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Home , Proteinoid, Protocell, Virus

The Theory of the Origin of

And Competing Ideas

SIDNEY W. FOX (-coded) Contemporary

ATP mechanisms

Genetic code

Model and theory Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021 complete to this b point by 1967- (Ultrastructured, selectively permeable, H20 reproductive) THE NUMBER OF REQUESTS I am receiving for photo- Protoprotein (Enzymic, micrographs suggests that we can expect dozens of ordered) new textbooks with passages or chapters on the -H20 origin of life. Dozens of textbooks of with a Amino acids such inclusions have indeed already appeared, most of them since 1971. The first textbook of to include a chapter on the origin of life was Albert I -H20 Lehninger's, published in 1970. Lehninger has ex- ( precursors) plained there that armchair speculation was being replaced in the 1960s by serious scientific investiga- I tion. Primordial organic (galactic organics, etc.) Late in the 1960s, in fact, we had one relatively Fig. 1. From organic matter to a reproductive protocell and complete theory (and physical model) of the origin beyond. Numerous experimental alternatives are available of a reproducing protocell, derived from geologically up to stage a but not above. Between a and b the steps are compatible sequentially, and the conditions are geologically relevant experiments. During the development of relevant. this theory, alternatives for individual component concepts have appeared. I have recently presented The extensive evidence (Kenyon 1973) for the elsewhere (Fox 1973a) a comparison of some of the proteinoid origin of life has been reviewed elsewhere alternative concepts. In responding to the present (Fox and 1972). The essential features of the invitation, I shall again outline the proteinoid theory theory, derived from the physical model, are seen in and then examine competing or alternative concepts fig. 1. Mixtures of a-amino acids are copolymerized in the light of our sequential experimental model. by heat. The resultant contain ordered My view of most of the competing concepts is sequences of amino acids and arrays of enzymelike expressed precisely by Florkin and Stotz (1972): activity. Upon contact with , the proteinoids

Accordingto [John] Northrop,there is a common patternin all ... . Sidney W. Fox is professor of chemistry and --- ... controversies "Thereis a compli- director of the Institute for Molecular and cated hypothesis, which usually entails an elemnent .... of mysteryand severalunnecessary assumptions. This Cellular at the University of a is opposedby a more simple explanation,which con- Miami, Coral Gables, Fla. 33134. His Ph.D. tains no unnecessaryassumptions. The complicated is in biology (biochemistry) from the Cali- fornia Institute of Technology. He has been one is always the popularone at first, but the simpler . one, as a rule, eventuallyis found to be correct.This U.S.-U.S.S.R. interacademy exchange lec- j processfrequently requires 10 to 20 years.The reason turer with A. I. Oparin (1969), and he is for this long lag was explainedby Max Planck. vice-president of the International Society He remarked that 'scientists never change their for the Study of the Origin of Life. He has received the minds,but eventually die."' Florida Academy of gold medal, the Priestman lectureship, the Iddles award, and a number of other honors specifically for his advances in the theory of the origin of In the case of the proteinoid theory, as in those life, in addition to recognition for his contributions to bio- cases described by Northrop, we have a relatively chemistry, especially amino acid sequence determination. He simple explanation, with no unnecessary assump- was recently presented with a festschrift; the authors in- tions. The clude Pauling, Szent-Gyorgyi, Calvin, Lipmann, Charles explanation emerged from experiments so Price, and Florkin. Fox is the author or coauthor of more simple that innumerable students at all levels have than 200 technical articles and several books. The present repeated many of them. paper was given at the 1973 NABT convention.

161 Fig. 2 is a view of proteinoid. Proteinoid is what is produced in the laboratory; protoprotein is the term for proteinoid that, according to interpretation, originated spontaneously on the . The theory has emerged from the preparation and properties of proteinoid. These polymers have molecular weights, typically, of 5,000-12,000; they contain some propor- tion of each of the amino acids common to contem- porary ; and qualitatively they have virtually all of the properties of contemporary protein (Fox and Dose 1972). The polymers are usually produced in substantial yield, such as 20-60%, from amino acids. The most striking feature of the process is its utter simplicity-a simplicity imputable to condi- tions at the surface of the Earth even now. According to our analyses, the principal reasons that our laboratory is the only one to have presented 41,~~~~~~~~~~ a comprehensive physical and conceptual model (in Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021 1967) for the origin of life are the following: 1. Life began in essentially only one way, al- though it may have done so innumerable . 2. We have studied processes in open systems, comparable to what has occurred in the geologic . Closed flasks used in the laboratory by others are far less relevant to the geologic realm. 3. We have discovered for each stage self-ordering phenomena leading to the next stage. 4. The proteinoid microsphere is found to have numerous properties of the contemporary cell and to be evolvable toward the latter. Such emergent functions had to be determined by experiment; they were not predicted. Some of the experiments, however, began as work- ing hypotheses. The organized body of interpretations is referred to as the proteinoid theory of the origin of life (Florida Academy of Sciences 1968); it is a sequentially coherent conceptualization. One of the earliest of the competing concepts is that of the famous chemist Emil Fischer (1906), who said (approximate ):

If today, througha lucky accident,with the help of a brutal reaction,for example, a melting of amino Fig. 2. Left:indiscriminately heated a -amino acids. Right: acids in the presence of a water-removingmedium, a mixture of a-ammno acids containing aspartic acid and there shouldsuccessfully be produceda true protein, glutamic acid, following purification by dialysis-the material and if it were furtherpossible, which is still unlikely, is called proteinoid. to identify the artificial product with the natural material-this would benefit protein chemistry little yield huge numbers of model cells. These lack some and biology not at all. of the properties of contemporary cells, but they possess others. The properties observed include a What was indeed true in 1906 is not true today; content of highly ordered, protometabolically active there is enough additional understanding to produce , ultrastructure, some kinds of selective true significance. Also, if Fischer had recognized the permeability, and the ability to reproduce by four ease with which material resulting from melted physical mechanisms. The properties found are those amino acids would easily form a kind of cell, he essential for further evolution. Up to the stage of might have seen a relationship to what is the first amino acids, many alternative explanations are avail- problem of biology: the origin of life. But his view able (Fox and Dose 1972; Fox et al. 1973). The sub- must have been in part the understandable myopia sequent two steps are without experimentally de- of one early chemist. , however, veloped alternatives. Those above the upper hori- imagined in a letter to a friend in 1871 "that a pro- zontal line in fig. 1 are based on partial experimental tein compound was chemically formed ready to demonstration (Fox 1974). undergo still more complex changes" (F. Darwin

162 THEAMERICAN BIOLOGY TEACHER, MARCH 1974 1896). Fischer's view and Darwin's earlier view seem Table 1. Principal advances in concept resulting from in- to me to have been truly competitive with each other. vestigation up to an act of , with However that situation is analyzed, I found, as late inhibitory assumptions. as 1963, a number of chemists and biochemists whose Advance Inhibitory assumption views were much like those of Fischer in 1906; in some cases it seemed to me that I was confronting an 1. Protein(oid) can be pro- Synthesis of protein re- duced acellularly quires a cell emotional as as a dialectic barrier. The principal advances in the art up to an act of 2. a-Amino acids can be a-Amino acids are decom- spontaneous generation of a (minimal) cell are seen thermally copolymerized posed by heating (Carothers and in table 1. Also included are competing assumptions nylon) that were prevalent at the time the experiments were 3. Heated a-amino acids Disordered polymers would designed. Had these negative assumptions and others themselves result (E. Fischer, A. I. Oparin, etc.). Nucleic acids been taken seriously, the experiments would not were needed have been performed. The idea that synthesis of protein requires a cell 4. Arrays of enzymic ac- Matter cannot organize it- 3 self is simply one of the later battles with , this tivity result from contest having often to be refought. The experimental 5. Proteinoids aggregate to advance number 1 is one modern equivalent of form minimal cells upon Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021 contact with water Wohler's synthesis of urea in the absence of a cell.

Ml 2 0 B1

B1 e

3 4

a t-2 ,B1 B2

U t , o~~~~~~~~~~~~~~

Fig. 3. of proteinoid microspheres through a cycle. M is microsphere; B is bud. Subscript denotes number of generation.

PROTEINOIDTHEORY 163 Another conceptual inhibition was that of fig. 2, on the left. Decomposition to tars was the usual expectation from heating a-amino acids. This view was fortified when W. H. Carothers (1936), instead A R IV of heating a-amino acids, H2NCHCOOH, heated w- Complete amino acids, H2N (CH2) nCOOH [n = 5] to produce a first nylon. In many respects, Carothers improved upon silk protein, a polymer of a-amino acids. He indicated, however, the difficulty or futility of heat- ing a-amino acids. The clue to overcoming this diffi- culty was the copolymerization of mixed a-amino acids in which nonneutral a-amino acids were pres- 3 B ent. 4 ~NoATP Emil Fischer, A. I. Oparin (1957), and numerous n

Table 2. Some salient properties of proteinoid microspheres. Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021 Limited range of size w~~~ Ultrastructure Types of semipermeability Ability of immature to grow heterotrophically Ability to reproduce (four modes) (Other properties in Fox and Dose 1972) No

g K'AA

0 Fig. 5. Synthesis of by models for protoribosomes. System consists of microparticles, ATP, and phenylalanine. I, III and IV are peptides of phenylalanine. II is unchanged phenylalanine.

0o others since have expected that a primordial poly- merization would yield disordered products. Yet others have assumed that nucleic acids would have been needed for such ordering. The idea that amino acids could order themselves comes the closest to being a prefatory assumption of any working hypo- thesis for our experiments. The concept of self- ordering was, however, not truly an assumption. We observed such action in experiments we 0O 0 were doing in the early 1950s (Fox et al. 1953). Once proteinoids had been produced and internal order of the amino acid residues had been demon- strated, the finding of enzymic functions was more Fig. 4. Model protoribosomes composed of basic proteinoid easily understood. and homopolynucleotide. These are smaller than proteinoid The concept of self-organization (table 1) is one microspheres. I shall take up later. In accord with the progression of fig. 1, the proto- cell-the product of "self"-aggregation of proteinoid Table 3. Experimental advances from reproductive proto- -is depicted next, in fig. 3. These are proteinoid cell toward contemporary cell. microspheres reproducing through a budding cycle. 1. Internucleotide bonds are synthesized by proteinoid They are approximately 15 ,um in diameter. Both the microspheres acting on ATP first-generation and second-generation (F1) micro- 2. Thermal polyamino acids and homopolynucleotides form spheres are highly uniform in diameter. The growth complexes (protosomes) selectively, in some cases codoni- process is accretive, or heterotrophic. cally or anticodonically The main properties found in the proteinoid micro- 3. Peptides are synthesized by nucleoproteinoid microparti- spheres are shown in table 2. (The microspheres have cles acting on ATP and amino acid many other properties of contemporary cells [Fox 4. Models of photophosphorylation have been demonstrated and Dose 1972].) They can be subtracted from the

164 THE AMERICANBIOLOGY TEACHER, MARCH 1974 functions of the contemporary cell to visualize what Table 4. vs. . was needed for evolution from a minimal cell to a maximal, or contemporary, cell. Progress has been Biogenesis Abiogenesis (and synonyms) made toward each of these goals (table 3). To treat Virchow (1858): "Omris Protobiogenesis these advances, we should first look at the models for cellula e cellula" ("All liv- Biopoesis the first ribosomes, the protein-synthesizingparticles ing cells arise from pre-ex- Spontaneous generation of the contemporary cell (fig. 4). This figure shows isting living cells") Self-assembly of a protocell complexes of basic proteinoid and polynucleotide. Pasteur (1864): "There is the question of so-called spon- These have several properties of ribosomes; we re- taneous generation. Can matter organize itself? In other gard them as models for protoribosomes.They have words, are there beings that can come into the world with- especially the ability to convert ATP and amino out parents, without ancestors? That is the question to be acid, in this case phenylalanine,into peptides (fig. 5). resolved." Work by John Jungck in our laboratory (Jungck and Fox 1973; Fox et al. 1974) has indicated how other microspheres of the right sort could convert ATP into small polymers of adenylic acid. This be- CELL gins to explain the origin of nucleic acids. Principal processes that need to be understood to Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021 bridge the gap from a model of a reproductive proto- cell to a contemporary cell (to summarize the anal- Naturalforces Vital force ysis) are cellular synthesis of protein, cellular syn- thesis of nucleic acid, a coding mechanism, and energetic of the collective biota with solar coupling Appropriatemacromolecules Appropriatemacromolecules . Having completed a description of the unifying proteinoid model, and having discussed some specific inhibiting assumptions for that model, we turn now I I to assumptionsnot directly related to concepts of the proteinoid or its evolution. For practical reasons I Naturalforces Vital force have selected fewer than all of those premises. What .1i Northrop referred to as "unnecessary assumptions" require only imagination and assertion; such un- Appropriatemonomers disciplined concepts can be virtually unlimited in Fig. 6. Natural forces vs. vital forces. number. Accordingly, I shall attempt to treat, espe- cially, competing concepts that are recent or have been prominent. One of the oldest of the conceptual conflicts is that Table 5. vs. . represented in table 4. The problem was stated by Pasteur. Although Pasteur held in disfavor the idea Molecular biology Molecular evolution of matter organizing itself into cells, he acknowl- Initial emphasis on con- Initial emphasis on primor- edged the conceptual possibility. Today's biochemist temporary dial or refers to such a heterotrophic act as self-assembly. What Pasteur needed was an under- Simple +- complex Simple complex standing of the and origin of matter of the Association of properties de- Some properties acquired appropriate kind. Now we can say in answer to stroyed suddenly gradually, some acquired Pasteur, a century later, "Yes, matter can organize suddenly itself into a cell without parents. We can furthermore specify the origin and nature of the necessary matter, plus many of the properties of the organized units." Many of the conceptual conflicts reflect choices of / appropriate experimental approach. Some of this Li~f e stems from the fact that leading molecular have concerned themselves with how life began. As table 5 proposes,the essential technique of molecular biology is not appropriate even to asking the question (as T. 0. Fox [personal communication 1972] was the first to insist), let alone answering it. Studies of assembly of components into systems, however, are in the same direction as evolution itself. The related -A.- LiiHTY-A X 'S - questions- (i) whether matter can organize itself and (ii) how one can study such processes-are fun- Fig. 7. . How did life begin on Mysticus?

PROTEINOIDTHEORY 165

/ ~~~~~~~~~~~~Aminoacids( Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021

Hot region Solfatara, etc. Low H20 % On hot locale, amino acids-- polyamino acids Fig. 8. Steps from aminoacids and concentrate; to microspheres. they are less volatile

Heat H20 damental to our entire discussion. I am not an anti- Amino acids - Proteinoid - Protocell reductionist, and before I conclude I hope to show that molecular evolution and molecular biology be- or long together. They need not be in competition. A very old competing concept, however, is that of Solutionof Heat Aminoacid Heat vitalism (fig. 6). The feeling that biologic phenomena amino acids --0- - mixture - Proteinoid cannot be fully understood in terms of physics and -H20 -H20 chemistry appears and reappears in many ways. As Fig. 9. Chemical reactions involved in the physical steps of stated earlier, vitalism was not simply and finally fig. 8. negated by Wohler's synthesis of urea, in 1828. I feel sure that the laboratory production of a model protocell was greatly delayed by the blanketing and recurring belief in vitalism. Fig. 7 deals with panspermia: the concept that seeds of life appeared on this planet from elsewhere. AMINOACIDS s I do not recall who said it, but this view simply "consigns the problem to a conveniently inaccessible corner of the ." It tells us nothing about how life originated there or here. In a recent revival of Arrhenius's old idea of panspermia, Crick and Orgel (1973) have suggested AMINOACIDS 0 "directed panspermia," with an extraterrestrial spaceship as the agent. Crick and Orgel acknowledge enzymes PROTEINS G the futility of transferring the problem elsewhere, except that another planet may contain a crucially ATP necessary mineral catalyst. In an earlier paper, Fig. 10. -bond synthesis in occurs to a Crick proposed that a geologically early mineral meaningful extent in the presence of ATP. permitted replication of nucleic acid; prior enzymic

166 THE AMERICANBIOLOGY TEACHER, MARCH 1974 protein was thereby unnecessary. We will return to Table 6. Amino acids protoprotein vs. HCN -. protopro- that problem. tein Fig. 8 deals with the recent criticism, allegedly by the late A. Katchalsky (1973), that our view is one Reactions that "life began in an oven." Katchalsky's paper was Amino acids HCN transcribed from a tape of a talk he gave before his untimely demise in the Lod airport; I am doubtful, Heat -H20 therefore, that the statement is quite like it sounds Polymer in print. However, in earlier years others made Proteinoid similar statements. What is here is important to H20 (H) separate two steps in our flowsheet. In the first, heat does , ovenlike, to polymerize amino acids. A second step is necessary. In the second step water Some amino acids triggers the aggregation of the special kind of matter, proteinoid, to a special kind of minimal cell. The Properties correct analogy therefore is that molecular pre- Peptide bonds No peptide bonds shown cursors of life began in an "oven," and that life All amino acids itself, by analogy, began in water. Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021 MW 5,000-15,000 MWs? (<1,000?) Moreover, we have shown that heat at a constant Ordered sequences No order shown temperature can promote two steps (fig. 9): the (i) Enzymic activities evaporation of water and (ii) the chemical condensa- Morphogenicity No biologic properties shown tion of the dried residues from a solution of amino acids into polymers of amino acids. This sequence meets all thermodynamic requirements (Fox and Dose 1972). Ordinarily, we polymerize dry amino Protocell acids above the contemporary boiling point of water. Cell Such temperatures are not necessary, however. I Water can certainly evaporate and does evaporate at lower temperatures; we showed, years ago, that the polymerization of amino acids would occur in the Descendantviruses Descendant cells presence of phosphates (Fox and Harada 1966) at temperatures well below 100 'C. (Young [1965] ac- Fig. 11. Protovirus vs. protocell. complished polymerization with phosphate at 25 'C.) An additional point derivable from this scenario is that, at temperatures that would cause fractionation, Table 7. Properties of proteinoids and competing concepts. the first materials to leave would be water and other Concept volatile organic compounds that arose in the syn- thesis, such as hydroxy acids. Amino acids would be Internal order Nucleic acid needed less volatile, and the polyamino acids that would re- Arrays of enzymic activity sult from them would be even less volatile. Concen- tration of the initial reactants would therefore be Microsystems Concept inevitable, and geologic fractionation would favor the Origin Nucleic acid needed reactions of our flowsheet up to the point of a change Compartmentalization in phase resulting from the entrance of water, as by Membranes needed rain or tidal wash. Associated enzymic The above explanation applies also to the question activities Nucleic acid needed Reproduction of how adequate concentrations of reactants would Growth Internal synthesis of occur. This question has been asked in several con- protein needed texts. The answer is simple, as indicated, being re- lated to the need to recognize processes in open sys- tems (Fox 1973b), as in the geologic realm. adenosine triphosphate (ATP). The thermodynamic Fig. 10 represents a statement that is found in a constraint is thus overcome, either in our model or in recently published book on the chemical origins of contemporary organisms, by ATP. Enzymes acceler- life (Ponnamperuma 1972a). The statement made ate reactions; they do not of themselves provide free there is that amino acids are converted to protein by energy for those reactions that require it. way of the enzymes, which help the molecules over Table 6 mentions a recently publicized idea: the the energy barrier. I find that principles of physical possibility that the first prebiotic proteins arose from chemistry and knowledge of biochemistry require precursors that were not amino acids. This idea was for peptide-bond synthesis a provision of free energy. originally suggested in principle by Akabori (1959), In contemporary organisms, as in our models for and it has been pursued by Matthews (1971). I see protoribosomes, the free energy is supplied by no reason, basically, to preclude the origin of pro-

PROTEINOIDTHEORY 167 replication in evolution. At first glance, molecular replication appears to be simpler than cellular evolu- tion. Operationally, however, primordial cells are seen to have arisen much more simply. Once thermal polyamino acids arose and water was present (as must have often occurred), minimal cells arose by aggregation of the polymer. These cells have been shown to have the ability to reproduce efficiently by budding and accretive growth of the separated buds. Recently, replication of proteinoid microspheres in four protoevolutionary modes has been described (Fox 1973c). Accordingly, the assumption that molecular replication preceded replication of micro- systems is not justified and might have interfered with the development of an understanding of the critical primordial sequence. Fig. 12. Closed flask vs. open system. Boiling points are as I would like now to return to competing concepts follows: H2, - 253 ?C; CH4, - 161 ?C; NH3, - 31 ?C; H20, Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021 100 OC. for earlier stages in fig. 1-especially to the question of the origin of amino acids, with which we begin teins via a non-amino-acid route as Matthews pro- the considerations that are presented here. In this poses, nor do I see that such a route precludes the conflict of concepts, we have experiments that are origin of proteins by combination of already formed interpreted on each side of the question. All of the a-amino acids. The two concepts need not be com- steps in the flowsheet of fig. 1 have been carried out petitive. We have preferred the amino acid route be- in open systems-a kind of situation that, as has been cause it is recapitulationist (Fox 1956; Brachet pointed out, is imperative for geochemical relevance. 1959), which is for us much more consonant with Many syntheses of amino acids and other simple evolutionary theory, and also because the recapitula- compounds have been conducted in simulation stud- tionist hypothesis has led, through experiments, to ies in closed flasks (fig. 12). The reactant gases are a comprehensive point of view. The main criticism of thus confined, and one can thereby obtain sub- the opposing concept in this instance is not of the stantial yields of products. Most significant is the concept but rather of the absence of supporting fact that reactions that would not go otherwise can evidence. The polymer obtained from HCN, which is be made to occur because of the retention of com- the one used, has not been shown to have even pep- pounds as fugacious as diatomic hydrogen, methane, bonds in it, as Ferris et al. (1973) have recently ammonia, and water. The historically influential emphasized. No evidence has been offered to show initial result in a closed flask was thus more one of that Matthews' polymer has any of the functions of stimulation than of simulation. proteins. This concept was in part put to the test in the Table 7 emphasizes the experimentally derived Apollo program during the past few years. fact that a protocell could have had both ordered Ponnamperuma (1972b) has pointed out that a molecules and some lipid quality. The absence of "bonanza of organic molecules" was expected in ad- need for nucleic acid is based on the finding that the vance from the analyses performed on samples from information could have arisen from diverse amino the surface of the Moon. When one traces the basis acids, as will be explained further. This basic idea for the high expectations through the nested bibli- is not new, having been expressed in various ways ographies, he finds that a considerable part of the by Lederberg (1959), Ehrensvard (1962), Com- basis existed in the closed-flask experiments of Miller moner (1964), Eigen (1971), and many others. Lipid (1955). At the end of some of those experiments, quality is found in the hydrocarbon sidechains of however, the hydrogen component was as high as many amino acids in proteinoids or proteins. A 75%. Such concentration could not have persisted minimal cell could, thus, have arisen very early. for a significant period in the open realm, lunar or Fig. 11 brings up a variation of the competing con- terrestrial. cept of nucleic-acids-first. The idea that a virus was Earlier, I emphasized that our reactions occur in the first living entity is not now widely held. We open systems, as does the subsequent aggregation of can examine this question on the basis of logic. the resultant macromolecules in the presence of Every virus known to us must, in order to multiply, water. Some investigators have taken the position have a cell. Accordingly, a cell had to precede that synthesis should occur in aqueous the first virus. If one argues that the protovirus had solution. For this purpose they have used various greater competence, not needing a host cell, he is agents, such as cyanamide, and ATP (fig. 13). A pyramiding Northrop's "unnecessary assumptions." reason that has been given for the use of aqueous A concept that competes in a subtle way with di- solutions for such syntheses is the alleged fact that rect inferences from constructionistic experiments is organisms are predominantly aqueous. Of course, the view that molecular replication preceded cellular organisms are predominantly aqueous, but the sur-

168 THE AMERICANBIOLOGY TEACHER, MARCH 1974 faces at which reactions are carried out are far dif- amino acids, by the side chains. These processes are ferent from dilute aqueous solution; I refer especially remarkably selective. The essential result has been to the surface of ribosomes. Another point that is established in a number of laboratories (Dose and missed by the proponents of carrying out such syn- Rauchfuss 1972), and in fact the ordering and theses in aqueous solution is that the origin of enzymic results from amino acids (Fox and Dose promoting agents would require even more drastic 1972) present by far the largest body of evidence on conditions than those used simply and directly to the consequences of thermal copolymerization of make polymers of amino acids by moderate heat. monomers. The opponents are therefore also pyramiding un- In my view, the principle of normal molecular necessary assumptions. Moreover, the thermal pro- interactions with stereochemical selectivity can now teinoids (see above) have a roster of properties (Fox be seen to be of fundamental significance in nearly and Dose 1972) not shown for the products of any all of evolution. Such forces underlie the origin of a of the peptides obtained in aqueous solutions or at first informational (protoprotein) , surfaces. and they underlie the origin of the . Our L. E. Orgel and associates (Fuller et al. 1972) original expectation of molecular selection was, how- have recently produced adenosine and other signifi- ever, not an assumption. It developed as a working cant biochemical substances in the absence of water. hypothesis from inferences that we drew fromn Orgel's use of solid-state synthesis is equivalent to Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021 the use of hypohydrous conditions, as we have long ATP advocated. These are neither competing nor alterna- Amino acids Peptides tive concepts. on surfacesin As another example of the utility of hypohydrous contactwith H20 conditions, Oro and Kimball (1961) produced adenine in aqueous solution from HCN and ammonia, but Wakamatsu et al. (1966) and associates multi- Cyanamide plied Oro's yield by carrying out the same synthesis Aminoacids Peptides under anhydrous conditions. in H20 In fig. 14 we come again to what I regard as a most fundamental aspect of molecular evolution and, therefore, of evolution as a whole. This is the concept Heat, -H20 that molecules react or interact selectively to yield largely ordered products. The competing concept is Aminoacids Peptides expressed in various ways: order out of chaos; ma- hypohydrous terial arising from a random matrix; and others. Our experiments in the thermal condensation of D;ry amino acids of different types and experiments in HCN - Adenine several other laboratories have demonstrated most vividly that the amino acids are ordered to a high heat degree in the resultant polymers. No prior nucleic Dry acid is necessary. As fig. 14 shows, in an oversimpli- Adenine + Ribose - Adenosine fied depiction of these effects, if one arranges three playing cards in all possible orders, he will get six heat sequences. This is a random array of the possible orders. Adenosine + PPA -* ATP The mistake has been to equate types of amino acids to playing cards or to colored beads, each of which is also of the same shape. Random arrange- 200 ?C t Heat ments can result with playing cards or colored beads simply because they are each of the same shape, ir- H3P04 respective of their labeled identities. Amino acids, however, are of different shapes. In the fig. 14 depiction of three amino acids we have 400 ?C selected shapes that show how three amino acids that 2 NaNH2 + C --* NaN-C-NNa are activated to react with each other would assume a configuration that is determined by those shapes. I __+ This is a stereochemical effect. The amino and H20 (H) carboxyl groups of the amino acids are represented by the hook-and-eye, in each case, and the coupling is the same as with amino acids. Because the shapes H2N-_CN differ, a unique order can result. The specific joining Fig. 13. Polymerizations of amino acids in water, near water, is determined by the superstructures or, for the and in relative absence of water.

PROTEINOIDTHEORY 169 enzyme experiments involving amino acids and pep- acids. It is not apparent to me that this kind of . Inasmuchas we had worked on terminal amino phenomenon could be identified by analytic, or re- acid assays since 1944 (before Sanger), we were ductionistic, studies. We would not have been able poised to apply Sanger's technique to the first pro- to infer it from sequence studies by us or by others- teinoids. This was the first evidence of nonrandom- only by synthetic or constructionistic experiments. ness within proteinoids; it was followed by many In fig. 15 the relationships of molecular selection other kinds of evidence from several laboratories. are developed further. Darwinian selection operates One of the most crucial concepts for the proteinoid on generated and evolved biosystems but not on theory is this one of order resulting from the stereo- those arising from a random matrix. Studies of chemical individualities of various types of amino molecular evolution have revealed vividly that con- straints in molecular possibilities have been domi- nant. This, then, is the matrix on which Darwinian A B C ABC selection had to operate. We are only beginning to 0 0 ACB obtain a quantitative evaluation of that matrix, Z) which by extrapolation must extend also into or- 0 _ BCA iXX g BAC ganismic evolution. The lesson of molecular selec- COOcM MIR. oO tion, as preceding and lying at the center of Dar- coos CAB Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021 102 I C BA winian selection, is a number 1 take-home lesson. Fig. 16 concerns a comparison between a random array droplets and proteinoid microspheres as models for the first cell. It would be difficult to find a definition "AMINOACIDS;WITH THE SHAPES OF of the coacervate droplet that would answer the PLAYINGCARDS question of whether the proteinoid microspheres are or are not themselves coacervate droplets. However, both in the Soviet Union and in this country I have encountered, many times, a question with respect to whether the typical coacervate droplet, as studied by A. I. Oparin and his school, is similar to or dif- ferent from the proteinoid microsphere. X ABC The type of coacervate droplet that Oparin usually product of works with is relatively unstable and nonuniform, as fig. 16 shows. The instability is revealed by the fact stereochemicalbias that a suspension of coacervate droplets will fall apart into two layers on standing for less than an UNITSOF DIFFERENTSHAPES, hour. The stability of proteinoid microspheres over- AS INAAMINOACIDS laps the stability range of cells. The crucial dif- Fig. 14. Random association of playing cards (all of same ference, however, is that the coacervate droplets do shape) vs. nonrandom association of amino acids (each of not answer the fundamental question of how cells unique shape). came into existence when there were no cells to sire them. This failure has its roots in the fact that the polymers used for coavervate droplets are ob- Evolving cells tained from cells that are already evolved. The pro- teinoid microspheres, however, arise from polymers obtained in turn from monomers polymerized under geologically relevant conditions. No cellular parent I Darwinian was needed. selection Another competing concept is that of the time re- quired for the origin of life (fig. 17). Again, I am using a model primordial reproductive cell as the Physical possibilities analog for the first life. I believe the fact that much time was available has been transmuted, in some way, to "much time was required." Perhaps much time was required to set the stage, but the experi- Molecular ments show that key events were rapid. The experi- selection ments suggest to us that these events occurred often, easily, and in many places on the planet. This view is reflected also in opposing inferences in table 8. I have recently read the argument that creationistic acts occurred innumerable times and at Fig. 15. Selection from conceptual possibilities vs. selection many stages; but this is not the Biblical view, and from physical possibilities. we can see, in the scientific context, that self-gen-

170 THE AMERICANBIOLOGY TEACHER, MARCH 1974 erating processes were sufficient to generate and maintain a sequence of events. It is easy to believe that natural processes had many repeat perfor- mances. If I, for example, had believed that life @0 arose only once, I would not have done protobio- genic experiments. Table 9 aligns the proteinoid theory against com- peting concepts in a broader way, and it serves as one kind of summary. Although this presentationhas concerned itself with individually competing con- cepts, the transcendental overview indicates that only one coherent scientific theory exists, and that Fig. 16. Coacervate droplets (left; X 320) vs. proteinoid it is a sequential continuity of steps based on experi- X ments carried out under geologically relevant condi- microspheres (right; 450). tions. In table 10 we come to the question of criteria of validity. A popular view is to state that our experi- occurrences. Table 8. Life only once vs. many protobiogenetic Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021 ments, or those of others, tell us not what did happen but what could happen. However, the view that Life only once Many occurrences chemistry is chemistry wherever and whenever it Divine creation Natural processes occurs seems axiomatic. Geologically relevant ex- periments say more than simply that it could have Fits best with a single event Not likely to occur a happened. It had to happen wherever the conditions single time were right. The question of the origin of life has been shown no longer to be imponderable. Analogously, we can now identify criteria of validity. Another criterion of validity of a total theory is Time compatibility of the steps in sequence. This in turn Required does require the premise that evolution is not one Gaseous random event arising out of another random event. reactants Experiments Conjeeture The validity of this underlying premise is judgeable only by overview and is therefore not easy to come 1 Minutes by. I believe the internally limited products from Amino acids heated a-amino acids are only one such manifesta- Hours tion, however dramatic this one is, of internal self- Eons limiting and self-generating processes in either pre- Protoprotein biotic or biotic evolution. If this view is correct, the criterion of sequential compatibility of individual I, Seconds steps appears to be a highly valid criterion of what Protocell did happen. With increasing frequency I have been asked if proteinoid microspheres are alive (table 11). In a Contemporary generally favorable report on my book with Klaus cell Dose on the origin of life, one reviewer (Sylvester- Bradley 1973) has pointed out that the units have Fig. 17. The time required. the properties of feeding, growth, and reproduction, and he asks "Is this not alive?" He then says, "The answer of course is very firmly in the negative," and he gives as a principal reason the fact that the Table 9. Proteinoid theory vs. competing concepts. microsphere does not have any DNA. This reason is Proteinoid Competing of course a reason for its not being alive in a con- Sequential compatibility Many reactions not temporary sense. We have not claimed that the disciplined by experiments proteinoidmicrospheres are alive. I believe, however, that one who claims that they are not alive should Geologic relevance; Some not geologically be required to present a definition of nonaliveness. reactions in open systems relevant The experimental results strongly suggest that the Unified theory to No complete sequence protocell arose and has gone through stages of alive- protocell ness and that the experiments should construct the definition-not the reverse. No unncessary assumptions Many unnecessary Finally, the experimentally derived concept of the assumptions protoribosome, mentioned earlier, provides a con- Unified laboratory model

PROTEINOIDTHEORY 171 Concepts of Concepts of molecular evolution molecularbiology

H20 ATP Protoprotein | Protocell - Nucleic acid Protoribosome

(Basic) Info. Info. \ ------Jnucleic /protein - H20 acid

Aminoacids ATP+ Amino acids

Fig. 18. Interdigitation of concepts of molecular evolution with concepts of molecular biology. Downloaded from http://online.ucpress.edu/abt/article-pdf/36/3/161/31762/4444705.pdf by guest on 24 September 2021

sequence of processes. Such trends can be seen in Table 10. Criteria of validity for "could" vs. "did." the evolution of organisms, but not so simply nor In realm Geologic relevance clearly as with molecules alone. the biologic the molecular processes operate in the ramified Sequential compatibility structures and functions of organisms. In my view, the most fundamental argument for not including ciliatory missing link between concepts of molecular creationist overviews in biology textbooks is that the evolution and of molecular biology. In my view, alone is sufficient to explain in outline what these two areas need not be regarded as competing is and has been going on, plus the subtle specificities (fig. 18). The processes of prebiotic molecular evolu- in the vast array of the biologic realm. No additional tion properly preceded the processes of molecular agent need be invoked to explain an . More specifically, the phenomena observed sequence that is proving to be self-generating at in our model protoribosomes are consistent with the every stage that has been studied. central dogma of molecular biology. The protoribo- somes require, however, a prior occurrence of pro- Acknowledgments.-Myresearch has been supported by grants NGR-10-007-08 and 10-007-088from the National Aeronauticsand Space Administration.This paper is con- Table 11. Aliveness vs. nonaliveness. tribution265 from the Institute for Molecularand Cellular Evolution. Aliveness Nonaliveness

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172 THE AMERICANBIOLOGY TEACHER, MARCH 1974 easily be replaced, or will he, too, have a meaningful VI. The influence of residue type on papain-catalyzed reac- role in the overall plan of the Creator? tions of some benzoylamino acids with some amino acid anilides. Journal of the American Chemical Society 75:5539. Man's life is a constant process of becoming. A et al. 1973. Accumulated analyses of amino acid human being is always emerging, and he is emerg- precursors in returned lunar samples. In Proceedings of ing from the initial genetic package that is, potentia- the Fourth Annual Lunar Science Conference, ed. by W. A. ly, all that he ever will be. Man is the body of his Gose et al. Pergamon Press, New York. Vol. 2, p. 2241. et al. 1974. From proteinoid microsphere to con- soul and the soul of his body. temporary cell: formation of internucleotide and peptide Man stands now at a doorway. We are all involved bonds by proteinoid particles. Origins of Life. In press, with the manner in which he crosses the threshold FULLER,W. D., et al. 1972. Studies in prebiotic synthesis VI. of science and technology. Beyond this portal, human Synthesis of purine nucleosides. Journal of Molecular Bi- existence will either ascend or descend, and it is the ology 67:25. JUNGCK, J. R., and S. W. Fox. 1973. Synthesis of oligonucleo- lot of our generation to make that choice. tides by proteinoid microspheres acting on ATP. Natur- Perhaps man's most profound progress will occur wissenschaften 60:425. to the extent that he will try to be more humane by KATCHALSKY,A. 1973. Prebiotic synthesis of biopolymers on refusing to do some of the things he could do. inorganic templates. Naturwissenschaften 60:215. KENYON,D. H. 1973. A theory of biogenesis [book review]. Science 179:789. References LEDERBERG,J. 1959. Ergebnisse und Probleme der Genetik.

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DIFFERENTPOINT OF VIEW 181