CONDENSATION OF GLYCYLGLYCINE TO OLIGOGLYCINES WITH TRIMETAPHOSPHATE IN AQUEOUS SOLUTION. II: Catalytic Effect of Magnesium Ion

The previously reported condensation reaction of glycylglycine with trimetaphosphate (Yamanaka et al., 1988) was reinvestigated and shown to be catalyzed by magnesium ion. Aqueous solutions containing glycylglycine (0.5 M), trimetaphosphate (0.5 M) and magnesium chloride (0.5 M) were incubated at 38 °C at pH 4, 5, 6, 7 and 8. After incubation for ten days at pH 5, the maximum yields of tetraglycine and hexaglycine as condensation products were found to be about 12 and 1.4%, respectively. This result indicated the presence of a considerable catalytic effect of magnesium ion compared with the maximum yield of about 2% for tetraglycine and 0% for hexaglycine in the absence of magnesium ion.     

Effect of polynucleotides on the dimerization of glycine

Polynucleotides were found to suppress the dimerization reaction of aqueous glycine with trimetaphosphate as the condensing agent. Small anions (chloride, acetate, and phosphate) did not show this effect. The reaction was studied at a pH of about 11.5 and at 70°C and room temperature with a 13 mM concentration of glycine and trimetaphosphate. Under these conditions, the effect of the polynucleotides was in the following order: polyguanylic acid < polycytidylic acid < polyadenylic acid < polyuridylic acid. The result may have a significant implication for the understanding of processes of chemical evolution.     

Oligo-glycine synthesis in an aqueous solution of glycine under oxidative conditions

Di-and tri-glycine were synthesized in 1M aqueous solution of glycine by bubbling for 90 hr with oxygen discharged in the path from an oxygen cylinder. The peptides were also produced by an incubation at 37°C of 2M glycine solution prepared with 75% hydrogen peroxide, and the yields were traced for 200 days. The final yields were about 0.25% and 0.01% for di-and tri-glycine, respectively. The solution at 166 days of incubation was applied to a Sephadex G 10 column, and the fractions around the top of the chromatogram were found to increase the intensity of ninhydrin color about 45 times after hydrolysis, indicating an existence of oligo-glycine. The solutions of 1M glycine and 0.5M diglycine prepared with 30% hydrogen peroxide were incubated at 37°C for 38 days, and di-and tetra-glycine were detected in the yields of 0.12% and 0.33%, respectively.     

Diketopiperazine-mediated peptide formation in aqueous solution II. Catalytic effect of phosphate

The previous paper (I) reported that DKP (glycine anhydride) spontaneously reacts with glycine (Gly) or oligoglycines (Gly _n ) to produce longer oligoglycines (Gly _n+2). This paper presents that phosphate catalyzes the condensation reaction quite effectively.Formation of Gly₄ from DKP (0.1 M) and Gly₂ (0.1 M) in phosphate solution of various concentrations was investigated at a neutral pH at 41 °C. The yields of Gly₄ increased almost linearly with the concentration of phosphate from 0.06 M to 0.24 M. The yield in 0.24 M phosphate solution was approximately one hundred times as high as that in the absence of the phosphate, whereas in the case of Gly₃ formation from DKP and Gly the effect of the phosphate was of ten times lower than in the former case. Orthophosphate was the most effective catalyst among the various kind of chemicals tried in the present investigation including polyphosphates.     

The formation of peptides from glycine thioesters

Summary The condensation products obtained from 0.01M S-glycyl-N-acetyl-cysteamine at different pH's were investigated. The highest yields of diketo-piperazine (approx. 50%) were observed in phosphate buffers between pH 7.5 and 8.5. The highest yields of diglycine (46%), triglycine (10%) and tetraglycine (2%) were observed in carbonate buffers at pH 9.5. At pH 8.0, over 90% of the glycyl residues of 0.15M S-glycyl-N-acetylcysteamine were incorporated into condensation products, mainly DKP (60–70%). The yields of products from the condensation of S-glycyl-ethanethiol under similar conditions closely re-sembled those obtained with S-glycyl-N-acetylcysteamine.Abbreviations Boc-gly N-tert-butyloxycarbonylglycine - Ac-cys N-acetylcysteine - csa cysteamine - Ac-csa N-acetylcysteamine - DKP diketopiperazine - (gly) ₂ diglycine - (gly) ₃ triglycine - (gly) ₄ tetraglycine - glySEt S-glycyl-ethanethiol - glyS-(Ac-cys) S-glycyl-N-acetylcysteine - glyS-(Ac-csa) S-glycyl-N-acetylcysteamine - Boc-glyS-(Ac-cys) S-(Boc-glycyl)-N-acetylcysteine - Boc-glyS-(Ac-csa) S-(Boc-glycyl)-N-acetylcysteamine - Boc-glySEt S-(Boc-glycyl)-ethanethiol - gly-bydrox glycine hydroxamate     

Diketopiperazine-mediated peptide formation in aqueous solution

Though diketopiperazines (DKP) are formed in most experiments concerning the prebiotic peptide formation, the molecules have not been paid attention in the studies of chemical evolution. We have found that triglycine, tetraglycine or pentaglycine are formed in aqueous solution of glycine anhydride (DKP) and glycine, diglycine or triglycine, respectively. A reaction of alanine with DKP resulted in the formation of glycylglycylalanine under the same conditions. These results indicate that the formation of the peptide bonds proceeds through the nucleophilic attack of an amino group of the amino acids or the oligoglycines on the DKP accompanied by the ring-opening.The formation of glycine anhydride, di-, tri- and tetraglycine was also observed in a mixed aqueous solution of urea and glycine in an open system to allow the evaporation of ammonia. A probable pathway is proposed for prebiotic peptide formation through diketopiperazine on the primitive Earth.     

Quantitative polyphosphate-induced "prebiotic" peptide formation in H2O by addition of certain azoles and ions

Aqueous solutions of 0.1 M amino acid and 0.1 M trimetaphosphate maintained at chosen pH values between 8.0 and 10.5 and at room temperature in the presence of imidazole or 1,2,4-triazole give rise after a few days to the corresponding peptides. Yields are highest when the pH is adjusted with concentrated NaOH or KOH instead of ammonia; in some cases glycine is quantitatively transformed within 10-15 days into peptides, mainly di-and tripeptides.     

Quantitative polyphosphate-induced “prebiotic” peptide formation in H2O by addition of certain azoles and ions

Summary Aqueous solutions of 0.1 M amino acid and 0.1 M trimetaphosphate maintained at chosen pH values between 8.0 and 10.5 and at room temperature in the presence of imidazole or 1,2,4-triazole give rise after a few days to the corresponding peptides. Yields are highest when the pH is adjusted with concentrated NaOH or KOH instead of ammonia; in some cases glycine is quantitatively transformed within 10–15 days into peptides, mainly diand tripeptides.     

Copper-catalyzed amino acid condensation in water — A simple possible way of prebiotic peptide formation

The recently reported condensation reaction of glycine to di- and triglycine in aqueous solution in the presence of higher concentrations of sodium chloride and copper ions has been investigated systematically and quantitatively using HPLC analytical methods. The influence of environmental factors (temperature, concentration, atmosphere) are discussed. Numerous other metal ions have been investigated with respect to similar catalytic effects, and molybdenum results as the only one inducing peptide condensation, although to a much lesser extent. Experiments based on evaporation of water and redissolution lead to peptide condensation up to (gly)₆ in concentrated solutions and produces peptides even starting from initially low concentrations.     

Transfer free energies of peptide backbone unit from water to aqueous electrolyte solutions at 298.15 K

Transfer free energies (ΔG_tr) of amino acids from water to aqueous electrolyte solutions have been determined from the solubility measurements, as a function of salt concentration at 298.15 K under atmospheric pressure. The investigated aqueous systems contain amino acids of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly₂), triglycine (Gly₃), and tetraglycine (Gly₄) and cyclic glycylglycine (c(GG)) with an electrolyte compound of potassium chloride (KCl), potassium bromide (KBr) or potassium acetate (KAc). The solubilities of glycine and diglycine in aqueous solution decrease with increasing the concentration of salts (salting-out effect), whereas those of triglycine and tetraglycine increase with increasing the concentration of salts (salting-in effect). Furthermore, salting-in effect was found in aqueous c(GG)/KBr system, while salting-out effect was observed in aqueous c(GG)/KCl or c(GG)/KAc system. The experimental results were used to estimate the transfer free energies (Δg_tr) of the peptide backbone unit (–CH₂CONH–) from water to the aqueous electrolyte solutions. We developed a new trail to determine the activity coefficients (γ) for aqueous and aqueous electrolyte solutions using an activity coefficient model, with which the total contribution of transfer free energy between solute and the solvent was calculated. We compared the difference between neglecting and using the activity coefficients term in predicting ΔG_tr. Since the transfer free energy contribution is negative, interactions between the ionic salts and the peptide backbone unit of zwitterionic glycine peptides are favorable and thus the ionic salts destabilize these amino acids. It was also found that KBr stabilizes c(GG), whereas KCl and KAc destabilize c(GG). These results provide evidence for the existence of interactions between the amide unit and ionic salts, in aqueous solution, which may be of importance in maintaining protein structure as well as in protein–solute and protein–solvent interactions.     

Reaction Behaviors of Glycine under Super- and Subcritical Water Conditions

The influence of temperature and pressure on the dimerizationand decomposition of glycine under simulated hydrothermal system conditions was studied by injecting a glycine solutioninto water in the sub- and supercritical state. The experimentsat five different temperatures of supplied water – 250, 300, 350, 374, and 400 °C – were performed at 22.2 and 40.0 MPa. At 350 °C, experiments under 15.0–40.0 MPa were conducted. Diglycine, triglycine (trace), diketopiperazine,and an unidentified product with a high molecular mass (433 Da) were the main products of oligomerization. The results show thattemperature and pressure influence the extent of dimerizationand decomposition of glycine. The maximum of dimers formationwas observed at 350 and 375 °C at 22.2 and 40.0 MPa,respectively, and coincided with a high rate of glycine decomposition. Glycine, alanine, aspartic acid, as well as otheramino acids, were obtained by injecting a mixture of formaldehydeand ammonia. The results support the oligomerization and synthesis of amino acids in a submarine hydrothermal system.     

Mineral Induced Phosphorylation of Glycolate Ion – a Metaphor in Chemical Evolution

Bilateral surface-active minerals with excess positive charge concentrate glycolate and trimetaphosphate ion from 10^–3 m aqueous solution to half-saturation of the internal surface sites, and induce phosphorylation of glycolate ion in the mineral with trimetaphosphate, sorbed from 10^–2 m solution. By utilizing reactants from dilute solution at near-neutral pH, and eliminating the need for participating organic nitrogen compounds, the reaction comprises several elements considered necessary for geochemical realism in models for molecular evolution.     

Specific effect of magnesium ion on 2′, 3′-cyclic amp synthesis from adenosine and trimeta phosphate in aqueous solution

Phosphorylation of adenosine by trimetaphosphate was investigated using various catalysts in aqueous solution under mild conditions at pH 7.0 and at 41 °C. The product was primarily 2,3-cyclic AMP together with smaller amounts of ATP. Magnesium ion was found to have a remarkable catalytic effect of approximately one hundred times greater than the other chemicals tested. The mechanism for the specific effect of magnesium ion is discussed.     

Optical activity of simple peptides. I. Glycine oligopeptides with internal L-alanyl or L-glutamyl residues

Oligopeptides containing glycine and one or two L -alanyl or L -glutamyl residues have been studied by circular dichroism (CD) and optical rotatory dispersion (ORD) in aqueous solution at pH 1.0, pH 6.0, and pH 10.0 and in aqueous ethanol. Two glycyl residues are required to remove effects of α-carboxyl or amino titration on the optical activity of the internal alanyl or glutamyl residues. The CD spectra of the alanyl and protonated glutamyl residues are similar, having two regions of negative ellipticity around 215 nm resulting in a spectrum reassembling that of poly-α-L -glutamic acid (PGA) at high pH. Another large positive band below 190 nm was observed for gly₂-glu₂-gly₂ in water at pH 6 and 10 and for several peptides in aqueous ethanol. Residue ellipticities were approximately additive in every case except for peptides containing intrenal glutamyl residu at pH 6.0.     

Thermodynamics of peptide bond formation at clay mineral surfaces

Summary The possibility of surface catalysed condensation of unsubstituted amino acids on kaolinite in aqueous systems at elevated temperatures was investigated; no evidence of clay catalysed polycondensation has been found. The thermodynamic feasibility of the hypothetical lysine/dilysine condensation reaction in the temperature-range up to 90° C was evaluated for a range of experimental conditions by the combination of measured free energies of lysine/dilysine cation exchange on kaolinite and on montmorillonite, and free energies for the analogous condensation reaction in homogeneous solution. The results indicate that, in spite of the high selectivity of the clays for the adsorption of cation dimers from dilute solutions, the thermodynamic barrier to the surface condensation of unsubstituted amino acids on clay minerals in aqueous systems up to 90° C is not lower than it is in homogeneous solution.     

Absorption of triglycine, diglycine, glycine or equimolar mixtures of diglycine and glycine in the perfused small intestine of rats

1. After a wash-out period of 20 min with Krebs-Henseleit-buffer a 20 cm segment of proximal or distal small intestine of nonanaesthesized rats got a bolus infusion of 3 ml either triglycine, diglycine, glycine or an equimolar mixture of diglycine and glycine, respectively, in a concentration range of 20 to 1000 mmol/l glycine equivalents. With 9 ml of a peptide and amino acid free solution the gut was perfused in a single-pass perfusion and the whole fluid recovered was investigated by quantitative thin layer chromatography for triglycine, diglycine and glycine. 2. In the concentration range up to 170 mmol/l glycine equivalents the nitrogen absorption is independent of the substrates perfused. In the highest concentration range an additional increased disappearance of triglycine could be found. This change cannot be observed to the same extent for diglycine, glycine or the mixture of both. In contrast, in the highest concentration range the competition between glycine and diglycine results in a decrease of absorption. 3. The proximal or distal nitrogen absorption of all substrates is similar. 4. In the perfusate the peptide and its splitting products were investigated. 5. Based on the discrepancy between the disappearance of triglycine and the appearance of its splitting products it is assumed. 1. that the transport of intact triglycine is of quantitative importance in the highest concentration range and 2. that in the lower concentration range membrane digestion followed the transport of the splitting products prevails. 6. Extending Ugoley's model for dipeptides three general variations for membrane digestion of tripeptides are proposed. One of this is likely for the triglycine absorption.     

The action of a water-soluble carbodiimide on adenosine-5''-polyphosphates.

When aqueous solutions of adenosine-5'-mono-, di-, or triphosphates are treated with a water soluble carbodiimide the major product is the expected diadenosine-5'-5'-polyphosphate. The yields of these pyrophosphates are greatly increased in the presence of the Mg2+ ion. Adenosine-5'-tetraphosphate behaves differently. The major product is adenosine-5'-monophosphate. We believe that this hydrolysis occurs via a cyclic trimetaphosphate intermediate.     

Phosphorylation of glyceric acid in aqueous solution using trimetaphosphate

The phosphorylation of glyceric acid is an interesting prebiotic reaction because it converts a simple, potentially prebiotic organic molecule into phosphate derivatives that are central to carbohydrate metabolism. We find that 0.05 M glyceric acid in the presence of 0.5 M trimetaphosphate in alkaline solution gives a mixture of 2- and 3-phosphoglyceric acids in combined yields of up to 40%.Abbreviations P3! trimetaphosphate - 2-P 2-Phosphoglycerate - 3-P 3-Phosphoglycerate - TSPS 3-trimethylsilyl propionic acid, sodium salt - NMR nuclear magnetic resonance     

DNA extraction from archival formalin-fixed,paraffin-embedded tissues: heat-induced retrieval in alkaline solution

Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5–12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.     

Heat capacity microcalorimetry of the in vitro reconstitution of calf brain microtubules.

The self-assembly of calf brain tubulin, purified by the modified Weisenberg procedure, was examined in an adiabatic differential heat capacity microcalorimeter. Tubulin solutions at concentrations between 6 and 17 mg/mL were heated from 8 to 40 degrees C at heating rates between 0.1 and 1.0 deg/min in a pH 7.0 phosphate buffer containing 1 X 10(-3) M GTP, 1.6 X 10(-2) M MgCl2, and 3.4 M glycerol. The heat capacity change, deltaCp of the microtubule growth reaction was found to be -1600 +/- 500 cal/(deg mol) per 110 000 molecular weight tubulin dimer incorporated into microtubules, in agreement with the reported van't Hoff deltaCp value of -1500 cal/(deg mol) [Lee, J.C., & Timasheff, S.N. (1977) Biochemistry 16, 1754-1765]. The assembly reaction is characterized by a complex heat uptake pattern comprising both endothermic and exothermic processes.