0
THE CHEMICAL PROBLEM OF SPONTANEOUS GENERATION' SIDNEY W. FOX The Florida State University, Tallahassee, Florida
Tm
concept of the spontaneous generation of l i e occupied much of the experimental and theoretical attention of some of the most pioneering biologists of the eighteenth and nineteenth centuries (Needham, Spallanzani, Pasteur, Tyndall, etc. (1)). When the appearance of maggots, mice, and microbes in the midst of decay was generally accepted as merely a manifestation of biological reproduction of living organisms, interest in this question lagged. The past few years have seen a vigorous revival of concern with the problem of spontaneous generation. This new look emphasizes, and can be explained as due to, the basically chemical nature of the problem. To one who examines the question in its broadest aspects, however, a purely chemical attack appears also to provide less than the most promising approach. To such a point of view i t seems instead almost axiomatic that this problem will be solved by primarily chemical techniques which are couched in recognition of principles of dynamic biochemistry as well as in the fundamental principles of an evolutionary and developmental biology. In other words, one may now expect t o discern in any valid matrix of prebiological chemistry the roots of phenomena which are known to govern a11 living matter. One of the great lessons of biochemical analyses and experimentation in the last three decades is that all life is remarkably alike in its chemical structures and processes (8), as contrasted to what one might expect from a review of the diversity of living forms. We are perhaps ignorant of the biological forms between the first organism and the most primitive type which has been found in fossils. Perhaps, also, the biochemical nature of those first and intermediate forms was markedly different from the chemical structures and reactions which are studied today. The fact that the biochemistry of all organisms is so nearly universal from the most ancestral to the most advanced permits us, however, to entertain seriously the thought that the first organism also fitted the general pattern closely. Accordins to this oremise. clues are abundantlv available for an understanding of the chemical nature of primordial life. The spontaneous generation of the most primitive type according to these specifications is an embarkation point for experimentation. With couching of the research in supported inferences from
-
Experimental vork in the author's laboratory was performed with the aid of grants from the Rockefeller Foundation, the National of Health of the U,S, Publio Health Service (Grants H-2314 and RG4666), and from the General2Foads
COT. 472
geology, biology, and biochemistry, the problem can be seen principally as a problem in organic and inorganic chemistry. The amount of archived writing on the origin of life is tremendous. The amount of experimentation is far less but is, in one sense, almost as difficult to identify. If the premise of the unity of biochemistry is correct, a large proportion of the vast literature of biochemistry and of rudimentary organic chemistry is traceable to prebiological chemistry. Accordingly, after one experiments in this field while simultaneously researching what appears to be pertinent literature, he may indeed easily develop the belief that the outlines of the answer to this most fundamental and provocative of problems are already in the journals. I n recent years, such experiments as imparting energy t o mixtures of presumably primitive gases have become a relatively respectable line of inquiry. The kinds of gaseous mixture inferred for the prebiological atmosphere are presented in Table 1. The evidence invoked has been assembled primarily by geologists from study of "juvenile gases" (current volcanic effluvia), from Precambrian rocks, from analyses of gases in stony and iron meteorites, from spectral analyses of planets and of stellar composition, and from equilibrium constants used jointly with knowledge of the abundances of the elements in the universe (5). What is known about terrestrial conditions today may, with modification by sufficient study and experimentation, reveal the nature of the prebiological world. The interdisciplinarian geologist, Rubey, has stated, "Everything considered, the composition of sea water and atmosphere has varied surprisingly little, a t least since early in geologic time" (4). In an environment of this degree of stability, the unity of biochemistry is more easily understood, a t least insofar as environmental changes might be expected to permit alterat,ions~ ... ... .
SUGGESTED PRIMITIVE ATMOSPHERES AND HYDROSPHERES
Inspection of Table 1 reveals areas both of difference and of agreement.~h~ most central point of agree. merit is, of course, the presence of ~ ~which 0 is , included both in atmospheres and hydrospheres and is the ~ 1 of 1 the DrinciDalconstituent of all lilivinesvstems. " carbon compounds proposed are methane, carbon dioxide, or carbon monoxide, the choice from this narrow range lies a principal disagreement among scientists dealing with primitive atmospheres. O ~ a r i n , Urey, and Miller prefer a reducing atmosphere of
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JOURNAL OF CHEMICAL EDUCATION
TABLE 1 Suggeeted Envelopes for Primitive Earth Atmosphere C R , NH., H20, HI Atmosphere CH4+C02, NH8+Nz, HsO, H2 Hydrosphere CO*, NH*, HB, HxO Atmosphere CO*, Nn, Has, Hz0 Hydrosphere CO,, NH,, HpS, Hs0 Atmosphere CO, Con, N1, HnS, HsO
Authors
References
Oparin,
(66)
TT.e.,
1
Bernal
(7)
Rubey Revelle
(5)
(8)
methane and hydrogen which was free of carbon dioxide. I n Miller's experiments, carbon dioxide was formed during sparking in which amino acids were produced. When this fact is coupled with Abelson's recent observations (9) that amino acids are formed by sparking various gaseous mixtures containing carbon dioxide, it can be seen that the problem of an original CO1-rich atmosphere (6) has not been resolved by experimentation. Although Rnbey (3) suggests trace amounts of carbon monoxide, Revelle (8)has argued for substantial proportions of this gas. Speculative organic chemistry is more facile with a reactive gas like carbon monoxide, which also would assure reducing conditions. The nitrogen is suggested as having been present as either diatomic nitrogen or ammonia; Bernal's outlook (7) visualizes both. In his atmosphere, ammonia and methane were slowly dehydrogenated and oxygenated to nitrogen and carbon dioxide, respectively. In relation to the nitrogen-ammonia equilibrium, it was shown long ago that the hydrolysis of nitrogen t o ammonia is thermodynamically unfavorable (10). Bayliss (11) has recently pointed out, however, that reaction of glucose with nitrogen can produce ammonia with a liberation of free energy. For such a process to operate, the participation of the hydrosphere is imperative and the early participation of the lithosphere is probable. One cannot, in any case, get very far biochemically in the atmosphere alone. Some of the proposed atmospheres include hydrogen. Bernal (7) considers its presence but believes it to have been lost from the earth's gravitational field too easily to have participated significantly as a gas in primordial biochemistry. Rubey (5) states that if either hydrogen or methane is allowed, the other should be present by virtue of a favorable equilibrium constant
P = sum of partial pressures of gaseous components participating in the reaction.
If one considers the primitive atmosphere as nonoxidizing or oxidized, rather than reducing or oxidizing, the disagreement in outlook is considerably dulled, and the evaluations are probably more sharply stated. All of the proposed atmospheres that recognize the importance of sulfur agree on hydrogen sulfide. The participation of sulfur atoms has been omitted from some considerations. I n view of the biochemical significance of sulfhydryl (18) groups and the fact that organisms are truly simultaneously anaerobic and aerobic t o some degree, such omission is not warranted. Some or all of the necessary reductive properties of VOLUME 34, NO. lo, OCTOBER, 1951
prehiological systems may have been underwritten by hydrogen sulfide in place of, for example, methane. Evidence for this is the fact that some sulfur bacteria rely on the reduction of COz with HzS (13). The similarities in various proposals for prehiological chemistry can be looked a t through either end of the telescope. Insofar as the atmosphere and hydrosphere are concerned, however, all lines of evidence and interpretation lead to proposals of systems which are not so widely differentthat it would not he possible to visualize the inclusion of all components which have received major attention. Bernal's picture (7), in fact, comes close to doing that. The thinking represented by Table 1 perhaps overemphasizes the atmosphere. I n attempting to overcome resultant difficulties, it has been suggested that amino acids that may have formed in the atmosphere would have fallen into the ocean. Another possible source of compounds is the lithosphere. I n this framework, Oparin has proposed the hydrolysis of metal carbides produced a t incandescent temperatures. This would yield acetylene which could be converted to such compounds as acetaldehyde and thence to acetic acid by some kind of oxidation. HnO CaCa-CH=CH
HIO H10 -CHICHO-CHaCOOH -(H)
Another point of view that deserves more attention is the Bernalian origin of many biochemical systems in the hydrosphere. Atmospheric, hydrospheric, and lithospheric origins can be inferred as reflected in metabolism of all organisms. A large part of the end-product of inammalian respiration, carbon dioxide and water, are components of some of the suggested primitive atmospheres. The original hydrosphere is reflected in the sap of plants and the blood of animals and is the basis for the frequent statement that we each carry a bit of the primeval sea within us. Our inherited dependence upon the lithosphere is particularly evident in our need for trace elements such as magnesium, zinc, and cobalt in the diet. PRODUCTION OF AMINO ACIDS UNDER HYPOTHETICALLY PRIMITIVE CONDITIONS
The experimental work which particularly stimulated interest in the origin of organic compounds on earth was that suggested by Urey (6) and carried out by Miller (14). The heart of the contribution was the discovery of amino acids among the products. It is necessary, however, to point out that although this discovery was indeed a stimulatory contribution the production of amino acids from mixtures of simple compounds containing atoms of carbon, hydrogen, oxygen, and nitrogen by lightning, heat., or other radiation is not surprising. The reason for this is simply that amino acids are relatively stable thermodynamically, on account of their zwitterion (dipolarionic) structure: R CHCOOI
They are not stable relative t o the elements nor to most simple gases like carbon dioxide and ammonia. If, however, mixtures of gases including representatives from such compounds as methane, carbon dioxide, water, nitrogen, and ammonia are brought to a high
energy level so that new organic compounds and radicals result, it is to be expected that these new compounds will be relatively unstable with respect t o the salt-like amino acids. Although this point of view was not widely recognized in 1953, by 1957 i t had already been learned that alanine could be produced in a t least seven different ways under conditions which could be considered to be prebiological. These experiments, from three different laboratories (9,14, t6), thus support the earlier judgment that amino acids could be formed easily. Perhaps the most significant inference from all of this is that in the spontaneous generation of a complex primordial life and its mutations from a relatively simple chemical matrix, the range of potential materials at any stage is set by a kind of thermodynamic predeterminism. Evidence for this is found not only in the observed range of prebiological types of synthesis of amino acids, but also in the range of structures of protein (16, 16) and in many instances of parallel evolution of organisms (17). However, evolution is slow enough so that we cannot yet accurately visualize the full potential scope of the entire process. BIOCHEMICAL STAPLES AND MACROMOLECULES
Table 2 lists the biochemical staples (key compounds), which have been shown virtually t o be common t o all organisms. The structures of the amino acids, vitamins, and monosaccharides reveal that these are essentially molecules which have been formed by loss of water and of hydrogen. Similarly the macromolecules of protein, nucleic acid, and polysaccharides are also molecules resulting from the schematic loss of water. This loss is referred to as schematic, partly because, as is well known, phosphoric acid residues appear t o be implicated. Exact mechanisms, both current and primordial, are in some doubt. The diversity of the protein molecules has been shown to be far from random (15). The interactions of these macromolecules furthermore provide a basis for an exponentially larger number of types of organisms. For such interactions and attendant phenomena t o yield organisms, water must enter the scene. All organisms are predominantly aqueous, and the event
of conversion of prebiochemical substances to a dynamic organism inferentially involved water in some fashion. The available clues suggest, then, that the generation of staples and macromolecules required a stage in which water was lost from reactants and that this was modulated to a hydrous stage in the origin of life. Evolution in Table 2 is chemical and biological a t different stages. Our primary concern here is with chemical = prebiological evolution. It is usually assumed that the Darwinian process of variation and selection does not apply to prebiological changes because they are not repeated by a controlling apparatus such as the gene. Variation in organisms can occur as a result of gene recombination. At the chemical level a comparable reshuffling would involve rearrangement of amino acid residues in protein and pyrimidine residues in nucleic acids. Changes in organisms might, however, also result from molecular alteration within the gene as well as from recombination of genes. During the continuing evolution through stages (1) through (6) the total number of types can theoretically increase. In this consideration, stage (2) and (3) belong together and the increase in total diversity through stage (5) is probably real. It can also be seen that as complexity and diversity increase, what we choose t o refer to as specificity also increases. As the absolute number of types in the spectrum is enlarged, individual types which are products of interaction are more easily differentiated. ORIGIN OF PROTEIN
The origin of protein was invoked in 1951 by Blum It is perhaps possible now to construct a fuller definition, based on the generation of a protein-containing system capable of initiating again the development of itself. Blum's point of departure is much closer, however, to the heart of spontaneous generation than is the interaction of primordial gases. In what follows, the concepts presented are to some degree compatible with any of the possible origins of organic compounds already discussed. (18) approximately to define the origin of lie.
TABLE 2 Biochemical Emergence (1)
-
Primitive dmosphee, hydrosphere, lithosphem
(8)
Simple organic compounds
-
COz or CH, NH. or N. Ha0 H9 HzS Metal atoms HQO, State of hydration
Malic acid, Urea, etc. HxPO,
Evolution
Chemical evolution
Diversity of type Specificity
-
474
-
(4)
Mmomolmles
Proteins, Nucleic acide.
Many of these cam- Formed by loss of Water has entered or pounds schematiwater from moncre-entered system cdly formed by mers loss of water and hydrogen + Amino acid and py- Generecombinstion rimidine recombination? Increasing in this direction >
Increasing in this direction
JOURNAL OF CHEMICAL EDUCATION
The research of our laboratory began with attempts to synthesize peptides by use of elevated temperatures in a presumably terrestrially primitive range. Preliminary indications of successful peptide formation (19) did not lead immediately to further experimentation because of unexpected results of a different, albeit probably related, sort. There was first observed the formation of additional amino acids (20) by thermal conversion of the original acids (analogous to what occurs biochemically). Exploration of these results led from one biochemical thing to another. Citric acid cycle intermediates, amino acids, peptides, vitamin intermediates, and the uucleic acid intermediate, ureidosuccinic acid, were all implicated. After several of these steps had been assembled and mapped (Fig. I), a striking parallelism suggested itself. These sequences were closely parallel to those of known biosynthetic pathways. This, in turn, could be seen to he a reduction to the chemical level of the biogenetic law (that development of the organism reflects its evolution). There was thus available a partial explanation of the formation of protein and nucleic acid consistent with biological principles. In addition, this picture, when combined in reverse order with Beadle's concepts of the relationships of gene, enzyme, and anabolism (dl), revealed how there might have arisen the universal anabolic reaction pattern and a material system for remembering and repeating those anabolic reactions leading again to the formation of the cyclical chemical memory system (Fig. 2). With this introduction, the development, interpretation, and discerned limitations of the thermal studies may be examined in more detail. THERMAL EXPERIMENTS
Application of the thermal conditions theoretically results in the overcoming of the energy barrier to peptide bond synthesis (22) by driving the reaction through removal of the water produced:
An experimental approach to this reaction focused on studying the thermal behavior of one or two amino acids. Aspartic acid was of particular interest because it is known to be formed early in biosynthesis (23). The literature indicates that the pyrocondensation of aspartic acid was accomplished by Schiff ($4) and reported in a paper dated 1897. Doubts as to the nature of Schiff's product have been expressed (25) and the possibility that diketopiperazine had been formed especially required scrutiny (26). Examination of this problem has shown that polyaspartic acid is distinct from the diketopiperadne, and is a biuret-positive material with average molecular weight about 10,OOW 20,000 (27). Kovacs and Koenyves (28) have indicated that polyaspartic acid has a polyimide structure which opens to a true peptide on alkaline treatment. A glutamic acid-glycine pyropolymer gives rich infrared indication of being a linear peptide, and such data for other polymers are a t hand (29). In studies of polymerization of aspartic acid alone and with other amino acids, it was discovered that thermal treatment of most of the reaction mixtures yielded more ninhydrin spots on paper chromatograms VOLUME 34, NO. 10, OCTOBER, 1957
than could be accounted for by the number of reactants. All these adventitious products have not yet been characterized, but a-alanine and palanine have been identified. In addition it was learned that one could start with the citric acid cycle intermediates, malic acid or fumaric acid, and that their ammonium salts under the same thermal conditions would yield aspartic acid and a polymer hydrolyzable to aspartic acid and alanine. Some aspartic acid was obtained when ammonia was replaced by urea for thermal reaction with malic acid, but the predominant product finally was identified as ureidosuccinic acid. The above represent all of the reactions for which the data have been quite fully published. Furthermore, unpublished experiments reveal additional reactions and intermediates that are also represented in Figure 1 by broken arrows. The conversion of malic acid to aspartic acid by heating with ammonia occurred also with fumaric acid hut not with succinic acid nor citric acid. The formation of asparagine from aspartic acid depends upon the
Prrmit8ve gases, Ihquids, + rolldr
Anoboliter such urea,
01
molic ocld, HIP%
Amino ocidr, Enzyme Krebs Cy> protein acids, formed in presence o f pyrimidines, vitamins, \anoboliter
,
Nucleic acid
Nucleaprote~n storing -cell imptes~ions of onobolile~
~i~~~
a. cy0lieal
daughter pel1 A \daughter cell B
__
nucleic ocid
+
enzyme
-
protein which cotolyzes
~ i ~ ~ t hE ~ ~ S tY t~ ~ ~md C. . ~~~i~ PPOC-. T~SS ul Having Been Formed i n the First
P..oc.ss Picturas the Firat Cell operetion of th. Cycle
475
presence of excess urea. The production of a-alanine and p-alanine was demonstrated in uncombined form, and also in the polymers formed, by recovery after hydrolysis. Lactic acid and urea give a-alanine (89), and glucose and urea give a-alanine and glycine (SO). Comparable to the conversion of malic acid to aspartic acid, a-hydroxyglutaric acid and urea yield glutamic acid (89, SO). Microbially positive tests for nicotinic acid have been found repeatedly in the thermal reaction product of glucose and asparagine. Clues to thermal syntheses of many other biochemicals are being investigated. The possibility of forming proteins by heating unsubstituted amino acids has received little but discouragement from reports in the literature. This kind of reaction was studied decades ago (25). At that time it was learned that heating of amino acids often led t o tars, other decomposition products, and diketopiperazines (85, 97). However, these early experimeqts were confined to a few amino acids heated singly. In our laboratory it has been learned that amino acids which alone do not form linear peptides do so when copolymerized with one of the few amino acids (aspartic acid, glycine) that has been recognized as probably forming linear peptides alone. Also, some linear ~ e ~ t i d are e s formed from reactants neither of which forms a peptide, such as glutamic acid and leucine (89). An additional aid has been the use of phosphoric acid (16), which speeds the copolymerizations to the degree that in one case it permits the formation of a linear copolymer which virtually does not result in the absence of phosphoric acid (89). Taken together, all of these results indicate in principle how a variegated peptide such as a typical protein could result from copolymerizations. At the biological level, interactions of biological units and of individuals are of particular significance. At this biochemical level, near the baseline of biology, the interactions (or molecular ecology) of amino acids introduce effects which cannot be visualized from the behavior of the individual monomers. I n attempting to assess critically the probability of correctness of any theory which is so wide-angled as to embrace the origin of life, it is especially imperative t o attempt to state what the theory does not include. The author, his associates, and others have so far found and voiced three types of limitation. The first of these concerned the fact that it has been recognized that many amino acids do not pyrocondense to linear peptides. The most recent research has shown, however, that there can no longer be any question that some of them do. Of more significance is the fact that such amino acids will in some cases copolymerize to linear molecules. The full scope of this phenomenon is yet to be assessed, but it represents a principle by which protein formation can be visualized. The second limitation is related t o the first. Production of ureidosuccinic acid is not the same as production of a nucleotide. One can also point out that the theory fails to account for the origin of malic acid or fumaric acid from which the pathways stem. The value of these criticisms is as yet not assessable, inasmuch as none of the experiments bearing on these questions have been performed. A third point ~ t e m sfrom the fact that although the
majority of the thermal pathways charted have their counterparts in biology, it is not clear as to whether one can or cannot closely relate the mode of peptide synthesis to the biosynthesis of protein. The difficulty here would seem t o be that our knowledge of protein biosynthesis is quite uncertain. Many mechanisms have been suggested, and one or more may be correct, but few disinterested viewers of this research scenery would seem t o care to designate which. The fact that many of the thermal syntheses of peptides are facilitated by the presence of phosphoric acid during heating may well lead in the direction of relating the primordial synthesis to current biosynthesis. One can also assume the over-all point of view that a prebiochemical flowsheet so like a current picture of biosynthesis is deceptive in its false simplicity and may merely represent the product of finding what is sought. It is however true that in this program the findings of adventitious amino acids, of ureidosuccinic acid, of virtually no unnatural products, of specific interaction effects of amido acids, and the linked nature of the reactions were all unexpected experimental results, the first three totally so. The interpretation evoked appears to he simpler than many would have presupposed, but unduly complicated presuppositions are not without precedent in the history of science. I n summary, all of these limitations t o interpretation are of the nature of a docket for further research. Such experiments are planned, but with a picture that appears more and more to be almost as large as biochemistry itself, each experiment will have t o wait its turn. ORIGIN OF OPTICAL ACTIVITY
The problem of the origin of optical activity, so long considered to be unique to living systems, has received consideration in these contexts. Of the many hypotheses advanced for the origin of optical activity, the thermal experiments have particularly led t o new emphasis on an old idea-that configurational one-sidedness may have arisen during the biological era rather than before it (91). The suggestions which have appeared for the origin of optical act,ivity include the polarizing effects of moonlight and of sunlight, the fact that the earth rotates in one direction only, the dissymmetry of the earth's magnetic field, the effect of d or 1 lattice (quartz) on primordial synthesis, and a few proposals which deserve fuller explanation. One might also add the idea that optical one-sidedness of molecules is related to the newly discovered nonparity of physical particles (98). The production of a single molecule of any of the presumably thousands of compounds in the original biosynthetic chain could explain the one-sidedness of molecules in nature (39). A single molecule containing one asymmetric center cannot, of course, he both D and L simultaneously. If but one form of any metabolite were produced, this dissymmetry should eventually affect all other molecules indirectly. A variation of this thinking is brought t o bear on the problem when one considers not merely the metabolites but rather a large, perhaps autocatalytic, macromolecule. In the initial formation of a protein containing 100 amino acid residues, for example, each residue would necessarily be of a single configuration, except for syrnmetJOURNAL OF CHEMICAL EDUCATION
rical glycine. Should one expect the production, then, of two enantiomorphic protein molecules, or would perhaps one be formed in advance of the other, and could this not be the point a t which Darwinian selection first occurs? This problem seems to merit extended theoretical consideration. Clues may be available from studies of physical properties of peptides (34). A related treatment is owed to Langenbeck (35), who suggested that a racemic world is therrnodynamically unstable and that any disproportionation between enantiomorphs would be progressively increased by evolution. The reaction of an enzyme with one form of a metabolite would be expected to be more effective than with its enantiomorph, in extension of Langenbeck's idea. One optical form would then have a selective advantage. A disproportionation in either metabolite molecule or macromolecule would then spread to all others in an evolutionary continuum. Another auxiliary idea here is that in the synthesis of a large number of DL molecules of one kind, the number of D and L would be expected not t o be exactly equal, although the minute percentage difference would not be discernible in a polarimeter. Any difference would then be subject t o enhancement by the Langenbeck evolutionary mechanism. Another kind of suggested solution is that of spontaneous resolution. This is an idea that many chemists find unacceptable a t first, despite a report of such a phenomenon by Pasteur, and of confirming work by others (36). A more readily welcomed notion stems from the related one of chance mechanical separation in space of crystals corresponding to D- and L- molecules (31). Virtually no one doubts that Pasteur was able to separate D- and 1.- sodium ammonium tartrates by the use of tweezers. With this as an accepted fact, one can visualize how ammonium hydrogen D- malate might crystallize alongside ammonium hydrogen L- malate in equal amount. If then, a primordial biosynthetic soup happens to touch the D crystal and not the other, only the D form would function as a seed and D would crystallize. This would leave a predominant L form in solution and various processes such as the Langenbeck evolution could continue the change. Eons of evolution would not be required. If the spontaneously separating compound happened to be an intermediate early in biosynthetic pathways, such as malate, all of the products derived therefrom (aspartic acid, ureidosuccinic acid, a-alanine, etc.) would be of the same configuration as the precursor left in the bi* synthetic solution or unit. Many of these various ideas can be seen to be applicable to the biological as well as to the prebiological era. The validation of the large theory can be visualized as arising in one of three or more ways: (1) the picture developed is found to be consistent with other biochemical knowledge, and leads t o new knowledge which is also consistent, (2) ultimately the accumulated information leads t o synthesis of a living unit, and (3) experimentation is carried through stage (8) and on into a primitive evolution. I t may well be that the final solution of the problem of optical activity in nature will require validation of type 3, which a t present promises to be the hardest to come by. Reasons for thinking in the context of the biological era are that VOLUME 34, NO. 10, OCTOBER, 1957
recently lower forms, bacteria, have been found to contain in their proteins D-amino acids as well as L-amino acids (Sf). BIOLOGICAL CONSIDERATIONS RELATIVE TO THERMAL ORIGINS
An explanation of biochemical origins which relies on thermal energy and can explain the generation of biosynthetic pathways is consistent with many other observations. Suitable thermal conditions such as those in the experiments must almost certainly have been available on the primitive earth and are abundantly in evidence even today, in volcanic regions. I n fact, biological research in Yellowstone National Park bears on the concept of thermal origins. I n 1936, Copeland (37) published the results of a study of the curious thermophilic blue-green algae of the Yellowstone area. Copeland decided, on the basis of botanical criteria, that the thermophilic blue-green algae are the evolutionary ancestors of the forms more characteristic of current terrestrial temperatures on most of this globe. He suggested from these studies "the probability of the origin of living organisms in the t.herma1waters." Copeland's interpretations thus point to elevated temperatures for the origin of life, as do the chemical experiments. This perspective will be discussed further near the end of this article. As indicated earlier, another area of inquiry with which the thermal experiments make contact is that of chemical genetics. The thermal experiments suggest the generation, in a sequence of overlapping order, of reactions, protein, and nucleic acid. If one substitutes, as he well may, the word anabolism for reactions, enzyme for protein, and gene for nucleic acid, he arrives at a sequence which is the reverse of the Bcadle picture (21) for the same materials, i.e., gene-enzyme-reaction. From a concerted interpretation of Beadle's concepts and those of the thermal experiments, a rational picture can be deduced (Fig. 2). The sequence proposed is a spontaneous generation in overlapping order of anabolic reactions, enzymic protein, and genic nucleic acid. The anabolic reactions, by virtue of the metabolites in the soup, are impressed upon the protein being formed, in a manner to be described as a substrate-enzyme interaction. The resultant enzyme proteins are then captured by spontaneous formation of the particulate organic salt of nucleoprotein, which is in effect an apparatus that captures the anabolic reactions. This, then, can be looked upon as a primitive, chemical mechanism for memory-to be invoked when the enzymes are released to act upon the nutrient substrates (Fig. 2). Among the reactions captured are those that lead directly to the formation of more enzymic protein and more genic nucleic acid. In this way it becomes possible to visualize grossly the basic process of selfreplication a t the chemical level. A place to search for the dim evolutionary origin of human memory is also indicated. These considerations bear on another esoteric problem of interest-the biochemist's equivalent of the old problem of which came first, the chicken or the egg? In this case the problem is: which came first, the autotroph or the heterotroph? In other words, was the first organism able t o synthesize its needed substances or did it find these in the environment? Was the first
organism, as all current ones are, somewhere in between these two extremes in synthetic ability? For a lonz time. the consensus of hioloeical belief held that the first brganism was autotroph; (predominantly hiosynthetic). I n recent years it has been visualized that the necessary chemical complexity arose in the environment over a geologically long period of time and that some heterotrophic (essentially nonhiosynthetic) type captured the resultant substances and gradually developed synthetic abilities as individual nutrients were exhausted from the primordial soup. Out of this situation eventually could evolve what would be an early autotroph. Each of these two explanations seeks to account for the presence of myriads of biochemical staples, hiochemical intermediates, and macromolecules. Without an attempt to assess the probable validity of propositions such as these, an alternative picture drawn from the thermal experiments will he presented. This picture visualizes that some combination of a few organic compounds, such as malic acid, urea, and a few others, with trace elements, phosphoric acid, etc., would give rise easily in a short period of time to many times as many compounds. If the energy source were continuous, e.g., thermal, these compounds would interact to form scores of new reactants, and these ~roductswould in turn react to form hundreds. The next stage could yield thousands of compounds, etc This picture makes unnecessary a slow evolution of biochemical substances. If macromolecules with the properties of an inheritance apparatus also arose through these pathways, the reactions (not merely the substances) leading to the macromolecules would he captured for reproduction. The thermal experiments indicate that all this could occur in less than a few hours, in fact, it would now he hard to visualize how a thermal environment would fail to yield a similar profusion of organic compounds in short order, once the obligatory reactants were present. Such an experimentally conditioned conclusion suggests an initial autotroph, a formation of this autotroph in a few small steps from the matrix which at that moment becomes environment, a dynamic biochemistry from the outset, an understanding of why only loss mutants2 are found, and the origin of a hiological development which reflects prebiochemical evolution. This picture appears as the most consistent in the light of the experimental results so far uncovered. Among the more compelling segments of evidence for the essential validity of the proposed picture is the manner in which it is consistent with the biological principles of evolution and development. The biogenetic law is based on the interpretation that the development of the embryo, in any animal, reflects the evolution of that form. Volumes can be written about this principle and its qualifications and, in fact, they have been. The modern outlook is that this generalization is more right than wrong (59). If the hiological principle is correct, then the mechanist expects that the phenomenon should he reducible to a chemical level. Inasmuch as the thermal pathways are so like the biosynthetic pathways, this requirement appears to he met. The concept of a biosynthetic reflection of 'In the work of Beadle and colleagues (?21), the biochemical mutants base lost synthetic abilities (compare 38). 478
prebiochemical evolution is an extension of Granick's similar generalization covering purely biochemical ~henomena(40). .. . CONCLUSION
Another biological principle that csn he discerned a t the chemical level is the Darwinian process of natural selection, which appears t o operate with macromolecules. Inasmuch as this concept has been quite fully treated elsewhere (16) only a few salient features will be mentioned here. Analyses of proteins have been interpreted t o suggest a nonrandom emergence and kinship of protein molecules (16). A random relationship has also been proposed (41) but the suggestion has been withdraw11 (43). The possibility exists, however, of a kind of intermediate situation in which there are closely homologous proteins (proteins of the same function from different species) (16, 43) evolving in a narrow range alongside other homologous groups to which each group hears a heterologous (proteins of diierent function) relationship. This question resolves to the question of whether the first protein was one or many. It will he of interest to determine whether properly devised thermal experiments will suggest an answer to this question. Although it is less risky to treat the problem of the origins of the biochemical world than that of the origins of life, certain aspects of the biochemical origins are more rational if they can be fitted into a larger picture which permits understanding also of how biological units might have originated. This was the impetus for Figure 2 geologically. Three possibilities seem at present to offer promise of fitting all of the facts. Each includes what is considered the necessary modulation from an almost anhydrous system to a hydrous unit. The first of these is essentially the Copeland picture of a hot springs origin. The organic material would presumably enter through the volcanic bed. The Bernalian picture (7) of dried-up lagoons of the ocean could give concentrations of organic compounds sufficient to suit the requirements of the organic chemist for reactions to proceed as rapidly as any kind of dynamic system might require. A third picture is that of a hot, almost anhydrous, organic magma under pressure escaping into a warm or hot primitive ocean through a fissure in the ocean floor. In the thermal pathways urea figures heavily. Urea can be produced a t temperatures such as 130°, in a closed system, from ammonia and carbon dioxide. Accordingly, a volcanic magma which could easily be at such temperature under pressure could provide conditions for original biochemical substances which would be extruded into marine waters. Under such conditions, protein formed in the anhydrous state could be expected to denature at the surface of small globules resulting from the extrusion a t a temperature close to the boiling point of water. This denatured protein could he the necessary membrane for the first cells. It is thus possible to see how chemical processes may lead to biochemical processes which in turn produce physiological phenomena. The accomplishments of food technology are testimony to the thermal stability of dry protein and the ease of denaturation of these substances when wet. Needless t o state, there are many gaps t o he filled in this yet highly tentative picture of spontaneous genJOURNAL OF CHEMICAL EDUCATION
eration. Attempts to fill in the outline will undouhtedly provide further tests of the essential ideas. Several colleagues have remarked on the extent of lay interest in research and interpretation of this sort. Particularly, this research has been found to be of a type which does not require justification to the public in terms of how it will cure one of their physiological ailments, improve their economic status, or otherwise contribute to technological advance. Virtually every thinking individual, scientist or nonscientist, is interested in his remote origin. If the hallmark of the scientist is truly curiosity, then everyone is a scientist to the extent that he dis~lavs " curiositv. The curiositv of the average nontechnical man in the answer to the problem of spontaneous generation has been quite evident. Increasingly, however, it appears that the answer to this age-old question will be provided by interdisciplinary interpretation and design modified by continual chemical experimentation.
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LITERATURE CITED (1) BAITBELL, G. A., "The Evolution of Earth and Man," Yale University Press, New Haven, 1929. (2) KLUYVER, A. J., AND C. B. VAN NIEL, "The Microbe's Contribution to Biology," Haward University Press, Cambridge, 1956. (3) RWEY, W. W., "Development of the Hydrosphere and Atmosphere, with Special Reference to Probable Composition of the Early Atmosphere," Geological Society of America Special Paper 62, 1955. (4) RUBEY,W. W., Bull. Geol. Soe. Amer., 62, 1111 (1951). "The Origin of (5) OPARIN,A. I., (trausl. by S. MONGULIS), Life," The Macmillan Co., New York, 1953. (6) UREY,H., "The Planets, their Origin and Development," Yale Univereitv Press. New Haven. 1952. BERNAL, 3. D., " h e ~hbsiealBsais oi Life," Routledge and Kegan Paul, London, 1951. REVELLE,R., J . Marine Res., 14, 446 (1955). AREISON,P. H., presented orally a t the meeting of the New York Academy of Science an Dee. 26, 1956. PARKS,G. S., AND H. M. HUFFMAN, "The Free Energies of Some Organic Communds," Chemical Catalog.Co.. Inc., New YO&, 1932. BAYLISS, N. S., Ausk. J. Biol. Sci., 9 , 364 (1956),
VOLUME 34, NO. 10, OCTOBER, 1957
E. S. G., Tezas Repts. Bid. filed., 11, 653 (1953). (12) BARRON, (13) VAN NIEL, C. B., Aduanss in Enzynol., 1, 263 (1941). (14) MILLER,S. L., Science, 117,528 (1953); J. Am. Chem. Soc., 77, 2351 (1955). Am. Naluralist, 89, 163 (15) Fox, S. W., A N D P. G. HOMEYER, 119.551. ~....,. (16) Fox, S. W., Am. Scientist, 44,347 (1956). G. L., E. MAYR,AND G. S. SIMPSON,"Genetics, (17) JEPSEN, Paleontology, and Evolution," Princeton University Press, Princeton, 1949. (18) BLUM,H. F., "Time's Arrow and Evolution," Princeton University Press, Princeton, 1951. (19) Fox, 6. W., AND M. MIDDLEBROOK, Federation Proc., 13, 211 119541. ~, Fox, S. W., JoHNsoN, J. E., AND MIDDLEBROOK, M.. J . Am. Chem. Soc., 77, 1048 (1955). BEADLE,G. W., The Harvey Ledures, 40, 179 (1944-45). HUFFMAN, H. M., J. Phys. Chem., 46, 885 (1942). CALVIN, M.. The Hamey Lectures, 46, 218 (1950-51). SCHIFF,H., Chem. Ber., 30, 2449 (1897). KATCHALSKJ, E., Advance8 in Protein Chem., 6, 123 (1951). K. HARADA. A N D P. D. HOAG, Fox.. S. W.. A. VEGOTSKY. LA&, A&. N . Y. Acad. s&, 69, 328 (1957). A,, K. HARADA, AND S. W. FOX, manuscript (27) VEGOTSKY, in preparation. (28) K o v ~ c sJ., , AND I. KOENWES,NaluTWi8~,14, 333 (1954). K., AND S. W. Fox, manuscript in preparation. (29) HARADA, (30) Fox, S. W., A N D J. E. JOHNSON, manuscript in preparation. (31) A N D A. VEGOTSKY. Science,. 124.. . . Fox. S. W.. J. E. JOHNSON. 923 (1956). L. S., AND V. F. WEISSKOPT, Science, 125, 627 (32) RODBERG, (19571. P., E. D. HUGHES, C. K. INGOLD, ANDP.A. D. S. (33) BREWS&, RAO,Nature, 166, 178 (1950). E., J . Cellula~and Comp. Physiol., 47, 151 (34) ELLENBOGEN, (Suppl. 1, 1956). W.. "Die Organischen Katalysataren," (35) LANGENBECK, Julius Springer, Berlin, 1935. (36) J. P.. Advances in Protein Chem... 9 ., 129 . . GREENSTEIN. (1954). J. J., Ann. N . Y. Acad. Sn'., 36, l(1936). (37) COPELAND, D. E., A N D S. W. FOX, A ~ e hBioehem. and Bio(38) ATKINSON, phya., 31, 220 (1951). N. J., "Man'8 Emerging Mind," Dodd, Mead and (39) BERRILL, Ca., New Yark, 1955. (40) GRANICK. S., The Harvey Lectures, 44, 243 (1948-49). (41) Gmow, G., Seientlfie American, 193, No. 4., 70 (1955). G., Seiatifie American, 193, No. 5, 4 (1955). (42) GAMOW, (43) PERRONE,J. D., D. PARREIRA, AND E. ToMALSQU~M, 3rd Congress International de Biochimie, Brussels, Resumes des Communications, 14 (1955).
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