CHEMISTRY AND THE ORIGIN OF LIFE Academician A. I. Oparin Director, Bakh Institute of Biochemistry Academy of Sciences of the USSR According to modern ideas the beginning of life on Earth is by no means the result of a lucky chance (as assumed until quite recently); it is a completely regular event and an integral component of the general evolutionary develop- ment of our planet. The basis of this event is the process of successive increase of the complexity of carbon compounds and of the polymolecular systems formed therefrom. The whole process, which has lasted for thousands of millions of years, can be subdivided into the following stages: 1. The first appearance of hydrocarbons, cyanides and their nearest derivatives in cosmic space and after the planet Earth was formed, during the formation of the Earth’s crust, atmosphere and hydrosphere.2. The conversion, on the Earth’s surface, of the initial carbon compounds to increasingly more complex organic substances-monomers and polymers. The emergence of the so-called ‘primordial broth’. 3. Generation, in this broth, of the open polymolecular systems capable of interacting with the ambient external medium, and thus of growing and reproducing themselves (formation of the so-called ‘protobionts’). 4. Further evolution of protobionts, the improvement of their metabolism and of the molecular and supramolecular structure on the basis of pre- biological selection. Emergence of primordial organisms. It is readily seen that the first two stages referred to above are of a clearly pronounced chemical nature.Their knowledge is based completely on the study of physical and chemical phenomena wholly unrelated to life and of a purely abiogenetic nature. However, during the later stages the objects undergoing evolution attain a very high complexity of organization, and their subsequent development begins to be determined by regularities of biological nature. Consequently at a certain stage of the development of matter towards the origin of life there takes place the transition from chemical to biological evolution, the latter being based on the interaction between organism and medium and on the Darwinian principle of natural selection. Life is material in its nature, but inherent in it are peculiar qualities which distinguish the organisms from the objects of the inorganic world.In the first place there is the exceptionally perfect adaptability or, as it is often called, ‘expediency’ of the entire organization of living creatures, which is directed towards their constant self-preservation and self-reproduction under the given conditions of environment, and the adaptation of the structure of the individual parts of the living organism (molecules, organoids, cells, tissues and organs) to the functions they perform in the process of life. This expe- diency is an obligatory property of any living organism that does not exist outside the world of living creatures under the natural conditions of inorganic Oparin 1 nature. For a long time its essence seemed to be mystical and supernatural.It was considered to be a purposeful fulfilment, by the living creatures, of the designs of a spiritual principle which governs life. Darwin supplied a rational explanation of the formation of this ‘expediency’ on the basis of natural selection, which is a specifically biological regularity. However Darwin’s teaching applied in principle only to the fully formed and relatively highly developed living creatures. At present we consider that Darwinism is the resplendent top of an iceberg, almost nine-tenths of which is hidden under water. The pre-biological and the initial biological evolution, during which the fundamental features and properties characteristic of every living thing were evolved, had lasted for a much longer period of time and had abounded in no less dramatic events than the evolution of the objects usually investigated by Darwinists.The emergence of substances which are essential to this initial pre-biological evolution, viz. of hydrocarbons, cyanides and their nearest derivatives, took place many thousands of millions years ago, a long time prior to the forma- tion of the solar system. Formation of these materials was caused both by the exceptional predominance of hydrogen in the universe and by the fact that carbon was formed before the heavy elements, that are indispensable to the emergence of planetary systems, in the stable process of stellar emission of radiation. Carbon is detected in the spectra of all star classes, inchrding the most ancient generations, the age of which is reckoned as 15-20 x 109 years. For this reason carbon-hydrogen compounds are extremely abundant in the universe; they occur both on the surface of stars with a temperature of several thousand degrees and with a very high gravitation, and in the inter- stellar gas-dust matter, at an extremely low gravitation and a temperature near absolute zero.This can be confirmed both by studying the present clouds of interstellar matter themselves, and by investigating the spectra of comets, cosmic bodies which form under conditions closely resembling these of interstellar space. These investigations demonstrate that comets abound in hydrocarbons and cyanogen. Of special interest are the data obtained from the investigation of meteorites, firstly because they are the only non-terrestrial bodies that can be directly analysed at present, and secondly because, with respect to their composition, they are very similar to the clusters of cosmic-dust matter-the planetesi- mals-from which Earth and the terrestrial-type planets were formed.In the composition of some meteorites, the so-called ‘carbonaceous chondrites’, not only were the initial simplest compounds of carbon and hydrogen detected, but also their much more complex derivatives, diverse organic substances, which arose here abiogenetically, independent of life. Along with the high molecular weight hydrocarbon polymers, which are sometimes very similar to animal or vegetable fats, carbonaceous chondrites also contain substances characteristic of the animal world such as amino acids and the nucleotides.Consequently, not only prior to the emergence of life, but even a long time before the formation of the Earth, the second stage of the evolution of carbon compounds began-their conversion to increasingly more complex organic substances. These conversions occurred abiogenetically on the surface of R.I.C. Reviews 2 cosmic-dust particles and planetesimals as a result of the action of short- wavelength ultraviolet radiation and cosmic rays. Thus the Earth obtained a certain quantity of these substances in a finished form during the process of its formation as a planet, and later it was ‘nourished’ with these substances when meteorites and comet material fell on its surface.Nevertheless the bulk of organic substances, indispensable for the emer- gence of life, seem to have appeared on Earth endogenetically, when the Earth’s crust, the hydrosphere and the secondary atmosphere were formed. According to current thinking, our planet was formed by the accumulation of cold solids (planetesimals). Gases such as molecular hydrogen, helium etc. could be preserved only when adsorbed by solid rocks, where they formed part of the primary Earth atmosphere. However, that atmosphere was not long lived, because its component gases were not retained by terrestrial gravitation. The remaining dense mass of the Earth continued its evolution, which was principally determined by the thermal history of the planet.Owing to the heat produced by gravitational energy and by the energy of decay of the radioactive elements, the primary rocks were partly melted, and the terrestrial crust, hydrosphere and atmosphere formed. Most of the water was originally bound in hydrated rocks. Consequently the amount of water on the Earth’s surface was originally much smaller than it is today, and the formation of the oceans took place only very gradually, in conjunction with the formation of the Earth’s crust. The generation of the secondary terrestrial atmosphere was also closely related to crust formation. The secondary atmosphere differed in essence from the present-day atmos- phere. It was reducing, lacked free oxygen (02) and, in addition to water vapour, contained such hydrogen compounds as gaseous hydrocarbons, ammonia and hydrogen sulphide.Owing to the absence of molecular oxygen no ozone shield could form so the atmosphere was completely permeable to short-wavelength ultraviolet rays. Having reproduced the conditions of the surface at that time on the laboratory scale, numerous investigators in various countries demonstrated convincingly the inescapability of synthesis, in the secondary atmosphere and in the Earth’s hydrosphere, of various organic substances-amino acids, sugars, purine and pyrimidine bases, nucleotides, organic acids, and various polymers, including protein-like and nuclein-like substances. These, however, lacked any adaptability of their intramolecular structure to the fulfilment of biological functions, a characteristic of the present-day proteins and nucleic acids.One can, at present, imagine and reproduce to a certain extent the sequence of emergence and the nature of transformation of the complex organic substances referred to above in the waters of the Earth’s primary hydrosphere, the so-called primordial broth. Of course the nature of these transformations differed in essence from the highly organized metabolism which takes place in present-day living organisms. In general outline it only corresponded to the sequence of phenomena which occur in a simple aqueous solution of organic substances, and of course one would not find any of the ‘expediency’ of phenomena characteristic of life.However, life is not simply dispersed in space like the substances of the Oparin 3 primordial broth. Life is represented by organisms-discrete systems which are spatially isolated from the ambient external medium and yet interact with this medium as open systems. The stability of such systems, the duration of their existence, is determined not by their immutability-quiescence-but, on the contrary, by the constant transformation of substances, by the regular combination of synthesis and decomposition, which form, in their totality, the biological metabolism in living organisms. As has already been said, the characteristic feature of biological metabolism is its purposefulness with respect to the constant self-preservation and self- reproduction of the entire living system as a whole under the given conditions of the ambient medium.This feature could not have arisen by chance. It could have been formed only in the process of gradual perfection of the initial polymolecular open systems, which were more primitive than the organisms. One can not only imagine, but also reproduce in an experiment a large number of such systems (bubbles of Goldacre, microspheres of Fox, coacer- vates of Bundenberg de Jong, and many others). For the further evolution of these systems it was of importance that they should interact with the ambient external solution as open systems, and that their stability should not be of a static, but of a dynamic steady-state nature. From this viewpoint the coacer- vate drops appear to be the most convenient, but of course not the only models possible for the reproduction of phenomena which took place in the remote past.In the formation of the coacervate drops molecules of various polymers which were previously distributed uniformly throughout the homogeneous solution begin to accumulate at definite points to form entire molecular swarms and clusters, separating out of the ambient medium in the form of drops visible under the microscope and ‘swimming’ in the original solution from which, however, they are now separated by a sharp boundary-the surface. In the drops the concentration of polymers may be 50 per cent or more, whilst the ambient medium is almost free of them. Experiments conducted at our laboratory in the Bakh Institute of Bio- chemistry demonstrated that the coacervate drops form when one mixes aqueous solutions of even non-specific or completely homopolymeric poly- peptides and polynucleotides (for instance, polyadenine and polylysine).The intramolecular structure of these polymers, which is of such great importance to present-day organisms, is of no significance in the formation of coacer- vates. Only the size of molecules is essential. Therefore, during the simul- taneous synthesis of polymers of a disordered structure (which should also occur in the ‘primordial broth’) coacervate drops emerge without fail as soon as a definite degree of polymerization of the substances referred to above is attained. The coacervate drops are capable of absorbing selectively from the ambient broth various low-molecular weight substances-amino acids, sugars, mononucleotides, diverse salts etc.If only some of these substances are capable of accelerating catalytically the chemical reactions which take place in the drops, the drops become open systems which react specifically with the external medium. In our model experiments, by incorporating into the drops the most diverse simple and complex catalysts (organic substances and inor- 4 R. I . C. Reviews ganic salts), we induced in the coacervate drops the reactions of synthesis and of decomposition of the component polymers. As an example I should like to describe the layout of one of our experiments. In this diagram the rectangular outline represents the coacervate drop which contains a catalyst that converts adenine to a homopolymer (Poly-A).The source of adenine is adenosine diphosphate (ADP). ADP enters the coacer- vate drop and is polymerized here to Poly-A, causing the drop to increase in volume and weight-it grows before our eyes-while inorganic phosphorus (Pi) separates out into the ambient medium, which previously had contained no such phosphorus. We also produced in the coacervates more complicated systems of meta- bolite flow, in which not one, but several reactions were combined. According to the combination, one obtained a more rapid or a relatively slow growth of the drops and, in other cases, their decomposition and disappearance. Open polymolecular systems endowed with a primitive metabolism and resembling our models should have appeared readily in the Earth’s primordial broth.While increasing their volume and weight, such systems (let us call them ‘protobionts’) should, under the conditions prevailing in the primordial broth, grow and then break up as a ksult of external mech’anical forces (e.g. surf or wave shock), just as the drops of an emulsion break up upon shaking. The daughter protobionts which emerged in this process would have retained to a certain extent the original protobiont’s interaction with the ambient medium, absorbing all the time certain catalysts from this medium and thus preserving the constant rate ratio and constant concordance of reactions occurring in them.Of course such constancy was highly imperfect compared with the self-reproducibility of present-day organisms. Upon this basis the ‘competition’ of the protobionts with respect to the rate of growth and reproduction could have originated, followed by the peculiar ‘pre- biological selection’ during which only the protobionts, which became increasingly more adapted to the conditions of the ambient medium, were preserved and kept on growing. In the model experiments with coacervates we demonstrated the possibility of ‘selection’ of this kind. For this purpose we incorporated into some drops a complex of catalysts which, under the given conditions of the ambient medium, led to a relatively rapid synthesis of polymers and to the growth of the entire system as a whole.On the contrary, in other drops this complex of catalysts was less perfect. It can be seen in Fig. 1 that the drops of the first kind grow rapidly, whereas the other kind show a suppressed growth. Thus, even at the relatively early stage of evolution which we have been examining there arose a new, previously absent, regularity which determined completely the trend of development of protobionts and of the subsequent biological systems. We can, to a certain extent, form the idea of the subsequent stages of this evolution on the basis of the comparative biochemical study of metabolism and of structures in the most primitive contemporary organisms. Oparin 5 2.0 - 1.8 - 1.6 - - 1.4 / Time (min) Fig. I.In the evolutionary development of the protobionts, their catalytic appara- tus was the most important factor of the organization of metabolism, based on the rate ratio of the component reactions. Of course, at the analysed stage of evolution the catalysts available to the protobionts could only have been the inorganic salts and organic substances present in the primordial broth, the catalytic activity of which is very low. However, given suitable mutual combination, this activity can be enhanced by a factor of many hundreds and thousands. We can imagine a colossal number of various atomic groupings and their combinations, which to some extent were endowed with the ability of catalys- ing the reactions indispensable for the existence of protobionts.However, as a result of the continual rejection by natural selection of the less perfect com- plexes, only very few have survived till now, viz. the co-enzymes. Their number is relatively small, but they are extraordinarily universal bio-catalysts, which points to their formation very early in the process of the origin of life. The required constancy of concentration of the co-enzymes in the growing and reproducing protobionts could have been sustained by the entry of these 6 R. I. C. Re views compounds or their components from the ambient medium. We also find something similar in present-day organisms which are obliged to obtain from the ambient medium vitamins which play the role of co-enzymes in their metabolism. However, of necessity the protobionts must have gradually generated the ability to synthesize the co-enzymes by themselves.This freed the progressing systems from their too great dependence on the ambient medium. Yet the gradual complication of the metabolism of protobionts necessi- tated a very distinct combination of a larger number of reactions to form long chains and cycles, an entire coordinated network of biochemical reac- tions. For a coordination of this kind the catalytic activity and specificity of co-enzymes was no longer sufficient and, therefore, in the process of subse- quent evolution, the co-enzymes were supplemented by an entire arsenal of much more powerful catalysts-enzymes, i.e. proteins whose secondary and tertiary structures are highly adapted to the functions they are to fulfil.Thus the era of co-enzymes gave place to a new era in which the decisive role was to be played by protein substances with intramolecular organization. The initially produced protein-like polymers with their random arrangement of the amino-acid residues could serve as the material for the formation of the coacervate drops and protobionts, but they were either very poor or not catalysts at all. Of course, during the polymerization of the amino acids, there could also form in the protobionts combinations of functional groups able to play the role of enzymes. Hdvever, in disordered polymerization this advantage was rapidly lost in the growing protobiont. Therefore the emergence of an organization that would fix the constancy of the secondary structure of the newly-synthesized polymers was of great importance.In this organization an exceptionally important role fell to the polynucleotides. In present-day organisms the synthesis of enzyme proteins is effected by means of a highly complex and perfect mechanism, with the aid of which the amino acids are consecutively ‘threaded’ onto the polypeptide chain, viz. precisely in the sequence required by the specific, strictly regular combination of the mononucleotide groups in the DNA and RNA molecules. Of course, a mechanism of this kind could arise only in the process of a prolonged evolution of the protobionts and living systems, but even at the much earlier stages of development the polynucleotides in the protobionts could have had an effect on the polymerization of amino acids which took place in these systems.The intramolecular structure of the primary polynucleotides them- selves was quite imperfect; during the growth process of protobionts it underwent considerable variations. Each variant thus produced could be fixed to a certain extent in the particular growing system owing to the com- plementary nature of polynucleotides; at the same time it could influence the order of arrangement of the amino-acid residues incorporated in the poly- peptide system. Oparin If the combination of amino-acid residues thus generated was convenient from the point of view of increased catalytic activity of the polypeptides, the system which gave rise to this combination obtained preference in its more rapid growth and reproduction.Otherwise it was destroyed by natural selection. In this way the intramolecular structure of the protein-like poly- 7 peptides, and at the same time of the polynucleotides which took part in their synthesis, became increasingly more ordered and more adapted to the functions which these polymers performed in the growing and reproducing systems. Nevertheless, it should be clearly understood that selection was applied not to a particular polynucleotide capable of replication, or to the polypeptides which arose under their influence and were already endowed with a certain sequence of the amino-acid residues, but to the entire systems, protobionts, with a primitive, but more or less perfect metabolism which did or did not correspond to the given conditions of existence. The role of the nucleic acids was that they fixed spatially the constancy of synthesis of catalytically convenient amino-acid combinations in the growing and reproducing systems, and served as a stabilizing factor in the process of their evolution.Thus, at a fairly late stage of evolution, a new era began; living systems rose to an unprecedentedly high level of exact self-reproduction, which is charac- teristic at present of the entire world of living creatures. Further development of living systems, and the perfection of their meta- bolism and of the supramolecular structures can be followed on the basis of more profound investigations in the field of comparative biochemistry. The data obtained here show clearly that some forms of organization of the metabolism, and some combinations of biochemical reactions made their appearance at the very beginning of life and are therefore found in all present- day organisms without exception, whereas others were formed considerably later as supplementary superstructures of the earlier metabolic mechanisms. At the outset, the only source of nourishment for the primary organisms was the organic substances of the primordial broth.Correspondingly, the ability to feed on organic substances is built into the very principle of life and is characteristic of all living creatures without exception. The absence of free oxygen in the secondary terrestrial atmosphere and in the hydrosphere caused the anaerobic nature of the energy exchange in primary organisms. Indeed, the data of comparative biochemistry show convincingly that anaerobic exchange is the basis of the energy of absolutely all present-day organisms, including the higher animals and plants capable of respiration.During the development of life the reserves of organic substances on the Earth’s surface, which were formed abiogenetically, gradually became exhausted because the development of life progressed faster than the genera- tion of these substances. This change in the conditions of existence brought to the forefront of development organisms capable, owing to the acquired ability to absorb light, of building organic substances from carbon dioxide in the atmosphere. It is true that, by analogy with what happened in the primordial broth, the coacervates, protobionts or primary organisms were able to some extent to synthesize organic substances under the conditions of reducing atmosphere by utilizing the energy of short-wavelength ultraviolet light.However, in the Earth’s atmosphere there was a very slow, but gradual formation of free oxygen by abiogenetic processes. This was accompanied by the formation of the ozone shield which barred the access of short-wavelength R.I.C. Reviews 8 ultraviolet rays to the surface. Because of this, the process of selection was necessarily associated with a transition from the utilization of the short- wavelength radiation by primary organisms to the utilization of the long- wavelength light which is shed so abundantly onto our planet by the sun.This transition encountered a major hindrance, in that the individual quan- tum of visible light carried a relatively small amount of energy. For this reason, it was necessary to use photosensitizers in order to accomplish biologically important photochemical reactions. On the strength of data from comparative biochemistry and from model experiments it is possible to follow the genesis of such photosensitizers- porphyrins and their magnesium derivatives-and their incorporation into photosynthetic systems. The paths of successive perfection of these systems passed, on the one hand, through the selection of increasingly more effective pigments (porphyrins) and, on the other, through the greater complexity and functional adaptation of the supramolecular structure of the photosynthetic apparatus.Thus photosynthesis originated-a new, highly perfect method of synthesis of organic compounds, which replaced the previous, very slow and imperfect abiogenetic synthesis. Consequently, in the further development of life, photosynthesis acquired a predominant, monopolistic significance in the formation of organic substances on the surface of the Earth. Its beginning changed the entire set of conditions for life. Some organisms began to build for themselves indispensable organic substances, whereas others preserved the previous heterotrophic forms of nutrition, using organic substances now formed biogenetically by photosynthesis.Thus, two branches of the living world were formed : plants and animals. However, the origin of photosynthesis not only created an abundance of organic substances, but also resulted in the rapid formation of free oxygen in terrestrial atmosphere. This changed the entire nature of chemical processes on the Earth, and enabled most of the living creatures to rationalize con- siderably their energy exchange : by adding to the previous anaerobic mecha- nism the superstructure of the new supplementary systems of oxygen breath- ing, it became possible to utilize completely the energy hidden in organic substances. Along with the perfection of metabolism, an evolution in the spatial organization of living organisms also took place.Its origin and perfection were closely related to the evolutionary development of the functions performed by structures. Anaerobic fermentation is possible in homogeneous solution, while photosynthesis and respiration require very complex structures. Apparently the most primitive structural formations were protein-lipid membranes. These can be detected as early as the stage of formation of coacervates, and they are found in all living creatures without exception. However, the formation of structures such as chloroplasts, mitochondria and cell nuclei was accomplished only during the gradual evolution of living systems. Oparin Thus we see that, for a very long time, the evolution of primitive organisms was in principle related to the perfection of metabolism and of intramolecular and subcellular structure.The emergence of the cell, which is usually con- sidered to be the most primary indivisible element of life, required immense 9 time intervals and a sequence of untold generations of pre-cellular living creatures. Some modern authors consider it even possible that, during the initial periods of the existence of life, the individual structural formations developed as independent protobionts or primitive organisms (‘organoids’), and only later combined to produce the intricate biological complex which we find in the cell. With the emergence of unicellular and, later, multicellular organisms it became possible to study the evolution of life by palaeontological methods, on the basis of investigations of fossil remains with a definitely biological structure.We have seen, however, that the initial steps of biological evolution were, on the whole, related to the perfection of metabolism and intramolecular structure, and this could not easily leave direct traces in the palaeontological record. Considerably more information about this period can be gained on the strength of the data obtained from comparative biochemistry. Similarly the possibility of preservation until now of the initially generated pre-biological and biological structures, which had the nature of colloidal clusters of organic polymers, hardly exists. Only in exceptionally rare cases were structures detected in the most ancient deposits which might have been of biological origin (but which could have been artefacts resembling the ‘organized corpuscles’ found in carbonaceous chondrites). Consequently, one should use great caution when dealing with such very old findings.An example of such findings is the calcareous secretions detected in South- ern Rhodesia, whose age is estimated at 2.7 x 109 years. They are often called ‘algal limestones’ and are considered to be the most ancient manifesta- tion of life. However, we lack genuine fossils with the structurally-preserved remains of early organisms; what we find here is a lamellar structure of limestone, which of course cannot easily be treated as a purely inorganic formation. At the same time, the producers of the lime secretions of Rhodesia might have lacked a biological structure; they could have been our hypotheti- cal protobionts or even clusters of the organic substances of the primordial broth.The oldest genuine fossils are considered nowadays to be the remains of organisms preserved in the iron ores of South Ontario (age, 1.6 x 109 years). However, even in this case it is difficult to identify these organisms and the type of their metabolism. Much more certain and well-defined are the remains of organisms which lived during the later periods of the Proterozoic era. We do not find there the resplendent diversity characteristic of the present-day flora and fauna. Life in the course of that era was on the whole represented by unicellular (or even pre-cellular) bacteria and algae, and only at the boundary of the Cambrian system do multicellular plants and animals begin to appear.One can assume, therefore, that the evolution of living creatures took place during this era at a much slower pace than during the post-Cambrian periods when increasingly more perfect organisms rapidly succeeded one another. Figure 2, which is of course only highly approximate, takes in at a glance the entire path of evolution referred to before. In this figure the vertical line plots the time (in thousands of millions of years) from the present to the event in R.I.C. Reviews 10 I Emergence of aerobes I Beginning of photosynthesis anaerobes Era of the beginning of enzymes and of the nucleic acid code 1 Era of co-enzymes Coace rvates I question.To the right of the line are marked the fundamental landmarks of the evolution of our planet; on the left, data are given on the evolution of carbon compounds during the emergence and development of life. Analysis of the diagram enables us, first of all, to realize the immensity of our ignorance and the vast horizons which open up before us for the future. On the basis of data from comparative morphology and palaeontology, we have at present a fairly good idea of the trend of biological evolution from the late pre-Cambrian until now. But this is only a small section of the dia- 11 5 Fig. 2. Oparin - - Beginning of the Cambrian period Formation of the oxygen- containing atmosphere of Stromatoliths present-day composition Ontario fossils A End of the reducing and beginning of the transitional atmosphere - Rhodesian limestone secretions Formation of global ocean completed Formation of secondary reducing atmosphere 8 4 Formation of the Earth's crust Formation of the Earth with present-day mass Beginning of solar system gram.On the basis of astronomical, geological and chemical data we can picture the first stages of the evolution of carbon compounds. However, between this stage and the topmost section of the diagram there is an immense period of evolution which lasted for thousands of millions of years. In the course of this expanse of time the fundamental and essential changes in the organization of systems which gave rise to living creatures were slowly achieved.We can only advance hypotheses with respect to the sequence and, also to some extent, the nature of these changes. Yet we are still unable to indicate the exact time when life emerged. The difference between the inorganic world and the world of living creatures, which is so easy to establish today, came into being only because all of the intermediate organizational forms had been destroyed by natural selection. But the evolutionary emergence and the subsequent development of life passed through a number of intermediate stages; therefore, the question posed can be answered only by deciding which of these stages we consider to represent the beginning of life : the emergence of protobionts endowed already with a perfect metabolism, the emergence of proteins and of the nucleic code or, finally, the emergence of the cell. Scientific knowledge of the periods of the formation and organizational perfection of life, which are not very clear to us, depends in the first place on the future powerful development of evolutionary biochemistry, biophysics, cytology and physiology. Even before Darwin a tremendous amount of morphological material had been accumulated in biology ; this acquired its scientific importance after having been generalized by means of the single idea of evolutionary development. By analogy, there is today in biology, thanks to the application of physical and chemical methods, a rapid accumu- lation of information about the organization of metabolism and the structure of the vitally important molecules, membranes and organoids in living organisms which stand at various levels of evolutionary development. This material will attain its own new, exceptionally high significance for the know- ledge of the essence of life only when we have united and systematized it on the basis of evolutionary theory. Only by using such an evolutionary approach shall we be able to recognize not only what occurs in the living creatures and how it occurs, but also to answer these ‘hundred thousand whys’ which arise unavoidably in the path of the true study of the essence of life. In particular, there is the question of why the entire organization of every living thing, from the molecular to the organizational level appears so ‘expedient’ and so adapted to constant self- preservation and self-reproduction under the extant conditions of the ambient medium. R. I. C. Reviews 12