by Thomas Hager
Hemoglobin could also be taken apart and examined in pieces. It was a conjugated protein, a protein linked to other structures, in this case a ringlike molecule called porphyrin, which in turn was attached to an iron atom, which in turn bonded somehow to the oxygen that hemoglobin carried throughout the body. Pauling had been interested in porphyrin since 1929 when he visited Harvard and James Bryant Conant had shown him some work he was doing with it. This molecular subunit was interesting not only because of its unusual shape—it appeared to be a ring made out of smaller rings—but also because it was found across nature, binding oxygen in the chlorophyll of plants as well as the hemoglobin of a variety of animals. Porphyrin seemed to epitomize the molecular biology idea of the commonality of life at the molecular level: It showed up almost everywhere there was life, playing a similar role in very different organisms.
Porphyrin was made of four subunits, called pyrroles, linked together to make the ring. Each pyrrole itself was a ring of atoms in which single and double bonds alternated, a "conjugated" structure, it was called, a type whose chemistry Pauling had described in one of his papers on the nature of the chemical bond. Pyrrole was a natural starting place for attacking hemoglobin. From there, Pauling could look up a ladder of increasing complexity: Four pyrroles linked together to make one porphyrin ring; one porphyrin ring plus one atom of iron to form a heme; each heme attached to a globular protein to make a heme-globin unit; four heme-globins joined together to make a molecule of hemoglobin. The final structure was almost too gigantic to think about: a roughly spherical mass containing thousands upon thousands of atoms. Pauling quickly concluded that it was far too complex to attack directly with x-ray crystallography, although some optimistic British researchers, funded with Rockefeller money, were trying to do just that. But perhaps he could break the hemoglobin molecule into its component pieces, solve the subunits, and then put it back together again.
Pauling started reading everything he could find on hemoglobin, including an in-depth review of how the molecule bound oxygen. Here was a mystery. Researchers had found that oxygen did not bind to the four hemes in hemoglobin as if they were independent of one another. Instead, the binding of the first oxygen atom made it easier for the remaining three to bind, and losing one oxygen atom made it easier to lose the rest. Somehow the hemes were communicating with each other. This explained in part how hemoglobin could pick up oxygen in the lungs and drop it off elsewhere in the body, but communication within a molecule was a difficult phenomenon to explain in chemical terms.
After a few weeks mulling it over, though, Pauling came up with an ingenious idea. He devised an equation that described the data others had gathered on oxygen binding, then made a mathematical analysis of various relationships among the four heme groups in space until he came up with an orientation that seemed to fit the binding curves. The most likely orientation for the four hemes, he said, was at the corners of a flat square. His idea was later proved wrong, but when it appeared in 1935, it excited discussion among the medical researchers and biochemists who formed the majority of hemoglobin researchers. They had not seen this kind of approach in their field. It was clear that a new talent had appeared with a new way of looking at things.
When Pauling published his idea, it showed Weaver that his commitment to a new research program was real. But Pauling's other work on the hemoglobin molecule was not going as well. The new x-ray technique he was trying on porphyrins quickly proved too cumbersome to provide quick results. Pauling dropped it, telling Weaver that he was not the kind of chemist who spent two years on the detailed crystal analysis of a single compound. Solving the structure of hemoglobin would take twenty years of grinding, repetitive x-ray work and would win someone else the Nobel Prize.
Pauling's one-year Rockefeller grant was due to expire, so he applied for a new grant for more basic research. Weaver could not promise any Rockefeller money for nonbiological work, but he had another idea. He suggested that Pauling use the potential of Rockefeller support as a lever to get Millikan to pitch in five thousand dollars or so of Caltech money for fundamental studies. A display of support like that from his institution plus the progress of Pauling's recent hemoglobin work might convince the Rockefeller board of trustees to extend its grant to three years. Pauling took Weaver's advice and added a threat of his own: He would accept an offer from another university if Millikan did not come through. He got his five thousand per year. Pauling telegraphed Weaver the good news; Weaver quickly wrote him back that the board voted to extend his grant of ten thousand dollars per year for another three years.
In three years, Weaver and Pauling had moved from patron and grantee to coconspirators and friends.
- - -
With his funding stabilized, Pauling was free to try other approaches to hemoglobin and indulge new interests. In 1935 he and a former student turned postdoctoral fellow, E. Bright Wilson, finished the three-year task of transforming the lecture notes from Pauling's wave mechanics course into a book, Introduction to Quantum Mechanics, with Applications to Chemistry. Although sales were less than spectacular for the first few years after publication—quantum mechanics wasn't yet accepted as standard fare for chemists—the book would prove both durable and influential, staying in print for three decades and acquainting generations of students with the importance of the new physics.
Also in 1935, Pauling had a flash of insight that led to a paper on "orientational disorder"—a theory concerning water molecules that explained the residual entropy of ice at absolute zero. It was purely theoretical work, harkening back to his days with Tolman. Thirty years later, when sophisticated computers were finally able to run the numbers thoroughly, Pauling's theory was proven right. Now called "proton disorder," the idea became, as one student of the field said, "the most important American contribution to the modern crystallography of water."
But those were side trips: Hemoglobin was the destination.
- - -
Pauling was now finding that biology could be almost as interesting as chemistry. He spent much of the summer of 1935 at the Caltech marine research facility at Corona del Mar, extracting hemocyanin, a hemoglobin relative, from the blue blood of keyhole limpets, and becoming friends with a young Caltech biology professor named Albert Tyler, who was trying to figure out the mechanism of self-sterility in sea urchins. Tyler's work increased Pauling's interest in another unexplained property of living systems, the ability to distinguish between self and nonself, to have molecules capable of reacting differently with one's own body than with others'. Perhaps there was a chemical connection there as well. Pauling, always looking for new ideas, filed that one away.
By the time he returned to Pasadena, Pauling had come up with a novel way to investigate hemoglobin by looking at its magnetic behavior. Pauling reasoned that when oxygen bound to the iron in hemoglobin, it probably did so covalently—the reaction was highly specific and fairly strong—which would mean that at least one of its unpaired electrons would be paired and its paramagnetism—a quality of molecules with one or more unpaired electrons—would decrease. If he could measure the change in paramagnetism, he might be able to answer the question of how oxygen bound to hemoglobin.
He already had a large, water-cooled magnet that he had borrowed for other work from Hale's private lab, and in the fall of 1935 he put Charles Coryell, an energetic, enthusiastic, newly minted Caltech Ph.D., to work with it. The experiment they set up was fairly simple: A small glass tube containing cow's blood was suspended between the poles of the magnet on a thread attached to the arm of a sensitive balance. When the magnet was switched on, a paramagnetic substance would be drawn in one direction; the balance would measure the degree of magnetic change.
They tried oxygenated blood and deoxygenated blood and appropriate controls and found that Pauling's prediction was right: The bound oxygen lost its unpaired electrons, which became involved in forming a covalent bond to the iron atom. This was an important step forward, proving that oxygen did not just a
dsorb nonspecifically, as some researchers had argued. But there were other surprises in the behavior of the hemoglobin molecule. Pauling and Coryell's experiments showed that the iron atom in hemoglobin underwent dramatic changes when it bound to oxygen as well, switching the bonds that held it in its porphyrin cage from ionic to covalent. "It is interesting and surprising that the hemoglobin molecule undergoes such an extreme structural change on the addition of oxygen," Pauling wrote. "Such a difference in bond type in very closely related substances has been observed so far only in hemoglobin derivatives."
The appearance of Pauling and Coryell's hemoglobin papers in 1936 continued to expand Pauling's renown. They had devised a very clever approach to solving an old problem and provided a clear indication that a physical chemist could do useful work in the field of biochemistry. He was now becoming known by researchers far removed from his original area of expertise. He had entered a new field and quickly begun to conquer it.
Hair and Horn
So far Pauling's work concerned the heme portion of the molecule, but at the same time, Pauling was thinking hard about the rest of it—the globin part, the protein. Protein chemistry was a large and somewhat disjointed field, and Pauling started teaching himself about it through his usual exhaustive process, digesting a vast review of the scientific literature while keeping an eye out for entry points, places where his knowledge of chemistry could offer an insight. He found that proteins were constructed out of a relatively small number of basic building blocks called amino acids, some twenty different ones, but all of them alike in one crucial respect: Each had an identical three-atom backbone, carbon-carbon-nitrogen. The end carbon was part of a carboxyl group, the end nitrogen part of an amino group. The only things that differed among the various amino acids were side chains that were attached to the middle carbon. The great German organic chemist Emil Fischer had shown just after the turn of the century how amino acids could link up into longer chains by joining the two ends, carboxyl group to amino group, through a covalent link that Fischer called a peptide bond. The resulting long molecules he called polypeptide chains. By the 1930s, while not everyone agreed that all proteins contained polypeptide chains, it was increasingly thought that at least some proteins did.
Fischer's work looked reasonable to Pauling, and he began thinking of proteins in Fischer's terms, as long chains of peptide-bonded amino acids. But how could this long-chain structure account for the incredible variety of proteins and the dizzying assortment of roles they played in the body? Were all proteins made of various arrangements of polypeptide chains, or were there other basic structures?
Structure, as always, was Pauling's focus. The way proteins were built, he believed, had to determine their activity. But discovering how they were built seemed an impossible task. Proteins were far too big and complex to solve directly with electron diffraction or x-ray crystallography. Recent work by Theodor Svedberg in Sweden, for instance, proved that hemoglobin was a molecular leviathan, a conglomeration of tens of thousands of atoms. Other proteins were almost as big.
Still, some laboratories were trying to use x-rays to get at least a preliminary idea of the structure of proteins. The two most important sites were in Britain. At Leeds, William Astbury was investigating the molecular structure of wool and other so-called fibrous proteins, hair, horn, feathers, and muscle fibers among them. His results—a surprise to many scientists—clearly showed a regular repeating structure, a crystalline structure, in these proteins.
One mystery Astbury thought he had figured out was wool's ability to stretch without breaking and then snap back to its original length. Wool, like horn, fingernail, and human hair, fell into a general class of proteins called keratin, and by the early 1930s, Astbury, too, was convinced that keratin was made of long, stringlike polypeptide chains. "Molecular yarn," he called it. His x-ray photos showed that when wool stretched, something changed at the molecular level. Although the photos were too fuzzy to allow him to place individual atoms, he saw enough to theorize that the stretched form of keratin—he named it the beta form—was like a polypeptide string pulled fairly straight, while the unstretched or alpha form was somehow puckered or folded. His measurements indicated that the folding occurred in two dimensions rather than three—as though in the alpha form the string were bent into a sawtooth pattern. Further studies showed this same basic pattern in muscle fibers.
This was an important step forward, a molecular explanation for the properties of a protein. Perhaps, Astbury thought, this molecular folding might also explain the contraction of muscle and chromosomes. He began to think of keratin as "the grandfather of all proteins," a basic structure that might explain the workings of all the others.
But he was getting carried away. X-ray diffraction was still inadequate to solve something as big as the polypeptide chain in wool keratin, with its thousands of atoms per strand. It was impossible to say precisely how keratin was built, and the forces that held it in place were still mysteries. It had not even been proved yet that keratin was made of long polypeptide chains—Astbury's work only indicated that it was probable.
Pauling made careful note of Astbury's work.
- - -
While Astbury concentrated on fibrous proteins like keratin, a second British group was focusing on the molecular structure of globular proteins, those that dissolve in the body's fluids, such as hemoglobin, antibodies, and enzymes. The problem here was getting good crystals. It was not that globular proteins did not crystallize—it had been known for years that hemoglobin, for instance, when dried, would form crystals—but when x-rayed, they gave nothing but vague blurs. This led some protein chemists to postulate that globular proteins had no internal structure but were more or less random aggregates of amino acids upon which the real players in the body—nonprotein molecules like vitamins and hormones—oriented themselves.
It was not until 1934 that the Cambridge crystallographer John D. Bernal proved differently. Bernal discovered that globular proteins were like jellyfish: They needed a liquid environment to retain their structure. When dried, they collapsed. By x-raying them suspended in liquids, Bernal was able to get usable patterns, and by the late 1930s, he was the center of a small group dedicated to cracking the structure of globular proteins. He and his coworkers—among them Dorothy Crowfoot and the young Vienna-trained chemist Max Perutz—purified, crystallized, and photographed a number of globular proteins: insulin, hemoglobin, chymotrypsin, excelsin. It was a heroic effort made in miserable surroundings. Perutz recalled their Cambridge laboratory as "a few ill-lit and dirty rooms on the ground floor of a stark, dilapidated grey building" transformed by Bernal’s brilliance into a "fairy castle." They found that all the globular proteins they looked at had an ordered structure and that all of them were terrifically complex—so complex that Pauling, when he saw the data coming from Cambridge, quickly decided that it would take decades to solve their structures through direct x-ray analysis, if it was possible at all. The work of Astbury and Bernal began to attract increasing attention, including the notice of Warren Weaver, who began giving research grants to the British groups in the mid-1930s. Fueled by Rockefeller money, three centers now began to coalesce around the question of precise protein structure. The first two, led by Astbury and Bernal, both of them physicists, worked from the premise that the secret of proteins could be unlocked only by painstaking, direct x-ray analysis of whole molecules, regardless of how huge they were. The third, in Pasadena, led by Pauling, began looking for theoretical approaches, shortcuts based on an understanding of structural chemistry. The difference in 1935 was that the British had generated a good deal of hard data from their x-ray work, while Pauling had yet to publish a single paper on the general topic of protein structure.
Hydrogen Bigamy
Pauling quickly recognized that if he wanted to perform experiments with these strange biomolecules, he would need help.
A group at the Rockefeller Institute for Medical Research had the expertise he lacked. Recent ex
periments by two Rockefeller scientists, Alfred Mirsky and Mortimer Anson, indicated that in some cases denaturation could be reversed; hemoglobin, for instance, could be heated enough to change its shape and lose its ability to carry oxygen, but if cooled carefully, at least some of the molecules could regain their original form and properties. This link between structure and function caught Pauling's eye, but more important was the Rockefeller team's laboratory expertise. On a trip to New York in the spring of 1935, Pauling visited Mirsky, and they hit it off. Mirsky was both intrigued and taken aback when Pauling asked him to come to Caltech for a couple of years. He stammered about how that might be a very nice idea but that his institute's president, Simon Flexner, would never allow him such a leave on such short notice. Pauling said he thought Flexner might. He found the president's office, talked his way in for a meeting, and asked Flexner not only for permission for Mirsky's long visit but also that the Rockefeller Institute pay for it. Flexner, a Rockefeller Foundation board member, had heard about Pauling from Weaver. Amused by the young scientist's brashness and intrigued by his application of chemical techniques to biology, he agreed.
Mirsky arrived in Pasadena at the beginning of the summer and began a further series of experiments on the denaturation of egg white, muscle, and other proteins. Pauling let Mirsky handle the laboratory work while they fed each other ideas about the chemical basis of denaturation—what the process actually did to the structure of proteins. Pauling was interested in the evidence Mirsky and Anson had gathered indicating that denaturation could be seen as a two-level process. The first, which could be caused by relatively mild heating or acid, was often reversible. The second level, caused by higher heat, harsher chemical conditions, or reaction with protein-destroying enzymes, was irreversible. Pauling quickly translated the data into chemical-bond terms. Two levels of denaturation could mean two kinds of chemical bonds, the first involving relatively weak bonds, easily broken and re-formed; the second, perhaps strong bonds that would be harder to break and more difficult to remake. The experimental evidence on the energy it took to break the strong, second-level bonds indicated they were covalent; this in turn could fit with the view of proteins as long chains of amino acids linked with covalent peptide bonds. Breaking such bonds between the links of the chain led to irreversible changes. Strong denaturation basically tore the protein into pieces.