Polymers thermal

S. W. Fox (from 1984, director of the Institute for Molecular and Cellular Evolution of the University of Miami) made the highly controversial suggestion that the amino acid sequences in the proteinoids are not random. Nakashima prepared a thermal polymer from glutamic acid, glycine and tyrosine the analysis showed that two tyrosine-containing tripeptides had been formed pyr-Glu-Gly-Tyr and pyr-Glu-Tyr-Gly (Nakashima et al 1977). The result was confirmed (Hartmann, 1981). A closer examination of the reaction mechanism showed that the formation of these two tripeptides under the reaction conditions used depends on three parameters ... 

Dose K, Rauchfuss H (1972) On the electrophoretic behavior of thermal polymers of amino acids. In Rohlfing DL, Oparin AI (Eds.) Molecular Evolution Prebiological and Biological. Plenum,... 

Equation of State of Thermal Polymer Solutions and Melts. 

Table I. Inspection of Typical Thermal Polymer Gasolines...

Modern techniques of mass spectrometry allow the determination of some polymer characteristics. The main studies are devoted to mass determinations (no standardization necessary) of polyethyleneglycols, polystyrenes and polymethylmethacrylates. However, a liquid secondary ion mass spectrometry (LSIMS) study [42] on a thermal polymer of a mononadimide model compound permits an approach to the determination of the polymer microstructure and goes some way to understanding the polymerization mechanism. 

Thermal polymer degradation is determined by the chemical structure and length of the polymer chain, by the presence of unstable structures (such as impurities or additives) and by the temperature level inside the reactor, which must be high enough to break the weakest, primary chemical bonds. Madorsky and Straus [39] found that some polymers (such as PMMA and PTFE) mainly revert to their monomers upon heating, while others (such as PE) yield a great many decomposition products. These two types of dominant thermal polymer degradation are called end-chain scission and random-chain... 

However, these added sulfonate groups are easily distinguished from sulfate groups obtained from OH, the latter ones being hydrolyzable, whereas the former ones not. The observed absorbance with thermal polymer could be taken as the blank reading and subtracted from the absorbance of the test samples before calculating for the concentration of sulfate end groups I38). 

It is interesting to note that in contrast to these results the thermal polymerization of DCH always proceeds heterogeneously with nucleation of separate polymer domains. The thermal polymer is polycrystalline with a fibrous texture. Lattice parameters are identical with those of the polymer obtained by irradiation. Observation of thermally polymerizing DCH crystals shows that the reaction starts at crystal... 

Polymerization of oil occurs under heat with or without the presence of oxygen. Heat can cleave the oil molecule or fatty acid. These cleaved compounds can then react with each other, forming large molecules. These polymers are referred to as thermal polymers. In the frying process, excessive fryer heat and excessive fryer down time can produce high levels of thermal polymers. Thermal polymers can be detected in the fresh product by expert panelists because they generally impart a bitter aftertaste to the fried food. 

In earlier work in this field, an excess of either basic or dicarboxylic amino acids in the reactants was thought to be essential for obtaining thermal polymers of reasonable molecular weight in substantial yield. Recently, however, three independent reports have appeared on the preparation of a third general type of proteinoid, termed neutral proteinoid. By using reactants that contained excess neutral amino... 

Proteinoids have been investigated as models for proteins. One may appropriately inquire to what extent the thermal polymers resemble, or differ from, contemporary protein. Some of the properties that these two types of polymer possess in common are summarized in Table I. Since a detailed description of these similarities has been presented elsewhere (7), only a brief treatment is given here. 

In a typical experiment, the thermal polymers were used at a concentration of 0.4 mg/ml. The substrate was present at 10 3 M in 0.067 M phosphate buffer, pH 6.2 or 6.8. These concentrations gave a substrate/polymer ratio of 2.5/xmoles/mg. Under these conditions at 30°, linear progress curves of p-nitrophenol produced were observed for at least 1 hoiH. Less than 10% of the NPA was hydrolyzed. Acetate was also identified as a product of the hydrolysis by a titrimetric procedure. Activity was expressed in units of micromoles of -nitrophenol produced per minute per milligram of polymer, or in a quantitative comparison to the activity of the equivalent amount of free histidine. All activity values were corrected for the rate of spontaneous hydrolysis of the substrate. 

From the available data, the action of the thermal polymer cannot be asserted to be truly catalytic in the studies of Usdin et al. The molar ratio of polymer residue/substrate (assuming an average amino acid residue weight of 100 for the polymer) was about 20/1 in the most favorable case, and hydrolysis of the substrate was followed to less than 5% completion. Moreover, consideration of the possibility of microbial contamination was not reported. 

The thermal polymer was inhibited by diisopropyl iluorophosphate (DFP Table V). The extent of inhibition was dependent upon the length of preincubation of polymer with DFP (20 mg of polymer per micromole of DFP) and was, e.g., 80% after 6 hoiurs of preincubation. The inhibition by DFP could be 80% reversed by 0.1 il/ 1,3-bis(iV-pyridinium aldoxime)propane. The authors conclude that the inhibition, reactivation, and Michaelis-Menten data obtained are indicative of enzyme-like properties. 

Noguchi and Saito (31) have tested a number of compounds on NPA, including thermal copolymers containing histidine and glutamic acid or aspartic acid residues. Their method of preparing thermal polymers differed considerably from the usual procedures of Fox and co-workers. 

Another substrate hydrolyzed by thermal polyanhydroamino acids is p-nitrophenyl phosphate (NPP). Oshima 25) has tested acidic proteinoids, a histidine-rich proteinoid, and proteinoids that contain a relatively high proportion of basic and neutral amino acids (p. 377). These polymers were fractionated in water and aqueous alkali, and by molecular sieve chromatography. Soluble fractions were used in most experiments on catalysis. The possibility of microbial contamination was obviated indirectly by virtue of several circumstances. These included using fractions with molecular weights between 2500 and 4000, and adding toluene to the test reaction mixtures. The reactions were carried out at 30° in 0.03 M tris buffer, pH 7.6, in the presence of 1 pmole/ml of NPP and 0.03-1.5 mg/ml of thermal polymer pM ZnCU and IQfiM MgCl2 were also present in the solutions. Truly catalytic... 

The optimal pH of the system was approximately 7 (Fig. 4) however, a definite shoulder was found at pH 8-9. Oshima suggests that at least two different catalytically active molecular species, with different ionic properties, are present. Michaelis-Menten kinetics were demonstrated (Fig. 5). Kra was 1.4 x 10 M, which is about 10 times greater than literature values for natural phosphatases. Digestion by pronase cleaved 10% of the peptide bonds and caused 30% loss of activity. 3-0-Methyl-fluorescein phosphate was hydrolyzed by the thermal polymers about one-sixth as fast as was NPA. 

In a typical experiment, 20 mg of thermal polymer in 20 ml of autoclaved 0.2 N tris buffer, pH 8.3, was incubated at 37.5° with 2.0 pCi of sodium l-i C-p3Tuvate (specific activity, 5/iCi//xmole, giving a substrate/polymer ratio of 0.02 /.imoles/mg). Incubation was usually for 24 hours, but occasionally for 48 or 72 hours, after which the liberated COa was assayed as barium carbonate. Similar results were obtained when radioactive acetate was counted. Aseptic precautions were taken during the preparation and assay of the polymers. Activities (cpm per 20 mg polymer per 24 hours. Table IX) were 30,000-33,000 for acidic 2 2 1-proteinoids, 33,000-41,000 for 1 1 1-proteinoids, and 33,000 for 1 1 3-proteinoid. A lysine-rich proteinoid gave a value of 15,800. Hydrolysates of polymers and unpolymerized mixtures of amino acids had activities of 4000-5000, only slightly greater than that of the spontaneous control (1000-3000). Enzymes other than carboxylase and other proteins gave values of 2000-5000. 

On a weight basis, thermal polylysine was three times as active as lysine-rich proteinoid. Acidic proteinoid showed little or no activity (Table X). Second-order rate constants were, respectively, 0.054, 0.012, and 0.0006 liter/gm per minute (these values being corrected for the spontaneous rate of 0.003 min i). Multiple samples of a particular kind of thermal polymer showed similar levels of activity ( 10 relative %). 

The action of the lysine-rich polymers was rather selective for OAA, in that pyruvic, malic, malonic, a-ketoglutaric, glucuronic, oxalic, or aspartic acid were not measurably decarboxylated under conditions in which OAA was 90% decarboxylated. Acetoacetic acid was decarboxylated about - 6 as fast as OAA. This selectivity is not in conflict with other reports of decarboxylation of some of these substrates, because conditions of assay have varied rather widely. The rate of decarboxylation may be essentially related to the relative stability of the substrate in question. Two reactions catalyzed separately by different types of thermal polymers describe a sequence, namely OAA pyruvate-> acetate. This sequence can be considered in the context of the beginnings of metabolism (p. 408). 

To date, four main types of catalytic activity have been reported in detail for thermal polyamino acids. These are (with the most studied substrates in parentheses) hydrolyses (p-nitrophenyl acetate, p-nitro-phenyl phosphate, ATP), decarboxylations (OAA, glucuronic acid, pyruvic acid), and aminations (a-ketoglutaric acid, OAA, pyruvic acid, phenylpyruvic acid). The fourth type is a deamination reaction yielding a-ketoglutaric acid (51). For some of the actions of the thermal polymers the products are identified quantitatively, and the kinds of amino acid side chain necessary for activity in the polymer elucidated. In others, products have yet to be fully identified. The activities of thermal polyamino acids are manifest on substrates which range from chemically labile to relatively stable. 

In each example, the activity of the thermal polymer is greater than that of the equivalent amount of free amino acid(s). In many cases, no measurable activity was shown by the monomers. Several examples have shown that the activity of the thermal polymers can be greater than that of similar polymers prepared from Leuchs anhydrides. In some cases involving metal-proteinoid, the metal was active only with proteinoid. In one case, zinc-proteinoid acting on ATP, the metal appeared to be responsible for all of the activity. With amination reactions, the simultaneous presence of both cofactor Cu + and thermal polymer was required for activity. 

The thermal polymers are incapable of mimicing a peptide or protein to the exact extent that the product of a stepwise synthesis does. An exact duplication of a functional protein, however, does little to elucidate the reason for its activity modification and study of the effect on activity are necessary. Systematic synthetic modification of polymeric models is easily achieved in the case of thermal polyamino acids. They are prepared with much ease and in large numbers, and their quantitative compositions can be regulated and controlled simply. Examples are already at hand to illustrate the use of the thermal method to evaluate qualitatively the kinds of amino acid residue that are necessary for, contributory to, or detrimental to, activity. Such studies augment information from enzymes and from nonthermal models. 

Proteinoids may more closely resemble prebiotic protein than they do contemporary protein. In the absence of rigorous evidence, the thermal polymers may be considered as the only representation of primitive proteins. Because, however, the exact nature of prebiotic proteins is unknown, the synthetic polymers are considered as models for, rather than models of, prebiotic protein. A significant consideration is that proteinoids have properties which permit them to be considered as evolvable through their tendency to yield proliferatable systems (65). 

Since several kinds of reaction have been shown to be catalyzed by thermal polyamino acids, the discovery of other kinds of reaction seems likely to occur on further examination. Those reactions already studied include hydrolysis, decarboxylation, amination, and one instance of deamination. Assemblage of some of the reactions catalyzed reflect metabolic sequences, as laid out in a flow sheet in the section on pathways (Section IV, E). Within these sequences, some specificity in reaction between proteinoid and substrate can be observed. As stated before, some activities of thermal polymers have been shown to be influenced by, or to be dependent on, cofactors. 

The class of polyamino acids includes both the thermal polymers and contemporary proteins (enzymes). The basis for this inclusive class is that both types of the polymer contain a variety of chemically functional groups. Because of their simultaneous occurrence in the same macromolecules, these functional groups provide a yet larger array of chemical reactivity. The association within a macromolecule provides a degree of fixed three-dimensional relationship, with some flexibility within this constraint. One of the crucial requirements of life, its origin, and evolution may well be, accordingly, the chemical polyfunctionality of polymers of amino acids. Selection and specialization was able to proceed from polymers composed of approximately 20 kinds of monomer. Special evolutionary advantage of catalytic polymers would result... 

Heating of D-glucose in the presence of acid catalysts has been investigated, and has been found to afford a polymeric product.Structural investigations have shown that this thermal polymer is highly branched and contains both (l- 4)- and (1—>6)-linkages. 

The above experiments clearly show that some organic volatiles must have infectious properties and act as pro-degradants that are carried across from the faster degrading polymer. A likely candidate for such infectious agents are peroxides or fragments thereof that are key intermediates in thermal polymer degradation. To test the hypothesis that even traces of peroxides would be sufficiently reactive and could act as remote initiators, a few simple overview experiments were conducted. 

The thermal polymers of amino acids reveal photo effects (Figure 1), which are almost certainly related to the photocatalytic activity that was reported in 1972 by Wood and Hardebeck. This property is due to the presence in the polymer of flavin and pterin derivatives.The pigments are formed during amino acid thermolysis. The chromophores appear to be covalently linked to thermal oligomers. One proposed chemical binding is... 

Thermal polymers of amino acids are able to make spherical and planar membranes. This gives us both theoretical and practical tools for studying the composition and functions of natural membranes. Having such models, in which we can manipulate amino acid composition, we may produce synthetically membranes resembling natural ones, and in some aspects surpass them in desired properties. 

K. Harada and S. W. Fox, Characterizations of Functional Groups of Acidic Thermal Polymers of a-Amino Acids, BioSysteins 7, 222-229 (1975). 

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