Amino Acid Analyses
Typical liquid chromatography UV fluorescence and TOF-MS mass chromatograms of the 6 M HCl-vapor hydrolyzed, hot-water extracts from the Tagish Lake 5b, 11h, and 11i meteorite specimens and the procedural blank show several peaks that were identified by comparison with amino acid standards, fluorescence, retention time, and mass (Figs. 1 and 2). We were able to obtain baseline separation of several α-H proteinogenic amino acids including aspartic and glutamic acids, serine, threonine, alanine, and valine and their enantiomers (Figs. 1 and 2), which was the primary focus of this study. The total procedural-blank-corrected amino acid abundances (free + bound) of identified C2 to C6 amino acids in the 6 M HCl-hydrolyzed, hot-water extracts of Tagish Lake were approximately 40 ppb for sample 11i, approximately 740 ppb for sample 5b, and approximately 5400 ppb for sample 11h (Tables 1 and 2). In contrast to samples 5b and 11h, the abundances of many amino acids in 11i were below analytical detection limits of <0.1–1 ppb. These results are consistent with a much higher degree of aqueous alteration experienced by 11i compared with 5b and 11h as inferred from variations in mineralogy, bulk isotopes, petrology, carboxylic acid abundances, and structure of the insoluble organic matter (Herd et al. 2011). The low total amino acid abundance in 11i is similar to the abundance (<100 ppb) previously found in another pristine Tagish Lake meteorite stone (Pizzarello et al. 2001). Overall, the amino acid abundances in the C2 Tagish Lake meteorite (approximately 40–5,400 ppb) are much lower than the levels measured in other less altered type 2 carbonaceous chondrites, but do fall within the range of amino acid concentrations measured for aqueously altered CI, CM, and CR type 1 carbonaceous chondrites (Glavin et al. 2010). The LC-TOF-MS instrument was optimized for the separation of the C5 acyclic amino alkanoic acids and the retention times were identified based on the analysis of standards (Fig. 3). Although complete separation of all 23 possible C5 amino acid isomers and enantiomers could not be achieved under the chromatographic conditions used, all of the C5 amino acids were accounted for, and we observed no interference or coelution of the d- and l-enantiomers of isovaline, norvaline, valine, and 3-aminopentanoic acid (3-apa) with other C5 amino acids (Fig. 3). Several C6 to C8 amino acid isomers were also detected by mass in the single ion chromatograms of the Tagish 5b and 11h meteorite extracts (Fig. 2). However, with the exception of the d,l-norleucine internal standard (Fig. 2, peak 33), the C6 to C8 amino acids could not be identified due to a lack of standards, low abundances, and poor chromatographic separation under the conditions employed.
Figure 1. The 5 to 23 min region of the LC-FD chromatograms. OPA/NAC derivatization (15 min) of amino acids in the standard mix and of the 6 M HCl-hydrolyzed, hot-water extracts of the procedural blank and Tagish Lake meteorite samples 11i, 5b, and 11h are shown. Similar chromatograms were also obtained for the nonhydrolyzed extracts. Separation was achieved for the first segment (5–15 min) of the chromatogram using a Waters BEH C18 column (2.1 × 50 mm, 1.7 μm bead) followed by a second Waters HSS T3 column (2.1 × 150 mm, 1.8 μm bead). For the second segment (15.3–23 min) of the chromatogram, the Waters HSS T3 column was replaced by a Waters BEH phenyl column (2.1 × 150 mm, 1.7 μm bead). The conditions for amino acid separations for the mobile phase at 30.0 °C for both segments were as follows: flow rate, 150 μL min−1; solvent A (50 mM ammonium formate, 8% methanol, pH 8.0); solvent B (methanol); gradient, time in minutes (%B): 0 (0), 35 (55), 45 (100). The peaks were identified by comparison of the fluorescence retention time and molecular mass to those in the amino acid standard run on the same day. Fluorescent peaks that did not have corresponding peaks in the single ion TOF-MS chromatograms (shown in Fig. 2) with m/z values of the OPA/NAC amino acid derivative in the standard were not identified and quantified. Peak identifications 1) d-aspartic acid; 2) l-aspartic acid; 3) l-glutamic acid; 4) d-glutamic acid; 5) d-serine; 6) l-serine; 7) d-threonine; 8) l-threonine; 9) glycine; 10) β-alanine; 11) γ-amino-n-butyric acid; 12) d-alanine; and 13) l-alanine. An unidentified fluorescent desalting artifact is labeled with an X.
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Figure 2. The 15–40 min regions of the LC-TOF-MS single ion chromatograms (C2: m/z = 337.09; C3: m/z = 351.10; C4: m/z = 365.12; C5: m/z = 379.13; C6: m/z = 393.15; C7: m/z = 407.16; C8: m/z = 421.18) in positive electrospray ionization mode. OPA/NAC derivatization (15 min) of amino acids in the 6 M HCl-hydrolyzed, hot-water extracts of Tagish Lake samples 11h, 5b, 11i, and a procedural blank are shown. Similar single ion chromatograms were obtained for the nonhydrolyzed extracts. Separation was achieved using the Waters BEH C18 column followed by the Waters BEH phenyl column using the same gradient as described in Fig. 1. Peaks were identified by comparison of the retention time and molecular mass with those in amino acid standards run on the same day. A nonfluorescent mass artifact that co-eluted with l-valine is labeled with an X. Peak identifications 9) glycine; 10) β-alanine (BALA); 11) γ-amino-n-butyric acid; 12) d-alanine; 13) l-alanine; 14) d-β-amino-n-butyric acid; 15) l-β-amino-n-butyric acid; 16) α-aminoisobutyric acid; 17) d, l-α-amino-n-butyric acid (ABA); 18–32) peak identifications for C5 amino acids given in Fig. 3; and 33) d, l-norleucine (internal standard). Asterisks designate nonfluorescent, noninterfering mass artifacts that could not be identified.
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Table 1. Summary of the average abundances (in ppb) of identified two- to six-carbon amino acids in the nonhydrolyzed (free) and 6 M HCl-hydrolyzed (total) hot-water extracts of the Tagish Lake meteorite measured by LC-FD/TOF-MSa.
|Amino acid||This study||Previous workb|
|Tagish Lake 11i||Tagish Lake 5b||Tagish Lake 11h||Tagish Lake 24-24|
| d-aspartic acid||<0.3||<1||1.7 ± 0.4||8.0 ± 1.8||13.6 ± 2.5||161 ± 14||11 ± 1|
| l-aspartic acid||0.7 ± 0.2||2.6 ± 0.5||8.7 ± 0.6||20.1 ± 3.3||55.0 ± 5.6||430 ± 65||83 ± 8|
| d-glutamic acid||<0.2||<0.2||1.6 ± 0.2||16.4 ± 0.5||41.2 ± 1.3||244 ± 23||16 ± 2|
| l-glutamic acid||0.2 ± 0.1||5.8 ± 1.6||2.7 ± 0.6||50.6 ± 2.3||53.5 ± 13.8||844 ± 89||306 ± 48|
| d-serine||<0.2||<0.2||1.3 ± 0.1||1.5 ± 0.1||23.6 ± 2.3||51.8 ± 7.3||n.d.|
| l-serine||1.8 ± 0.1||2.1 ± 0.9||17.3 ± 1.5||13.9 ± 4.2||124 ± 23||181 ± 29||n.d.|
| l-threonine||0.9 ± 0.3||1.3 ± 0.4||7.4 ± 1.8||3.5 ± 1.6||55.2 ± 5.3||97.3 ± 17.8||n.d.|
| glycine ||2.4 ± 0.4||9.7 ± 4.2||90.0 ± 6.4||129 ± 21||619 ± 184||987 ± 257||147 ± 17|
|β-alanine||0.1 ± 0.1||13.5 ± 0.7||70.4 ± 14.1||82.3 ± 9.5||107 ± 19||150 ± 30||64 ± 10|
|γ-amino-n-butyric acid||0.1 ± 0.1||<1||7.2 ± 1.0||216 ± 24||41.1 ± 2.8||374 ± 50||77 ± 10|
| d-alanine||<0.1||<0.5||25.7 ± 0.7||54.1 ± 3.3||252 ± 32||387 ± 25||20 ± 5|
| l-alanine||1.2 ± 1.0||1.6 ± 0.5||25.1 ± 0.7||49.7 ± 4.3||240 ± 33||363 ± 41||75 ± 18|
| d-β-amino-n-butyric acid||<0.1||<0.1||13.3 ± 2.0||11.5 ± 1.2||19.0 ± 3.8||36.0 ± 3.9||<26d|
| l-β-amino-n-butyric acid||<0.1||<0.1||12.5 ± 2.0||12.8 ± 1.4||17.4 ± 4.8||38.8 ± 3.0|
|α-aminoisobutyric acid (α-AIB)||0.3 ± 0.1||1.3 ± 0.2||9.2 ± 0.8||20.7 ± 2.1||161 ± 29||179 ± 23||<27|
| d, l-α-amino-n-butyric acidc||<0.1||<0.2||10.2 ± 0.1||24.2 ± 3.9||62.1 ± 16.5||82.2 ± 15.2||84 ± 40|
|ε-amino-n-caproic acid (EACA)||<0.2||<0.2||<0.2||<0.2||<0.3||<0.3||n.d.|
|C5 amino acids (from Table 2)||<0.7||<1||∼40||∼210||∼190||∼790||n.d.|
Table 2. Summary of the average five-carbon amino acid abundances (in ppb) in the nonhydrolyzed (free) and 6 M HCl-hydrolyzed (total) hot-water extracts of the Tagish Lake meteorite measured by LC-FD/TOF-MSa.
|C5 Amino acid detected||Tagish Lake 11i||Tagish Lake 5b||Tagish Lake 11h|
|α|| d-norvaline (d-2-apa)||<0.2||<0.4||2.2 ± 0.2||2.6 ± 0.2||5.4 ± 0.2||6.8 ± 0.4|
| l-norvaline (l-2-apa)||<0.2||<0.3||1.9 ± 0.1||2.7 ± 0.1||5.6 ± 0.2||7.5 ± 0.4|
| d-isovaline (d-2-a-2-mba)||<0.2||<0.5||2.5 ± 0.2||6.6 ± 0.2||42.7 ± 1.6||43.7 ± 1.8|
| l-isovaline (l-2-a-2-mba)||<0.2||<0.5||3.1 ± 0.2||7.6 ± 0.2||41.9 ± 1.5||43.7 ± 1.7|
| d-valine (d-2-a-3-mba)||<0.2||<0.4||1.9 ± 0.1||5.9 ± 0.1||7.1 ± 0.2||16.4 ± 0.5|
| l-valine (l-2-a-3-mba)||<0.7||<1||<4b||<9b||<37b||<86b|
|β|| d, l-3-apac||<0.2||<0.4||12.7 ± 0.5||10.6 ± 0.4||13.4 ± 0.4||12.7 ± 0.7|
| d, l- and allo-3-a-2-mbac||<0.1||<0.2||2.1 ± 0.2||2.6 ± 0.2||4.3 ± 0.2||5.4 ± 0.7|
|3-a-2,2-dmpa||<0.1||<0.1||1.5 ± 0.1||3.8 ± 0.1||9.2 ± 0.3||17.7 ± 0.6|
| d, l-3-a-2-epae||<0.5||<1||2.7 ± 0.2||10.7 ± 1.6||6.1 ± 0.2||10.2 ± 0.4|
|γ|| d, l-4-apac||<0.1||<0.1||1.3 ± 0.2||24.5 ± 0.8||5.3 ± 0.7||112 ± 6|
| d, l-4-a-2-mbae||<0.3||<0.5||1.7 ± 0.2||34.2 ± 0.7||4.5 ± 0.3||191 ± 7|
| d, l-4-a-3-mbae||<0.2||<0.3||1.0 ± 0.1||42.9 ± 1.3||4.3 ± 0.2||183 ± 5|
|δ||5-apa||<0.1||<0.2||1.2 ± 0.1||46.3 ± 1.2||2.7 ± 0.1||53.6 ± 1.1|
| ||Total (ppb)|| || ||∼40||∼210||∼190||∼790|
Figure 3. The 20–42 min region of the LC-TOF-MS single ion chromatograms of the C5 amino acids (m/z = 379.13 ± 0.02) in positive electrospray ionization mode. OPA/NAC derivatization (15 min) of amino acids in the standard mix and of the 6 M HCl-hydrolyzed, hot-water extracts of the procedural blank and the Tagish Lake meteorite samples 11i, 5b, and 11h. Similar LC-TOF-MS single ion chromatograms were obtained for the nonhydrolyzed extracts. The peaks were identified by comparison of the retention time and exact molecular mass with those in the C5 amino acid standard run on the same day. Separation was achieved using a Waters BEH C18 column followed by a second Waters BEH phenyl column. The conditions for amino acid separations for the mobile phase at 30.0 °C were as follows: flow rate, 150 μL min−1; solvent A (50 mM ammonium formate, 8% methanol, pH 8.0); solvent B (methanol); gradient, time in minutes (%B): 0 (15), 25 (20), 25.06 (35), 44.5 (40), 45 (100). The peaks were identified by comparison of the retention time and molecular mass with those in the C5 amino acid standard run on the same day. Peak 24 (d, l-4-a-2-mba) was not present in the standard shown, but was run separately. Peak identifications: 18, 3-amino-2,2-dimethylpropanoic acid (3-a-2,2-dmpa); 19, d, l-4-aminopentanoic acid (d, l-4-apa); 20, d, l-4-amino-3-methylbutanoic acid (d, l-4-a-3-mba); 21, d, l-3-amino-2-methylbutanoic acid (d, l-3-a-2-mba); 22, d, l-3-amino-2-ethylpropanoic acid (d, l-3-a-2-epa); 23, 5-aminopentanoic acid (5-apa); 24, d, l-4-amino-2-methylbutanoic acid (d, l-4-a-2-mba); 25, 3-amino-3-methylbutanoic acid (3-a-3-mba); 26, d-2-amino-2-methylbutanoic acid (d-isovaline); 27, d, l-3-aminopentanoic acid (d, l-3-apa); 28, l-2-amino-2-methylbutanoic acid (l-isovaline); 29, l-2-amino-3-methylbutanoic acid (l-valine); 30, d-2-amino-3-methylbutanoic acid (d-valine); 31, d-2-aminopentanoic acid (d-norvaline); 32, l-2-aminopentanoic acid (l-norvaline); and X, nonfluorescent mass artifact.
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A large increase in amino acid abundance (>100% increase) in all three meteorite extracts was observed after acid hydrolysis, which indicates that most of the amino acids in the Tagish Lake specimens are present in an acid-labile or bound form (Tables 1 and 2). This observation is consistent with our amino acid analyses of CI, CM, and CR carbonaceous chondrites (Glavin et al. 2010) and was also noted by Engel and Nagy (1982) in a previous study on the isotopic composition of amino acids in Murchison. The most abundant amino acids detected and quantified by LC-FD/TOF-MS in the Tagish Lake 5b and 11h extracts were d- and l-aspartic acid, d- and l-glutamic acid, d- and l-alanine, d- and l-serine, l-threonine, β-alanine (BALA), d, l-α-amino-n-butyric acid (ABA), d- and l-β-ABA, γ-ABA, α-aminoisobutyic acid (AIB), d- and l-valine, d, l-norvaline, and d- and l-isovaline (Figs. 1 and 2; Tables 1 and 2). Only trace levels (<10 ppb) of l-serine, l-threonine, glycine, β-alanine, l-alanine, and l-valine were measured by LC-FD/TOF-MS in the procedural blanks, indicating that very little amino acid contamination of the samples occurred during sample processing (Figs. 1 and 2). However, the low amino acid abundances in the procedural blanks do not rule out the possibility of amino acid contamination of the meteorites from the Tagish Lake ice, or during collection, storage, and handling of the samples. The Tagish Lake meteorite samples 11i, 5b, and 11h investigated in this study were collected at the same time directly from the surface ice within days after the fall and have all been kept at temperatures below 0 °C prior to extraction (Herd et al. 2011); in contrast to these specimens, a nonpristine Tagish Lake fragment (sample 24-24) that was collected from lake meltwater 3 months after the fall was found to contain a significant amount of terrestrial amino acid contamination from the lake meltwater itself (Table 1, Kminek et al. 2002).
The distributions of amino acids measured in the Tagish Lake meteorite samples 5b and 11h are clearly distinct from the Tagish Lake ice meltwater, which had higher relative abundances (normalized to glycine) of l-aspartic and l-glutamic acids, l-serine, l-alanine and d, l-α-amino-n-butyric acid (Fig. 4). The abundance of d-serine in the Tagish Lake ice meltwater was not reported by Kminek et al. (2002) and therefore was not included in Fig. 4. In addition, several nonprotein amino acids that are not common in terrestrial samples, including α-aminoisobutyric acid (AIB), d- and l-β-ABA, d- and l-isovaline, and d- and l-norvaline, were identified above background levels in both Tagish 5b and 11h (Tables 1 and 2), but have not been reported in the Tagish Lake meltwater or previous analyses of the Tagish Lake meteorite (Pizzarello et al. 2001; Kminek et al. 2002). The differences in relative amino acid abundances between the Tagish Lake meteorite samples 5b and 11h (Fig. 4) and absolute abundances of both protein and nonprotein amino acids that increase in order 11i ≪ 5b < 11h (Tables 1 and 2) provide additional support that most of these amino acids are indigenous to the meteorites, as all three fragments were collected, stored, and processed in parallel under identical conditions and therefore exposed to the same terrestrial contamination environments (Herd et al. 2011). Herd et al. (2011) previously argued that differences in the absolute abundances of amino and carboxylic acids among the Tagish Lake samples are best explained by differences in the extent of aqueous alteration in the meteorite fragments on the parent body, and not terrestrial contamination.
Figure 4. A comparison of the relative molar abundances of several amino acids (glycine = 1.0) in the 6 M HCl-hydrolyzed, hot-water extracts of the Tagish Lake meteorite samples 11h and 5b and a 250 mL sample of Tagish Lake meltwater. The relative abundance data for the Tagish Lake meteorite samples were determined from the absolute abundances measured in this study. Relative amino acid abundances for the Tagish Lake water were calculated from the absolute concentrations reported in Kminek et al. (2002).
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Amino Acid Isotopic Composition and Enantiomeric Measurements
Carbon isotope measurements were made for the most abundant amino acids in the Tagish Lake meteorite extracts 5b and 11h (11i had insufficient amino acids for isotope measurements). The GC-MS/IRMS technique employed provides simultaneous compound-specific structural and carbon isotopic information from a single injection, which permitted three replicate analyses of glycine, d- and l-alanine, and β-alanine and two replicate analyses of d- and l-aspartic acid, l-glutamic acid, and γ-ABA in the meteorite extracts. We previously reported a δ13C value of +19 ± 4‰ for glycine (Herd et al. 2011), the most abundant amino acid in Tagish Lake 11h, which is similar to the values previously obtained for glycine (δ13C ∼ +22‰, Table 3) in the CM Murchison (Engel et al. 1990) and CI Orgueil meteorites (Ehrenfreund et al. 2001). The corrected δ13C values for d- and l-aspartic acid peaks in 11h were +24‰ and +29‰ and were similar within a measurement error of ±4‰ (Table 3). These values fall well outside of the typical terrestrial range for organic carbon of −6‰ to −40‰ (Bowen 1988) and for aspartate (aspartic acid and asparagine) in a variety of microorganisms (−54‰ to 0‰; Scott et al. 2006) and indicate an extraterrestrial origin for both d- and l-aspartic acid. The GC-MS/IRMS data for the d- and l-aspartic acid and d- and l-alanine peaks in the combined hydrolyzed and nonhydrolyzed Tagish Lake 11h hot-water extracts are shown in Fig. 5. The abundances of amino acids in the nonhydrolyzed water extracts of the Tagish Lake meteorite were insufficient to make carbon isotope measurements of the free amino acids only. The retention times and mass spectra for both peaks in the 11h extract closely match those for the TFAA/IPA derivatives of the d- and l-aspartic acid peaks in the racemic standard (Fig. 5). We found no evidence of additional mass fragments in the mass spectra obtained for the d- and l-aspartic acid peaks in the Tagish Lake 11h extract compared with the mass spectra of the racemic aspartic acid standard that would suggest the presence of any co-eluting or interfering compounds (Fig. 5).
Table 3. Summary of the δ13C values (‰, VPDB) of amino acids in the 6 M HCl-acid hydrolyzed extracts of the Tagish Lake meteorite samples 5b and 11h compared with the Murchison meteoritea.
|Amino acids||This study||Previous work|
|Tagish Lake 5b||Tagish Lake 11h||Murchison|
| d-aspartic acid|| n.d.||+24 ± 4||+25b|
| l-aspartic acid|| n.d.||+29 ± 4||−6b|
| d-glutamic acid|| n.d.||n.d.||+29b, +32d|
| l-glutamic acid|| n.d.||−4 ± 9||+7b, +6c, +15d|
| glycine ||+39 ± 6||+19 ± 4||+41b, +22c, +13e|
| d-alanine||+67 ± 7||+6 ± 3||+52b, +30c|
| l-alanine||+55 ± 3||+16 ± 4||+38b, +27c, +41e|
|β-alanine||+30 ± 6||−5 ± 4||+5b|
|γ-ABA|| n.d.||+4 ± 3||+18b|
Figure 5. Gas chromatography separation and mass spectrometry analysis of d- and l-aspartic acid (A) and d- and l-alanine (B) of the TFAA/IPA-derivatized combined 6 M HCl-hydrolyzed and nonhydrolyzed extracts of the Tagish Lake 11h meteorite and the procedural blank, and a racemic standard. The traces on the left show the m/z 44 (12CO2) peak produced and measured from GC-IRMS for the peaks assigned to d- and l-aspartic acid. The traces on the right show the simultaneously collected mass spectral fragmentation pattern for these peaks in the Tagish Lake meteorite and standard. GC separation used a 5 m base-deactivated fused silica guard column (Restek) coupled with four 25 m Chirasil l-Val columns (Restek) and the following temperature program: initial oven temperature 50 °C, ramped at 10 °C min−1 to 85 °C, ramped at 2 °C min−1 to 120 °C, ramped at 4 °C min−1 to 200 °C, and held for 10 min. Peaks were identified by comparison of retention time and mass spectral fragmentation with the amino acid standard run on the same day. The asterisk marks a derivatization artifact with parent mass m/z 97 that could not be identified.
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A corrected D/L aspartic acid ratio of 0.26 ± 0.02 was determined by GC-MS at the same time from the integrated d- and l-aspartic acid peak areas in 11h compared with the racemic aspartic acid standard and corresponds to an l-enantiomeric excess (lee = l%–d%) of 58.7 ± 1.8%. A similarly high lee in the total aspartic acid of 45.5 ± 5.2% was determined independently from the absolute abundances of d- and l-aspartic acid measured by LC-FD/TOF-MS (Table 4). If the observed l-excess for aspartic acid was due to terrestrial contamination, the measured carbon isotope value of l-aspartic acid should have been less enriched in 13C compared with d-aspartic acid. For example, Pizzarello et al. (2004) measured the l- and d-aspartic acid carbon isotope ratios on a sample of Murchison with an l-enantiomer excess and reported a δ13C value of −6‰ for l-aspartic acid and +25‰ for d-aspartic acid (Table 3). In that case, the depletion in δ13C for the l-aspartic acid provided a clear indication that the Murchison sample contained both extraterrestrial and terrestrial sources of l-aspartic acid. This was not observed in the Tagish lake meteorite sample 11h, where l-aspartic acid nominally had a higher δ13C value compared with d-aspartic acid (Table 3), which cannot be explained by terrestrial l-aspartic acid contamination. No carbon isotope fractionation or changes in the enantiomeric composition were observed in a racemic aspartic acid standard carried through the same extraction and analytical procedure. The carbon isotope values (δ13C) for the d- and l-aspartic acid racemic standard were identical within measurement error, as expected from a standard made from d, l-aspartic acid.
Table 4. Summary of the l-enantiomeric excesses measured for several amino acids in the 6 M HCl-hydrolyzed hot-water extracts of Tagish Lake meteorites 11h and 5b and racemic standardsa.
|Amino acids||Tagish Lake 5b||Tagish Lake 11h||Racemic Standards|
| l ee (%)||δx (n)|| l ee (%)||δx (n)|| l ee (%)||δx (n)|
| Aspartic acid ||43.1||±8.6 (8)||45.5 58.7b||±5.2 (8) ±1.8 (3)b||6.6 4.7b||±2.9 (9) ±2.3 (3)b|
| Glutamic acid ||51.0||±1.5 (6)||55.1||±3.6 (6)||−2.6||±2.1 (9)|
| Alanine ||−4.8||±5.5 (9)||−3.2 7.5b||±6.7 (9) ±1.1 (3)*||1.0 1.0b||±1.6 (9) ±1.4 (3)b|
| Serine ||80.5||±3.9 (6)||55.5||±3.6 (6)||3.3||±1.5 (9)|
| Threonine ||89.2||±4.9 (6)||99.4||±0.3 (6)||0.3||±2.1 (9)|
| Valine ||<19.2||±7.1 (6)||<68.2||±2.2 (8)||0.0||±0.4 (14)|
|β-ABA||5.3||±7.2 (6)||3.7||±6.4 (6)||0.8||±1.1 (9)|
|Norvaline||1.9||±4.2 (6)||4.9||±3.8 (6)||1.0||±0.8 (8)|
|Isovaline||7.0||±1.9 (8)||0.0||±2.8 (8)||−2.3||±1.3 (14)|
The measured δ13C values for glycine, d- and l-alanine, l-glutamic acid, β-alanine, and γ-ABA in the Tagish Lake meteorite samples range from −5‰ to +67‰ (Table 3), which all fall outside of the typical terrestrial range for these amino acids. We were unable to determine the δ13C value for d-glutamic acid in the Tagish Lake samples due to low d-glutamic acid abundances and a chromatographic interference. A d/l glutamic acid ratio of approximately 0.3 (lee ∼ 55%) was measured by LC-FD/TOF-MS in Tagish Lake 5b and 11h, which was similar to the values measured for aspartic acid in the same meteorite extracts (Tables 4 and 5). On the basis of the δ13C value of −4 ± 9‰ measured for l-glutamic acid in Tagish 11h, we cannot rule out the possibility that some terrestrial l-glutamic acid contamination of the meteorite occurred, although in the absence of the d-glutamic acid carbon isotope value, we have no basis for comparison. However, if we assume that the level of l-glutamic acid contamination in 11h is similar to the total amount of l-glutamic acid measured in 11i (approximately 6 ppb, Table 1), which is a reasonable assumption as both meteorite specimens have been exposed to the same contamination environments since the time of their fall, then terrestrial l-glutamic acid contamination would represent <1% of the total l-glutamic acid in 11h (Table 1).
Table 5. Amino acid enantiomeric ratios (d/l) measured in the nonhydrolyzed (free) and 6 M HCl-hydrolyzed (total) hot-water extracts of the Tagish Lake meteoritea.
| ||Tagish Lake 5b||Tagish Lake 11h||Tagish Lake 24-24|
|This study||Previous workb|| |
| Aspartic acid ||0.20 ± 0.05||0.40 ± 0.11||0.25 ± 0.05||0.37 ± 0.07 0.26 ± 0.02c||0.13 ± 0.02|
| Glutamic acid ||0.59 ± 0.15||0.32 ± 0.02||0.77 ± 0.20||0.29 ± 0.06||0.05 ± 0.01|
| Serine ||0.08 ± 0.01||0.11 ± 0.03||0.19 ± 0.04||0.29 ± 0.06||n.d.|
| Threonine ||<0.01||<0.06||<0.01||<0.01||n.d.|
| Alanine ||1.02 ± 0.04||1.09 ± 0.12||1.05 ± 0.20||1.06 ± 0.14 0.86 ± 0.02c||0.27 ± 0.09|
| Valine ||> 0.48||> 0.65||> 0.19||> 0.19||n.d.|
|β-ABA||1.06 ± 0.23||0.90 ± 0.35||1.09 ± 0.37||0.80 ± 0.31||n.d.|
|Norvaline||1.16 ± 0.12||0.96 ± 0.08||0.96 ± 0.05||0.91 ± 0.07||n.d.|
|Isovaline||0.81 ± 0.08||0.87 ± 0.03||1.02 ± 0.05||1.00 ± 0.06||n.d.|
A similarly low d/l ratio (d/l ∼ 0.3) for glutamic acid has previously been measured in the Murchison meteorite and was argued to be indigenous based on nonterrestrial nitrogen isotopic values for both d- and l-enantiomers that were similar (Engel and Macko 2001). However, another study of Murchison found a large lee (approximately 16–47%) of pyroglutamic acid, but with lower δ13C values for the l-enantiomer compared with the d-enantiomer, pointing to a significant terrestrial contribution to the l-excesses (Pizzarello and Cooper 2001). Similar d/l ratios of aspartic and glutamic acids (d/l ∼ 0.1–0.4) have been reported in oceanic dissolved organic matter in which the d-amino acids were attributed to peptidoglycan remnants derived from bacterial cell walls (McCarthy et al. 1998). The d/l alanine ratios measured in the same seawater samples ranged from d/l ∼ 0.3 to 0.6 (McCarthy et al. 1998) and the Tagish Lake meltwater and a nonpristine Tagish lake meteorite sample were both reported to have d/l alanine ratios of approximately 0.2–0.3 (Kminek et al. 2002). All of these values are much lower than the racemic d/l alanine ratios found in the Tagish Lake 5b and 11h meteorite extracts measured in this study (Table 5). Therefore, it is difficult to reconcile how bacterially derived terrestrial amino acid contamination of the Tagish Lake meteorite samples 5b and 11h from the Tagish Lake ice meltwater or other sources is consistent with both the low d/l ratios of aspartic and glutamic acids and the racemic d/l alanine ratios measured in Tagish 5b and 11h (Table 5). The measured d/l ratios for aspartic and glutamic acids in the Tagish Lake meteorite are not due to the extraction procedure or LC-FD/TOF-MS analytical biases, as we observed no change in the enantiomeric ratios of a racemic aspartic and glutamic acid standard taken through the identical procedure. In addition, both aspartic and glutamic acids were found to be racemic (d/l = 1) within analytical error in the Antarctic CR meteorites EET 92042 and QUE 99177 and a sample of the CM2 Murchison meteorite using the same extraction and analytical technique (Glavin et al. 2010).
The δ13C values for d- and l-alanine in the Tagish Lake meteorite were measured to be +6 ± 3‰ and +16 ± 4‰, respectively, for sample 11h and +67 ± 7‰ and +55 ± 3‰, respectively, for sample 5b (Table 3). The d/l alanine ratio of sample 11h was also measured independently by GC-MS and found to be nearly racemic (d/l ≈ 0.9). On the basis of observation that alanine is racemic in both meteorite samples with carbon isotope values that indicate an extraterrestrial origin, we would have expected the δ13C values of d- and l-alanine in each sample to be similar. It is possible that the slightly 13C-depleted value for d-alanine in 11h is due to the isotopically light peak (indicated by an asterisk in Fig. 5) that cannot be completely resolved from the tail of the d-alanine peak. The main mass fragment of this isotopically light compound is at m/z 97, and we were unable to find a good match for this peak in the NIST mass spectral library. Although we did not observe m/z 97 mass fragments in the d- or l-alanine mass spectra of the racemic standard and Tagish Lake 11h extract (Fig. 5), it is possible that the difference in baselines between the d- and l-alanine peaks in Tagish 11h due to the presence of the artifact could explain the difference in the δ13C values measured. Unfortunately, the unidentified artifact is a by-product of the TFAA/IPA derivatization reaction (see procedural blank trace in Fig. 5); therefore, additional purification of the meteorite extracts would not remove it from the sample. The d/l alanine ratio of the Tagish 11h extract and the racemic standard measured by GC-MS were not affected by the m/z 97 mass artifact as the m/z 140 mass trace was used to quantify the d- and l-alanine peak areas.
The carbon isotope values measured in Tagish Lake sample 5b for glycine, d- and l-alanine, and β-alanine were all enriched in 13C compared with the same amino acids in 11h, with δ13C values ranging from +30 to +67‰ (Table 3). Carbon isotope values for aspartic and glutamic acids in 5b could not be obtained due to low amino acid abundances; however, low d/l ratios (approximately 0.3–0.4) were also measured for aspartic and glutamic acids in 5b by LC-FD/TOF-MS, corresponding to large lee of approximately 43–51% (Table 4). The relatively high δ13C values in 5b indicate that these amino acids and/or their precursor materials retained a more primitive carbon isotopic signature compared with 11h, consistent with mineralogical differences and isotopic measurements of the insoluble organic matter, demonstrating that 5b has experienced less aqueous alteration compared with 11h (Herd et al. 2011). As most of the organic carbon in the Tagish Lake samples 5b and 11h is depleted with an average bulk δ13C of −14‰ (Herd et al. 2011), the lighter carbon isotope values measured for the amino acids in 11h could be explained by their formation from 13C-depleted precursor material during a secondary aqueous alteration stage in the parent body. We believe that the elevated abundances of α-amino acids in 11h with depleted carbon isotope ratios compared with 5b (Table 4) are best interpreted as reflecting a secondary pulse of amino acid formation in 11h during parent body alteration from 13C depleted precursors by Strecker cyanohydrin synthesis (Peltzer and Bada 1978; Peltzer et al. 1984; Herd et al. 2011), although other formation mechanisms for α- and other amino acids before their incorporation in the parent body have been suggested (Elsila et al. 2007). Another possibility is that the elevated amino acid abundances and lower δ13C values for the amino acids in 11h compared with 5b is an indication that 11h experienced a higher degree of terrestrial amino acid contamination than 5b. However, if we consider the differences in total abundances and carbon isotope values for l-alanine between samples 5b and 11h (Tables 1 and 4), a mass balance calculation indicates that the additional l-alanine in 11h compared with 5b (=313 ppb, Table 1) must have an average δ13C value of +10‰ to reduce the l-alanine δ13C value from +55‰ in 5b to +16‰ in 11h (Table 3). These δ13C values are all well outside of the carbon isotope range (−3‰ to −54‰) that has been measured for alanine in a diverse set of terrestrial microorganisms (Scott et al. 2006); therefore, the elevated levels of l-alanine in 11h compared with 5b is highly unlikely to be due to terrestrial biological contamination.
Enantiomeric measurements were obtained by LC-FD/TOF-MS for several other α-H amino acids in Tagish Lake 5b and 11h including serine, threonine, and valine with lee values that range from approximately 19% for valine to >99% for threonine (Table 4). These amino acids were not reported in previous amino acid analyses of the Tagish Lake meteorite (Pizzarello et al. 2001; Kminek et al. 2002; Herd et al. 2011). As the total abundances of these amino acids increase in the order 11i << 5b < 11h (Tables 1 and 2), it is possible that some fraction of these amino acids and their lee were formed during parent body aqueous alteration and are indigenous to the Tagish Lake meteorite. However, due to insufficient amino acid concentrations in the samples, we could not measure their carbon isotope ratios. In particular, due to the poor TFAA/IPA derivatization efficiency and higher GC-MS/IRMS detection limit for serine compared with other amino acids in the Tagish Lake meteorite present at similar abundances, we were unable to obtain the carbon isotope value for l-serine in the Tagish Lake 11h meteorite extract. In the absence of isotopic data, a case for the l-excesses of serine, threonine, and valine being extraterrestrial in origin cannot be made. In addition, due to a possible interfering mass peak in the procedural blank at the same retention time as l-valine (Figs. 2 and 3, peak X), the reported lee for valine may have been overestimated and are given as upper limits in Table 4.
In contrast to most of the α-H protein amino acids in the Tagish Lake 5b and 11h meteorite specimens that displayed large lee ranging from approximately 19 to 99%, only very small enantiomeric excesses (lee ∼ 0–7%) were observed for the chiral nonprotein amino acids β-ABA, norvaline, and isovaline and most were racemic within analytical error (Tables 4 and 5). Although the α-methyl amino acid isovaline is highly resistant to racemization (Pollock et al. 1975; Bonner et al. 1979), β-ABA and norvaline will readily racemize on geologic time scales under aqueous conditions; therefore, it is not surprising that no enantiomeric enrichment was observed for these two nonprotein amino acids in the Tagish Lake meteorite. A slight l-isovaline excess (lee = 7.0 ± 1.9%) was measured in the Tagish 5b sample, but no l-isovaline enrichment was observed in the more aqueously altered 11h sample (lee = 0.0 ± 2.8%). These results are consistent with small l-isovaline enrichments (approximately 0–3%) that have been reported in pristine Antarctic CR carbonaceous meteorites (Pizzarello et al. 2008; Glavin and Dworkin 2009). It is possible that some radioracemization (≤5%) of isovaline from ionizing radiation produced by radioactive decay in the Tagish Lake meteorite parent body (Bonner et al. 1979) could have reduced the original l-isovaline enrichments in both Tagish Lake meteorite samples, or that a secondary pulse of amino acid formation during aqueous alteration in 11h could have overprinted any original l-isovaline excess with a racemic mixture (Herd et al. 2011). However, it is surprising that secondary aqueous alteration leading to the formation of racemic amino acids and higher amino acid abundances observed in 11h did not also reduce the l-enantiomer excesses measured for several α-H amino acids including aspartic and glutamic acids, threonine, and valine. In fact, the l-excesses measured for these amino acids were slightly higher in 11h compared with 5b (Table 4). Given that all of these α-H amino acids will racemize under aqueous conditions, another explanation is needed for the presence of these large l-excesses, particularly for l-aspartic acid shown to be indigenous to the Tagish Lake meteorite.
Enantioenrichment by Racemization during Parent Body Alteration
The α-H amino acids alanine and aspartic acid in Tagish Lake 11h both have extraterrestrial carbon isotopic values, indicating they are indigenous to the meteorite; however, alanine is racemic, while aspartic acid shows a significant l-enantiomeric excess. One possible explanation for this disparity exists in the crystallization behavior of these two amino acids. It has been shown experimentally that aspartic acid and alanine have very different crystallization behaviors in saturated solutions—alanine preferentially forms racemic crystals (Klussman et al. 2006), while aspartic acid can form metastable conglomerate crystals in addition to racemic crystals (Viedma 2001; Viedma et al. 2008). It has also been demonstrated that saturated solutions of aspartic acid with slight enantiomeric excesses can be converted to enantiopurity under solution-phase racemizing conditions such as aldehyde-catalyzed racemization (Viedma et al. 2008). A conglomerate-forming phenylglycine derivative was also converted to enantiopurity from a slight initial excess using base-catalyzed racemization, indicating that this phenomenon is not limited to a specific racemization method (Noorduin et al. 2009). While we want to make it clear that we do not have the necessary carbon isotope data to firmly establish an extraterrestrial origin for the l-excesses measured for other α-H amino acids in Tagish Lake 11h, the tendency to form conglomerate crystals has also been observed for glutamic acid (Viedma 2001; Viedma et al. 2008) and threonine (Rodrigo et al. 2004), making it possible that l-glutamic acid and l-threonine excesses could have been formed by the same mechanism proposed for aspartic acid. It has even been suggested that spontaneous resolution of conglomerates is the most likely terrestrial mechanism for the origin of homochirality on the early Earth (Bonner 1972).
The conglomerate crystal amplification process is illustrated in Fig. 6 and is based on the observation that larger crystals are more stable than smaller crystals that preferentially dissolve in solution and begin to disappear. Over time, the major enantiomer (present in excess) will accumulate more material in the solid phase than does the minor enantiomer. This is because the minor enantiomer tends to form smaller crystals that dissolve more rapidly than larger crystals. Racemization of the minor enantiomer in solution will favor production of the major enantiomer, which will eventually precipitate out and help build even larger crystals of the major enantiomer (Fig. 6A). If we assume that there was a slight initial bias toward l-aspartic and l-glutamic acids on the Tagish Lake meteorite parent body generated via any of the proposed methods of breaking symmetry in amino acids or their precursors, racemization of aspartic and glutamic acids during aqueous alteration could have resulted in a large net enrichment of l- over the d-enantiomers as is observed for these two amino acids in the meteorite. The extent to which the enantioconversion occurs would be dependent on the duration of parent body aqueous alteration and the rate of racemization for the individual amino acids, with a theoretical maximum conversion of 100%, based on laboratory experiments (Viedma 2001; Viedma et al. 2008). It is likely that the interaction of amino acids with other organic and inorganic species in the parent body would also have an effect on amplification. This is a line of research that should be explored in greater detail through laboratory crystallization experiments of free amino acids and their acid-hydrolyzable precursors under relevant parent body conditions.
Figure 6. Schematic illustrating the solid–liquid phase behavior of amino acids that form conglomerate (A) and racemic (B) solid crystals. For the conglomerates, amplification of the major enantiomer (in this case, the l-enantiomer) will occur through racemization and crystallization, assuming that there is a slight initial excess. For racemates, any initial excess (in this case, the l-enantiomer) will decrease over time through racemization.
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Although the temperature and duration of aqueous alteration on the Tagish Lake meteorite parent body remain poorly constrained, Mn-Cr isotopic analyses of carbonate in Tagish 11h indicate that this sample experienced extensive aqueous alteration in a similar setting and timescale to the CI meteorite parent body (Blinova et al. 2011). Temperatures ranging from approximately 50 to 150 °C have been estimated previously for CI meteorites (Clayton and Mayeda 1984; Leshin et al. 1997), and aqueous alteration periods of approximately 102 to 104 yr have been estimated for CM meteorites (Zolensky and McSween 1988; Browning et al. 1996), with recent models suggesting that liquid water could have been present in asteroids for up to millions of years on CI and CM meteorite parent bodies (Cohen and Coker 2000; Palguta et al. 2010). Under these conditions, racemization of aspartic acid would have been especially rapid. For example, at a temperature of approximately 25 °C under aqueous conditions at neutral pH, the racemization half-life of aspartic acid in natural samples ranges from approximately 103 to 104 yr, and at a temperature of 100 °C, the racemization half-life of free aspartic acid in aqueous solution is only 30 days (Bada 1991). However, we note that the racemization rate of free glutamic acid is four times slower than free aspartic acid under the same conditions, and would be even slower with the formation of pyroglutamic acid (Smith and Reddy 1989). It should also be acknowledged that for metastable conglomerates such as aspartic and glutamic acids, changes in conditions on the Tagish Lake parent body such as rapid changes in temperature could trigger a shift from conglomerate crystals to racemic ones, thus stopping enantioenrichment via crystallization. This may explain why the l-aspartic and l-glutamic acid excesses measured in the Tagish Lake meteorite did not reach 100%.
For compounds that preferentially form racemic crystals, such as alanine and the majority of the chiral α-H proteinogenic amino acids (Klussman et al. 2006), as well as metastable conglomerates such as aspartic and glutamic acids that have switched to racemic crystals, racemization in the solution phase works in the opposite direction, resulting in an increase in the amount of racemic solid phase and an overall reduction in enantiomeric excess (Fig. 6B). This is because an enantiomeric excess in solution will drive racemization toward a racemic solution phase, causing more racemic crystals (with lower solubility) to precipitate. As enantiopure crystals are more soluble and will dissolve more rapidly than racemic crystals, preferential dissolution of the major enantiomer in the solid phase will drive racemization to the minor enantiomer, resulting in a net conversion of the major to the minor enantiomer and formation of racemic solid crystals. This hypothesis is consistent with racemic alanine measured in the Tagish Lake meteorite and the large lee values measured for aspartic acid, provided that there was a slight initial l-bias of aspartic acid. We also note that similarly high l-aspartic acid and l-glutamic acid excesses (lee ∼ 32–61%) were measured in the aqueously altered type 1 CI meteorite Orgueil, while alanine was also found to be racemic (Ehrenfreund et al. 2001). Although isotopic measurements were not made for these amino acids in Orgueil, the conglomerate crystallization behavior of aspartic and glutamic acids could explain the observed l-aspartic and l-glutamic acid excesses provided that there was a slight initial l-excess.
It has been suggested previously that some amino acids formed by Strecker synthesis in meteorites could have inherited their asymmetry directly from asymmetric photolysis of aldehyde or ketone precursors that were exposed to UV circularly polarized radiation in the solar nebula prior to their incorporation inside the meteorite parent body (Pizzarello et al. 2008). For example, slight (approximately 1%) enantiomeric excesses in alanine have been produced directly from laboratory interstellar ice analogs exposed to circularly polarized UV light (De Marcellus et al. 2011). However, aspartic acid would probably not have obtained its initial asymmetry via this mechanism as its most plausible aldehyde precursor is 3-oxopropanoic acid, which is achiral. Aspartic acid could also have formed from Michael addition of ammonia to fumaric or maleic acid, although both of these precursor molecules are also achiral. Finally, the greater abundance of aspartic acid in the more aqueously altered 11h sample (approximately 600 ppb) compared with 5b (approximately 30 ppb) suggests that most of the aspartic acid was formed inside the parent body during aqueous alteration, thus shielded from any UV CPL.