Potassium Ions are More Effective than Sodium Ions in Salt Induced Peptide Formation
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Prebiotic peptide formation under aqueous conditions in the presence of metal ions is one of the plausible triggers of the emergence of life. The salt-induced peptide formation reaction has been suggested as being prebiotically relevant and was examined for the formation of peptides in NaCl solutions. In previous work we have argued that the first protocell could have emerged in KCl solution. Using HPLC-MS/MS analysis, we found that K+ is more than an order of magnitude more effective in the L-glutamic acid oligomerization with 1,1'-carbonyldiimidazole in aqueous solutions than the same concentration of Na+, which is consistent with the diffusion theory calculations. We anticipate that prebiotic peptides could have formed with K+ as the driving force, not Na+, as commonly believed.
KeywordsPotassium Sodium Prebiotic Peptide formation Origin of life
Since Oparin’s ideas (1924; 1938) and Miller-Urey’s famous experiment (1953) on the prebiotic synthesis of amino acids, one of the main problems of prebiotic chemistry is to “re-invent” the plausible range of indispensable physical-chemical boundary requirements that would enable the emergence of stable and replicable molecules on the primordial Earth (Eschenmoser 2003). According to almost all discussions of the prebiotic soup theory, biological precursor molecules might have formed and evolved in the context of the sodium chloride ocean on primordial Earth (Fox 1960; Rode 1999; Zimmer 2009). Salt-induced peptide formation reaction has been suggested to be prebiotically relevant for the very first steps of chemical evolution (Schwendinger and Rode 1989). Based on Monte Carlo computer simulations, Rode and co-workers found that sodium chloride at concentrations above 3 M effectively acts as a dehydrating agent to overcome the thermodynamic barrier of peptide bond formation in aqueous solutions, and the first hydration shell of the sodium ion was assumed to no longer be saturated with water molecules (Jakschitz and Rode 2012). Furthermore, using HPLC-MS/MS analysis, a high concentration of sodium chloride was found to significantly enhance the formation of peptides from L-glutamic acid (L-Glu) in homogenous water solutions (Wang et al. 2005).
All the references we have found that discuss the presence of other mono- and divalent inorganic cations in prebiotic peptide formation speculate that these ions support the dehydrating effect of sodium chloride. However, the level of potassium exceeds that of sodium by more than an order of magnitude inside all living cells (Aronson et al. 2009), and the ion ratio is actively preserved with Na+/K+ pumps in the cell membrane, which suggests that potassium is more essential for life. The physical-chemical differences between Na+ and K+ are small (Freedman 1995), although the bio-directed activity of these ions differs dramatically; for example, K+ is required for ribosomal peptide synthesis (Spirin and Gavrilova 1971) and the amplification of DNA with thermostable Taq polymerase (Saiki et al. 1988), whereas Na+ attenuates these processes. The contradiction between the Na+ and K+ compositions of seawater and living cell cytoplasm led to the hypothesis that the first protocell could have emerged in KCl solution (Natochin 2007; Natochin 2010). However, the hypothesis of the K+-driven emergence of prebiotic peptides remains to be tested. Here we investigate the relative effects of Na+ and K+ in a model peptide synthesis reaction.
L-glutamic acid and 1,1′-carbonyldiimidazole (CDI) were obtained from Sigma-Aldrich Co. LLC (St. Louis, USA). In total, 10 mmol KCl or 10 mmol NaCl was added to reaction mixtures containing 3 mmol L-Glu in 5 ml distilled water. The mixture was diluted to 10 ml and cooled on a crashed ice-NaCl mixture, and 6 mmol CDI was added into each mixture and incubated at room temperature for 24 h. A 10 μl sample was loaded onto a Zorbax SAX (4.6 mm × 250 mm, 5 μm) column using an autosampler.
Peptide separation was performed at a flow rate of 0.5 ml/min using an NaCl gradient (2–80 % B for 80 min; buffer A: 20 % acetonitrile in 0.020 M NaH2PO4 at pH 7.0; buffer B: 2.0 M NaCl in buffer A) using an Agilent 1100 nano-HPLC system (Agilent Technologies Inc., USA). LC analysis of the peptides was performed by an established procedure (Ishihama et al. 2002) on a homemade 12-cm reverse-phase spraying fused silica capillary column (75 μm i.d. × 12 cm) with a 3-μm ReproSil-Pur Basic-C18 (Dr. Maisch HPLC GmbH, Germany). Peptide fractions were collected for further analysis.
MS/MS analysis of the samples was performed using a 7-Tesla LTQ-FT Ultra mass spectrometer and Xcalibur software in data-dependent mode (Thermo Fisher Scientific Inc., USA). The precursor ion MS spectra were acquired in the ICR trap with a resolution of 50,000 at m/z 400. The three most intense ions were isolated from MS/MS spectra and fragmented in LTQ. Oligomers from 2- to 9-mers were identified with ESI-MS. Other oligomers were assigned based on the one-charge increase in oligomers on HPLC traces.
We used the basic theories of catalytic reactions and nucleation (Dubrovskii and Nazarenko 2010) to model the ion-mediated condensation of amino acids in the liquid phase.
Liquid Chromatography and Mass Spectrometry
Chromatography and mass spectrometry data for Na+- or K+ - catalyzed peptides
Number of residues
L-Glu oligomers + 1.0 M NaCl
L-Glu oligomers + 1.0 M KCl
Mass spectrometry [M + H]+ ([M + Na]+)
Mass spectrometry [M + H]+ ([M + K]+)
Relative area, %
Relative area, %
C10H17O7N2 277.104 C10H16O7N2Na (299.086)
C10H17O7N2 277.104 C10H16O7N2K (315.059)
C15H24O10N3 406.146 C15H23O10N3Na (428.128)
C15H24O10N3 406.146 C15H23O10N3K (444.102)
C20H31O13N4 535.189 C20H30O13N4Na (557.171)
C20H31O13N4 535.189 C20H30O13N4K (573.145)
C25H38O16N5 664.231 C25H37O16N5Na (686.213)
C25H38O16N5 664.231 C25H37O16N5K (702.187)
C30H45O19N6 793.274 C30H44O19N6Na (815.256)
C30H45O19N6 793.274 C30H44O19N6K (831.230)
C35H52O22N7 922.317 C35H51O22N7Na (944.298)
C35H52O22N7 922.317 C35H51O22N7K (960.272)
C40H59O25N8 1051.359 C40H58O25N8K (1089.315)
Our experimental results demonstrate that K+ has a 3-fold to 10-fold greater catalytic effect than the same concentration of Na+ on the reaction peak of 5-mer to 8-mer L-Glu condensation in aqueous solutions. Computations and blackbody infrared radioactive dissociations have shown that Na+ is coordinated to the nitrogen and carbonyl oxygen atoms (NO coordination) of amino acids, whereas K+ is coordinated to both oxygen atoms (OO coordination), with lower binding energy (Jockusch et al. 2001). This finding allows us to suggest that NO coordination decreases the reactivity of amino acids for each subsequent peptide bond formation, as it begins with a nucleophilic attack on the lone electron pair of the N atom, in full agreement with our obtained experimental data showing the weaker Na+-mediated formation of L-Glu peptides.
Taken together, our experimental and theoretical evidences show that K+ predominates over Na+ ions in the formation of peptides. This allows us to suggest that the high K+/Na+ ratio in any prebiotic water reservoir could accelerate the first step in the chemical evolution of self-assembling organic molecules. Geochemically, a high K+/Na+ ratio in aqueous solution could also have formed during the differentiation of primary chondritic material into the Earth’s core and mantle (Galimov et al. 2011). It was also suggested that the ion composition required for the initial environment for the first cells could have emerged in inland geothermal ponds (Mulkidjanian et al. 2012). Although this assumption has been criticised (Switek 2012), from a biological point of view, the “modern” cytoplasm of the living cells might represent the same functional conditions that determined the first protocell’s chemical content. Thus, if the emergence of the ancient metabolic and information systems of the protocells occurred in potassium-rich habitats, it seems evident that all the living cells would have evolved to preserve the initial ion gradients by using energy-dependent membrane pumps in sodium aqueous media.
In conclusion, we hypothesise that the following conditions could have led to the prebiotic polymerisation of amino acids: (1) aqueous media contained the building blocks of organic matter and positive inorganic ions, which are geochemically abundant; (2) binding reversibility to amino acids and the moderate hydration energy of the ions in liquid phase at 0–100 °C; and (3) high diffusion and specific ion coordination to oxygen atoms of amino acids in zwitterion form, which enhances the ion-dependent yields of oligomerization. We propose that K+ complies with all the above-listed requirements, which is unique in contrast to other mono- and divalent metallic ions (Fig. 3). Further peptide evolution at later stages could have occurred due to the presence of other abundant cations, e.g., Na+, Mg2+ and Ca2+, which may have resulted from their lower diffusion and higher hydration energy. The elongation and functionalization of the peptides might also have been driven by other inorganic cations or clays or minerals (Ferris et al. 1996; Hill and Orgel 1999; Rode et al. 1999; Rees and Howard 2003) because they form more stable complexes with biomolecules.
We assume that our findings could be useful not only for discussions of the origin of life but also for more sophisticated research on the role of the physical-chemical properties of inorganic ions, biomolecules and nanoparticles in molecular physiology. The data on the difference in K+versus Na+ coordination- and diffusion-controlled condensation of amino acids may be of particular interest in understanding ion-exchange regulation by the membrane Na+/K+-ATPase pump.
We are grateful to Prof. Yuri V. Trushin and Prof. Vladimir G. Dubrovskii for helpful discussions of the physics of diffusion, Dr. Viktor G. Zgoda for his discussions of mass spectrometry and PhD student Ivan N. Terterov for his technical assistance. This work was performed under a grant from the Presidium of the Russian Academy of Sciences.
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