An Interview with Manuel and Ann Varela: Dorothy Crowfoot Hodgkin and the Structures of Penicillin and Insulin

Apr 6, 2020 by

Michael F. Shaughnessy –

Dorothy Crowfoot Hodgkin

1) First of all, who was Dorothy Hodgkin, and where was she born and educated, and why does that name sound so familiar?

Dr. Dorothy Crowfoot Hodgkin was a world-renowned biochemist and 1964 Nobel Laureate in chemistry. Dorothy was born on the 12th of May in 1910 in the city of Cairo, Egypt. Her parents were John Winter Crowfoot and Grace Mary Crowfoot. John was employed in the Egyptian Education Service and then moved to Sudan to pursue his interest in archaeology. Her mother, Mary, was an expert weaver and a gifted illustrator of the official flora of Sudan.

Dorothy and her three sisters were transported to England for their education; therefore, separating them from their parents. Her fascination with chemistry began at the age of eight. During this period, a family friend, Dr. A.F. Joseph, allowed her to examine and study some chemicals in his laboratory located in Sudan. She was privileged to be one of two females allowed to study chemistry with the boys.

The Great War affected her early education. Dorothy and her three sisters were temporarily moved to England for their schooling, thus being apart from their parents. Consequently, she was repeatedly transferred between a variety of small private schools.

Between the ages of 11 and 18, Dorothy attended Sir John Leman Grammar School. As a student in the new school, Dorothy quickly encountered two serious problems. She had been extremely behind her peers in mathematics, and girls were not allowed to study chemistry, which had been a passion for the child. Fortunately, the school’s chemistry teacher, Ms. Deeley, made a unique accommodation for Hodgkin and was permitted to enroll in the chemistry course. After graduation from secondary school, she learned that she was not qualified for college at Oxford, because she lacked courses in Latin and other science disciplines. She also lacked funds for Oxford’s tuition. So she studied Latin and was tutored in botany by her mother. Mercifully, her generous aunt Dorothy Hood provided the required Oxford tuition.

In addition to studying and tutoring, Hodgkin spent the summer with her parents excavating and drawing. This experience was an influential one for Hodgkin, as she seriously contemplated pursuing archaeology for a career.

In 1928, she enrolled in Somerville College in Oxford, where Hodgkin studied chemistry and X-ray crystallography, graduating with a B.A. degree in 1932. Next, she entered graduate school in the Ph.D. program at the University of Cambridge, working under the famous John Desmond Bernal, as her graduate advisor. Hodgkin’s Ph.D. thesis project involved X-ray crystallography and chemistry of sterols, where she crystallized cholesterol and related steroid molecules.

Dr. Dorothy C. Hodgkin took her Ph.D. at Cambridge in 1937. Her Ph.D. project proved to be of great importance for the protein structure world. She performed many calculations of three-dimensional structures, as opposed to those for only two dimensions. This methodological approach permitted her to elucidate the stereochemical relationships of the carbons in cholesterol and other steroids. Her approach allowed her and others to study much larger and medically necessary molecules.

No doubt, you have recognized the name Hodgkin because of Sir Alan Lloyd Hodgkin, who in 1963 shared a Nobel Prize with Andrew Huxley for their discoveries on the flow of ions across the neuronal membrane and their effects on the nerve action potential. Dorothy Crowfoot married Thomas Lionel Hodgkin in 1937. Both Alan and Thomas belong to the 5th generation of the famous Hodgkin family.

In 1957, Dorothy Hodgkin became an independent investigator at the University of Oxford, establishing a research laboratory. On her own, she became scientifically productive. She was promoted, in 1960, with an endowed appointment to the Royal Society, to Wolfson Research Professor. In 1977, Hodgkin became a Fellow of Wolfson College, Oxford, until 1983.

2) Many things happen by chance, luck, and fate—what happened to Dorothy on a train ride?

The fateful meeting on a train ride that altered Hodgkin’s life occurred between Dr. Joseph, the friend and neighbor of the Crowfoot family, and an eminent professor of chemistry by the name of Thomas Martin Lowry, who was famous for his studies of optical stereochemistry and the behavior of acids and bases. Presumably, during the train-ride meeting between Joseph and Lowry, the promise of Hodgkin and the work of J.D. Bernal were mentioned in the same conversation. While Bernal did not get a Nobel (though many prominent biochemists thought he was deserving of one), he was a pioneer in the field of X-ray crystallography.

Before the history-changing train ride, Hodgkin, who had just graduated from college, could not secure employment. Therefore, after the introduction of Bernal to Hodgkin, she managed to scrape enough funding from a research grant funded by Cambridge University and from another generous gift from her doting Aunt Dorothy to pay for graduate tuition and lab work. Taken together, chance, luck, and fate permitted Hodgkin to enter college and later Bernal’s laboratory to pursue her graduate degree.

3) Oxford and the spires of Oxford—how did it impact Dorothy?

The early 20th century years were tumultuous ones at Oxford and similar educational institutions. A sort of battle of the sexes was at hand, and it reached a peak in 1927. Oxford reacted by relentlessly limiting the numbers of women who could enroll in higher education. The new policy aimed to maintain a male-dominated climate, and such was the case at Oxford. The system would be invoked well into the late 1960s, when Oxford became co-educational.

Meanwhile, the strict enrollment policy instilled at Oxford in the late 1920s also meant that women students like Hodgkin would experience tighter scrutiny on campus. Such restriction meant constant chaperoning of female students during campus events and separate rooms for cadaver dissections for men and women, not to also mention exclusion from college societies (for men only). Even the campus choir was off-limits to women students. Particularly hurtful for Hodgkin was the fact that she was barred from Oxford’s chemistry club!

The Oxford spires also signified a sort of coming of age for Hodgkin, who was then in her teens and 20s while in college and graduate school and generally considered a responsible adult. Her education at Oxford proved to be an era in which she flourished both academically and scientifically.

Her undergraduate research supervisor and later graduate advisor, Professor J.D. Bernal, quickly learned that compared to her peers in the laboratory, Hodgkin stood out as not only an exceptionally brilliant student but also dedicated to learning physical chemistry. Hodgkin, now a bona fide investigator in training, acquired a significant quantity of X-ray diffraction data on pepsin, an enzyme involved in the digestion of nutrients in the gastrointestinal tract. In the end, Dr. Dorothy Crowfoot Hodgkin would successfully defend her Ph.D. thesis project. Her protein work at the graduate level was to be the first case of a three-dimensional examination of crystals at the macromolecular protein level. She also demonstrated at Oxford that protein crystals could develop uniformly and that such structures could be evaluated in a liquid solution, rather than in dried forms, as had been done before her studies.

4) Why is it essential for scientists to know about the structure of penicillin? And can you give us a pictographic example?

In 1946, Dr. Dorothy Hodgkin determined the structure of penicillin, a vitally imperative molecule. The antibiotic penicillin became widely prescribed to treat a variety of bacterial infections.

Knowledge of the penicillin structure was vitally important. Hodgkin’s molecular structure permitted one to determine its active site, the atoms of penicillin that acted to kill bacteria. The known penicillin structure also allowed one to discover how it worked, that is, its mode of action on exploding a bacterium to pieces. Furthermore, with the knowledge of Hodgkin’s penicillin structure in hand, one could chemically modify it to produce more effective derivatives that could be used in the medical world to treat infections caused by antibiotic-resistant bacteria.

As you requested, below is her historical representation of the deduced penicillin structure. The ball-and-stick model shown embodies the atoms and bonds, respectively, of her penicillin structure. The two walls shown behind the penicillin model depict the electron density maps, also known as Patterson or contour maps, that were generated by the X-ray diffraction data and after thousands of calculations were performed by Hodgkin. It was arduous work.

The electron density maps generated by Hodgkin pointed to regions of the crystallized penicillin molecule where electrons were packed. The contour maps can be viewed as sort of a topological model showing hills but differing in that they were hills of concentrated electrons. These hilly regions of electrons, especially their peaks, predicted that atoms were to be found there!

In contrast, Hodgkin’s electron density map showed valleys, regions devoid of electrons, indicating areas where atoms were not to be found. Soon after obtaining her X-ray diffraction data and her corresponding electron density maps, Hodgkin became astute in interpreting the data to elucidate molecular structural data. Hodgkin’s intellect in conducting such ingenious work became legendary.

https://upload.wikimedia.org/wikipedia/commons/6/6e/Molecular_model_of_Penicillin_by_Dorothy_Hodgkin_%289663803982%29.jpg

https://upload.wikimedia.org/wikipedia/commons/6/6e/Molecular_model_of_Penicillin_by_Dorothy_Hodgkin_%289663803982%29.jpg

Figure Structure of Penicillin as Determined by Dr. D.C. Hodgkin, 1947

The penicillin structure work by Hodgkin was a monumental task. What is more, the labor-intensive work was performed during the Second World War! She began the project in 1940, after a chance encounter with Ernst Chain, who would later share the Nobel Prize with penicillin discoverer Alexander Fleming and Howard Florey, who, along with Chain, would mass-produce the renowned antibiotic. Professor Chain had promised to deliver penicillin crystals, which were need for X-ray diffraction analysis, to Hodgkin once he (Chain) made them. It was reported that Hodgkin was quite pleased when Chain made good on the delivery of his promised crystals.

Once the promised penicillin crystals arrived at the Hodgkin laboratory, the project appeared to be doomed from the start. First, there were in existence several structural shapes to penicillin, and it was unclear, which was the most critical one. Second, it was difficult to ascertain the necessary chemical conditions that were needed to obtain properly packed penicillin crystals. The worst problem, however, was that no one knew what chemical groups made up the penicillin molecule. Hardly anyone had studied much of penicillin’s chemistry. What little penicillin chemistry that was known turned out later to be inaccurate. Thus, Hodgkin was starting the project entirely from scratch!

Nevertheless, Hodgkin persisted in preparing the penicillin crystals with tightly packed atoms, conducting the X-ray diffractions, generating the electron density maps, and calculating the locations of the atoms. In the end, she prevailed and constructed a molecular model, for the first time in history, of the famous penicillin.

Startlingly, Hodgkin’s penicillin structure had an interesting chemical part, called the β-lactam ring. The β-ring structure was penicillin’s active site! Yet, almost immediately, the work was intensely criticized as being wrong! It was not. Hodgkin was correct all along in showing that penicillin had the β-lactam ring!

Before Hodgkin elucidated the penicillin structure, she had already established herself in the research community by determining the structures of pepsin (graduate work), cholesterol, and other steroids. She would later go on to discover the molecular structures of vitamin B12 and insulin!

5) Now insulin—what is it about its chemical structure and annotation—and by the way—when do those boxes and lines (and what do you call them) first start to appear and who is responsible for those things?

The boxes that you referred to first appeared as Hodgkin’s remarkable invention for visually displaying her molecular structures; see the figure above for penicillin. These boxes appeared with penicillin when Hodgkin discovered its molecular configuration. The lines indicate the atomic bonds that connected the atoms to form the larger molecule.

As a tribute to Hodgkin, these boxes became a permanent fixture in the Science Museum Group, in London, England. Her box displays of penicillin and insulin are featured alongside the DNA model display of Watson and Crick and other famous artifacts of scientific breakthroughs.

Hodgkin’s work with insulin started in 1935 by growing crystals in Bernal’s lab as a graduate student. At first, the insulin turned out to be too large for molecular analysis, and she instead focused on penicillin, a much smaller molecule. Hodgkin would not return to insulin for another 20 years.

Compared to penicillin, insulin was a large molecule. The molecule of insulin was thought to harbor over 777 distinctive atoms, with two polypeptide chains, called A and B, 51 amino acids (21 and 30 amino acids in the A and B chains, respectively) and molecular weight of 5,808 Daltons. Thus, studying the atomic structure and its configurational shape was a massive undertaking. Determining the molecular structure of insulin, in 1969, a full 34 years after she first encountered its crystals, was a profound success.

The historical insulin work was published in Nature in November of 1969. Surprisingly, because the order of authors in the paper was alphabetical, Hodgkin, who was the principal investigator of the project, was listed in the sixth position of eight authors.

6) Now back to Dr. Hodgkin: X-ray crystallography—what is it, and how does it fit into the big picture?

As an undergraduate student, Hodgkin acquired a formal education in X-ray crystallography, a method developed by pioneer Dr. Max von Laue and expanded upon by father and son William Lawrence and William Henry Bragg, respectively. In 1914, von Laue received the Nobel Prize, and the Braggs would acquire the Nobel during the following year. Incidentally, Hodgkin, at 16 years of age, was given a book written by W.H. Bragg about crystals and the famous X-ray crystallography method.

https://upload.wikimedia.org/wikipedia/commons/1/11/X-ray_Crystallography.jpg

https://upload.wikimedia.org/wikipedia/commons/1/11/X-ray_Crystallography.jpg

Figure: X-ray crystallography

The technique of X-ray crystallography and diffraction became vitally significant for determining the molecular structures of essential molecules, especially larger ones, like proteins. In the method, X-ray beams are fired at a crystallized unit. The X-rays will diffract (deflect) away as they bounce off the crystal, exposing a sheet of photographic film, which is sensitive to the diffracted X-rays. The resulting spots produced on the film sheets are reflective of the angles of the deflected X-ray beams. These angles shed information regarding the shapes of the molecular crystals. The work required large amounts of X-ray diffraction films and mathematical calculations to produce electron density maps, from which one could deduce the locations of individual atoms within the molecules. It was arduous work. It also required a significant degree of comprehension. Hodgkin was widely known as outstanding in this field.

7) She was the third woman to win the Nobel Prize (Who were the first two?), and for what did she win it?

Hodgkin earned the Nobel Prize in chemistry in 1964 for her work in elucidating structures of vitally critical biomolecules, such as penicillin, cholesterol, and insulin, but especially for her vitamin B12 structure determination, completed in 1955. The first two women to win the Nobel Prize in chemistry before Professor Hodgkin were Dr. Marie Sklodowska Curie and her daughter Dr. Irène Joliot-Curie.

Hodgkin’s Nobel-worthy discovery of vitamin B12 structure started in 1948, after the end of World War II. An investigator, Dr. Lester Smith, from a pharmaceutical company called Glaxo, provided some intriguingly deep-red crystals to Hodgkin. The pure crystals were derived from a substance called cobalamin, which is now known as vitamin B12.

Compared to the penicillin structure, with less than a dozen atoms, vitamin B12 was massively complicated, as you will see below. She took excellent photographs using her X-ray diffraction technique in one night of work, refined the crystal growth, and repeated the X-ray analysis. In the end, she took over 2,500 X-ray photographs of vitamin B12. Hodgkin then spent the next six years working through the massive amount of electron density map data. The work was an astounding success.

During this painstaking work, a curious episode occurred. An unnamed collaborator in Hodgkin’s laboratory was so dispirited by the considerable crystal work, that he emptied all the solvents and reagents he could find into a large vat, and he went on a vacation. In his absence, a tar-like mass of rock developed and was waiting. Hodgkin was said to have chipped away at the tar rock, and at the bottom of the mess, she found a useful vitamin B12 crystal.

The dispirited collaborator was never able to reproduce the method.

8) 0kay—below is B-12—or, I am told—for the layperson—what does this mean, why is it important, and how is it linked to Dorothy?

https://upload.wikimedia.org/wikipedia/commons/7/79/Cyanocobalamin-b12.png

https://upload.wikimedia.org/wikipedia/commons/7/79/Cyanocobalamin-b12.png

Figure Cyanocobalamin – vitamin B12

As you now know, Dr. Dorothy Hodgkin earned the Nobel for elucidating structures of medically and biologically important molecules. Her elucidation of the vitamin B12 structure was to garner much attention, especially that of the Nobel commission. The structure of vitamin B12, above, is, as you can see, quite complicated in its structural nature. You will no doubt notice the cobalt atom, Co, complexed in the center of the molecule.

The cobalamin, or vitamin B12, is indispensable as a co-factor for enzymes that undergo alkylation. Medically speaking, the molecule is needed for alleviating the detrimental effects of pernicious anemia, which is the result of a deficiency in dietary vitamin B12.

Before the discovery of vitamin B12 started by the clinical observation by Drs. George Minot and William Murphy that raw liver could treat the terrible pernicious anemia. The fresh liver was said to harbor the anti-pernicious anemia factor, which we now know as the vitamin B12.

Vitamin B12 is not synthesized by plants or animals. The co-factor is “vital” for alkylation. Some organisms acquire vitamin B12 by diet, like humans, or by gut bacteria, which make it for their hosts, like herbivores.

The prime source of vitamin B12 for humans is meat. Once a person acquires the vitamin B12, another protein called intrinsic factor, made by the stomach, binds the vitamin to form a complex of the two molecules. The vitamin B12-intrinsic factor complex is then absorbed from the gut and transported to the blood. The vitamin B12 is later released from the complex and taken up by blood plasma proteins called transcobalamins, which then carry the vitamin B12 to the tissues and cells, where it serves as a critical co-enzyme for a host of biochemical reactions.

9) She published something about steroids—What did she find out about steroids at that time—and what do we know about them now in terms of their use as a medical treatment.?

Dr. Hodgkin started work with the steroids, namely cholesterol, in the late 1930s. Until her involvement in the project, many had failed to learn the structure of cholesterol. Several investigators had proposed incorrect molecular models of cholesterol. The field had become a quagmire.

Hodgkin and her student lab assistant, Harry Carlisle, were the first to deduce the cholesterol structure correctly. It was a magnificent discovery, and it became a sort of triumph for Hodgkin. She chose to investigate cholesterol precisely because it was known to be complicated. Furthermore, she was able to shed light on the controversy over its various proposed structures. All the previously known structures were wrong, and hers in 1940 was correct.

In today’s modern world of cholesterol studies, while it can otherwise serve useful purposes like as a precursor to vitamin D production or in regulating membrane fluidity, many medicines are devoted to the inhibition of its manufacture in the body. When cholesterol is complexed to individual fatty acids, its transport to the tissues and their cells, is unhealthy, as these complexes can build up in arteries that supply oxygen to the heart, causing coronary artery disease. Such conditions are associated with a higher propensity for stroke and heart attacks.

10) What have I neglected to ask?

Dr. Dorothy Crowfoot Hodgkin’s life and scientific career might best be described as trailblazing, if not extraordinary. She was truly remarkable in many of these respects.

Unfortunately, at the age of 28, Hodgkin acquired an early case of rheumatoid arthritis, presenting primarily in her hands, and the ailment would affect her tremendously for the remainder of her life. Nevertheless, she persisted in conducting her X-ray crystallography experiments. Hodgkin painstakingly took thousands of photographs and vigorously performed a vast array of calculations to generate contour maps for crystal structure elucidation.

During the penicillin structure investigations in the early 1940s, Hodgkin became a pioneer in the use of the world’s first IBM computers, which she used to help with the necessary calculations. She started with rudimentary computers, which at the time consisted of mechanical devices aimed at merely conducting the requisite calculations. She used the punch cards, which at the time were considered innovative. As the computer technologies improved, Hodgkin advanced with them. She was to use computers for the remainder of her scientific career.

Hodgkin was gifted in mathematics and data analysis, and she was talented in overcoming entrenched sexism to establish her dedicated research laboratory and contribute multiple astonishing scientific discoveries. These scientific achievements have had relevance in many medical and scientific avenues, such as in diabetes, coronary artery disease, pernicious anemia, antibiotic medicines, metabolism of disorders, and in fundamental biochemistry.

Meanwhile, she was able to meet the demands of motherhood, successfully raising four children with her husband, Thomas. Such accomplishments were genuinely extraordinary as she triumphed over adversity on many fronts. As a trailblazer, Dorothy Hodgkin became vanguard, inspiring many women to pursue science and serving as a key role model for brilliant young scientists to follow her example.

In 1965, Hodgkin was awarded the Order of Merit by Queen Elizabeth and given a private audience with the Queen. Hodgkin was the second woman in history to receive the honor, with Florence Nightingale having first receiving it, in 1907. During the award banquet dinner for Hodgkin, Henry Moore, an artist, sat adjacent to her. Moore noticed Hodgkin’s severely deformed hands and fingers, which were terribly gnarled by her rheumatoid arthritis and was so touched by her affliction that he asked her if he could make a sketch of her hands. The drawing of Hodgkin’s hands hangs alongside her portrait in the halls of the Royal Society. She later reported that of the two works of art, the drawing of her hands had become her favorite.

Hodgkin was also acutely involved in politics of the day, starting in the 1960s and being active well into the 1980s. She had been a member of the labor party. Towards this, she wrote extensively on the issues of war, peace, the nuclear arms race, economics, and of science policy. She became an influential force as chancellor of Bristol University.

An interesting fact about Hodgkin was that as a chemistry instructor in the 1940s, one of her students, Margaret Roberts, later became prime minister of England. She is better known as Margaret Thatcher. In the 1980s, Hodgkin was to write to Thatcher and implore her to visit the Soviet Union before she (Thatcher) criticized it. Thatcher did precisely that, visiting Mikhail Gorbachev and improving the international relations between the Soviet Union and England.

On the 29th of July in 1994, at the age of 84 years, Hodgkin passed away after suffering a stroke.

For additional information about this truly extraordinary and inspiring scientist, visit:

Video: https://www.bbc.co.uk/programmes/m0008wkk

Video: https://www.bbc.co.uk/programmes/b04lc6h3

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