ICARUS 6~, 176-184 (1986) Amino Acids Derived from Titan Tholins BISHUN N. KHARE AND CARL SAGAN Laboratory. for Planetary Studies, Cornell University, Ithaca, New York 14853 HIROSHI OGINO,' BARTHOLOMEW NAGY, AND CEVAT ER Department of Geosciences, University qf Arizona, Tacson, Arizona 85721 KARL H. SCHRAM Department of Pharmaceutical Science, University of Arizona, Tucson, Arizona 85721 AND EDWARD T. ARAKAWA Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Received October 24, 1985; revised May 22, 1986 An organic h e t e r o p o l y m e r (Titan tholin) was produced by continuous dc discharge through a 0.9 N J 0 . 1 C H 4 gas mixture at 0.2 m b a r pressure, roughly simulating the cloudtop a t m o s p h e r e of Titan. T r e a t m e n t of this tholin with 6 N HC1 yielded 16 amino acids by gas c h r o m a t o g r a p h y after derivatization to N-trifluroacetyl isopropyl esters on two different capillary columns. Identifications were confirmed by G C / M S . Glycine, aspartic acid, and c~- and fl-alanine were produced in greatest a b u n d a n c e ; the total yield of amino acids was - 1 0 2 approximately equal to the yield of urea. The presence of "nonbiological" amino acids, the absence of serine, and the fact that the amino acids are racemic within experimental error together indicate that these molecules are not due to microbial or other contamination, but are derived from the tholin. In addition to the H C N , HC2CN, and (CN)2 found by Voyager, nitriles and aminonitriles should be sought in the Titanian a t m o s p h e r e and, eventually, amino acids on the surface. T h e s e results suggest that episodes of liquid water in the past or future of Titan might lead to major further steps in prebiological organic chemistry on that body. ~,~1986AcademicPress. Inc. INTRODUCTION When cosmically abundant reducing gases are irradiated, a class of dark reddishbrown tarry organic solids are produced, which have been called tholins, a modelindependent designation deriving from the Greek word for muddy (Sagan and Khare, 1979; Sagan et at., 1984a). When nitrogen is mixed with 10% methane, or less, and irradiated at low pressures in experiments Present address: D e p a r t m e n t of Industrial Chemistry, T o k y o Metropolitan Univesity, Setagaya-Ku, Tokyo 158, Japan. simulating conditions in the atmosphere of Titan, a category of tholin is produced that seems to resemble closely the reddish aerosol (Smith et al., 1981) which is the most striking characteristic of Titan's atmosphere. The complex refractive index of Titan tholin has been measured from X-ray to microwave frequencies (Khare et al., 1984), and seems able to account for Earth orbital ultraviolet and ground-based visible spectroscopy of the integrated disk of Titan (Sagan et al., 1984b; 1985); Voyager measurements of limb darkening in the visible (Squyres et al., 1984; Thompson et al., 1984); and for the Voyager infrared continuum (Thompson and Sagan, 1984). Nine or176 0019-1035/86 $3.00 Copyright © 1986by AcademicPress. Inc. All rights of reproduction in any form reserved. TITAN THOLIN AMINO ACIDS ganic molecules ranging in complexity up to butadiyne and propynenitrile have been identified in the gas phase by Voyager infrared spectroscopy (Hanel et al., 1981; Maguire et al., 1981; Kunde et al., 1981). Many of these molecules are produced directly by the irradiation of simulated Titan atmospheres (Balestic, 1974; Toupance et al., 1975; Scattergood et al., 1975; Scattergood and Owen, 1977; Gupta et al., 1981; Raulin et al., 1982). Much more complex organic matter is to be expected in Titan tholins. The detached limb hazes seen by Voyager in the visible, and the high-altitude ultraviolet haze detected by Voyager instruments seem also to require more complex organic molecules (Sagan and Thompson, 1984). Preliminary pyrolysis/gas chromatography/mass spectrometry (py GC/MS) of Titan tholins reveals more than 75 products, including saturated and unsaturated aliphatic hydrocarbons, substituted polycyclic aromatics, amines, pyrroles, pyrazines, pyridines, pyrimidines, and abundant nitriles (Khare et al., 1985). We recognize that some products may be synthesized during pyrolysis (Irwin, 1981) in a py GC/ MS analysis, but argue that many of the products formed are true fragments of the complex organic heteropolymeric material that constitutes Titan tholin (see also Khare et al., 1981; Ogino and Nagy, 1981). The abundance and variety of nitriles ( C ~ N ) in the pyrolysis products, the prominent 4.6/xm nitrile (or isocyanide) absorption feature in the untreated tholin (Khare et al., 1984), and the strong N - - H absorption near 3.0 ~m in the untreated tholin (Sagan et al., 1984a) naturally raise the question of whether amino acids can be produced from Titan tholin. The mean density of Titan is 1.88 g c m 3, implying--in conjunction with currently fashionable models of the origin of the solar system--that the interior of Titan contains several tens of percent of water ice (cf. Hunten et al., 1984); some models of the interior structure of Titan propose water ice 177 at the surface and layers of water-containing liquids -100 km subsurface. Since moons in the outer solar system, including both Ganymede (of roughly the same radius and density as Titan) and the much smaller Enceladus, show clear signs of comparatively recent surface melting, it would be unwise to exclude at least episodic surface melting on Titan. High-temperature tholinwater solution reactions are expected from impact processes. At present production rates, the quantity of tholin accumulated on the surface of Titan over its history amounts to a layer -100 m thick if only short-wavelength ultraviolet irradiation is employed in the synthetic process, and kilometers thick if longer wavelength photons can also be utilized (Sagan and Thompson, 1984). This material is denser than the putative deep hydrocarbon ocean on Titan (Lunine et al., 1983), and would thus accumulate as a submarine deposit. Thus episodic melting events would subject Titan tholin to liquid water, and solid state convection on Titan may carry tholins down to liquid depths. Hydrolysis of proteins (e.g., Nagy, 1975) and other polymeric substances (e.g., Er et al., 1986) can be accomplished in eit h e r acidic or basic media and a similar reaction is possible for the heteropolymeric tholins. Under present Titanian conditions, liquid water would form NH4OH, in which would be dissolved a variety of other organic compounds; a subsurface liquid water-ammonia ocean is considered "conceivable" by Hunten et al. (1984). Finally, somewhat similar tholins synthesized from CHa/NH3 gas mixtures (with a few percent H20) are known, in aqueous media, to be fully sufficient carbon and nitrogen sources for both aerobic and anaerobic miroorganisms (Boston et al., 1986), and tholin-rich surface deposits on the primitive Earth may have provided an important environment for the origin of life. Accordingly, experiments on amino acid production by acid (or base) treatment of Titan tholin seem relevant on three independent accounts: 178 KHARE ET AL. (1) for the light they may cast on the composition of Titan tholin; (2) as relevant to possible episodic water-ice melting events on the surface and solid state convection in the immediate subsurface of Titan; and (3) as possibly relevant to the primitive Earth and the origin of life in aqueous media. Synthesis o f Titan Tholin rity of gases used are given elsewhere (Khare et al., 1984). Acid Treatment o f Titan Tholins We here describe the techniques used for the acid treatment and analysis by gas chromatography/mass spectrometry (GC/MS) of Titan tholins, following the procedures of Gil-Av (1975), Engel et al. (1977), Zumberge et al. (1980), and Nagy et al. (1981). Tholins deposited on the glass wall of the reaction vessel were scraped off. A 3.9-rag portion of this tholin was placed into a glass tube (Pyrex, 150 mm long × 15 mm internal diameter) with 2 ml of double distilled 6N HCI. The glass tube was sealed under N2 and heated to 100°C for 20 hr, after which the solution was passed through a fine frit glass filter. (All glassware was cleaned prior to use with an 85/15 mixture by volume of hot concentrated H2SO 4 and HNO3). Then, the filter was washed repeatedly with triple, glass-distilled H20 and the filtrate and the washing water were combined. One quarter of this solution was used for the quantitative determination of amino acids, the remainder for the qualitative analysis. A 0.2 /xl solution of the amino acid threonine of known concentration was added to the solution to serve as an internal standard for the quantitative analysis. Then the solutions, both for the quantitative and the qualitative analysis, were evaporated to dryness under a stream of N2. Titan tholin was generated from a gas mixture of 0.9 N2 and 0.1 CH4 by volume at a total pressure of 0.2 mbar. This corresponds to a radial distance from the center of Titan of about 2825 km, just at the top of the main cloud deck viewed by Voyagers 1 and 2, and below most of the visible aerosol haze. The solar ultraviolet flux at X < 900 A, Saturn magnetospheric electrons and protons, solar wind electrons, and cosmic rays are all able to break N2 chemical bonds and synthesize nitrogenous organics from N2/CH4 atmosphere (cf. Strobel, 1982); ultraviolet photons at X < 1450 A generate higher hydrocarbons, and longer wavelength photons may be involved in secondary reactions, both those including and those excluding nitrogen (Sagan and Thompson, 1984). The experimental apparatus essentially consists of two horizontal aluminum electrodes of diameter -~7.6 cm, separated by a vertical distance of 5.7 cm, enclosed within a vertical glass cylinder of interIon-Exchange Clean-Up nal diameter =10 cm. The 0.9 N2/0.1 CH4 Cation exchange columns were packed gas mixture continuously flowed through the l-liter chamber at a rate -~0.05 ml/sec. with 5 ml Bio-Rad AG 50W-X8, 50-100 A 15-mA direct current electrical discharge mesh resin, a procedure commonly used to was maintained by a 200-V potential differ- purify amino acids after hydrolysis. Before ence between the electrodes. The tholin each sample application, the columns were products presumably form by quenching preconditioned by washing 10 times, reand the reaction of the ionization and disso- peatedly and consecutively with 50 ml of ciation products in the region of the dis- water, 50 ml of 2N N a O H , 50 ml of water, charge, and then diffuse outward, where 10 ml of 1.5N HC1, 50 ml of 6N HCI, and 50 they are deposited on the inner walls of the ml of water. Next, the evaporation residues vertical glass cylinder. Further details of were redissolved in 1 ml of 0.06 N HCI the experimental setup, the electrode and and were placed on the cation exchange field configurations, and the source and pu- columns. Then, 30 ml of water, containing TITAN THOLIN AMINO ACIDS three drops of phenolphthalein solution, was added onto the columns. The samples were eluted with 15 ml of 2N NH4OH and were collected at the NH4OH eluent front. Again, the solutions were evaporated to dryness. The residue contains pure amino acids free from other compounds available on acid treatment of Titan tholin. Derivatization of Amino Acids to N-Trifluoroacetyl Isopropyl Esters Pure amino acids are nonvolatile and therefore are not suitable for direct GC/MS analysis. Accordingly, we volatilized them by first esterifying the amino acid to isopropyl ester hydrochlorides: HCI N H 2 - - C H - - C O O H + (CH3)2CHOH + 179 100°C 3 hr I ) R Amino acid Isopropyl alcohol Hydrogen chloride CI-NH~--CH--COOCH(CH3)2 + H20 (i) I R Isopropyl ester hydrochloride We acidified isopropyl alcohol with dry HCI gas to 3N, its normality determined by weight, and then added it to the vial containing dry tholin residue. After the cap on the vial was closed, the samples were heated to 100°C for 3 hr. After this esterification step, the unreacted isopropyl alcohol, hydrogen chloride, and the liberated CI-NH~--CH--COOCH(CH3)2 + I Water water were evaporated under a stream of dry nitrogen leaving behind isopropyl ester hydrochloride in the vial. The second step involved acylation of isopropyl ester hydrochloride with trifluoroacetic anhydride to form volatile Ntrifluoroacetyl isopropyl esters suitable for GC/MS analysis: CH2CI2 (CF3C0)20 room letup 2 hr R Isopropyl ester hydrochloride Trifluoroacetic anhydride C F 3 C O - - N H - - C H - - C O O C H ( C H 3 ) 2 + CF3COOH + I HCI R N-Trifluoroacetyl isopropyl ester We added 0.2 ml of trifluoroacetic anhydride (TFAA) and 2 ml of methylene chloride (CH2CI2) to the evaporated residues to allow the acylation reaction to proceed at room temperature for 2 hr after the cap on the vial was closed. Closing the vial during both esterification and acylation serves to avoid contamination by airborne contami- Trifluoroacetic acid Hydrogen chloride nants and loss of reagent mixture by evaporation. After acylation, the samples were again evaporated to dryness under nitrogen to remove excess TFAA, CH2C12, and liberated trifluoroacetic acid and hydrogen chloride, all being more volatile than the N-trifluoroacetyl isopropyl esters. Finally, the 180 KHARE ET AL. c (.9 "o u c ~ a. ~g ~ 1 , ~- ~,,.~ o _~ c c c - : 7,0 5 w c -oJ :_= = 5- o ~o k 9p ~o ,~o t~o -c 15 3O 45 60 rain FIG. 1. Amino acids derived from Titan tholin and derivatized as their N-trifluoroacetyl isopropyl esters injected on ChirasiI-Va130m × 0.2 mm i.d. fused quartz capillary column. Carrier gas is helium. residues left as N-trifluoroacetyl isopropyl esters of amino acids were redissolved in 10-20 p.I CH2C12 to be injected into the GC/ MS. Gas Chromatography~Mass Spectrometry Initial separation and identification of the amino acids derived from Titan tholin were made using a Hewlett Packard 5880A gas chromatograph fitted with an optically active phase Chirasil-Val 30 m × 0.2 mm i.d. fused quartz capillary column. Aliquots of 0.5 /zl volume were injected. Unknown samples were also run on an SP-2100, 50 m × 0.2 mm i.d. fused quartz capillary column. Retention times of the unknown on both columns were compared with standards. Finally, standards were coinjected with the unknowns to confirm the identity of the amino acids. Procedure blanks, encompassing the entire analytical scheme without samples, were handled identically to tholins and examined by quantitative and qualitative analysis. Finally, mass spectra of the unknowns were obtained using a Varian-MAT 311A gas chromatograph/mass spectrometer under control of a Varian SS-200 data system. Operating conditions of the mass spectrometer were: ionization energy, 70 eV; source temperature, 190°C; interface temperature, 170°C; resolution, 1000; and scan speed, 2.5 sec/decade over the mass range 200-475 daltons with an interscan time of 0.5 sec. The operating conditions of the coupled gas chromatograph using two different columns were as follows: the first column was a fused silica quartz capillary column 30 m × 0.2 mm i.d. carbowax; the second, a fused silica quartz capillary column 30 m × 0.2 mm i.d., SP-2100. The initial temperature in both cases was 100°C for 15 rain followed by a programmed heating at I°C/ min to a final temperature of 190°C. The carrier gas of the column directly coupled to the mass spectrometer was helium with a flow rate maintained at 1 STP cmVmin. RESULTS Figure 1 displays a chromatogram of the derivatized amino acids, obtained from the hydrolysis of Titan lholin, as the N-trifluoroacetyl isopropyl esters, on a ChirasilVal column that contains an optically active phase. The most abundant products on this column as well as on the SP-2100 column are glycine, /3-alanine, and aspartic acid. All identified products are tabulated in Table I, where their abundance per gram of TITAN THOLIN AMINO ACIDS TABLE I AMINO ACIDS AND UREA IDENTIFIED IN ACID-TREATED THOLIN 181 The identification of aspartic and glutamic acids as examples are illustrated in Figs. 2 and 3, where they are compared with standards. mg/g CONCLUSIONS Glycine Alanine ~x-Amino-n-butyric acid Valine Threonine Aspartic acid Glutamic acid fl-Alanine fl-Amino-n-butyric acid B-Aminoisobutyric acid y-Amino-n-butyric acid a - A m i n o i s o b u t y r i c acid a - M e t h y l - a - a m i n o - n - b u t y r i c acid (Isovaline) a,B-Diaminopropionic acid a o / - D i a m i n o - n - b u t y r i c acid N-Methylglycine Urea 5.30 0.70 0.10 t t 1.10 0.40 1.20 0.20 0.13 0.30 0.06 t 0.10 0.02 0.18 10.30 A m i n o acids Urea 9.79 10.30 Note. t = trace a m o u n t s . initial Titan tholin is shown. The yields by mass of all amino acids and of urea are almost the same, each approximately 10-2 . The Chirasil-Val column permitted the separation of the D and L enantiomers. The D/L ratio of a few well-determined amino acids, produced from hyrolysis of the tholin, are shown in Table II. These values are racemic within experimental error. This fact, together with the presence of "nonbiological" amino acids, the clean procedure blanks, and the absence of the common contaminant serine on either column, indicates that the amino acids are not contaminants from microorganisms or from handling ("fingerprint" amino acids) during tholin synthesis and/or analysis. Threonine, another such indicator, occurred only in trace quantities, permitting the use of threonine as an internal standard for quantitative analysis. All amino acids listed in Table I are confirmed by their mass spectra on GC/MS. The tholin produced by irradiation of an Nz/CH4 atmosphere results in an approximately 1% yield of amino acids on HC1 treatment. Very crudely, the yield of amino acids, discussed in this paper, in the acid treatment of Titan tholin is an order of magnitude less than the yield of alkyl nitriles in the pyrolysis of Titan tholin (cf. Khare et al., 1985). The synthesis of amino acids from Titan tholin may involve a mechanism in which some tholin moieties, such as the abundant nitriles, are attacked by free radicals and are converted to aminonitriles; for example, the nitrene radical (NH), produced by high-frequency electrical discharge through N2 and CH4, may be inserted into any of the C - - H bonds of an alkyl chain (Gilchrist and Rees, 1969) and yield after acid treatment a, fl, y . . . . amino acids after hydrolysis. The present results seem consistent with the conclusion that the reddish Titan aerosols contain the nitrile functional group, and suggest the importance of infrared spectroscopic searches for the 4.6-p.m absorption feature. Infrared properties of nitriles more complex than HCN, which are expected to exist freely in the Titanian atmosphere (Sagan and Thompson, 1984; Thompson, et al., 1986) T A B L E I1 STEREOSPECIFICITY OF AMINO ACIDS PRODUCED FROM ACID-TREATED TITAN THOLINS A m i n o acid Alanine c~-Amino-n-butyric acid Aspartic acid Diaminopropionic acid D/L 0.96 1.07 0.95 1.00 -+ 0.03 -+ 0.06 -+ 0.04 _+ 0.01 Note. Errors represent m a x i m u m deviations from the m e a n in s u c c e s s i v e trials. 182 KHARE ET AL. TITAN THOLIN ~oo F 60 Electron Impact Asparlic Acid ~6 1%40 262 [ 20F 0 .I 200 2i8 ,11 i 220 I I, , 240 |l J ~fo 260 J~l I 280 500 320 290 [%60f L JC6I 340 Electron Impact Aspartic Acid Standard 262 z,z 20 0 200 ,. , .i I i 220 .1 ,ll 2~o 260 280 , 240 j,8 [ 3j, I .I 300 . 520 340 m/e FIG. 2. Mass spectrum of a GC peak identified as the N-trifluoroacetyl isopropyl ester of aspartic acid. Sample injected is the derivatized amino acid from Titan tholin. The bottom spectrum is a standard, authentic aspartic acid, similarly derivatized. The ordinate is the intensity, a m e a s u r e of a b u n d a n c e , normalized to the most prominent peak. have been tabulated by Cerceau e t a l . (1985). Future Titan entry probes should be capable of detecting aliphatic and aromatic nitriles, including aminonitriles, preferably up to m / e ~ 400; landers might be equipped ':jr with specific analytic protocols for detecting surface amino acids. Exposure of tholins on the primitive Earth to treatment by acids or bases may have provided a copious source of free amino acids and their poly- ZJO T I T A N THOLIN ,,, Electron Impact Glutamic Acid I% 6 0 f 30¢ 40 r 2O o .e, 200 220 zsz F I 275 I Ih I I., 260 280 300 , 240 33t I i * 320 340 I I I I 360 380 400 420 Electron Impact Glutomic Acid Standard Jo4 I% 6° 40 I 20 0 2~7 ,, 200 220 . , .h.,,/ 240 260 2z5 h., , 280 300 I 320 m/e .21 J,o • I I 340 I 360 I rio, 1580 400 I 420 FIG. 3. Mass spectrum of a GC peak identified as the N-trifluoroacetyl isopropyl ester of glutamic acid. Sample injected is the derivatized amino acid from Titan tholin. The bottom spectrum is a standard, authentic glutamic acid, similarly derivatized. The ordinate is the intensity, a m e a s u r e of a b u n d a n c e , normalized to the most prominent peak. TITAN THOLIN AMINO ACIDS mers and, thereby, a significant source via an indirect route (besides direct Strecker synthesis from free aminonitriles) of building blocks for the origin of life. While life on Titan is hardly to be expected at an ambient surface temperature of 95°K, episodic melting events, endogenous or exogenous, and solid state convection may have led to important further organic synthetic steps on Titan, beyond the generation of the tholins themselves. Several times 10 9 years from now, standard theories of solar evolution predict that the Sun will enter its red giant stage, and for - I 0 8 years or more the surface temperature on Titan should be above the freezing point of water. In this remote era, surface liquid water (probably containing NH4OH) may be abundant, and chemical evolution on Titan (until now frozen at what for the Earth would be a comparatively early stage) is then likely to take further steps. ACKNOWLEDGMENTS We are grateful to Tylon O. Willingham and Peter F. Baker for assistance, to W. Reid Thompson and Pradyot Patnaik for helpful discussions, and to Francois Raulin for a review of the manuscript. This research is supported in part by the National Aeronautics and Space Administration, Grants NGR 33-010-101, NGR 33-010-220, and NGR 03-002-171, and by the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc. REFERENCES BALESTIC, F. S. (1974). Synthese Abiotique d'Acides Aminds par Voie Radiochirnique. Doctoral thesis, Universit6 Paris Sud, Orsay, France. BOSTON, P. J., C. STOKER, W. D. SEGAL, B. N. KHARE, AND C. SAGAN (1986). Microbiol Metabolism of Spark Tholin. In preparation. CERCEAU, F., F. RAULIN, R. COURTIN, AND D. GAUTIER (1985). Infrared spectra of gaseous mononitriles: Application to the atmosphere of Titan. Icarus 62, 207-220. ENGEL, M. H., J. E. ZUMBERGE,AND B. NAGY (1977). Kinetics of amino acid racemization in Sequoiadendron giganteum Heartwood. Anal. Biochem. 82, 415-422. ER, C., B. NAGY, E. C. RISER, K. H. SCHRAM, AND P. F. BAKER(1986). Analysis of muramic acid in Holocene microbial environments by gas chromatogra- 183 phy, electron impact and fast atom bombardment mass spectrometry. Geomicrobiol. J., in press. GIL-Av, E. (1975). Present status of enantiomeric analysis by gas chromatography. J. Mol. Evol. 6, 131-144. GILCHRIST, T. L., AND C. W. REES (1969). Carbenes, Nitrenes, and Arynes, p. 131. Appleton-CenturyCrofts, New York. GUPTA, S., E. OCHIAI, AND C. PONNAMPERUMA (1981). Organic synthesis in the atmosphere of Titan. Nature 293, 725-727. HANEL, R., B. CONRATH, F. M. FLASAR, V. KUNDE, W. MAGUIRE, J. PEARL, J. PIRRAGEIA, R. SAMUELSON, L. HEARTH, M. ALLISON, D. CRUIKSHANK, D. GAUTIER, P. GIERASCH, L. HORN, R. KOPPANY, AND C. PONNAMPERUMA(1981). Infrared observations of the Saturnian system from Voyager 1. Science 212, 192-200. HUNTEN, D. M., M. G. TOMASKO, F. M. FLASAR, R. E. SAMUELSON,D. F. STROBEL, AND D. J. STEVENSON (1984). Titan. In Saturn (M. S. Matthews and T. Gehrels, Eds.), p. 753. Univ. of Arizona Press, Tucson. IRWIN, W. J. (1982). Analytical Pyrolysis. Marcel Dekker, New York. KHARE, B. N., C. SAGAN, E. T. ARAKAWA,F., SUITS, Z. A. CALLICOTT, AND M. W. WILLIAMS (1984). Optical constants of organic tholins produced in a simulated Titanian atmosphere: From soft X-ray to microwave frequencies. Icarus 60, 127-137. KHARE, B. N., C. SAGAN, W. R. THOMPSON, E. T. ARAKAWA, F. SUITS, T. A. CAELCOTT, M. W. WILLIAMS, S. SHRADER, H. OGINO, T. O. WILLINGHAM, AND B. NAGY (1985). The organic aerosols of Titan. Adv. Space Res. 402), 59-68. KHARE, B. N., C. SAGAN, J. E. ZUMBERGE, D. S. SKLAREW, AND B. NAGY (1981). Organic solids produced by electrical discharge in reducing atmospheres: Tholin molecular analysis. Icarus 48, 290297. KUNDE, V. G., A. C. ALDEN, R. A. HANEL, D. E. JENNINGS, W. C. MAGUIRE, AND R. E. SAMUELSON (1981). C4H2, HC3N and C2N2 in Titan's atmosphere. Nature 292, 686-688. LUNINE, J. L., D. J. STEVENSON, AND Y. L. YUNG (1983). Ethane ocean on Titan. Science 222, 12291230. MAGUIRE, W. C., R. A. HANEL, D. E. JENNINGS, V. G. KUNDE, AND R. E. SAMUELSON(1981). C3H8 and C3H4 in Titan's atmosphere. Nature 292, 683-686. NAGY, B. (1975). Carbonaceous Meteorites, pp. 434435. Elsevier, New York. NAGY, B., M. H. ENGEL, J. E. ZUMBERGE,H. OGINO, AND S. Y. CHANG (1981). Amino acids and hydrocarbons -3800-M yr old in the Isua rocks, southwestern Greenland. Nature 289, 53-56. OGINO, H., AND B. NAGY (1981). Pyrolysis of Transvaal kerogens. II. An evaluation of vacuum pyrolysis with polyethylene, polystyrene and their mixtures with minerals. Precambrian Res. 15, 113-130. 184 K H A R E ET AL. RAULIN, F., D. MOUREY, AND G. TOUPANCE (1982). Organic synthesis from CH4-N2 atmospheres: Implications for Titan. Orig. Life 12, 267-279. SAGAN, C., AND B. N. KHARE (1979). Tholins: Organic chemistry of interstellar grains and gas. Nature 277, 102-107. SAGAN, C., AND W. R. THOMPSON (1984). Production and condensation of organic gases in the atmosphere of Titan. Icarus 59, 133-161. SAGAN, C., B. N. KHARE, AND J. S. LEWIS (1984a). Organic matter in the Saturn system. In Saturn (M. S. Matthews and T. Gehrels, Eds.), pp. 788-807. Univ. of Arizona Press, Tucson. SAGAN, C., W. R. THOMPSON, B. N. KHARE, AND E. T. ARAKAWA(1984b). Titan: Multiple light scattering by organic tholins and condensates. Bull. Amer. Astron. Soc. 16, 665. SAGAN, C., W. R. THOMPSON, S. SQUYRES,AND B. N. KHARE (1985). Photometry, multiple light scattering, and lab simulations: Contraints on the structure of Titan's haze/cloud. Bull. A m e r . Astron. Soc. 17, 700. SCATTERGOOD, T., P. LESSER, AND T. ()WEN (1975). Production of organic molecules in the outer Solar System by proton irradiation: Laboratory simulations. Icarus 24, 465-471. SCATTERGOOD, T., AND T. OWEN (1977). On the sources of ultraviolet absorption in spectra of Titan and the outer planets, h'arus 30, 780-788. SMITH, B., L. SODERBLOM, R. BEEBE, J. BOYCE, G. BRIGGS, A. BUNKER, S. A. COLLINS, C. J. HANSEN, T. V. JOHNSON, J. L. MITCHELL, R. J. TERRILE, M. CARR, A. F. COOK II, J. CuzzL J. B. POLLACK,G. E. DANIELSON, A. INGERSOLL, M. E. DAVIES,G. E. HUNT, H. MASURSKY, E. SHOEMAKER, D. MORRISON, T. OWEN, C. SAGAN, J. VEVERKA, R. STROM, AND V. E. SUOMI (1981). Encounter with Saturn. Voyager 1 imaging sciences results. Science 212, 163-190. SQUYRES, S. W., W. R. THOMPSON, AND C. SAGAN (1984). Voyager imaging observations of Titan's atmosphere. 1. Disk-resolved photometric properties. Bull. Amer. Astron. Soc. 16, 664. STROBEL, D. F. (1982). Chemistry and evolution of Titan's atmosphere. Planet. Space Sci. 30, 839-848. THOMPSON, W. R., AND C. SAGAN (1984). Titan: Farinfrared and microwave remote sensing of methane clouds and organic haze. Icarus 60, 236-259. TttOMPSON, W. R., T. HENRY, B. N. KHARE, AND C. SAGAN (1986). Charged particle organic synthesis in low and moderate pressure N2-CH4 atmospheres: Implications for Titan and Triton. Bull. Amer. As'tron. S o t . , in press. THOMPSON, W. R., S. W. SQUYRES, AND C. SAGAN (1984). Voyager imaging observations of Titan's atmosphere. II. Variation of particle properties with altitude and latitude. Bull. A m e r . Astron. Soc. 16, 664. TOUPANCE, G., F. RAULIN, AND R. BUVET (1975). Formation of prebiochemical compounds in models of the primitive Earth's atmosphere. 1. CH4-NH~ and CH4-N2 atmospheres. Orig. Lift" 6, 83-90. ZUMBERGE, J. E., M. H. ENGLE, AND B. NAGY (1980). Amino acids in bristlecone pine: An evaluation of factors affecting racemization rates and paleothermometry. In Biochemistry ~ f A m i n o Acids (P. E. Hare, T. C. Hoering, and K. King, Jr., Eds.), pp. 503-525. Wiley, New York.