Chemiosmotic Energy for Primitive Cellular Life Andres Acosta and Stefan Paula, PhD Department of Chemistry California State University, Sacramento Abstract Methods Results Conclusions The purpose of this study was to create a Liposome Preparation: Control: • The control trial was successful, and a proton chemiosmotic energy system that could have gradient was obtained, maintained, and diffused existed on prebiotic Earth. A transmembrane proton Ascorbate as Electron Donor to restore initial liposome pH gradient was created using a model liposome 22000 Ascorbate Triton X-100 • Thiosulfate was able to induce a proton gradient system. Internal ferricyanide served as an oxidant, 16500 • It was not fully understood why the addition of phenazine methosulfate acted as an electron carrier, Triton X-100 did not restore the initial facilitating the transport of protons and electrons 11000 fluorescence intensity for the thiosulfate system within the liposome membrane, and external • Future work will be to replace the current ascorbate provided a source of electrons for 5500 Intensity (A.U) Intensity electron carrier, PMS, with a quinone derivative exchange with electron carriers. Sodium thiosulfate 0 that is found in meteorites was substituted for ascorbate to achieve a more Figure 2: Basic liposome composition consisting of a 0 125 250 375 500 • More sulfur-based electron donors will be tested feasible mechanism that coincided with the types phospholipid bilayer with hydrophilic heads and Time (s) of compounds that would have been found on hydrophobic tails. Figure 3: The fluorescence intensity (A.U.) vs time (s), upon the prebiotic Earth. 9-aminoacridine fluorescence was • Liposomes were prepared from a 10:1 mole addition of ascorbate to induce a proton gradient. The proton used to measure the internal pH of the liposome gradient was diffused with the addition of Triton X-100. system to indicate the formation of a proton ratio mixture of 1,2-dioleoyl-sn-glycero-3- References phosphocholine (DOPC) and gradient. Triton X-100 was used to disrupt the lipid 1. Milshteyn, D., Cooper, G. & Deamer, D. phosphatidylglycerol (PG) membrane which caused the diffusion of protons to Sodium Thiosulfate: Chemiosmotic energy for primitive cellular life: restore initial pH. The study found that the • Chloroform was added to the lipid mixture and Proton gradients are generated across lipid ascorbate liposome model induced a proton the solution was dried using a gentle stream of membranes by redox reactions coupled to gradient which ensured that the mechanism nitrogen until a lipid film was formed Thiosulfate as Electron Donor 650000 meteoritic quinones. Sci Rep 9, 12447 (2019). worked. The thiosulfate system achieved the • A 37.5mM potassium ferricyanide in 10mM Thiosulfate https://doi.org/10.1038/s41598-019-48328-5 formation of an internal proton gradient, as seen by phosphate buffer (7.4 pH) solution was added 487500 2. Deamer, D. W., Prince, R. C. & Crofts, A. R. a change in internal pH, however, reversal of the to the lipid film 24 hours later to rehydrate proton gradient was not fully obtained, alluding to 325000 The response of fluorescent amines to pH lipids to a final concentration of 1mg/mL external factors also influencing results. Instead, Triton X-100 gradients across liposome membranes. Biochim. • The rehydrated liposome solution was 162500 Biophys. Acta. 274, 323–335 (1972). confirmation of the thiosulfate system was done by (A.U.) Intensity examining the leaking of protons which was seen extruded ten times to ensure uniformed vesicle 3. Meteoritical Society Bulletin database, https:// 0 in the 9-aminoacridine fluorescence analysis. diameter (100-200 nm) 0 63 125 188 250 www.lpi.usra.edu/meteor/metbull.php. • To remove external ferricyanide, gel filtration Time (s) 4. Juntunen, H. L. et al. Investigating the Introduction chromatography was used Figure 4: Fluorescence shows that a pH gradient was obtained kinetics of Montmorillonite clay-catalyzed • Liposome fractions were collected within using thiosulfate. However, the addition of Triton X-100 did not return initial intensity. conversion of anthracene to 9,10-anthraquinone All cellular life depends on the energy provided 3-5mL of eluted volume in the context of prebiotic chemistry. Orig Life from electrochemical proton gradients (Milshteyn Evol Biosph 48, 321–330 (2018). et al., 2019). This energy is utilized to allow energy Sodium Thiosulfate (zoomed): dependent reactions to occur. In order to understand 9-Aminoacridine Fluorescence: how life may have began, this study aims to provide a feasible mechanism by which a proton Thiosulfate as Electron Donor Acknowledgements • Set ƛex = 326 nm, ƛem = 455 nm gradient may have developed on prebiotic Earth in • Add liposomes, 9-AA, and PMS 130000 a freshwater environment. • Record for 60 seconds • Dr. Stefan Paula who provided me with his 120000 expertise and guidance • Our collaborator Dr. David Deamer from the Theory 110000 Department of Biomolecular Engineering, University of California, Santa Cruz, CA • Add electron donor 100000 • Record for 180 seconds (A.U.) Intensity • Department of Chemistry at Sacramento State 90000 55 89 123 156 190 Time (s) • Add Triton X-100 Figure 5: A magnified view of fluorescence after the addition of Figure 1: The proposed redox reaction using ascorbate as an • Record for 60 seconds thiosulfate. The gradual increase of fluorescence during this time electron donor, phenazine methosulfate as an intermembrane • Compile data is caused by a small leak of protons. This leaking is expected and electron carrier, and internal ferricyanide as an electron acceptor. can indicate the formation of a proton gradient..