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Notes on the Early History of the Interaction between Physical Chemistry and Biochemistry: The Development of Physical Biochemistry Mercedes Guzmán-Casado and Antonio Parody-Morreale* Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, 18071 Granada, Spain; *[email protected]

The interaction mentioned in the title has led to the discipline that we know today as physical biochemistry.1 What is physical biochemistry? If we accept the definition of physical chemistry as “the part of science that describes the world in terms of atoms, molecules and energy” (1), then we could define physical biochemistry as the part of science that describes the biological world in terms of atoms, molecules, and energy. This means the study of the structure and the physicochemical properties of molecules and molecular aggregations that are biologically functional, and the use of structural and physicochemical knowledge to explain the biological function. Physical biochemistry forms part of the curriculum of university students, mainly biochemists, throughout the world. The first course in physical biochemistry by that name began in the Department of Physical Chemistry in the Harvard School of Medicine toward the end of the 1940s. It is striking that this department of Physical Chemistry belonged to the School of Medicine, a rare situation even in the United States at the time of the school’s inception in 1919. The department changed its name to the Department of Physical Biochemistry in 1950. The professors of the course in question were John Edsall and Jeffries Wyman, who in 1958 compiled their teaching work in the book Physical Biochemistry, the first textbook for such a course (2). Although they initially planned for two volumes, only the first was published. It could be said that the 1950s saw the birth of physical biochemistry as a university discipline. By then, several courses had been taught, a department had been created, and the first textbook had been published. This resulted from powerful currents in the world of biological research moving toward a physicochemical explanation of reality. The same decade marked the appearance of molecular biology (the product of, among other factors, the interest in biology of physicists such as Schrödinger, Szilard, and Delbrück and physical chemists such as Bernal and Pauling), one of the most momentous scientific and intellectual movements of the 20th century. In what follows, an overview of some of the salient points in the emergence of physical biochemistry is given. I have used it as an introduction to the discipline in the first lecture of a yearlong course for chemistry and biochemistry students that I teach at the University of Granada. Historical Notes (3)

Early Studies In 1887, J. van’t Hoff and W. Ostwald founded the journal Zeitschrift für Physikalishe Chemie. This event marked the appearance of physical chemistry as an independent area of science, implying a new way of viewing nature, in the sense of using the principles of physics to describe chemical systems.

We can consider that physical biochemistry emerged as a subdiscipline of physical chemistry as a result of the application of the same intellectual attitude in the study of molecular biological systems. In any case, long before this conceptual revolution, when science was regarded as an undivided whole, certain biological phenomena that today are the object of intense physicochemical study attracted considerable attention from philosophers of natural history. One of the first of these phenomena was bioluminescence, and one of the first studies on the emission of light by animals was made in the 17th century by the German Jesuit Athanasius Kircher, who devoted two chapters of his book Ars Magna Lucis et Umbrae to this topic. Curiously, in this work he discarded as invalid the common belief of his time that an extract from glowworms could serve to illuminate houses. The relationship between electricity and biology also drew speculation in the 17th century, and intense experimental work during the 18th and 19th centuries. At the end of the 18th century, the field was dominated by the controversy between Luigi Galvani and Alessandro Volta on the correlation between electric impulse and the contraction of a frog’s leg. Galvani and his disciples held that the current generated by the animal caused the muscular contraction, whereas Volta’s contended that the leg of the frog served only to measure very small external power differences. Volta’s work led him to invent the electric battery, an event of paramount importance, which obscured the controversy with Galvani. Ultimate acceptance of Galvani’s hypothesis began when the 19th-century German philosopher Du Bris-Reymond constructed a galvanometer sensitive enough (5.1 km of wire wrapped in 24,000 coils) to detect the current generated by animal tissues. This illustrates the importance of developing the proper instrument to achieve significant advances in any experimental science. Other phenomena that in the 19th century prompted strong interest were diffusion through natural membranes and osmotic pressure; experiments on osmotic pressure were invariably conducted with biological membranes. The phenomenon of osmotic pressure was first described by J. A. Nollet, a professor of experimental physics. Adolf Fick, studying diffusion, published Die medizinishe Physik—perhaps the first textbook of biophysics—in 1856. Curiously, Fick did not formulate the laws of diffusion from experimental data, but rather by analogy with the laws that govern heat conduction. Later experiments demonstrated that this analogy was appropriate. Above I have sketched some scientific problems that were multidisciplinary in the sense that we use the word today. From a formal perspective, the analysis of the physicochemical aspect of these problems is striking inasmuch as physical chemistry probably did not exist as a distinct discipline at that time. Only as this field began to delineate itself at the end of

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the 19th century do we find in both the conceptual and the practical treatment of certain biological problems a methodology we can genuinely call physicochemical. The phenomenological solution to the problem of the transport of oxygen by hemoglobin is one of the first cases of this.

Proteins At the beginning of the 20th century in Copenhagen, Böhr, Hasselbalch, and Krogh performed perhaps the first quantitative measurements of the functional properties of a biological macromolecule: the saturation of hemoglobin by oxygen in the presence of variable quantities of carbon dioxide. Figure 1A shows the graph from their 1904 publication (4). It reveals what today is known as the homotropic cooperative character of the oxygen binding to hemoglobin, and the heterotropic control exerted by carbon dioxide, which diminishes the affinity for oxygen. From a functional standpoint, the importance of the obvious sigmoidal response was immediately recognized. This type of behavior enabled a maximum discharge of oxygen into the muscle capillaries from a maximum

saturation of the macromolecule at the partial oxygen pressure in the lung alveoli. Although at that time the structure of the hemoglobin molecule was not known, it was assumed that there was one heme group per functional unit (the molecular weight of which has been determined very precisely by O. Zinoffsky in Zurich as 16,669 g mol᎑1) and that an oxygen molecule was bound to each heme group. If this were so, however, and the oxygen binding followed the law of mass action, the binding curve should be hyperbolic and not sigmoidal, as found experimentally. One of the few scientists who saw this problem and proposed a solution (in 1910) was A. V. Hill, who, after finishing his studies in mathematics at Cambridge, joined the laboratory of physiologist Joseph Barcroft. In 1922, Hill and Otto Meyerhoff would receive the Nobel Prize in Medicine for their discoveries, Meyerhoff in the chemistry of muscle contraction and Hill for correlating that chemistry with heat production. Hill postulated that hemoglobin monomers associated into aggregates and that the differential oxygen binding to these aggregates was the basis for the sigmoidal behavior observed.

Figure 1. A: Oxygen binding curves of hemoglobin at different CO2 partial pressures. Redrawn from the paper by Bohr and colleagues (4). B: Changes of the heme site upon binding of oxygen to hemoglobin according to Perutz’s mechanism based upon X-ray structural studies (5). C: Quantitative thermodynamic model for Perutz’s mechanism (6). The binding partition function is shown. Its expansion leads to a fourth-power polynomial in x, the oxygen pressure, in which the terms represent the relative population of the different ligation states of the protein referred to the unligated one. The polynomial coefficients are the Adair constants for the binding equilibria.

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His hypothesis for the reaction of oxygen with hemoglobin (Hb) postulated two steps nHb (Hb)n (HbO2)n (Hb)n + nO2 and led to an expression for the saturation (θ) of hemoglobin with oxygen as θ = kpn/(1 + kpn) where k is a constant and p is the partial pressure of the gas. Hill evaluated n values of between 2.5 and 3.0, which accounted rather well for the experimental data except for the end points, which in any case were difficult to obtain experimentally. The non-whole value for n was explained by assuming that hemoglobin was a mixture of different states of aggregation. The model explained the sigmoidal nature of the saturation curve and was accepted as the solution to the problem. Apparently everyone overlooked the fact that the existence of the monomer n-mer equilibrium implies a hemoglobin concentration dependence of its equilibrium with oxygen, which in fact was not observed experimentally. In 1919, Adair (according to Edsall, one of the few biochemists who at that time had read the work of J. W. Gibbs and understood it), also at Cambridge, began to perfect the techniques for measuring osmotic pressure. In 1925 Adair reported that the weight of hemoglobin was four times greater than the weight previously determined by Zinoffsky for a functional unit containing one heme group. Adair then postulated the existence of four functional subunits in hemoglobin, all having the same molecular weight. In this formulation the equilibrium of association with oxygen was based on the existence of intermediate saturation states and four equilibrium constants for the successive steps of the reaction. Adair’s work helped in understanding, at least phenomenologically, the biological functioning of hemoglobin and may be the first example of an approach to understanding a biological problem from a physicochemical perspective—from the standpoint of both experimental technique (measurement of osmotic pressure) and applied concepts (thermodynamic formulation of the problem). When Adair published his evaluation of the molecular weight of hemoglobin, his results were received with skepticism by the scientific community until shortly afterward Svedberg, without knowing of Adair’s work, determined the same value from measurements of sedimentation equilibrium. Svedberg, another key scientist in the development of what we now know as physical biochemistry, was a professor at the University of Uppsala and received the 1926 Nobel Prize for his work. He developed the ultracentrifuge, which could subject samples to several hundred thousand times the force of gravity. This instrumentation, though conceptually very simple, presented imposing technical challenges in its construction. The ultracentrifuge and its associated optical system made it possible to determine, with great precision, molecular weights of soluble proteins. This technique was of enormous importance in the structural study of biological macromolecules in the 50 years following its development. Suffice it to say that the 1956 experiment of Meselson and Stahl, which demonstrated the semiconservative nature of DNA replication, was performed in an ultracentrifuge.

Of additional importance to our history is the fact that the first experiments utilizing the ultracentrifuge provided exact and reproducible molecular weights and thus contributed to establishing the concept that macromolecules have defined chemical structures—in contrast to their being nonspecific aggregates of small molecules, as would result from the hypothesis of the colloidal state of aggregation of matter. This hypothesis had already received the coup de grâce in the early 1920s in Staudinger’s work with synthetic “colloids”. A disciple of Svedberg whose work is also of prime importance in the history of the study of biological macromolecules was Tiselius, who too was a professor at Uppsala and received the Nobel Prize (1948). Tiselius developed electrophoretic techniques for the characterization and separation of proteins and was instrumental as well in illustrating the non-“colloidal” nature of macromolecules. By the beginning of the 1930s, the enzyme pepsin had been crystallized by accident in Svedberg’s laboratory. With such crystals, John Bernal and Dorothy Crowfoot Hodgkin, in Cambridge, obtained the first clearly defined X-ray diffraction patterns of a protein. Their results, published in Nature in 1934, marked the birth of protein crystallography. The technique was developed from the observations of Max von Laue in 1912 and the work of Lawrence Bragg and his father William Henry Bragg and was first applied successfully to the study of minerals, which have a low number of atoms per unit cell. Proteins, which are extraordinarily complex, would not yield successful results until some 20 years later, with the resolution of the structures of myoglobin by Kendrew (1957) and hemoglobin by Perutz (1959), both of whom received the Nobel Prize in 1962 for this work. Perutz began to work in the laboratory of Bernal in 1936 and was able to choose between hemoglobin and chymotrypsin for his studies. He chose the former. In his own words, he could later have justified the choice because at the time hemoglobin was the protein whose function was known in most detail. For Perutz the problem of the moment was only to establish the structure of the protein. Bernal, however, viewed the study of structure as the most direct path to an explanation of biological function.

Pauling’s Work It is perhaps the work of Linus Pauling in which we can best appreciate Bernal’s idea put into practice. By the beginning of the 1930s, Pauling had formulated the theories of valence bond and resonance that, in a spectacular way, introduced the principles of quantum mechanics to explain the nature of chemical bond. Pauling began to orient his work toward the biological world partly because Warren Weaver, director of the program of grants for research in the natural sciences of the Rockefeller Foundation (which began financing Pauling’s work in 1933) informed him that the Foundation would be more interested in his work if he could use biological substances. Weaver’s idea was that the moment had come to take a more basic (i.e., molecular) approach to the problem of life. Pauling began by working with hemoglobin in 1935. At first, his intent was to establish whether the bond of hemoglobin with oxygen involved a specific reaction with iron or a nonspecific adsorption to the surface of the macromolecule. He attacked the problem by measuring the magnetic properties of hemo-


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globin, oxyhemoglobin, and carbomonoxyhemoglobin. In addition to establishing that the bonding was due to a direct interaction with iron, a most important finding was that an appreciable change in macromolecule structure resulted from the binding of oxygen and carbon monoxide. During the same period, Pauling studied the function of hemoglobin and published the first model to explain the sigmoidal binding curve for oxygen according to molecular parameters. This model would be generalized by Koshland, Nemethy, and Filmer in the 1960s to explain the cooperative interaction of any macromolecule with a ligand. The validity of a model explaining a biological function cannot be tested without structural information, and from this standpoint Pauling approached the study of protein structure. Unlike Perutz, he began with the simplest systems (an approach Bernal considered slow and indirect), analyzing the structure of amino acids and the peptide bond. After 10 years of investigation Pauling and his collaborator Corey had gathered enough information to postulate, from the construction of molecular models, a structure for the helical disposition of amino acids in a protein: the α-helix. Pauling’s hypothesis explained the experimental data from the crystallography group of Cambridge while at the same time questioning their approach. According to Pauling, it was the lack of detailed knowledge of the principles of structural chemistry—that is, of the nature of chemical bond—that hindered Bragg, Kendrew, and Perutz from correctly interpreting their own results. Pauling received the Nobel Prize in 1954 “for his work on the nature of chemical bond and its application to the elucidation of the structure of complex substances.” Although contributing to its eventual solution, Pauling fell short of his own personal goal of determining the complete three-dimensional structure of a protein.

Harvard Medical School Special consideration in relation to the early development of physical biochemistry is owed to the Physical Chemistry Department of the Harvard Medical School. In the 1920s, under the direction of E. Cohn, the research of this department centered on the study of protein solubility, with the idea of establishing the general rules for protein separation (“salting out”) and crystallization. Cohn understood the importance of the electric charge of proteins as a determinant of their thermodynamic activity and therefore of their solubility. The impossibility of applying the new theory of Debye and Hückel for electrolyte solutions to molecules as complex as proteins steered the research of the group toward the study of physical chemistry of the constituent amino acids (fundamentally, their behavior as electrolytes in solution). In this sense, it could be said that Cohn’s attitude coincided with Pauling’s: to ascertain the properties of the whole from the properties of the parts. Wyman’s Work The work with amino acids, largely carried out in the 1930s, benefited greatly from the collaboration of physical chemists such as Scatchard and Kirkwood of MIT. Wyman was of particular note in determining the dielectric constants of amino acids in solution. Gradually, Wyman’s attention would veer toward larger molecules—specifically, hemoglobin. His first work in this area appeared at the end of the 1930s and subsequently his entire scientific life was devoted to the study 330

of the biological function and regulation of hemoglobin. It might be said that his professional life paralleled that of Perutz. However, Perutz’s interest in hemoglobin was structural, whereas Wyman’s was in the thermodynamic description of the protein’s behavior. In 1951, Wyman introduced the concept of allosterism to explain the cooperativity in the binding of oxygen to hemoglobin: allosterism involves specific conformational change in the macromolecule induced by the presence of the substrate. In 1964, this concept was generalized by Monod, Changeux, and Wyman himself to include the effect of different ligands on the functional activity of a biological macromolecule. The idea of allosterism in the context of the regulation of enzymatic activity had a strong influence in establishing the basis of a connection between independent metabolic circuits. It is noteworthy that once the structure of hemoglobin was resolved, Perutz began working on the explanation of its biological behavior from the standpoint of structural studies and in 1970 published a mechanism for oxygen–hemoglobin interaction. Figure 1B shows the heme stereochemistry of oxygen binding in Perutz’s mechanism, in which two allosteric forms of the protein (R and T) are considered, according to Wyman’s ideas. These ideas are taken into account in a thermodynamic model shown in Figure 1C, a quantitative interpretation of Perutz’s mechanism. It is interesting that in this model, the interaction between sites, according to Pauling, is also considered in the binding of oxygen to the allosteric T form of the protein.

Nucleic Acids The long scientific journey of Perutz, from studying the structure of hemoglobin to explaining its biological function on the basis of its structure, spanned some 30 years. For Watson and Crick, in the study of DNA, the journey was much shorter. The description of this event has been left to the end of this historical overview because it is the cornerstone of molecular biology. Like Perutz, Watson and Crick worked in the Cavendish laboratory, where they analyzed the X-ray diffractograms produced by Franklin and Wilkins. This led to the publication in 1953 of an article in Nature, which postulated a structure of DNA as a double helix stabilized by the pairing of specific bases. The repetitive pattern of the double helix, as revealed in the diffractograms, facilitated the resolution of its structure in a very short time compared to the time required to evaluate the overall structure of proteins, which have an irregular structure. To interpret the experimental data, Watson and Crick constructed molecular models, as had Pauling with the αhelix. In fact, according to Watson in his book The Double Helix, Pauling also competed to resolve the structure of DNA, but the Cambridge group was successful first. Pauling, slightly ahead of Watson and Crick, postulated a structure for DNA that was helical but had three chains instead of two; the phosphate groups were arranged toward the interior, requiring hydrogen in the connection P–O…H–O–P; and the charges were neutralized, whereupon the DNA would not be an acid. The significance of the discovery of the structure of DNA was that the structure contained an explanation for the genetic code and immediately suggested the possible copying mechanism. This is then a prime example of biological function absolutely correlated to the chemical structure. It is also striking

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that it fulfils the principles expressed by Erwin Schrödinger in his book What Is Life?, published in 1944, in which he predicted that the gene is a physical substance that, while completely stable, is capable of expressing immense variety. Schrödinger believed that the gene had to be an “aperiodic crystal” in the sense that, unlike an inorganic crystal (NaCl, for example), it would not have a repeated elemental unit. On the other hand this aperiodic crystal would have to contain certain fixed molecular structures, the association of which could be used to construct an alphabet in the same way as points and dashes constitute the Morse alphabet. The point and the dash of the genetic code are represented by 4 bases (adenine, thymine, guanine, and cytosine), and after the resolution of the structure of DNA, it was established that the letters of the alphabet (each of which would correspond to a specific amino acid of a protein sequence) would be formed by particular combinations of three of these four bases. Schrödinger’s book, in its effort to achieve a unified and universal vision, exerted powerful influence on the scientists who later led the revolution in molecular biology. Closing Remarks This historical account is neither exhaustive nor complete, but it does provide a sample of the progressive interest that certain chemists, physical chemists, and physicists showed for the study of systems of biological interest throughout the 20th century. The story ends in the 1950s, when the first structures of nucleic acids and of proteins were resolved by means of X-ray diffraction. This more or less isolated interest has only increased in the last 50 years, and today physical biochemistry can be considered an established discipline (in the mid-1970s the journal Biophysical Chemistry was first published, and a permanent biophysical section was recently established in The Journal of Physical Chemistry). The molecular size span of molecules of biological interest is exceedingly broad, ranging for example from an amino acid such as glycine, molecular weight 75, to a nucleic acid with a molecular weight of several million. Physical biochemistry is restricted to the study of biological macromolecules. It is with these high-molecular-weight molecules and their aggregates that the biological dimension of the discipline acquires meaning,

since in these molecules the characteristic phenomena of the chemistry of life are made possible: self-replication and the ability to control complex metabolic phenomena, through the linkage of interactions. Note 1. The term “biophysical chemistry” is also used to name the discipline.

Literature Cited 1. Eisenberg, D; Crothers, D. Physical Chemistry with Applications to Life Sciences; Benjamin/Cummings: Menlo Park, CA, 1979; p xvi. 2. Excellent textbooks are now available, among them: Van Holde, K. E., with Johnson, W. C.; Ho, P. S. Principles of Physical Biochemistry, 3rd ed.; Prentice-Hall: Upper Saddle River, NJ, 1998. Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry; Freeman: San Francisco, 1980. Bergethon, P. R. The Physical Basis of Biochemistry ; Springer: New York, 1998. Two other textbooks: Bull, H. B. An Introduction to Physical Biochemistry; Davis: Philadelphia, 1971. Marshall, A. G. Biophysical Chemistry. Principles, Techniques and Applications; Wiley: New York, 1978. 3. I consulted the following sources in preparing this paper. Judson, H. F. The Eighth Day of Creation. The Makers of the Revolution in Biology; Simon and Schuster: New York, 1979. Edsall, J. Fed. Proc. 1980, 39, 226–235. Edsall, J. In Selected Topics of the History of Biochemistry: Personal Recollections; Comprehensive Biochemistry Vol. 36; Semenza, G., Ed.; Elsevier: Amsterdam, 1985; pp 99–195. Watson, J. D. The Double Helix; Atheneum: New York, 1968. Schrödinger, E. What Is Life? Cambridge University Press: London, 1967. I also consulted the 1986 edition of the Encyclopaedia Britannica. 4. Bohr, C.; Hasselbalch, K. A.; Krogh, A. Skan. Arch. Physiol. 1904, 16, 401–412. 5. Perutz, M. F. Nature 1970, 228, 726–739. Figure adapted from Perutz, M. F.; Fermi, G.; Luisi, B.; Shaanan, B.; Liddington, R. C. Acc. Chem. Res. 1987, 20, 309–321. 6. Di Cera, E.; Robert, C. H.; Gill, S. J. Biochemistry 1987, 26, 4003–4008.


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