Abstract: The unambiguous detection of amino acids on Mars would be of pivotal importance in the understanding of the processes involved in the origin of life. Homochirality would be the best indicator of whether any detected martian amino acids were biotic or abiotic in origin. Several possible methods could be used for in situ amino acid analyses on Mars, but capillary electrophoresis would likely be the most suitable because it can be easily miniaturized and has small reagent and power requirements. Returned samples could be analyzed by any method then in routine laboratory use, but terrestrial contamination could interfere with the detection of trace levels of endogenous martian amino acids.
Introduction
Understanding the events that led to the origin of life on Earth is complicated by the lack of geological evidence around 4 billion years (4 Gyr) ago when the transition from prebiotic chemistry to biochemistry likely took place. Although erosion and plate tectonics have erased the terrestrial geological record at the time of the origin of life, there is a possibility that information about this period of Earth history may be still preserved on Mars. Compared to Earth, Mars is a much more placid planet; there is no known plate tectonic activity, and surface alteration rates are minimal. Extensive areas of the martian surface may date to >4 Gyr ago (1, 2). Geomorphological evidence suggests that liquid water existed on the martian surface at some point in the past and early Mars may have had an atmosphere similar to that of the early Earth (3). If this is the case, then at least some of the steps leading to the origin of terrestrial biochemistry may have also taken place on Mars (4). Thus, traces of prebiotic chemistry, or biochemical evidence associated with extinct martian biota, could be present on Mars. Though deemed unlikely, life may even still exist today on Mars in some protected subsurface environments (5, 6).
The processes thought to be involved in the origin of life on Earth are summarized in Figure 1. The first requirement is the presence of a prebiotic soup consisting of a rich variety of organic compounds, although at this point we do not know the soup composition necessary for the origin of life. The components of the soup may have been made directly on Earth, or supplied from space by comets, asteroids, micrometeorites or interplanetary dust particles (7). A large variety of organic compounds, including those which play a major role in biochemistry such as amino acids, purines, pyrimidines, etc., have been identified in one class of meteorites, the carbonaceous chondrites (Figure 2). Besides demonstrating that important biomolecules can be produced abiotically in extraterrestrial environments, their presence also suggests that exogenous compounds should be periodically delivered to the surface of the Earth (and other planetary bodies as well) by various delivery processes (7). The subsequent transition from the abiotic chemistry of the primitive Earth to the first self-replicating molecular systems capable of Darwinian evolution marked the point of the origin of life. On the Earth, subsequent evolution of the first self-replicating molecules then gave rise to the RNA world and finally the DNA/protein world characteristic of all life today.
The surface of Mars could hold clues about the various processes and stages involved in the origin of life. A major goal of the NASA Space Exploration Program over the next several decades is to search for evidence of extinct and extant life, and abiotic chemistry, on Mars. During the next decade, spacecraft will orbit Mars and land on the surface. Within 15 years, sample return missions are planned that will provide scientists with material to analyze directly in the laboratory. An important consideration of these efforts is what compounds do we search for, either directly on Mars or in martian samples returned to Earth, that will answer unambiguously whether abiotic and/or biotic organic molecules are present.
Previous organic analyses of Mars
The detection of organic material on the martian surface was attempted by the Viking 1 and 2 landers in 1976. These spacecraft each carried a gas chromatograph coupled to a mass spectrometer (9-11). No organic compounds were detected above the part per billion (ppb) level in the upper few cm of the martian surface. The results of other experiments aboard the landers, however, led to the conclusion that the martian surface is saturated with an oxidant of unknown type, and thus any organics deposited in the martian surface layer would be destroyed on short time scales (12). The oxidizing layer may only extend a few meters below the surface, however, (13), so the preservation of martian organics in the sub-surface is possible (14).
Aside from these Viking missions, the only other opportunities to directly analyze martian samples have come from the SNC meteorites, which are fragments of the martian crust ejected by impact events that eventually found their way to Earth (15). The Antarctic shergottite EET A79001 has been of considerable interest because it contains a carbonate component with 600-700 ppm combustible carbon which has been suggested to be endogenous martian organic material (16). Analyses of a small fragment of the EET A79001 carbonate material detected only the L-enantiomers of the amino acids found in proteins (17). There is no indication of the presence of a-aminoisobutyric acid (Aib). Aib is a common amino acid in carbonaceous meteorites and is readily synthesized in laboratory-based prebiotic experiments, but is not one of the amino acids found in the proteins of terrestrial organisms (18). The amino acids in this martian meteorite are thus terrestrial contaminates derived from Antarctic ice meltwater which had percolated through the meteorite (17). Failure to detect extraterrestrial amino acids in this martian meteorite does not rule out the possibility of endogenous amino acids on Mars because the severe conditions experienced during impact ejection should have destroyed any amino acids which were originally present. These results do suggest, however, that the transfer of organic material from Mars to Earth, or vice versa, by impact ejecta appears unlikely.
What molecules should we search for during
future Mars missions?
Any strategy for investigating whether organic molecules are present on Mars should focus on compounds which are readily synthesized under plausible prebiotic conditions, are abundant in carbonaceous meteorites and play an essential role in biochemistry. One of the few classes of molecules that fulfill all these requirements are amino acids, although we do not know whether amino acids were a component of the first self-replicating systems, or even required for the origin of life. Amino acids are synthesized in high yields in prebiotic experiments (18), are one of the more abundant types of organic compounds present in carbonaceous meteorites (see Figure 2) and are the building blocks of proteins and enzymes. Amino acids are ubiquitous molecules on the surface of the Earth (18), and it is likely that regardless of whether they are of abiotic or biotic origin, they would be widespread on the surface of Mars as well.
A central problem in future organic analyses of martian samples is not only identifying and quantifying organic compounds that may be present, but also distinguishing those molecules produced abiotically from those synthesized by extinct or extant life. Terrestrial biology uses only a small subset of the large variety of amino acids, nucleic acid bases, and sugars that can be synthesized abiotically and would have thus possibly been present in the prebiotic soup. The detection on Mars of a limited number of the total array of possible organic molecules of biological important could be suggestive of biochemistry, but that criteria alone would be weak evidence. The most reliable indicator of the biological vs. abiotic origin of organic molecules is molecular homochirality (19). Terrestrial organisms use almost exclusively L-amino acids (the L- enantiomer) in protein biosynthesis, and D-ribose and D-deoxyribose in nucleic acids. The structural principles on which biomacromolecule activity is based lead us to believe that any functional biochemistry must use a single enantiomer of any molecule which possess a chiral carbon. In contrast, all known laboratory abiotic synthetic processes result in racemic mixtures of organic compounds with chiral carbons, and the amino acids in carbonaceous chondrites are also racemic (19).
Amino acid homochirality provides an unambiguous way of distinguishing between abiotic vs. biotic origins (Figure 3). In terrestrial organisms, amino acid homochirality is important because proteins cannot fold into bioactive configurations such as the a-helix if the amino acids are racemic. Enzymes likely could not have been efficient catalysts in early organisms if they were composed of racemic amino acids. However, enzymes made up of all D-amino acids function just as well as those made up of only L-amino acids, but the two enzymes react with the opposite stereoisomeric substrates (20). There are no biochemical reasons why L-amino acids would be favored over D-amino acids. On Earth, the use of only L-amino acids by life is likely simply a matter of chance. We assume that if proteins and enzymes were a component of extinct or extant life on Mars, then amino acid homochirality would have been a requirement. However, the possibility that martian life was (or is) based on D-amino acids would be equal to that based on L-amino acids.
As can be seen in Table I, the detection of a non-racemic mixture of amino acids in a martian sample would be strong evidence for the presence of an extinct or extant biota on Mars. The finding of an excess of D-amino acids would provide irrefutable evidence of unique martian life that could not have been derived from seeding the planet with terrestrial life. In contrast, the presence of racemic amino acids, along with abiotic amino acids such as Aib, would be indicative of an abiotic origin, although we have to consider the possibility whether the racemic amino acids where generated from the racemization of biotically produced amino acids (18).
When an organism dies, its amino acids begin to racemize at a rate which is dependent on the particular amino acid, the temperature, and the chemical environment (18). Racemization reactions are rapid on the terrestrial geologic time scale and even at deep ocean temperatures of 2°C, amino acids are totally racemized (e.g., D/L = 1.0) in about 5-10 million years. Using kinetic data, the racemization half-lives and times for total racemization of aspartic acid, a common protein amino acid, under conditions relevant to the surface history of Mars have been estimated (see (21) and Figure 4). Amino acids from an extinct martian biota maintained in a dry, cold (<250°K) environment would not have racemized significantly over the lifetime of the planet (4.5 Gyr). Racemization would have taken place in environments where liquid water was present even for time periods of only a few million years following biotic extinction. The best preservation of amino acid homochirality associated with extinct martian life would be in the polar regions. When biogenic amino acids are completely racemized, they would be indistinguishable from a chirality point-of-view from the racemic amino acids produced by abiotic organic synthesis or those derived from exogenous sources. Although a-dialkyl amino acids with a chiral center, which are common in carbonaceous meteorites (8), are very resistant to racemization (18), these amino acids are not generally found in the proteins of terrestrial organisms. However, we can not exclude the possibility that a-dialkyl amino acids might not be used by life elsewhere. The finding on Mars of racemic amino acids of the type found in the proteins of terrestrial organisms along with the presence of non-racemic a-dialkyl amino acids would suggest that life did or still does exist.
Amino acid detection methodologies on Mars
In Table II, we have evaluated the spacecraft worthiness of various amino acid analytical methods in routine use in the laboratory today that might be used to carry out in situ analyses on the surface of Mars. In general the three methods, gas chromatography coupled with mass spectrometry detection (GC/MS), high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), appear to be about equally suitable for spacecraft instrumentation. However, the prospects for miniaturization make CE probably the best choice.
GC/MS is an obvious method for molecular organic analysis from a landed martian spacecraft, because of the success with similar instrumentation during the Viking missions. Any GC/MS spacecraft system for future missions, however, must be able to distinguish abiotic vs. biotic origin through enantiomeric resolution. Either chemical derivatization procedures that produce diastereomeric derivatives, or a chiral stationary phase that can separate derivatized enantiomers, would be required. These procedures require additional hardware such as reaction chambers, valves, and pumps, and can greatly increase the size, weight, and mechanical complexity of the GC/MS system. Commonly used GC detectors besides MS such as thermal conductivity detectors likely lack the sensitivity needed to detect amino acids at the sub-ppb level, a necessity that should be a requirement for exobiological analysis on Mars. Flame ionization detectors have greater sensitivity, but would probably be too unstable and dangerous for spacecraft use.
HPLC is somewhat more suited to chiral amino acid analysis. Simple chiral derivatization procedures exist for HPLC, and fluorescence detection can be used to achieve sensitivities of well below the ppb level. Reverse-phase HPLC with o-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC) derivatization and fluorescence detection has been used to search for extraterrestrial amino acids in meteorites (17) and lunar samples (22), in sediments from the Cretaceous/Tertiary boundary (23), and in polar ice core samples (24). HPLC hardware, however, is heavy and mechanically complex and requires large volumes of solvents. These are all disadvantages when designing instrumentation for spacecraft use.
A relatively new technology which shows promise for spacecraft-based amino acid analysis is microchip-based capillary electrophoresis (25, 26). CE can use the same chiral derivatization reagents (such as OPA/NAC) and sensitive detection techniques (such as laser-induced fluorescence) as HPLC. The actual separation hardware, including buffer reservoirs and derivatization reaction chambers, can be etched onto glass microchips with dimensions on the order of cm. Such a system has great advantages over GC or HPLC systems in weight and size. The reagents, sample and solvents can also be manipulated using the electro-osmotic forces that effect the separation, with no need for mechanical pumps or valves. Sensitive detection systems such as laser-induced fluorescence or electrochemical detection (27, 28) can be used in a microchip CE system to achieve sub-ppb detection limits. Microchip capillary electrophoresis appears to be the best currently available technique for the in situ enantiomeric resolution of optically active compounds in extraterrestrial samples.
Amino acid detection in martian samples returned to Earth
A complete evaluation of the inventory of the organic compounds that may be present on Mars will require returned samples, especially if prior in situ analyses yield any positive results. Future martian samples returned to Earth could be analyzed, in theory at least, buy any suitable analytical technique then in existence. However, there are limitations in returned sample analyses. The cost of a sample return mission may limit at least initially sampling to only a few geologically distinct sites on Mars. The size of sample that can be returned using presently available space transportation technology may limit the number of laboratory based analyses than can be performed, and may even eliminate techniques with large sample requirements. Compound-specific organic analyses of a returned martian sample might be limited to techniques that are compatible with other areas of investigation, such as mineralogy and stable isotope analyses.
However, the main limitation of organic analyses of samples returned from Mars will be the omnipresent problem of terrestrial contamination. Even the best and most sensitive analytical methodologies used today must deal with contaminates in reagents, etc. that limit the detection of extraterrestrial organic compounds. Although this could also be a potential problem for in situ martian analyses, there are ways that this might be minimized. Any spacecraft landed on the martian surface would be required to undergo rigorous decontamination in order to ensure that the planet is not inoculated with terrestrial organisms. Reagents used for in situ analytical systems would thus be extensively purified prior to the mission and probably transported dry. Water required for aqueous buffers and sample processing could be made, or condensed from the atmosphere, directly on the martian surface.
Terrestrial contamination has limited the detection of Aib in lunar soils to about 0.1 ppb (22) and to around 1 part-per trillion (ppt) in polar ices (24). As a example of the contamination problem, consider the analyses of small samples of the organic-rich Murchison meteorite shown in Figure 5. Although with a 10 mg sample, the extraterrestrial amino acid Aib is clearly detectable, in the 100 mg sample Aib is almost completely obscured by interfering peaks. Using a value of 10 ppm for the Aib content of Murchison (22), the Aib detected in the 100 mg sample corresponds to about 1 ng (10-9 g). Thus, in order to detect extraterrestrial amino acids such as Aib at the ppb level would require at least 1 g of a martian sample. This is would likely be considered a " large" sample, and samples could be restricted to much smaller amounts. Thus, in order to detect trace levels of amino acids in samples of Mars returned to Earth, the background contamination from terrestrial amino acids and other interfering compounds would need to be greatly reduced. Because any returned sample from Mars would be quarantined in order to ensure that any martian organisms present did not contaminate the Earth, facilities could be setup as well to prepare super clean reagents, etc. that would be necessary to reduce terrestrial back ground organic levels.
Future Prospects
The next couple of decades will be exciting times with respect to the question of whether life existed or exists elsewhere in the solar system and the resolution of the problem of how life originated on Earth. The exploration and the organic analyses of the surface of Mars will undoubtedly be of pivotal importance. State-of-the-art analytical chemical techniques will play a major role in these endeavors. Finding evidence of extinct life on Mars would be, to put in mildly, sensational. The presence of extant life on Mars could be even more so, and would revolutionize our understanding of the chemistry of life. We can hardly wait!
References:
(1) Tanaka, K. L., Scott, D. H. and Greeley, R. In "Mars" (Kieffer, H. H.; Jakosky, B. M.; Snyder, C. W.; Matthews, M. S., Eds.), Univ. Arizona Press: Tucson, AZ, 1992; p. 345.
(2) Ash, R. D., Knott, S. F. and Turner, G. Nature 380, 57
