From Friedrich Wöhler’s Urine to Eduard Buchner’s Alcohol

Francis Crick is quoted as having changed from physics to biology because he was impatient to throw light into the remaining shadows of vitalist illusions (Judson, 1979, 1996). In a series of lectures delivered in 1966 Crick examined various examples of vitalist writing from the preceding few years and concluded, reluctantly, that we are far from having seen the last of such ideas (Crick, 1966). Indeed, Michael Polanyi subsequent to these lectures forcefully argued for a vitalist type of approach to an understanding of biological phenomena (Polanyi, 1967, 1968). Jacques Monod (1970), again, finds vitalism not dead at all:

Certain schools of thought... challenge the value of the analytical approach to systems as complex as living beings. According to the holist schools which, phoenix-like, spring up anew with every generation, only failure awaits attempts to reduce the properties of a very complex organization to the sum of the properties of its parts. A most foolish and wrongheaded quarrel it is [C’est là une très mauvaise et très stupide querelle].

In a highly perceptive article on the mechanism-vitalism controversy, Hilde Hein (1972) also concludes that this controversy is unending, but she refrains from taking sides. In her opinion the point of view taken by any one individual on this issue — and she quotes eminent contemporary biologists on both sides of the fence — is indeed determined not by the examination of scientific evidence, but by attitudes and prejudices prior to inquiry. She contends that the mechanism-vitalism dispute is but one of a number of... fundamental disagreements which will be perpetuated as a long as people ask questions and seek rational answers. It should be stressed that the notion of a vital force has had ramifications far beyond our rather confined biological focus. In the quotations from Bishop Berkeley and from William Cowper at the beginning of this chapter the connotations of the terms vital flame and vital energy include those that were assumed to hold for biology, but they are far broader. At the beginning of our century, the philosopher Henri Bergson’s élan vital, the centrepiece of his theory of creative evolution, enjoyed a long vogue without any obvious profound effects on biological thinking.

With the rejection of a vitalist approach to a study of living phenomena, one does two things: one expresses a particular mental attitude, and one embarks on an experimentally feasible course of investigation. As a result of these two contributing factors, the phenomena assume a new, broad and, as pointed out, a self-consistent and cross-fertilizing meaning that could not have been predicted in every instance and in every detail. The physicochemical approach inevitably merges with the biological approach. A fresh and at times unexpected view of biological processes is obtained. The study of enzymes — an important exercise in this approach — has provided potent examples not just of the discovery of facts but of the accompanying change in attitudes, initiated by Buchner’s work.

What was new with Buchner’s discovery was not just the demonstration that a process previously considered indissoluble (word chosen deliberately) from living processes could be demonstrated to survive the intact organism and therefore be amenable to study of a kind impossible with the living cell (an insight that leads to the not always accurate but useful approximation in vitro = in vivo). What was new was the wider meaning of this relationship in terms of the implicit recognition that extract repeats or mirrors the living system, i.e. extract repeats or mirrors process. The notion of extraction for the preparation of natural products is not at all new. In this sense the preparation of enzyme extracts continues an old-established tradition. What was new with Buchner was the elevation of this time-honoured procedure to a demonstration that tissue extracts could provide not just what have been called natural products, i.e. compounds synthesized in tissues, but in addition natural processes. In the sense that extracted enzymes are necessarily also natural products — although never designated as such — their ability to survive the extraction process tells us not only something about their properties as chemical entities, but teaches us a far more important lesson, namely that we have here an illustration of the scientific principle, going back at least as far as Galileo, that abstraction from the whole is necessary for understanding of the whole. Enzyme extracts had, of course, been obtained earlier [according to Malcolm Dixon (1971, p. 16), as late as 1920 only about a dozen enzymes were known] and Buchner’s active material, centred on the mystery of fermentation, had been looked for before. However, Buchner’s discovery had a far greater impact on biological thinking and on the development of biochemistry than the discoveries of any of the previous enzymes because its basis had been the subject of a lengthy and acrimonious dispute between two scientific giants, Pasteur and Berthelot, with important views expressed by the likes of Traube and Hoppe-Seyler. Buchner’s work was a lucky break not only because he happened, unlike Pasteur and others, to use a yeast that yielded active extracts, not only because he stumbled upon a discovery more or less by accident, but, far more important, because it so happens that the living cell can yield some of its most important secrets since destruction of the inner organization of the cell is not, as we now know, associated with destruction of the conformation of most of the proteins that constitute fundamental functional and structural components of the cell.

The equivalence between in vivo and in vitro aroused the opposition of the vitalistically inclined critics, and was accepted by the ever growing number of mechanistically inclined admirers. Interest in function predominated, at least initially, over interest in structure which was completely inaccessible and of very minor relevance. Classical biochemistry, the topic of metabolic maps that forms its core, developed without the detailed knowledge of protein structure that has become a major ingredient of what has come to be called structural biology. Biochemistry soon developed the use of homogenates (in the first 50 years of this century the biochemical literature was full of the word Brei), tissue slices, membrane fractions and components of organelles such as mitochondria, but these later studies were initiated with extracts, and such studies still continue. The in vitro – in vivo correlation, which refers not to compound but to activity, is now absolutely taken for granted as one of the paradigms or principles of the field. Any apparent exceptions are explained not, as they would have been tempted to be a hundred years ago, as a consequence of interference with a vital force, but rather to interference with in vivo controls at different organizational levels such as membrane structure, the interplay between organelles, the function of the endoplasmic reticulum and of the Golgi apparatus, removal of channelling, and so on. A recent book (Young et al., 1997) enquires into these correlations.

Buchner’s experiment had a double impact: it decreased the vitalist contribution to biochemical thinking by establishing that a fundamental cellular in vivo process could be studied in vitro, and it initiated a study of the chemical nature of this and of many other cellular processes. And thus the Janus-like nature of Buchner’s work was driven home, since these two results are related: vitalist persistence would have made in vitro studies impossible, and, in a complementary fashion, in vitro studies would have no in vivo significance. The abolition of vitalist perspective abolished backward-looking dogma, while the in vitro – in vivo correlation opened the view to the future. We can now see that it was the solubility and the persistence or relative stability of intercellular enzymes that abolished notions of a mysteriously guiding, essential vital force and the associated idea of a special kind of intracellular chemistry. The vital force, which had beaten a reluctant retreat into biosynthetic versus biodegradative events (Bernard), into insoluble versus soluble material (Kühne) had finally succumbed to experimental inevitability. Vague notions of protoplasm, comparable to that of the ether in physics, were abolished, and ferment in the sense of insoluble material whose activity was life-dependent was replaced conceptually and experimentally by enzyme. An amazing byproduct of in vitro enzyme persistence was the fact that organized function continued in extracts.

We have to return to Wöhler’s urine and to examine its relation to Buchner’s alcohol. Both the synthesis of urea and the demonstration of cell-free fermentation were accidental. It bears to examine the impact on the dominance of vitalism of these two very different discoveries. Friedrich Wöhler’s 1828 synthesis of urea in the test tube is widely (not universally, a point that is not central here) represented as marking the beginning of organic chemistry, just as Eduard Buchner’s test tube conversion of glucose to ethanol is recognized to mark the beginning of modern biochemistry.

Wöhler was clearly aware of the fact that his synthesis had overthrown an assumption, that a special vital force had to participate in the formation of an organic compound. There were two consequences, the first well recognized, the second usually ignored: (i) the impressive 19th century growth of wrongly named organic chemistry, begun by Wöhler’s work, followed his initiative by emphasizing synthesis (cf. the title of Marcellin Berthelot’s influential textbook (1860b)Organic Chemistry Founded on Synthesis); (ii) organic chemistry developed by deliberately and consciously doing without biological processes, since the very nature, the properties as it turns out, of organic molecules made this possible: any resemblance of organic synthesis to synthesis in organic material was ignored as irrelevant and, if one had asked a practicing organic chemist, as undoable.

The development of organic chemistry did not abolish vitalism, it simply confined the vital force to processes in the living cell rather than assigning it as a necessary participant in the formation of these molecules. The matter is murky, since biological processes include the synthesis of biological molecules. Organic synthesis was just a small step in the direction of removing from organic molecules an aura of vitalist mystery. Seventy years had to elapse before Buchner’s work lifted vital processes from the cell into the test tube, thus initiating what organic chemistry had not attempted: the duplication of a cellular chemical process away from an intact biological system. Buchner’s discovery of cell-free fermentation near the end of the 19th century bears a strange analogy to Wöhler’s 1828 synthesis of urea: both Wöhler’s and Buchner’s work demonstrated that no vital force is needed to form so-called organic compounds. The approach of Wöhler’s work, which set the tone for the development of organic chemistry, simply demonstrated that the chemistry of organic compounds was such that they could be made outside tissues by more or less drastic methods that clearly had no relevance to vital processes. The approach of Buchner’s work initiated the complementary approach, that the organic among the organic compounds could be made, again in the test tube, by the very chemical approaches that were used by the living cell. The vital force was shown to be irrelevant to the operation of those cellular chemical processes to which it had willy-nilly been consigned by the flowering of organic chemistry. It was at long last recognized that chemical influences brought about chemical reactions where, before, vitalist forces were considered at play.

It is the sense of surprise about a newly demonstrated halfway house between the living cell and its products that as we saw permitted the likes of Duclaux and Roux to proclaim that a vitalist contribution remained as long as the synthetic methods of organic chemistry could not reproduce the components and thereby the activities of cell extracts. Although very few enzymes have been synthesized since Duclaux and Roux raised their objections, and certainly none of the enzymes of glycolysis, no such vitalist considerations are resuscitated a hundred years later by our inability to synthesize enzymes in the test tube, or by the requirement for the ribosomal machinery to participate in the synthesis of proteins altered via site-directed mutagenesis. Today’s unanticipated success can afford to move yesterday’s attitude to a sidetrack of triviality. Buchner’s experiment, exactly 20 years after Kühne introduced a distinction between enzymes and ferments, dealt the death knoll to this distinction by showing that it did not exist, that in fact all ferments were enzymes. By then Liebig (1803–1873) had been dead over twenty years, and Pasteur (1822–1895), Traube (1826–1894) and Hoppe-Seyler (1825–1895) had all as it were bowed out within a year of each other before the curtain opened on a new, hitherto nebulous land, and Kühne (1837– 1900) and Berthelot (1827–1907) were near the ends of their careers. A new era had dawned, and a word, in yeast, had come into its own as a reminder of contentions centred on an age-old concern with alcoholic fermentation, and after 1837, on the agent necessary for this process.

The scientific study of fermentation has an old progeny. Thus the dissertation by the later mathematician Johann Bernoulli, submitted for public defense at Basel University in 1690 had the title Dissertatio de Effervescentia & Fermentatione (On Effervescence and Fermentation). It was reviewed by no less a person than Gottfried Wilhelm Leibniz (see Maquet, 1997). Numerous references to wide and to our mind confusing ancient uses of the word fermentation can be found in Multhauf (1966). The central early interest in fermentation has been admirably surveyed by Fruton (1972, pp. 23–42). As an example, Zymotechnica Fundamentalis was the title of an important book (1697) by Georg Ernst Stahl, the person remembered for using phlogiston to form the core of his system of chemistry (Fruton, 1972, p. 33). A hundred and forty years later, right from the demonstration in 1837 that yeast was alive, yeast became the touchstone of vitalist contention, not only because it was readily available, not only because of its intrinsic interest as an agent directly related to the formation of alcohol, but because it was so very easy to look for fermentation. So it was used by Lüdersdorff (1846) and others, by Berthelot, by Pasteur, by many others, and eventually by Buchner. If Pasteur or others had succeeded in obtaining a cell-free active extract from some other microorganism, most of which, as we now know, are easier to open up than yeast, the impact would have been much less dramatic. Buchner’s work continued to pursue vitalism’s first retreat, from the presumed organic properties of organic molecules, to its second refuge, the presumed inscrutable mode of chemical synthesis and behavior in living cells. His work, in turn, set the tone for the development of modern biochemistry that has proved ever more fruitful and ever more valid as the 20th century is proceeding towards its end.

The development of biochemistry was very much slower than that of organic chemistry. Thirty five years had to elapse after Buchner’s discovery, and more than a hundred years after Wöhler, before Krebs and Henseleit (1932) showed how urea is made in living tissues. At that time work was proceeding on the unravelling of the details of the chemistry of many other biochemical processes as well. Buchner’s zymase, in particular, had undergone a lengthy and at the time not quite completed dissection into unanticipated components, including, as it turned out, as many as twelve enzymes, various essential ions, a coenzyme and a cofactor, and remarkable ramifications into the process of so-called glycolysis.

It is symptomatic of the divergence of biochemistry from organic chemistry that in the classic paper on the urea cycle there is no mention of the much earlier and biologically irrelevant first organic synthesis of urea. By 1932 it had become abundantly clear that the study and understanding of the various biochemical processes had a direct lineage to Buchner’s work. More time had to elapse, however, before any remaining beliefs in a vital force could receive yet a further push into the netherworld of irrelevance: by a supreme irony the unraveling of organic chemical mechanisms, achieved independently of biological systems, has demonstrated to the point of obviousness, or if you like of paradigmatic certainty, that biochemical mechanisms are organic chemical mechanisms. Chemistry, and especially organic chemistry, has come full circle. Wöhler’s urine has, as it were, come home to roost, and it is Buchner’s work that began to build its biological home. Buchner’s observation, fortified by the organic chemistry initiated by Wöhler, was a prelude to the immense edifice of biochemistry that has sprouted throughout the 20th century and that continues to sprout with unallayed vigour and unbounded impact on understanding and on application.

A short, lucid, remarkable address from our own days puts Eduard Buchner’s achievement into a luminous perspective. This address, given by the 85 year old Nobel Laureate Otto Loewi as President of the Honorary Committee of the Fourth International Congress of Biochemistry, Vienna, September 1958 serves as a bridge between our days and those of Eduard Buchner. At the time of Buchner’s discovery Loewi was just finishing his medical studies:

I was already 24 years old when Eduard Buchner in 1897 showed that sugar could be fermented by a cell-free yeast extract, from which he then prepared zymase. I vividly remember the tremendous sensation [ungeheures Aufsehen] that this discovery elicited, far beyond the circle of biologists and chemists, for instance also in the field of the philosophers, for it once and for all contradicted the thesis of the great Pasteur, that a chemical process as complex as fermentation would only be possible in the living cell. I began [my address] with Buchner’s discovery because it, and the method used by him, in the words of a Stockholm committee, opened the way to exact ferment research. The effect on the development of biochemistry was and remains powerful. This is understandable when one agrees with Frederick Gowland Hopkins that the final aim of biochemical research is a sufficient, acceptable description of the dynamics of the living cell. In order to approach this goal it was first of all necessary to isolate and to analyse the individual cell components, especially those that serve as substrates and the factors that act on them, especially the ferments, and to elucidate their successful activity [Wirkungserfolg] and the mechanism of their activity. These experiments could of course not be performed with living cells; hence one had to use cells whose struc ture had been destroyed, extracts obtained from them and of late apparently intact cell constituents such as mitochondria and their colleagues. Undoubtedly the countless observations obtained with such materials also apply to the living cell, and so one obtained from the beginning of our century a comprehensive and deep insight into the kinds of many fundamental chemical processes in the cell, and into their reciprocal relationships.

LOEWI (1958, my translation)

How beggarly appear arguments before a defiant deed!

WALT WHITMAN, Leaves of Grass, Song of the Broad-Axe