Organic Microstructures and Terrestrial Protocells Sidney W. Fox Institute for Molecular and Cellular Evolution. University of Miami, Coral Gables, FL 33134 This letter concerns several issues in Folsome's recent paper on "Synthetic Organic Microstructnres and the Origins of Cellular Life" [1]. Thereismuch agreement [2, 3] that facile early formation of cell-like structures from (appropriate) nonbiological polymers is a crucial question [4]. However: 1. The answer to Folsome's question of "Why Haven't Microstructures Been Seen Before in Spark-Discharge Experiments?" is that they have been. Such publication is by Grossenbacher and Knight, in a volume which Folsome cites for other reasons [5]. The Grossenbacher-Knight microstructures contain more than 10% carbon. Those authors infer special properties for the" organic content,. What makes the proteinoid microspheres of overall interest is their many functions [6] beyond their varied complex morphologies. 2. Several chemical statements of Folsome's are already answered in a paper in Naturwissenschaften [7] cited by him for other purposes. Any evaporative process would necessarily result in concentrating amino acids and also their thermal polymers [7] since both are of very low volatility. The reason for low volatility is, of course, that e-amino acids, H3+NCHRCOO -, are virtually the only ampholytic micromolecules found from either lunar or meteoritic material, or in heuristic laboratory experiments [6]. Polyamino acids obtained by heating ofc~-amino acids are also nonvolatile. If one assume other compounds such as urea, hydroxy acids, nitriles, etc. to have been formed, those compounds were bound to have been more volatile than ampholytes of either low or high molecular weight. Experiments done with such volatile organic compounds have accordingly seemed not to require publication, although results with minerals, which are nonvolatile, have merited reporting [8]. Folsome's imputation that dicarboxylic amino acids must be present in excess over other amino acids in amino acid reaction mixtures is false [9-13]. Folsome's imputed "overheated" locales, such as lava flows, are likewise falsely based. Temperatures as low as 65 ~ have been demonstrated for polymerization of amino acids [13-16] and are plausible for the early Earth. 380
Folsome's comments on fi and c~ linkages in protein and proteinoids are unsupported for protobiogenesis; reasons why the comments of Andini [17] and Temussi [18] are indefensible have been explained [13, 19]. The presence of seawater salts can help, rather than hinder, the formation of meaningful polymers [15]. In the light of selectively low volatility of amino acids and their polymers, one can add to the concentrative mechanisms proposed by Dose [20], the conditions of fresh water and estuarine events as well. A wide range of ratios of amino acid: mineral saltswould thus be possible. 3. The ungeologicality of spark-discharge experiments in gases within closed flasks, as conducted by Folsome et al. [21], has been emphasized and explained in recent years [13, 22, 23], especially for the high yields found in closed flasks. As Florkin [22] indicated, the 75% I-I2 in gases in flasks energized by electric discharge are without terrestrial counterpart. For this and other reasons, the alleged potential of Folsome's organic microstructures seems to me to be poorly supported; until functions are demonstrated, their occurrence is only a priori assumption for any given microstructure. Folsome's "faint hint" [21] of amino acid contents suggests contamination; his failure to find the kind of catalytic activities that is known to be abundant in thermal copolyamino acids [6] raises serious questions about what he is looking at. Experiments yielding activity [6] and those not doing so [211 contribute to the inference that protoenzymic activity on the early Earth was supported by copolyamino acid structure. Basically, Folsome's "polymers" are uncharacterized; nor have they been shown to be polymers. In summary, Folsome's paper fails to report on much that has been done and inferred [6, 24] and, in my judgment, it extrapolates from inadequate evidence and overstates the significance of his products for a context of protobiogenesis.
Acknowledgment: I thank Drs. Klaus Dose and D.L. Rohlfing for reviewing an earlier version of this manuscript.- The work done was supported by Grant No. NGR 10-007-008 of the
National Aeronautics and Space Administration. Contribution No. 350 of the Institute for Molecular and Cellular Evolution.
Received November 22, 1976 and February 28, 1977
1. Folsome, C.E.: Naturwissenschaften 63, 303 (1976) 2. Fox, S.W.: Origins of Life 7, 49 (1976) 3. Zuckerkandl, E. : J. Mol. Evolution 7, 39 (1975) 4. Fox, S.W.: Naturwissenschaften 56, 1 (1969) 5. Grossenbacher, K.A., Knight, C.A.: Origins of Prebiological Systems and of their Molecular Matrices, p. 173 (Fox, S.W., ed.). New York: Academic Press 1965 6. Fox, S.W., Dose, K. : Molecular Evolution and the Origin of Life. San Francisco : Freeman 1972 7. Fox, S.W. : Naturwissenschaften 60, 359 (1973) 8. Rohlfing, D.L., MeAlhaney, W.W. : BioSystems 8, 139 (1976) 9. Harada, K., Fox, S.W. : Arch. Biochem. Biophys. 109, 49 (1965) 10. Rohlfing, DL. : Nature 216, 657 (1967) 11. Oshima, T. : Arch. Biochem. Biophys. 126, 478 (1968) 12. Fox, S.W., Waehneldt, T.V. : Biochim. Biophys. Acta 160, 246 (1968) 13. Fox, S.W. :J. Mol. Evolution 8, 301 (1976) 14. Harada, K., Fox, S.W. : The Origins of Prebiological Systems and of their Molecular Matrices, p. 289 (Fox, S.W., ed.). New York: Academic Press 1965 15. Snyder, W.D., Fox, S.W.: BioSystems 7, 222 (1975) 16. Rohlfing, D.L. : Science 193, 68 (1976) 17. Andini, S., et al. : Origins of Life 6, 147 (1975) 18. Temussi, P.A., et al. : J. Mol. Evolution 7, 105 (1976) 19. Fox, S.W., Suzuki, F. : BioSystems 8, 40 (1976) 20. Dose, K.D.: ibid. 6, 227 (1975) 2l. Folsome, C.E., et al. : Precambrian Res. 2, 263 (1975) 22. Florkin, M. : Comprehensive Biochemistry, Vol. 29 B, p. 231. Amsterdam: Elsevier 1975 23. Keosian, J. : The Origin of Life and Evolutionary Biochemistry (Dose, K., etal., eds.). New York: Plenum Press 1974 24. Fox, S.W. : Int. J. Quant. Chem. QBS2, 307 (1975)
Namrwissenschaften 64 (1977)
O by Springer-Verlag 1977
Reply Clair E. Folsome Laboratory for Exobiology, Department of Microbiology, University of Hawaii, Honolulu, Hawaii 96822 My work dealt with organic microstructures [2]. Grossenbacher and Knight [4] observed spherules of 800 A to 50 A with a density of 1.8 and a "low carbon content". These workers concluded that their spherules possibly were silicates derived from borosilicate glass upon continuous exposure to ammonia (refer to p. 182). These spherules would not be visible by light microscopy and are not within the size domains of cells or of presumed protocells. One test of the fitness of the organic microstructures I have described as models of protobionts is to look for instances of independent synthesis by natural means. Carbonaceous chondrites [7], organic inclusions in quartz crystals [5], and the organic structures of the Isua micaceous metaquartzites [6] are such instances. In all these cases one is presented with evidence of natural non-biological synthesis of organic microstructures which markedly resemble the spark-discharge structures that can be made in the laboratory. Lack of catalytic activity appears a problem. Either spark-discharge organic microstructures do in fact have none, or it has yet to be observed. It is by no means requisite that such activity be manifest in this aspect of my protocell model. Catalysis might be a property of microstructure-cation complexes, of microstructurepolypeptide complexes, etc. Certainly more work needs to be done. One can imagine a primitive world in which microstructures accumulate. Fraser and Folsome [3] have shown that these structures self-assemble autocatalytically. During later stages in protobiout evolution catalytic activity could have become of predominant import. Fox states that the presence of seawater salts helps, rather than hinders the formation of meaningful (i.e. proteinoid) polymers [8]. His experimental procedure reveals that 15 g total weight of 18 amino acids were mixed in 375 ml artificial seawater to make proteinoid microstructures. We are presented with an incredible reaction mixture of about 13 g salts plus 15 g amino acids; a molar ratio of about 1 to 8 (amino acid to salt). Calculations regarding formation rates, concentrative Naturwissenschaften 64 (1977)
rates, thermal and photochemical decomposition rates [1] point to an abundance of amino acids in seawater of 10-TM, a molar ratio of amino acids to salts 10 million times less in the geologically plausible world. Indeed, one can create special case concentrative mechanisms as fresh water evaporites nearby volcanoes and ocean shores, etc. In these instances there is no reason to think Nature would have been single-minded regarding amino acids. Assume we can drastically concentrate in special locales the sum total of all likely non-volatile monomers found in reconstruction experiments. This mixture, prior to anhydrous condensation, will contain hydroxy acids, diacids, sugars, nitriles, hydrocarbons, ureas, guanides, heterocyclic compounds, etc. as well as '~ and "non-biological" amino acids. To my knowledge no one has attempted condensation reactions upon such a complex mixture. In my opinion, until one performs such experiments and demonstrates significant yields of, say, thermal proteinoids, the plausibility of any concentrative scenario remains a matter of wish and not of fact. Fox also missed the point of quenched spark-discharge reactions. In these, discharge occurs directly upon a water phase which serves effectively to trap reactive intermediates. A surface film of hydro-
phobic material rapidly accumulates and attains more stable states as spheres when the water surface is disturbed. One could consider the whole of the primitive earth to be one immense closed flask: the question Fox raised of "closed" or "open" systems in this context seems more a point of view of size. I do agree with Fox that many protobiont experiments must have occurred in the dawn of the Archean. I think that to require a protobiont to have catalytic activity, or even protein (why not polynucleotides?, lipids?) structure because cells now evolved possess these characteristics is arbitrary attribute listing and not a reasonable scientific approach. Received January 24 and March 9, 1977 1. Dose, K. : BioSystems6, 224 (1975) 2. Folsome, C.E. : Naturwissenschaften 63, 303 (1976) 3. Fraser, C.L., Folsome, C.E. : Origins of Life 6, 429 (1975) 4. Grossenbacher, K.A., Knight, C.A. : Origins of Prebiological Systems and of their Molecular Matrices, p. 173 (Fox, S.W., ed.). New York: Academic Press 1965 5. Mneller, G. : Nature 235, 90 (1972) 6. Nagy, B., Zumberge, J.E., Nagy, L:A.: Proc. Natl. Acad. Sci. USA 72, 1206 (1975) 7. Rossignol-Strick, M., Barghoorn, E.S.: Space Life Sci. 3, 89 (1971) 8. Snyder, W.D., Fox, S.W. : Bio Systems 7, 222 (1975)
Uber Sterane und Triterpane in Erd61en und ihre phylogenetische Bedeutung A. Hollerbach und D.H. Welte Lehrstuhl fiir Geologie, Geochemie und Lagerst/itten des Erd61s und der Kohle, Rheinisch-Westffilisehe Technische Hochschule, D-5100 Aachen Die gltesten Hinweise auf fossile OrgaNsmen kennen wit aus Gesteinen, die mehr als 3.109 Jahre alt sind, z.B. Mikroorganismen in siidafrikanischen Hornsteinen [1, 2]. Neben morphologisch dokumentierten Zeugnissen vergangeneu Lebens finder man auch biogene, molekulare Bausteine als chemische Zeugnisse ftir vergangenes Leben, die h~ufig ,,Chemofossilien" genannt werden.
9 by Spriuger-Verlag 1977
Typische Chemofossilien sind terpenartige Kohlenwasserstoffe, wie Sterane und pentacyclische Triterpane, deren Kohlenstoffgerfiste nur biologisch entstanden sein k6nnen. Verbindungen dieser Art wurden zwar bereits im 2,7.109 Jahre alten Soudan-Schiefer gefunden [3], aber ihr autochthoner Ursprung ist zweifelhaft [4]. Es gilt als nahezu sicher, dab die biologische Evolution, die im wesentlichen im Palfiozoi381
Folsome's comments on fi and c~ linkages in protein and proteinoids are unsupported for protobiogenesis; reasons why the comments of Andini [17] and Temussi [18] are indefensible have been explained [13, 19]. The presence of seawater salts can help, rather than hinder, the formation of meaningful polymers [15]. In the light of selectively low volatility of amino acids and their polymers, one can add to the concentrative mechanisms proposed by Dose [20], the conditions of fresh water and estuarine events as well. A wide range of ratios of amino acid: mineral saltswould thus be possible. 3. The ungeologicality of spark-discharge experiments in gases within closed flasks, as conducted by Folsome et al. [21], has been emphasized and explained in recent years [13, 22, 23], especially for the high yields found in closed flasks. As Florkin [22] indicated, the 75% I-I2 in gases in flasks energized by electric discharge are without terrestrial counterpart. For this and other reasons, the alleged potential of Folsome's organic microstructures seems to me to be poorly supported; until functions are demonstrated, their occurrence is only a priori assumption for any given microstructure. Folsome's "faint hint" [21] of amino acid contents suggests contamination; his failure to find the kind of catalytic activities that is known to be abundant in thermal copolyamino acids [6] raises serious questions about what he is looking at. Experiments yielding activity [6] and those not doing so [211 contribute to the inference that protoenzymic activity on the early Earth was supported by copolyamino acid structure. Basically, Folsome's "polymers" are uncharacterized; nor have they been shown to be polymers. In summary, Folsome's paper fails to report on much that has been done and inferred [6, 24] and, in my judgment, it extrapolates from inadequate evidence and overstates the significance of his products for a context of protobiogenesis.
Acknowledgment: I thank Drs. Klaus Dose and D.L. Rohlfing for reviewing an earlier version of this manuscript.- The work done was supported by Grant No. NGR 10-007-008 of the
National Aeronautics and Space Administration. Contribution No. 350 of the Institute for Molecular and Cellular Evolution.
Received November 22, 1976 and February 28, 1977
1. Folsome, C.E.: Naturwissenschaften 63, 303 (1976) 2. Fox, S.W.: Origins of Life 7, 49 (1976) 3. Zuckerkandl, E. : J. Mol. Evolution 7, 39 (1975) 4. Fox, S.W.: Naturwissenschaften 56, 1 (1969) 5. Grossenbacher, K.A., Knight, C.A.: Origins of Prebiological Systems and of their Molecular Matrices, p. 173 (Fox, S.W., ed.). New York: Academic Press 1965 6. Fox, S.W., Dose, K. : Molecular Evolution and the Origin of Life. San Francisco : Freeman 1972 7. Fox, S.W. : Naturwissenschaften 60, 359 (1973) 8. Rohlfing, D.L., MeAlhaney, W.W. : BioSystems 8, 139 (1976) 9. Harada, K., Fox, S.W. : Arch. Biochem. Biophys. 109, 49 (1965) 10. Rohlfing, DL. : Nature 216, 657 (1967) 11. Oshima, T. : Arch. Biochem. Biophys. 126, 478 (1968) 12. Fox, S.W., Waehneldt, T.V. : Biochim. Biophys. Acta 160, 246 (1968) 13. Fox, S.W. :J. Mol. Evolution 8, 301 (1976) 14. Harada, K., Fox, S.W. : The Origins of Prebiological Systems and of their Molecular Matrices, p. 289 (Fox, S.W., ed.). New York: Academic Press 1965 15. Snyder, W.D., Fox, S.W.: BioSystems 7, 222 (1975) 16. Rohlfing, D.L. : Science 193, 68 (1976) 17. Andini, S., et al. : Origins of Life 6, 147 (1975) 18. Temussi, P.A., et al. : J. Mol. Evolution 7, 105 (1976) 19. Fox, S.W., Suzuki, F. : BioSystems 8, 40 (1976) 20. Dose, K.D.: ibid. 6, 227 (1975) 2l. Folsome, C.E., et al. : Precambrian Res. 2, 263 (1975) 22. Florkin, M. : Comprehensive Biochemistry, Vol. 29 B, p. 231. Amsterdam: Elsevier 1975 23. Keosian, J. : The Origin of Life and Evolutionary Biochemistry (Dose, K., etal., eds.). New York: Plenum Press 1974 24. Fox, S.W. : Int. J. Quant. Chem. QBS2, 307 (1975)
Namrwissenschaften 64 (1977)
O by Springer-Verlag 1977
Reply Clair E. Folsome Laboratory for Exobiology, Department of Microbiology, University of Hawaii, Honolulu, Hawaii 96822 My work dealt with organic microstructures [2]. Grossenbacher and Knight [4] observed spherules of 800 A to 50 A with a density of 1.8 and a "low carbon content". These workers concluded that their spherules possibly were silicates derived from borosilicate glass upon continuous exposure to ammonia (refer to p. 182). These spherules would not be visible by light microscopy and are not within the size domains of cells or of presumed protocells. One test of the fitness of the organic microstructures I have described as models of protobionts is to look for instances of independent synthesis by natural means. Carbonaceous chondrites [7], organic inclusions in quartz crystals [5], and the organic structures of the Isua micaceous metaquartzites [6] are such instances. In all these cases one is presented with evidence of natural non-biological synthesis of organic microstructures which markedly resemble the spark-discharge structures that can be made in the laboratory. Lack of catalytic activity appears a problem. Either spark-discharge organic microstructures do in fact have none, or it has yet to be observed. It is by no means requisite that such activity be manifest in this aspect of my protocell model. Catalysis might be a property of microstructure-cation complexes, of microstructurepolypeptide complexes, etc. Certainly more work needs to be done. One can imagine a primitive world in which microstructures accumulate. Fraser and Folsome [3] have shown that these structures self-assemble autocatalytically. During later stages in protobiout evolution catalytic activity could have become of predominant import. Fox states that the presence of seawater salts helps, rather than hinders the formation of meaningful (i.e. proteinoid) polymers [8]. His experimental procedure reveals that 15 g total weight of 18 amino acids were mixed in 375 ml artificial seawater to make proteinoid microstructures. We are presented with an incredible reaction mixture of about 13 g salts plus 15 g amino acids; a molar ratio of about 1 to 8 (amino acid to salt). Calculations regarding formation rates, concentrative Naturwissenschaften 64 (1977)
rates, thermal and photochemical decomposition rates [1] point to an abundance of amino acids in seawater of 10-TM, a molar ratio of amino acids to salts 10 million times less in the geologically plausible world. Indeed, one can create special case concentrative mechanisms as fresh water evaporites nearby volcanoes and ocean shores, etc. In these instances there is no reason to think Nature would have been single-minded regarding amino acids. Assume we can drastically concentrate in special locales the sum total of all likely non-volatile monomers found in reconstruction experiments. This mixture, prior to anhydrous condensation, will contain hydroxy acids, diacids, sugars, nitriles, hydrocarbons, ureas, guanides, heterocyclic compounds, etc. as well as '~ and "non-biological" amino acids. To my knowledge no one has attempted condensation reactions upon such a complex mixture. In my opinion, until one performs such experiments and demonstrates significant yields of, say, thermal proteinoids, the plausibility of any concentrative scenario remains a matter of wish and not of fact. Fox also missed the point of quenched spark-discharge reactions. In these, discharge occurs directly upon a water phase which serves effectively to trap reactive intermediates. A surface film of hydro-
phobic material rapidly accumulates and attains more stable states as spheres when the water surface is disturbed. One could consider the whole of the primitive earth to be one immense closed flask: the question Fox raised of "closed" or "open" systems in this context seems more a point of view of size. I do agree with Fox that many protobiont experiments must have occurred in the dawn of the Archean. I think that to require a protobiont to have catalytic activity, or even protein (why not polynucleotides?, lipids?) structure because cells now evolved possess these characteristics is arbitrary attribute listing and not a reasonable scientific approach. Received January 24 and March 9, 1977 1. Dose, K. : BioSystems6, 224 (1975) 2. Folsome, C.E. : Naturwissenschaften 63, 303 (1976) 3. Fraser, C.L., Folsome, C.E. : Origins of Life 6, 429 (1975) 4. Grossenbacher, K.A., Knight, C.A. : Origins of Prebiological Systems and of their Molecular Matrices, p. 173 (Fox, S.W., ed.). New York: Academic Press 1965 5. Mneller, G. : Nature 235, 90 (1972) 6. Nagy, B., Zumberge, J.E., Nagy, L:A.: Proc. Natl. Acad. Sci. USA 72, 1206 (1975) 7. Rossignol-Strick, M., Barghoorn, E.S.: Space Life Sci. 3, 89 (1971) 8. Snyder, W.D., Fox, S.W. : Bio Systems 7, 222 (1975)
Uber Sterane und Triterpane in Erd61en und ihre phylogenetische Bedeutung A. Hollerbach und D.H. Welte Lehrstuhl fiir Geologie, Geochemie und Lagerst/itten des Erd61s und der Kohle, Rheinisch-Westffilisehe Technische Hochschule, D-5100 Aachen Die gltesten Hinweise auf fossile OrgaNsmen kennen wit aus Gesteinen, die mehr als 3.109 Jahre alt sind, z.B. Mikroorganismen in siidafrikanischen Hornsteinen [1, 2]. Neben morphologisch dokumentierten Zeugnissen vergangeneu Lebens finder man auch biogene, molekulare Bausteine als chemische Zeugnisse ftir vergangenes Leben, die h~ufig ,,Chemofossilien" genannt werden.
9 by Spriuger-Verlag 1977
Typische Chemofossilien sind terpenartige Kohlenwasserstoffe, wie Sterane und pentacyclische Triterpane, deren Kohlenstoffgerfiste nur biologisch entstanden sein k6nnen. Verbindungen dieser Art wurden zwar bereits im 2,7.109 Jahre alten Soudan-Schiefer gefunden [3], aber ihr autochthoner Ursprung ist zweifelhaft [4]. Es gilt als nahezu sicher, dab die biologische Evolution, die im wesentlichen im Palfiozoi381
