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The 'growth' of organic matter is seen forcibly in the almost endless carbon chains with their most varied arrangements, as they are formed in the bodies of plants. These chains have originated from quite separate carbon atoms, which earlier were present in carbonic acid. Thus carbon has a great tendency in living molecules to cause growth by chain formation. Cyanogen also has this tendency in a high degree, especially for [other molecules of] cyanogen. The important elements of living protein [cyanogen radicals] thus have the most marked tendency to attract radicals of the same kind and in this way to produce ever larger molecules, i.e., to grow.
(Pflüger, 1875, 342)
... since the molecules of organized substances contain on an average about fifty of the more elementary atoms, we may assume that the smallest particle visible under the microscope contains about two million molecules of organic matter. At least half of every living organism consists of water, so that the smallest living being visible under the microscope does not contain more than about a million organic molecules. Some exceedingly simple organisms may be supposed built up of not more than a million similar molecules. It is impossible, however, to conceive so small a number sufficient to form a being furnished with a whole system of specialized organs.
Thus molecular science sets us face to face with physiological theories. It forbids the physiologist from imagining that structural details of infinitely small dimensions can furnish an explanation of the infinite variety which exists in the properties and functions of the most minute organisms.
(Maxwell, 1875, 42)
[Walter Morley] Fletcher had a bias towards chemistry, which he had acquired from his father. He asked Michael Foster whether he thought that there was any future in the application of chemistry to physiology. At this enquiry, Foster rolled his large beard over his mouth with both hands, to smother his hilarity.
(Crowther, 1968, 313)
A very distinguished organic chemist, long since dead, said to me in the late eighties: 'The chemistry of the living? That is the chemistry of protoplasm; that is superchemistry; seek, my young friend, for other ambitions'. (Hopkins, 1933, 245)
One reason which has led the organic chemist to avert his mind from the problems of biochemistry is the obsession that the really significant happenings in the animal body are concerned in the main with substances of such high molecular weight and consequent vagueness of structure as to make their reactions impossible of study by his available and accurate methods. There remains, I find, pretty widely spread, the feeling—due to earlier biological teaching—that, apart from substances which are obviously excreta, all the simplest products which can be found in cells or tissues are as a class mere dejecta, already too remote from the fundamental bio-chemical events to have much significance. So far from this being the case, recent progress points in the clearest way to the fact that the molecules with which a most important and significant part of the chemical dynamics of living tissues is concerned are of a comparatively simple character.
(Hopkins, 1913, 144)
No upper limit is usually assigned to molecular magnitude. E. Fischer has synthesized a polypeptide with the molecular weight 1212, and in the case of colloids, molecular weights of the order 104, and even 105, are commonly spoken of. A difficulty arises, however, in admitting that molecular weights can exceed a certain value, unless the density increases as the molecular weight increases.
For suppose that a compound can exist, such as a protein, with a density at 0º not much greater than that of water, and with a molecular weight of rather more than 30 000, the grammolecule of such a compound at 0º would occupy about 30 000 cc. The gram-molecule of a perfect gas under the standard conditions occupies only 22 400 cc, and we should therefore have a solid compound, at 0º and under a pressure that cannot be less than one atmosphere, occupying a greater molecular volume than that of any gas.
That the molecules of liquids and solids should occupy greater volumes than those of gases under similar conditions, seems at first contrary to the usual conceptions of the gaseous, liquid, and solid states. It is true that at sufficiently low temperatures this condition must arise for all substances, but a simple calculation shows that for the majority of chemical compounds it would only occur at temperatures not far removed from the absolute zero.
Two suggestions appear to be indicated. The first is that under the ordinary conditions there is an upper limit to molecular magnitude, and that for most substances, more especially colloids, the molecular weight cannot exceed a value of about 20 000. The second is that our ordinary kineto-molecular conceptions no longer apply when for a given temperature the molecular magnitude exceeds a certain critical value. The latter view seems most in keeping with our present knowledge, and perhaps serves to throw some light on the behaviour of colloids.
(Crompton, 1912, 193–194)
... Of course for oxyhaemoglobin, which crystallizes in the well-known beautiful manner, a molecular weight of 16 000 has been deduced from the iron content, but against such calculations the objection can be raised that the existence of crystals in no way guarantees by itself chemical individuality; on the contrary one can have to do with an isomorphous mixture such as the mineral kingdom offers us in such variety in the silicates. Such doubts cease in the case of synthetic products whose production by analogous reactions can be controlled.
(Fischer, 1913, 3288)
... In the application of the condensation principle of albuminous compounds, to molecules of the order of magnitude of 4000 and far higher, chains of truly fantastic proportions would be yielded, the existence of which can be assumed very improbable according to our ideas.
(Hess, 1920, 232)
The concept of very large molecules is not a twentieth century idea. It arose in the previous century from the consideration of the chemistry of carbon compounds and from the attempt to describe physiological processes in chemical terms. In 1857 the quadrivalency of carbon had been evident to Kekulé in the case of methane, and in 1858 he made it a law for all carbon compounds (Kekulé, 1858, 152). At the same time he arrived at the conception of the carbon—carbon link. Carbon atoms, he said, "are themselves joined together, so that naturally a part of the affinity of one for the other will bind an equally great part of the affinity of the other" (Kekulé, 1858, 154). This idea was, he declared, distinct from the earlier picture of atoms associated in a centric or mutually connected pattern. As he later recalled: "The separate atoms of a molecule are not connected all with all, or all with one, but, on the contrary, each one is connected only with one or a few neighbouring atoms, just as in a chain link is connected with link" (Kekulé, 1878, 212). Such linkages when made between polyvalent atoms could lead to the production of "net-like" and "sponge-like" "molecular masses which resist diffusion, and which, according to Graham's proposition, are called colloidal ones" (Ibid, 212). The same hypothesis, he argued, could be used as Pflüger had done, to produce the elements of the form of living organisms.
The Application of Kekulé's Polymer Concept
This concept was used by Kekulé's colleague, Eduard Pflüger, in his theory of intracellular respiration. Pflüger believed that the energy released during respiration was liberated by the decomposition of the peculiar, highly unstable, polymeric, living protein molecules of protoplasm (Pflüger, 1875). W. Pfitzner applied the polymer concept to the chromosome structures which he called "chromomeres" in 1882. Edmund Montgomery in 1885 and George Hörmann in 1899 applied it to a number of morphological structures. All these authors saw the polymeric state as distinct from that of the compounds analysed by the organic chemists. In Pflüger's opinion, living polymeric compounds were to the molecules of the chemists as the sun was to the smallest meteor. In studying albumin extracted from the organism "one has to do mostly with torn-off fragments of those vast molecules, which may well be as large as an entire creature" (Pflüger, 1875, p. 343). At the close of his life he commented on Emil Fischer's synthesis of polypeptides: "in spite of the great exploits of Emil Fischer the synthesis of protein will take up another century and the synthesis of living protein is hardly likely ever to succeed" (Cited in Cyon, 1910, 1).
Emil Fischer's attitude to the polymer concept was, no doubt, influenced by the vitalism it had acquired. But it was also natural that he should try to construct his version of the chemistry of life only on the solid foundation of organic chemistry. He was able to characterize some small polypeptides by the demanding criteria of this science; therefore such chains of up to thirty amino acids were acceptable to him. Of such, he thought, were proteins constituted. Native proteins were mixtures of these. To support this view he worked out the potential isomerism of a polypeptide consisting of thirty different residues [today only 20 are recognized in the amino acid alphabet]. It came to 2.635 × 1032 (Fischer, 1916). What irony that Fischer's recognition of the sequence hypothesis for proteins served to consolidate the case against their macromolecular nature, for, he implied, if the isomerism of small polypeptides was so great, what need had nature for giant polypeptides!
The Colloid-Aggregate Theory
The fact that the polymer theory enjoyed some popularity at the close of the nineteenth century was due rather to the lack of a clear alternative than to the cogency of the arguments in its support. When the aggregate theory of colloidal particles was developed in the early part of the twentieth century the polymer theory was quickly dropped. Most so- called polymeric molecules (with molecular weights above 5000) were then regarded as aggregates of much smaller molecules. Haemoglobin, for instance, was probably an aggregate of four globin molecules of molecular weight ~ 4000, which, when combined with the prosthetic group gave a particle with the well known empirical weight of 16 700.
There were three developments which made this suggestion plausible. Alfred Werner, founder of the co-ordination theory, had introduced the concept of two kinds of combining forces in chemical compounds—Hauptvalenzen or primary valency forces, and Nebenvalenzen or secondary valency forces (Werner, 1902, 268). He claimed that atoms united by primary valency forces [covalent bonds] still possessed varying degrees of "residual affinity" whereby several molecules became united into "compound molecules" or aggregate molecules. This idea was applied by Karrer and Hess to starch and cellulose, by Pummerer and Harries to rubber, by Bergmann to proteins, and by Hammarsten to thymonucleic acid or DNA.
A second support came from the estimates of the unit cells of these substances made by the X-ray crystallographers. Many such scientists claimed that the molecule could not be larger than the unit cell, and since their unit cells were small, so were the molecules (see Chapter 2).
The third and most influential support was provided by the promising young subject of colloid science. This bridge subject between chemistry, physics and biology achieved such recognition that many reputable textbooks on physiology and biochemistry took the principles of colloid science for their foundations. This tendency has been labelled "Biocolloidology", a "dark age", the "age of micellar biology" (Florkin, 1972, 279). This was no crank science, but a subject which fascinated the most powerful minds. Was it not from colloidal particles that Jean Perrin gained his evidence for the reality of the molecule? It was, surely, through the paths of colloid science that Svedberg was led to design his ultracentrifuge and demonstrate the unique character of macromolecular species of proteins.
The chief axiom of colloid science was that there is a state of matter, the colloidal state, to which the ordinary laws of chemistry—the laws of constant and multiple proportions (Hardy, 1903, p. xxix) and the law of mass action—are not applicable. This is because the particles are large aggregates of molecules, many of which are not therefore accessible to the other reactants. The surfaces of such large particles show special properties: they adsorb ions, double electrical layers form around them, they often function in catalysis. Their "autoregulative properties" were attributed to "the surface field around the particle" (Svedberg, 1928, 17). Colloidal solutions showed abnormally high viscosities and gave abnormally low osmotic pressures. Some of these properties were shown by the thymonucleic acid or DNA extracted by Einar Hammarsten in 1924.
Not surprisingly, experimentally-minded cytologists fastened upon colloid science in their biophysical attack on the living substance of the cell. Protoplasm was a colloidal system. What organic chemistry could not explain about the behaviour of the proteins of the living substance colloidal science would clarify. In the absence of the electron microscope and the technique of differential centrifugation and with the neglect of the ultraviolet microscope there was a "neglected dimension" which colloid science, it seemed, could fill. There thus arose in opposition to the old morphological cytology a new experimental cytology founded upon colloidal biophysics.
The Debate over Macromolecules
This debate came to a head in the period 1926 to 1930. The man who coined the term Macromolecule was the German chemist, subsequently Nobel Laureate, Hermann Staudinger. He began his opposition to the aggregate theory by devising a crucial experiment to test the theory as it had been applied to rubber (Harries, 1904), and by the time of his farewell lecture to the Zürich Chemical Society in 1926 he was convinced that Kekulé's polymer concept was right and the aggregate theory wrong. Chain molecules held together by Kekulé [co-valent] bonds could be of fantastic lengths without the need to invoke secondary valency forces. This was the import of his lecture, but it was not well received, as the following report of a witness shows:
I remember Staudinger's lecture to the Zürich Chemical Society in 1925 on his high polymer thread molecules with a long series of Kekulé valency bonds. It was impossible to accommodate his view in the unit cell as established by X-ray analysis. All the great men present: the organic chemist, Karrer, the mineralogist, Niggli, the colloidal chemist, Wiegner, the physicist, Scherrer and the X-ray crystallographer (subsequently cellulose chemist), Ott, tried in vain to convince Staudinger of the impossibility of his idea because it conflicted with exact scientific data.
The stormy meeting ended with Staudinger shouting 'Hier stehe ich, ich kann nicht anders' in defiance of his critics.
(Frey-Wyssling, 1964, 5)
Another witness of this meeting was Rudolph Signer, now professor of organic chemistry at Bern. At that time (1925) he was working under Staudinger for his doctoral thesis. Throughout his stay in Zürich, Signer
... was very impressed by Staudinger. He was completely sure that his idea of the existence of macromolecules was right and he had practically all his colleagues against him and his opinions. And so it was a very interesting situation to see this man already having a great experience in this field, having a special conviction, and having against him all his colleagues.... The crystallographer Niggli said that each substance in the pure state should crystallize and if these materials of polystyrenes and other polymers which Staudinger had synthesized were pure they should form crystals. After the meeting Wieland told Staudinger that in his opinion molecules with more than forty carbon atoms should not exist.
According to Staudinger's own recollections, Niggli's contribution to the discussion was simply to oppose Staudinger's suggestion of the macromolecule with the words: "Such a thing does not exist!" (Staudinger, 1961, 85). And his friend Wieland had advised him "at the end of the 1920s", presumably by letter to Freiburg:
Dear Colleague, leave the concept of large molecules well alone: organic molecules with a molecular weight above 5000 do not exist. Purify your products, such as rubber, then they will crystallize and prove to be lower molecular substances.
(Staudinger, 1961, 79)
Excerpted from The Path to the Double Helix by Robert Olby. Copyright © 1994 Robert Olby. Excerpted by permission of Dover Publications, Inc..
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