“You know, hemoglobin is a wonderful substance. I like it. It’s a red substance that brings color into the cheeks of girls, and in the course of my hemoglobin investigation I look about a good bit to appreciate it.”
– Linus Pauling, March 30, 1966
Seventy-five years ago, in 1935, Linus Pauling began publishing his research on the protein hemoglobin with a set of papers titled “The oxygen equilibrium of hemoglobin and its structural interpretation” appearing in Science and the Proceedings of the National Academy of Science .
In the fall Pauling extended this work and began collaborating with newly minted Caltech Ph. D. Charles Coryell, on the problem of the binding of oxygen to hemoglobin in the formation of the compound oxyhemoglobin. In April 1936, the duo published a paper specifically devoted to the subject, “The magnetic properties and structure of hemoglobin, oxyhemoglobin, and carbonmonoxyhemoglobin,” an important article which appeared in PNAS.
In order to better understand this early hemoglobin work, it is important to first discuss some of the basics of the hemoglobin molecule. Hemoglobin is a major protein component in the cytoplasm of red blood cells, and is made up of two distinct parts – the heme and the globin. Its primary function is to facilitate gas exchange: it picks up oxygen in the lungs, carries it to the tissues, and returns to the lungs in order to expel the carbon dioxide produced in the tissues.
There are four hemes per hemoglobin molecule, and each is made up of a single iron atom surrounded by a porphyrin ring. Each heme has the ability to bind to a single oxygen dimer, therein giving hemoglobin the capacity to bond with four molecules of O2. The globin is the main protein component of the molecule. Carbon dioxide, rather than competing with oxygen for a binding site at the heme, instead binds to the globin.
In their 1936 paper, Pauling and Coryell tackled the question of how oxygen binds to hemoglobin by looking at the molecule’s magnetic behavior, using an experiment involving bovine blood and magnets. In a 1976 interview, Pauling provided this description of their experimental design.
It occurred to me that the same magnetic methods that we had been using to study simple compounds of iron, in order to determine the bond type, could be used to study the hemoglobin molecule. One of my students, Charles Coryell, and I, then got some blood, cattle blood, and put it into an apparatus. It consisted of a balance, which we had fitted out in such a way that a wire was suspended from one arm of the balance through a hole in the base of the cabinet, and held a tube. This tube was placed between the poles of an electromagnet. We filled it with blood, oxygenated blood, and balanced it to measure its weight. Then we passed an electric current through the coils of wire and the apparent weight changed.
From the experimental results, the pair found that oxyhemoglobin contains no unpaired electrons, although free oxygen molecules contain two, and each heme contains four. This was something of a surprise as, quoting from the paper, “It might well have been expected, in view of the ease with which oxygen is attached to and detached from hemoglobin, that the oxygen molecule in oxyhemoglobin would retain these pair of electrons.”
In spite of this possibly more intuitive expectation, Pauling had earlier theorized that oxygen binds to hemoglobin covalently, a prediction which the experiment confirmed. Indeed, it was found that “the oxygen molecule undergoes a profound change in electronic structure on combination with hemoglobin,” and binds to the iron atom in the heme covalently.
This was, however, only one of the striking discoveries that surfaced out of this research. In a deoxygenated hemoglobin molecule, the bonds between iron and the four porphyrin nitrogen atoms surrounding it are ionic. Nonetheless, upon the binding of oxygen, these bonds become covalent, a rather dramatic change. Pauling and Coryell were keen to point this out:
It is interesting and surprising that the hemoglobin molecule undergoes such an extreme structural change on the addition of oxygen. Such a difference in bond type in very closely related substances has been observed so far only in hemoglobin derivatives.
Clearly something of consequence was being observed. In their conclusion, the authors noted as much.
It is not yet possible to discuss the significance of these structural differences in detail, but they are without doubt closely related to and in a sense responsible for the characteristic properties of hemoglobin.
Linus Pauling’s work with hemoglobin continued on and off until his death in 1994, and led to a number of important discoveries – most prominent among them the molecular basis of sickle cell anemia. For more information on Linus Pauling’s hemoglobin research, please visit the website It’s in the Blood! A Documentary History of Linus Pauling, Hemoglobin, and Sickle Cell Anemia.