Roughton, Francis John Worsley
ROUGHTON, FRANCIS JOHN WORSLEY
(b. Kettering, England, 6 June 1899; d. Cambridge, England, 29 April 1972)
Roughton came of a line of physicians in Kettering; his father was the fifth consecutive Roughton to practice medicine there, and the family had many connections in the area. He had one sister, five years older than himself, to whom he was greatly attached. He himself might have gone into medicine, but he was subject to attacks of paroxysmal tachycardia, and his solicitous mother regarded him as a cardiac invalid. Later he came to realize that his condition was a disability, not an illness. Because of the tachycardia, Roughton was not called up for military service in World War I. He went to school at Winchester and proceeded to Cambridge in 1918. Though he planned at first to study medicine, his aptitude in basic science soon became apparent, and the inspiration and encouragement of Joseph Barcroft drew him to work on the physiology of oxygen and carbon dioxide transport in blood. Roughton’s aptitude for mathematics and his training in physical chemistry enabled him to deal with problems arising from Barcroft’s work concerning the respiratory function of the blood and the mechanisms involved in the absorption, transport, diffusion, and release of oxygen and carbon dioxide. Roughton could deal with these at a more advanced level than was possible for Barcroft or his contemporaries. Such problems occupied Roughton throughout his entire research career.
In 1923 Roughton became a fellow of Trinity College, a connection that he valued greatly, and took an active part in the affairs of the college. In 1925 he married Alice Hopkinson, whose father was a professor of engineering at Cambridge and whose mother was a Siemens of the German electrical firm. She herself became an active physician with numerous patients. They had a son and a daughter.
One important problem that soon concerned Roughton was the rate of uptake of oxygen by hemoglobin as the blood flows through the lung capillaries, and the release of oxygen from hemoglobin in the tissue capillaries. The time required for an individual red blood cell to pass through a capillary is on the order of one second. Was this long enough to allow these reactions to come virtually to completion, so that the blood could be considered to attain oxygen equilibrium with the lungs and tissues? Although it was known that these processes were rapid, it was not clear whether they were rapid enough to meet this requirement.
The new technique that permitted a quantitative approach to this problem was primarily the creation of another remarkable member of Barcroft’s department, Hamilton Hartridge, who, as Roughton once said, was gifted with “almost diabolical technical ingenuity” in devising new approaches to difficult experimental problems. The apparatus he devised contained two large reservoirs that held two liquids that were to undergo a chemical reaction on mixing. The two liquids flowed out under pressure through tubes that led to a mixing chamber with carefully designed intake jets that achieved complete mixing within a tenth of a millisecond. The mixing chamber was Hartridge’s crucial contribution. After mixing, the resultant solution moved rapidly, in turbulent flow, along an observation tube of known cross section. Knowing the rate of outflow of the liquid, one could calculate its velocity as it moved along the tube. The hemoglobin solutions under study showed strong absorption bands for visible light that shifted in position when hemoglobin combined with oxygen or carbon monoxide. Measurements of light absorption, at a suitable wavelength and at various points along the tube, permitted calculation of the extent of chemical reaction at each point as a function of distance and, therefore, of time.
Before the work of Hartridge and Roughton, accurate rate measurements of chemical reactions in solutions were possible if their times of half completion were on the order of a few minutes or more. Their new method reduced this time to about one millisecond, thus making possible the study of reactions some 50,000 times as rapid as those that could be studied before. The method, however, required large volumes of the reacting liquids in the reservoirs and was most effective for reactions, like those of hemoglobin, that could be followed spectroscopically.
Hartridge and Roughton, in the period between 1923 and 1926, showed that the reactions involving hemoglobin and oxygen were quite rapid enough to go essentially to completion in the short time required for flow through the capillaries. Roughton also showed in 1934, to the surprise of many, that carbon monoxide (CO) combines with hemoglobin some ten times as slowly as oxygen does, although at equilibrium it binds some two hundred times as strongly as oxygen. This comes about because CO, once bound, is released at a rate only about 1/2000 the rate of oxygen release.
The continuous-flow techniques of Hartridge and Roughton were forerunners of such later developments as Quentin H. Gibson’s stopped-flow technique, which permitted accurate study of rapid processes with very small quantities of liquid. G. A. Millikan, who took his Ph.D. with Roughton, developed photoelectric techniques for recording rapid reactions in work published in 1936. Electronic methods of rapid recording, nonexistent in the 1920’s, were essential for later developments. Britton Chance, who also took his Ph.D. with Roughton, was a major figure in these and later developments. Quentin H. Gibson, another leader in the field, also worked closely with Roughton.
Roughton soon became concerned with the kinetic problems relating to carbon dioxide (CO2) transport in blood. Whereas oxygen is transported almost entirely in combination with hemoglobin in the red cell, CO2 is mostly converted to bicarbonate ion; and both CO2 and bicarbonate are distributed between the cells and the surrounding plasma, passing readily back and forth across the cell membrane. Some early authors had suggested that part of the CO2 in the cells is directly bound to hemoglobin, but by 1920 this view was largely discredited. Lawrence J. Henderson at Harvard and Donald D. van Slyke at the Rockefeller Institute, the principal architects in the 1920’s of a comprehensive picture of blood as a physicochemical system, disregarded possible CO2 binding to hemoglobin. Roughton considered the question to be still open; moreover, he noted that no one had considered the kinetics of the uptake and discharge of CO2 in blood. New evidence made this a crucial question. A. Thiel in 1913, and especially Carl Faurholt in 1925, had shown that the hydration of CO2 to form carbonic acid and the reverse dehydration process were relatively slow reactions that could be accurately measured, although the ionization of H2CO3, and the reverse reaction, were far too rapid to be measured by techniques then available:
Roughton’s calculations from Faurholt’s kinetic data led him to believe that the first step in the above reaction would go far too slowly, in the absence of a catalyst, for CO2 in blood to equilibrate with its surroundings while flowing through the capillaries. Yet the physiological evidence indicated that equilibrium was in fact attained. To explain these facts there appeared to be two obvious possibilities, not mutually exclusive: the blood might contain a catalyst to speed up the reaction, or some of the CO2 might exist in a bound form that could be rapidly released to the lungs. O. Henriques in Copenhagen was also studying the problem, and in 1928 showed that some CO2 was indeed rapidly released when the blood was exposed to a partial vacuum; the rest was released much more slowly. The slow release, he inferred, represented CO2 derived from the ion, whereas the rapid release could come from CO2 bound directly to hemoglobin. Roughton, reinvestigating these findings, found that all the CO2 released from normal blood came off rapidly; he could duplicate the phase of slow release, reported by Henriques, only by adding cyanide or some other chemical inhibitor. He concluded that there must indeed be an active catalyst in blood to accelerate the uptake and discharge of CO2 that could be poisoned by cyanide.
With N. U, Meldrum, Roughton achieved the separation of the catalytic enzyme from mammalian red cells (there was none of it in plasma) in 1932 and 1933. The enzyme was christened carbonic anhydrase, and the amount present in the cells was shown to be far more than sufficient to produce rapid equilibration of CO2 in the capillaries. Roughton and others later showed the presence of carbonic anhydrase in many other organs of vertebrates and invertebrates; it is also found in plants, and even in certain bacteria. In 1940 and 1941 David Keilin and Thaddeus Mann at Cambridge showed that it contained zinc, which proved to be essential for its catalytic activity. The detailed chemistry and catalytic action of carbonic anhydrase have been widely studied by many investigators, especially since 1960.
With J. K. W. Ferguson, Roughton investigated the evidence for direct binding of CO2 to hemoglobin. There was a well-known chemical reaction that might well explain the facts: proteins contain amino groups (RNH2) that in their basic form can bind CO2 directly and reversibly to form carbamates: RNH2 + CO2 ⇌ R · NH · COO− + H+. Ferguson and Roughton concluded that this reaction did occur with hemoglobin, and also inferred that oxygenation of the hemoglobin must cause release of much of the CO2 transported as carbamate, perhaps as much as 20 percent of the total CO2 discharged in the lungs. Jeffries Wyman, a leading worker in the field, questioned the latter conclusion, arguing that a small change of blood pH in the lungs might explain the facts without assuming any change in carbamate content of the blood. Roughton was unwilling to accept Wyman’s view and finally, with Luigi Rossi-Bernardi of Milan (1967), developed greatly improved methods for the study of carbamates that gave evi-dence for the essential validity of his earlier work, Rossi-Bernardi, with J. V. Kilmartin of Cambridge, in 1969 produced decisive evidence that the carbamate formation was located on the amino terminal αamino groups on the four peptide chains of the hemoglobin molecule.
Over many years Roughton studied the equilibrium in the reaction of hemoglobin with oxygen (and with other gases). Hemoglobin, as Roughton’s colleague G. S. Adair had shown, contains four oxygen binding sites (heme groups) and the binding is cooperative; that is, the oxygen affinity increases as binding proceeds. In 1955, following many earlier studies, Roughton and his co-workers, A. B. Otis and R. L. J. Lyster, carried out the most accurate measurements made up to that time on the successive binding constants under various conditions. They showed that the fourth oxygen bound, with all the other three sites occupied, combined with an affinity more than one hundred times as great as that for the first oxygen to be picked up. Since the hemoglobin in blood is carried in the erythrocytes, Roughton was also much concerned with the process of diffusion of oxygen across the cell membrane, which imposed an added delay on the total course of the hemoglobin-oxygen reactions. He studied this and other problems of diffusion, both experimentally and mathematically, in extensive calculations. With his considerable mathematical ability, Roughton was very much a physiologist; he was concerned with gas transport in the blood in its relation to the total functioning of the organism, not merely with the reactions of hemoglobin in solution.
At Cambridge, Roughton was lecturer in biochemistry (1923-1927) and then in physiology (1927-1947). In 1947 he succeeded E. K. Rideal as professor of colloid science, in which post he presided over a group of investigators working on diverse problems. In his personal research he continued to work on the same problems as before, and much of his most important work was done in these later years. Roughton frequently visited the United States, and during World War II he worked extensively in the Fatigue Laboratory at the Harvard Business School, with David B. Dill and his colleagues, on problems related to carbon monoxide in blood and other problems related to the war. Indeed, his work was perhaps somewhat more widely known in the United States than in England. In any case, Roughton’s leading place among workers on blood and hemoglobin was clear. In May 1971 he was to have been the central figure in a conference in Copenhagen on oxygen affinity of hemoglobin and red cell acid-base status (Fourth Alfred Benzon Symposium). At the last moment illness prevented his attendance, but four papers he coauthored were presented there. He continued his work for nearly another year, until his sudden death.
I. Original Works. Roughton published about two hundred papers. On his collaboration with Hartridge on the kinetics of hemoglobin reactions, see “Velocity with Which Oxygen Dissociates from Its Combination with Hemoglobin,” in Proceedings of the Royal Society of London, A104 (1923), 395-430, written with Hartridge; and “Diffusion and Chemical Reaction Velocity as Joint Factors in Determining the Rate of Uptake of Oxygen and Carbon Monoxide by the Red Blood Corpuscle,” ibid., Bill (1932), 1-36. His studies on the combination of carbon monoxide with deoxyhemoglobin are in the series “The Kinetics of Hemoglobin, IV, V, and VI,” in Proceedings of the Royal Society of London, B115 (1934), 451-464, 464-473, 473-495. See also “The Origin of the Hartridge-Roughton Rapid Reaction Velocity Method,” in Britton Chance, R. H. Eisenhardt, Q. H. Gibson, and K. K. Lonberg-Holm, eds., International Colloquium on Rapid Mixing and Sampling Techniques in Biochemistry: Proceedings (New York, 1964), 5-13.
For Roughton’s early work on oxygen equilibrium in blood, “The Equilibrium between Oxygen and Haemoglobin. I. The Oxygen Dissociation Curve of Dilute Blood Solutions,” in Journal of Physiology, 71 (1931), 229-260; and the later, much more advanced study, “The Determination of the Individual Equilibrium Constants of the Four Intermediate Reactions Between Oxygen and Sheep Haemoglobin,” in Proceedings of the Royal Society of London, B144 (1955), 29-54, written with A. B. Otis and R. L. J. Lyster. On his kinetic studies in this period, see “The Kinetics of Human Haemoglobin in Solution and in the Red Cell at 37° C,” in Journal of Physiology, 129 (1955), 65-89, written with Q. H. Gibson, F. Kreuzer, and E. Meda; and “The Kinetics and Equilibria of the Reactions of Nitric Oxide with Sheep Haemoglobin, ibid., 136 (1957), 507-526, written with Q. H. Gibson.
The discovery of carbonic anhydrase is described in “Carbonic Anhydrase. Its Preparation and Properties,” in Journal of Physiology, 80 (1933), 113-142, written with N. U. Meldrum. His work with J. K. W. Ferguson on “carbamino-bound” CO2 in blood is in “The Direct Chemical Estimation of Carbamino Compounds of CO2 with Haemoglobin,” in Journal of Physiology, 83 (1934), 68-86, and “The Chemical Relationships and Physiological Importance of Compounds of CO2 with Haemoglobin,” ibid., 87-102. A survey of the field is “Recent Work on Carbon Dioxide Transport in the Blood,” in Physiological Reviews, 15 (1935), 241-296. A major advance on earlier work is “The Specific influence of Carbon Dioxide and Carbamate Compounds on the Buffer Power and Bohr Effects in Human Haemoglobin Solutions,” in Journal of Physiology, 189 (1967), 1-29, written with Luigi Rossi Bernardi. Investigations on the Bohr effect—the oxygen-linked ionizations of hemoglobin—both by calorimetry and by the temperature variation of the ionization constants are reported in “Direct Calorimetric Studies on the Heats of Ionization of Oxygenated and Deoxygenated Haemoglobin,” in Journal of Biological Chemistry, 242 (1967), 777-783, and “The Effect of Temperature on the Oxygen-Linked Ionization of Haemoglobin,” ibid., 784-792, written with Luigi Rossi-Bernardi and J. R. Chipperfield. For a view of his thinking on all these subjects near the end of his life, see Roughton’s Hopkins Memorial Lecture, “Some Recent Work on the Interactions of Oxygen, Carbon Dioxide and Haemoglobin,” in Biochemical Journal, 117 (1970), 801-812 (with portrait).
Roughton published no books, but he was coeditor with J. C. Kendrew of Haemoglobin: Barcroft Memorial Conference (London and New York, 1949) and with Robert E. Forster, John T. Edsall, and A. B. Otis of Carbon Dioxide: Chemical, Biochemical and Physiological Aspects (Washington, D.C., 1969). He contributed important papers to both these volumes.
Roughton’s personal scientific papers, including correspondence, laboratory notebooks, diaries, unpublished speeches and lectures, manuscripts of published papers, reprints, and referee reports, have been deposited by Dr. Alice Roughton in the American Philosophical Society Library in Philadelphia. The collection is a large one: over three hundred boxes, with a finding aid. See D. Bearman and John T. Edsall, “Archival Sources for the History of Biochemistry and Molecular Biology” (Philadelphia, 1980), p. 84, collection 40154.
II. Secondary Literature. The principal source of information on Roughton’s life and work is Quentin H. Gibson, “Francis John Worsley Roughton (1899-1972), in Biographical Memoirs of Fellows of the Royal Society, 19 (1973), 563-582, with portrait. There is also a brief, unsigned article in Nature, 238 (1972), 297.
John T. Edsall