CHEM-342 INTRODUCTION TO BIOCHEMISTRY

Background for the Article by T. Svedberg and R. Fåhraeus (1926)

A New Direct Method

for the Determination of the Molecular Weight of the Proteins

J. Am. Chem. Soc. 48, 430 - 438


Background

Lord Kelvin, close friend, fellow physicist, and frequent correspondent of G. G. Stokes (1), defined science in terms of measurement. Kelvin once said, "If you can't measure something in numbers, your knowledge of it is not really scientific." He also said, "I can't really understand something unless I can make a mechanical model of it." By those criteria, Kelvin must have considered his friend's article on cruorine pure fluff. That article by Stokes, which we read at the beginning of the semester, dealt with the effect of various reducing agents and carbon dioxide on the absorption spectrum of oxyhemoglobin (2). It lacked numerical measurement and used only chemical analogies. Despite its qualitative nature, the article presented significant scientific observations. It initiated a trail of research that involved many physical, chemical, and mathematical models.

From our perspective, Stokes had a primitive understanding of oxidation and reduction that prevented him from sorting out the reason that reducing agents and carbon dioxide had similar effects on the spectrum of oxyhemoglobin. It took more than 60 years until Conant (3, in your course reader), using quantitative electrochemical methods and mathematical models (The Nernst Equation), showed clearly that oxygenation and oxidation of hemoglobin were chemically distinct processes. Stokes had observed deoxygenation, not reduction, of oxyhemoglobin. Conant (4), the future president of Harvard University, U. S. ambassador to Germany, and author of many influential books on American education, showed that a one electron oxidation produced methemoglobin (=ferrihemoglobin) which cannot bind oxygen. Still later Linus Pauling (5) showed that the spectral differences between oxy- and deoxyhemoglobin that Stokes attributed to oxidized and reduced states were due respectively to the low and high-spin states of FeII (ferrous iron with and without coordination to molecular oxygen). Such a conclusion required sophisticated physico-chemical models of the electronic states of atoms and molecular orbitals and ways to measure quantitatively the paramagnetic properties of molecules.

Both the Zinoffsky (6) and the Douglas et al. (7) articles used quantitative measurements to evaluate chemical models of hemoglobin expressed mathematically. Zinoffsky reasoned that if hemoglobin were a discrete chemical substance, it should contain stoichiometric ratios of iron and sulfur. Douglas et al. used the mathematical models of Hill (8) and others to evaluate quantitatively the competitive binding of oxygen and carbon monoxide to hemoglobin in the presence of various amounts of carbon dioxide. They could see that carbon dioxide altered the binding affinity of hemoglobin for oxygen and carbon monoxide in ways that had escaped Stokes. While the Hill model fit the data, their physical model of hemoglobin proved to be wrong. They had accepted a molecular weight of 16,670 for hemoglobin, a value calculated from the empirical formula of Zinoffsky (6) and others before them and used by Conant (3) after them. Our next article uses the physical behavior of hemoglobin in very large centrifugal fields to determine its molecular weight.

Proteins, like grains of sand, are more dense than water yet, unlike sand, they remain suspended in solution because gravity is much too weak to counteract the effects of diffusion on such small particles. Theodor Svedberg who was interested in the properties of colloidal suspensions, designed and built an ultracentrifuge that could generate forces over 100,000 times gravity (9). At a radius of several inches the outer surface of the centrifuge rotor moved at twice the velocity of a commercial jet airplane. Under those conditions proteins do sediment. Many factors such as viscosity, density, shape, and mass affect the rate at which a particle moves in solution. Interestingly, these were precisely issues that Stokes had addressed in 1856 for spheres moving through viscous media (10). Svedberg applied this theory to macromolecules and developed a way to determine their molecular weight. For this work, Svedberg received the 1926 Nobel Prize in Chemistry.

In this article Svedberg and Fåhraeus describe the technique of equilibrium ultracentrifugation in which the rate of sedimentation is exactly compensated by diffusion in the reverse direction. Under defined conditions the resulting stable concentration gradient is directly related to the molecular weight.

While Svedberg had developed a new way to determine the molecular weights of proteins, Adair (11) had carefully refined the classic osmotic methods and had shown shortly before Svedberg that hemoglobin had a molecular weight of about 67,000. Thus in a short time two independent physical methods gave consistent values for the molecular weight of hemoglobin that were four times the previously accepted empirical weight. The prevailing physical model of hemoglobin changed as did the chemical and mathematical models for cooperative binding of oxygen (12-14).

On Saturday October 16, 1937, Svedberg was the distinguished featured speaker at the dedication of Brown Laboratory at the University of Delaware (15). At that time the du Pont Experimental Station was the only other place in the world besides Upsala, Sweden (where Svedberg worked) that had an ultracentrafuge (16). The Biochemical Research Institute (located in what is now Penny Hall) also became a site of reseach on the use of ultracentrifugation.

References

1. Wilson, D. B. (ed.) (1990) The Correspondence Between Sir George Gabriel Stokes and Sir William Thomson, Baron Kelvin of Largs, Cambridge University Press, Cambribde, U. K.

2.* Stokes, G. G. (1864) On the Reduction and Oxidation of the Colouring Matter of the Blood, Proc. Royal Soc. London 13, 355 - 364.

3.* Conant, J. B. (1923) An Electrochemical Study of Hemoglobin, J. Biol. Chem. 57, 401 - 414.

4. Hershberg, J. (1993) James Bryant Conant and the Birth of the Nuclear Age: From Harvard to Hiroshima, Knopf

5. Pauling, L. and Coryell, C. D. (1936) The Magnetic Properties and Structure of Hemoglobin, Oxyhemoglobin, and Carbonmonoxyhemoglobin, Proc. Natl. Acad. Sci. USA 22, 210 - 216.

6.* Zinoffsky, O. (1886) Ueber Die Grösse des Hämoglobinmolecüls, Hoppe-Seyler's Zeitschrift für Physiologische Chemie 10, 16 - 34.

7.* Douglas, C. G., Haldane, J. S., and Haldane, J. B. S. (1912) The Laws of Combination of Hæmoglobin with Carbon Monoxide and Oxygen, J. Physiol. 44, 275- 304.

8. Hill, A. V. (1913) The Combinations of Hæmoglobin with Oxygen and Carbon Monoxide, Biochem. J. 7, 471 - 480.

9. Gray, G. W. (1951) The Ultracentrifuge, Scientific American 184(June), 42 - 51.

10. Stokes, G. G. (1856) Trans. Cambridge Phil. Soc. 9, 8.

11.*Adair, G. S. (1925) A Critical Study of the Direct Method of Measuring the Osmotic Pressure of Hemoglobin, Proc. Royal Soc. London 108A, 627 - 637.

12. Adair, G. S. (1925) The Osmotic Pressure of Hemoglobin in the Presence of Salts, Proc. Royal Soc. London 109A, 292 - 300.

13. Schejter, A. and Margoliash, E. (1985) The Adair Hypothesis, TIBS 10, 490 - 492.

14. Pauling, L. (1935) The Oxygen Equilibrium of Hemoglobin and Its Structural Interpretation, Proc. Natl. Acad. Sci. USA 21, 186 - 191.

15. The Review (Undergraduate weekly of the University of Delaware)(Oct. 15, 1937), 56(4), 1 & 4.

16. McHugh, F. D. (ed) (1936) 250,000 Times Gravity, Scientific American 154(6), 329 - 330.


* Indicates articles that are in the course reader.


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