Compartmentalization in proteinoid microspheres

Proteinoid microspheres with stable internal compartments and internal structure are made from acidic proteinoid and basic proteinoid with calcium. The populations of microspheres are characterized by a wide diversity of structure. A model of primitive intracellular communication is suggested by the observed movement of internal particles between compartments of a multicompartmentalized unit. Differential response to pH change and to temperature change has been demonstrated within one population and suggests one mode of adaptive selection among primordial cell populations.     

A model for the origin of stable protocells in a primitive alkaline ocean

When a mixture of the eighteen proteinous amino acids are suitably heated in the dry state with seawater salts, a copolyamino acid results. One fraction of this polymer is found, through isoelectric focusing, to consist of a mixture of acidic and basic proteinoids, each of sharply limited heterogeneity. When one fraction of the seawater proteinoid is dissolved in hot water, and the solution is cooled, proteinoid microspheres result. These have properties in common with simpler types, but are also stable at pH values to 9, in common with microspheres prepared by mixing acidic and basic proteinoids. These processes thus constitute a simple model for the origin of a protocell stable in a primitive alkaline ocean.     

Interactions between diverse froteinoids and microspheres in simulation of primordial evolution

Experiments demonstrating an incorporation of different enzymelike activities into a single preparation of proteinoid microspherss provide a conceptual basis for the primitive lengthening of protometabolic pathways. An enhancement of one enzymelike activity by another proteinoid in the same microsphere has been found. This effect, plus the pathway-lengthening propensity of combinations of microspheres, indicates selective advantages contributing to adaptive protoselection.Data reported in this paper also bring into purview the concept of internally controlled variation. Inferences are derived for the origin of protosexuality in protocells.When allowance is made for a closer relationship to the environment than that needed in contemporary selection, the fundamental mechanistic requirements of protoevolution are regarded as met by the proteinoid microsphere.     

Coacervate-like microspheres from lysine-rich proteinoid

Microspheres form isothermally from lysine-rich proteinoid when the ionic strength of the solution is increased with NaCl or other salts. Studies with different monovalent anions and with polymers of different amino acid composition indicate that charge neutralization and hydrophobic bonding contribute to microsphere formation. The particles also form in sea water, especially if heated or made slightly alkaline. The microspheres differ from those made from acidic proteinoid but resemble coacervate dtoplets in some ways (isothermal formation, limited stability, stabilization by quinone, uptake of dyes). Because the constituent lysine-rich proteinoid is of simulated prebiotic origin, the study is interpreted to add emphasis to and suggest an evolutionary continuity for coacervation phenomena.     

Formation of proteinoid microspheres under simulated prebiotic atmospheres and individual gases.

The formation of microspheres from acidic and basic proteinoids was attempted under simulated prebiotic atmospheres and constituent gases thereof. Both types of proteinoid yielded microspheres under carbon dioxide, carbon monoxide, methane, hydrogen sulfide, hydrogen, nitrogen, and oxygen (tested separately) and also under nitrogen-carbon dioxide atmospheres; higher proportions of carbon dioxide resulted in fewer spheres from basic proteinoid. Neither type of proteinoid formed spheres on 10-minute exposure to ammonia or methane-hydrogen-ammonia atmospheres. (Brief exposure resulted in spheres from basic proteinoid.) The effects, both qualitative and quantitative, were indicated by control experiments to be due to pH, rather than to the specific gas (or ion). The results suggest that the proteinoid microsphere model for protocells is applicable under a variety of possible prebiotic atmospheres, with some restrictions imposed by pH.     

From proteinoid microsphere to contemporary cell: Formation of internucleotide and peptide bonds by proteinoid particles

Proteinoid microspheres of appropriate sorts promote the conversion of ATP to adenine dinucleotide and adenine trinucleotide. Other microparticles composed of basic proteinoid and enzymically synthesized poly A cause the conversion of ATP and phenylalanine to various peptides of phenylalanine. When viewed in a context with the origin and properties of proteinoid microspheres, these results model the origin from a protocell of a more contemporary type of cell able to synthesize its own polyamino acids and polynucleotides. Related earlier experiments explain in part the origin of the genetic code and mechanism.     

Formation of peptides from amino acids by single or multiple additions of ATP to suspensions of nucleoproteinoid microparticles

When lysine-rich proteinoid, which catalyzes the formation of peptides from amino acids and ATP, is complexed with acidic proteinoid to form microspheres of mixed constitution, the normal synthesis by basic proteinoid alone is multiplied several-fold. The product consists not only of small peptides but also of a high-molecular-weight fraction of substituted proteinoid.Suspensions of particles of lysine-rich proteinoid complexed with polyadenylic acid catalyze the synthesis of peptides from each of the amino acids tested with ATP. When equimolar solutions of mixtures of glycine and phenylalanine with ATP are tested in suspensions of complexes of lysine-rich proteinoid and each of various polyribonucleotides, both homopeptides and heteropeptides are produced. Glycylphenylalanine or phenylalanylglycine is the principal product; the preference is related to which polyribonucleotide is in the complex.The rate of conversion of amino acid to peptide is a function of whether ATP is added in a single batch or in repeated amounts adding to the same amount as in the single batch. Related experiments indicate a relatively rapid initial rate of decay of ATP in this system. These results are discussed relative to the mechanisms for continuous generation in modern organisms, as are the results in peptide formation.     

Activation of glycine by ATP,a divalent cation,and proteinoid microspheres

The activation of glycine to yield glycyl hydroxamate has been studied in the absence of enzymes. Activation with ATP in aqueous solution requires only a divalent metal cation. ATP is far more active than other nucleoside triphosphates; AMP and pyrophosphate are inactive. The pH optimum is 4 to 5; activation at pH 7 is most enhanced in the presence of proteinoid microspheres.     

Assembly of microspheres from acidic proteinoids and histones or histone-like proteinoids

Basic polyamino acids, whether organismic or synthetic in origin, alter the morphology of proteinoid microspheres in which they are included. The particles obtained with histone are essentially indistirguishable under the light microscope from those obtained with proteinoids of histone-like composition (histonoids). In each instance, structures of morphological complexity similar to those of some microfossils are obtained.     

Proteinoid microspheres and the process of prebiological photophosphorylation

A chemical model of prebiological photophosphorylation with participation of hemoproteinoid microspheres, mixed microspheres containing bonded riboflavin and microspheres obtained from glycine rich proteinoids was studied. The illumination of aqueous solutions containing microspheres, K₂HPO₄, ADP and electron acceptor leads to an increase of ATP concentration and to a decrease of concentration of inorganic phosphate. Initial photochemical reactions with participations of proteinoid microspheres could have evolved in the course of chemical evolution and led to the emergence of the photophosphorylation in its modern biochemical form.     

Electrical excitability of proteinoid microspheres composed of basic and acidic proteinoids

Material flow equilibration, an endogenous interaction adjustment process underlying fulfillment of material flow continuity during material self-assembly, also underlies electrical excitability observed in the proteinoid microspheres as simulated models of the protocell. The spikings of the membrane potentials are attributed to a singular character of the interaction rate coefficients measuring the strengths of the coupling between basic and acidic proteinoids, in which the rates change singularly with time due to material flow equilibration.     

Synthesis of life in the lab? Defining a protoliving system

The synthesis of a living system in the lab has been judged by a number of critics as partly attained by the proteinoid microsphere because of its primitive properties of metabolism, growth, and reproduction. These same critics, however, judge the organism as not alive, or as being 50 to 75 percent alive (Baltscheffsky and Jurka, 1984), owing to the absence of a nucleic acid genetic coding mechanism. The experiments in retracing evolution suggest, however, that the self-sequencing of amino acids was the evolutionary precursor of modern nucleic acid templating; the genetic memory is the molecule. The proteinoid microsphere is not a modern living system, but does represent at least a protoliving system (Fox and Dose, 1972). Berra (1990, p. 75) has commented on other difficulties in defining a protoliving system. In Berra's opinion, metabolism, reproduction, responsiveness to stimuli, and cellularity constitute or describe aliveness. These properties characterize proteinoid microspheres. A number of experiments demonstrate that amino acids in aminoacyl adenylates yield specific products, whereas nucleotides are without effect. For this and related reasons, especially the demonstrated self-sequencing of amino acids when they are warmed, resultant bio-functional properties of self-assembled microstructures, and demonstrated self-sequencing of amino acids in modern systems, the results appear to bridge from the chemical era to the biological period. All the above emerges from a departure in style of research (Young, 1984; Pauling and Zuckerkandl, 1972). The latter authors said, "It appears likely that biogenesis is the passage from a 'non-living system' existing in a large number of states to a 'living' system also existing in a large number of states."(ABSTRACT TRUNCATED AT 250 WORDS)     

Protocell-like Microspheres from Thermal Polyaspartic Acid

One of the most prominent amino acids to appear in monomer-generating origin-of-life experiments is aspartic acid. Hugo Schiff found in 1897 that aspartic acid polymerizes when heated to form polyaspartylimide which hydrolyzes in basic aqueous solution to form thermal polyaspartic acid which is a branched polypeptide. We recently reported at the ISSOL 2005 Conference that commercially made thermal polyaspartic acid forms microspheres when heated in boiling water and allowed to cool. In a new experiment we heated aspartic acid at 180°C for up to 100 h to form thermal polyaspartylimide which when heated in boiling water without addition of base hydrolyzed to form thermal polyaspartic acid which upon cooling formed microspheres. Thermal polyaspartic acid microspheres appear protocell-like in the sense of being prebiotically plausible lattices or containers that could eventually have been filled with just the right additions of primordial proteins, nucleic acids, lipids, and metabolites so as to constitute protocells capable of undergoing further chemical and biological evolution. Thermal polyaspartic acid microspheres are extremely simple models of protocells that are more amenable to precise quantitative experimental investigation than the proteinoid microspheres of Sidney W. Fox. We present here scanning electron microscope images of such thermal polyaspartic acid microspheres. Figure 1 shows thermal polyaspartic acid microspheres from l-aspartic acid heated at 180°C for 50 h, at a magnification of 3,500×. Figure 2 shows thermal polyaspartic acid microspheres from the same sample at a magnification of 7,000×. The thermal polyaspartic acid microspheres have a diameter of approximately 1 μm These images were viewed with a Hitachi S2460N scanning electron microscope at 20 kV acceleration voltage. Figure 1 Thermal polyaspartic acid microspheres from l-aspartic acid heated at 180°C for 50 h, at a magnification of 3,500×.

---

Figure 2 Thermal polyaspartic acid microspheres from l-aspartic acid heated at 180°C for 50 h, at a magnification of 7,000×.

---

Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

Protoporphyrin ix as a possible ancient photosensitizer: Spectral and photochemical studies

Due to the potential special position of protoporphyrin IX in the evolution of photosynthesis, the absorption and fluorescence characteristics of this pigment and its complexes with human serum albumin (HSA) and basic proteinoid have been studied in parallel with their photochemical activity. The most significant change in the absorption spectrum of PP IX was the appearance of a new maximum at 455 (or 461) nm in the presence of HSA or proteinoid respectively. Some changes in the physicochemical properties of PP IX in different microenvironments have been detected by changes in fluorescence emission and excitation spectra (intensity, quantum yields, position of maxima). The increase of fluorescence quantum yield resulting from the formation of PP IX complexes with HSA or proteinoid correlates with the increase of their photochemical activity. Results obtained are discussed from the point of view of the early evolution of the photosynthetic apparatus.     

Binding constants of phenylalanine for the four mononucleotides

Summary Earlier work has shown that several properties of amino acids correlate directly with properties of their anticodonic nucleotides. Furthermore, in precipitation studies with thermal proteinoids and homopolyribonucleotides, an anticodonic preference was displayed between Lys-rich, Pro-rich and Gly-rich thermal proteinoids and their anticodonic polyribonucleotides. However, Phe-rich thermal proteinoid displayed a preference for its codonic nucleotide, poly U. This inconsistency seemed to be explained by a folding in of the hydrophobic residues of Phe causing the proteinoid to appear more hydrophilic. The present work used nuclear magnetic resonance techniques to resolve a limited question: To which of the four nucleotides does Phe bind most strongly? The results show quite clearly that Phe binds most strongly to its anticodonic nucleotide, AMP.     

Coprecipitation of thermal lysine-rich proteinoids with polyribonucleotides.

Major variables in interactions between basic thermal proteinoids and homopolyribonucleotides were magnesium concentration in solution (0–40 mM) and mol% lysine in the proteinoid (16–55%). The formation of microparticles was monitored both by the turbidity and by the mass of precipitate formed. Under some conditions, only, was the turbidity reading a reliable indication of the amount of precipitate. Increasing concentration of Mg²⁺ tended to displace proteinoid from the complex with polynucleotide. Of 4 polynucleotides, only polyguanylic acid showed an enhanced precipitation of proteinoid in the presence of Mg²⁺, and then only with those having high lysine contents. At high lysine contents, the amount of proteinoid in the precipitate was inversely proportional to the lysine content of the proteinoids, probably due to decreased sidechain interactions. The precipitation with polynucleotides is partly a function of the amino acid composition of the proteinoid; therefore the interaction of thermal proteinoids with polynucleotides appears to be a tool that can be used to study specificities of interactions between proteins and nucleic acids.     

Proteinoids as complexes of polyamino acids with melanoidins

Proteinoids have been demonstrated as complexes of amino acid polymers with melanoidin pigments. Some physico-chemical properties of proteinoid pigments were studied in comparison with the standard melanoidins. Proteinoid pigments were able to enhance oxidoreduction and hydrolysis reactions, and their activity was comparable with the activity of the corresponding polyamino acid components or even of the entire proteinoids. The pigmented proteinoids had relatively strong ESR signal indicating the presence of free radicals into melanoidin components. Hypothetical participation of proteinoid melanoidin pigments in prebiotic evolution is discussed.     

The evolutionary significance of phase-separated microsystems

The source, preparation, and properties of phase-separated systems such as lipid layers, coacervate droplets, sulphobes, and proteinoid microspheres are reviewed. These microsystems are of interest as partial models for the cell and as partial or total models for the protocell. Conceptual benefits from study of such models are: clues to experiments on origins, insights into principles of action and, in some instances, presumable models of the origin of the protocell. The benefits to evolution of organized chemical units are many, and can in part be analyzed. Ease of formation suggests that such units would have arisen early in primordial organic evolution. Integration of these various concepts and the results of consequent experiments have contributed to the developing theory of the origins of primordial and of contemporary life.Invited paper. Presented at the International Seminar Origin of Life, 2–7 August 1974, Moscow, U.S.S.R.     

Electrical membrane phenomena in spherules from proteinoid and Lecithin

Spontaneous and induced electrical phenomena resembling membrane and action potentials in natural excitable cells have been observed in artificial cells. These artificial cells were made from thermal proteinoid and lecithin in a solution of potassium acid phosphate with glycerol.