differences that Pauling and Zuckerkandl were interested in when they compared the fingerprint patterns of different species, and their research led to an important breakthrough. As the duo compared more and more fingerprint patterns in a wider range of combinations, they observed that the number of differences between fingerprints lessened as the two species became more closely related. Pauling later stated that

[Zuckerkandl] found that in the beta chain of the human and the beta chain of the horse, for example, 20 of the 146 amino acids are different; but with human and gorilla, only one is different. It is the same amount of difference, just one amino acid residue, as between ordinary humans and sickle cell anemia patients, who manufacture sickle-cell-anemia hemoglobin.

From there Pauling and Zuckerkandl proposed that the comparative-fingerprinting method could be used to speculate as to how long ago any two species deviated from a common ancestor. Even more specifically, they reached the conclusion that one amino acid would be substituted every eleven to eighteen million years for any given species.

The evolutionary theory of the Molecular Clock was not readily accepted by scientists because it proposed a constant rate of evolution. However, it’s importance has now been noted and more research has been done on Pauling and Zuckerkandl’s original work. See, for instance, the very thorough examination conducted by Dr. Gregory J. Morgan in his 1998 paper “Emile Zuckerkandl, Linus Pauling, and the Molecular Evolutionary Clock, 1959-1965.” [pdf link], as well as Naoyuki Takahata’s “Molecular Clock: An Anti-neo-Darwinian Legacy,” [Genetics, (May 2007) 176: 1-6; not freely available online] which concludes that “a molecular clock is a most remarkable manifestation and a tribute from nature to anyone who studies evolutionary biology.”

For more information on Pauling’s hemoglobin work, please visit the website It’s in the Blood!  A Documentary History of Linus Pauling, Hemoglobin and Sickle Cell Anemia, and for more on Linus Pauling, check out the Pauling Online portal.

Pauling’s Theory of Sickle Cell Anemia

It's in the BloodWe owe to Pauling and his collaborators the realization that sickle cell anaemia is an example of an inherited ‘molecular disease’ and that it is due to an alteration in the structure of a large protein molecule, an alteration leading to a protein which is by all criteria still a haemoglobin.
– Vernon M. Ingram, 1957.

Of the four Documentary History websites that the OSU Libraries Special Collections has produced, “It’s in the Blood!  A Documentary History of Linus Pauling, Hemoglobin and Sickle Cell Anemia” is, in certain respects, the most unique.

For one, “the blood site” — its usual in-house appellation — is the only of our Documentary Histories not to have been written by Pauling biographer Tom Hager.  On the contrary, the idea for the blood site arose out of a history of science master’s thesis that Melinda Gormley — then a graduate student and now a professor at OSU — developed from research done in the Ava Helen and Linus Pauling Papers. As Dr. Gormley documented in this article (PDF, see pp. 8-9) it took the better part of two years to repurpose the text of her dissertation into a format suitable for the web.

Gormley’s thesis topic was relatively broad — “The Varieties of Linus Pauling’s Work on Hemoglobin and Sickle Cell Anemia,” (PDF, 1.8 MB) — and, as a result, the swath of content covered in the website is similarly wide.  The website begins its narrative in 1930, ends it in 1994, and along the way discusses Pauling’s contributions to areas ranging from immunology to Scientific War Work to evolutionary theory to orthomolecular psychiatry.  All of these topics will be addressed in future posts on this blog.

The heart of the blood site, however, is Pauling’s research on sickle cell anemia. Sickle cell anemia is a terrible disease that predominantly effects inhabitants of sub-Saharan Africa or those who can trace their lineage to that region.  The disease is a painful one, characterized by drastically-malformed red blood cells, and manifesting itself in a host of health maladies and, often, shortened lifespans.

Many folks who are semi-acquainted with the Pauling legacy know that he was, in some way, important to the modern understanding of sickle cell anemia.  But how? Well, Linus Pauling was the first individual to correctly theorize that sickle cell anemia is a disease that locates its source to the molecular level — in the process Pauling likewise became the first individual to postulate the concept of a molecular disease.

What then, exactly, was Pauling’s theory of sickle cell anemia?  That is the question that we aim to explore in this post.

Linus Pauling probably wasn’t a true freak-of-nature genius in the manner of an Einstein or a Mozart.  On the contrary, the likely secret of his profound success as a scientist was at least threefold in nature: 1) he possessed a relentless work ethic; 2) he was a very clear and concise thinker who conceptualized his ideas well and understood the efficiencies inherent to leading teams of researchers as opposed to going it alone; 3) and most importantly, he was deeply interested in, and capable of concretely understanding, radically-disparate areas of scientific study.  All three of these traits reveal themselves in the sickle cell anemia story.

Pauling first encountered the problem of sickle cell anemia rather by accident.  At a dinner in 1945, Pauling sat in the audience of an informal presentation by physician Dr. William Castle, wherein it was noted that the shape of red blood cells in sickle cell patients varied depending on whether the blood was venous or arterial —  normal in arterial blood, sickled in venous blood.  Clearly this suggested that the oxygen content in sickle cell blood played a major role in its molecular architecture. By his own recollection, “within two seconds,” Pauling concluded that the oxygen piece of the equation suggested that hemoglobin must be involved in the sickling mechanism — a conclusion that he could reach because of his keen understanding of the structural chemistry of hemoglobin.

In 1960, Pauling provided this description of his initial thoughts on how malformed hemoglobin could lead to sickled red blood cells.

…immediately I thought, “could it be possible that this disease, which seems to be a disease of the red cell because the red cells in the patients are twisted out of shape, could really be a disease of the hemoglobin molecule?” Nobody had ever suggested that there could be molecular diseases before, but this idea popped into my head. I thought, “could it be that these patients can manufacture a special kind of hemoglobin such that the molecules are sticky and clamp on to one another to form long rods, which then line up side by side to form a long needle-like crystal, which as it grows inside of the red cell becomes longer than the diameter of the cell and thus twists the red cell out of shape?”

From here, Pauling delegated many of the details necessary to verifying his thinking on the sickle cell problem to a team of Caltech graduate students led by Harvey Itano.  (This was common practice for Pauling, and helps explain how he was able to generate over 1,100 published papers in ninety-three years of living)  Using a variety of methods including electrophoresis, the Itano team, in the words of a 1950 Caltech press release

found a difference – slight but still unmistakable – between normal hemoglobin and that of a sickle-cell anemia patient.  Sickle-cell hemoglobin proved to have a greater positive electrical charge, under the proper chemical conditions, than did the hemoglobin from a normal person.  Such a difference in electrical properties can only mean a difference in molecular architecture, in the way in which the hemoglobin molecules are constructed.

In other words, Pauling was right: sickle cell anemia was a molecular disease and malformed hemoglobin was the cause.

In 1956, an English chemist named Vernon Ingram, using a new technique called fingerprinting, (Pauling provides a rather technical description of the method here) proved conclusively that sickle cell anemia was an inherited disease as well.  Moreover, sickle cell anemia was found to be caused by an astonishingly small change at the molecular level.  Physicist John Hopfield described it this way

On the surface of the ten-thousand atom molecule, there is a slight change. A small group of a few atoms on the edge of the molecule is replaced by another small group of atoms. That’s all that happens – an exchange of a few atoms. Yet it’s enough to make people very ill. The effect of the change is to create a sticky point between an abnormal molecule and its neighbor, causing molecules to pile up on each other.

Just as Linus Pauling predicted, after dinner, in 1945.