I. General A.Time range
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1 I. General A.Time range Depends on: temperature, amino acid, taxon For isoleucine in the Arctic = 3-5 Ma For aspartic acid in tropics = few 1000 yr with annual resolution Resolution is higher during early stage of the equilibrium reaction
2 I. General B. Materials Molluscs most commonly used Also other carbonates: braciopods, eggshells, oolites, whole rock beach sand, otoliths, ostracodes, speleothems, corals, foraminifera Has also been tried on wood, soils, meteorites
3 II. Principles A. Amino acids All living organisms contain amino acids (~20 common in protein) Molecule contains an amino group (NH 2 ) attached to central C Amino acids link together by peptide bonds to form proteins
4 Decalcified eggshell
5 II. Principles A. Amino acids (cont.) Amino acids are asymmetric or chiral D and L are isomers (same compound; different configuration) Exist in two different configurations which are mirror images = enantiomers Amino acids are optically active (= rotate polarized light) Levorotary = rotate light to left (L) Dextrorotary = rotate to right (D)
6 II. Principles B. Racemization All amino acids in living organisms are Levo (except in some bacteria) After death, L is converted to D by reaction called racemization Extent of reaction is defined by D/L ratio (ranges from 0 to 1.0) Rate of reaction depends on amino acid Aspartic acid is among fastest; isoleucine is relatively slow
7 II. Principles C. Epimerization Several amino acids contain two chiral C atoms (isoleucine) Racemization about one atom produces a new, nonstereo image isomer = diastereomer D-alloisoleucine is formed by epimerization of L- isoleucine A/I ratio equilibrium = ~1.3
8 II. Principles D. Temperature Rate of racemization is highly dependent on temperature Rate approximately doubles for each 10 C increase
9 III. Age determinations A. Relative ages: aminostratigraphy D/L ratios are used as a relativeage index Used for correlation between sites Cluster of ratios (aminogroups) imply intervals of deposition Also used for general age approximation A/I Number of samples A (1) C (5a) E (5e II)? (5e I) F (7) G/H (9/11) A/I Latitude ( N) From Hearty and Kaufman, 2000 I (>13) correlated marine oxygen-isotope stage 9/11 7 5eI 5eII 5a 1/mid Holocene modern
10 III. Age determinations A. Relative ages: aminostratigraphy (cont.) 0 Extent of racemization (D/L) for aspartic acid (Asp) and glutamic acid (Glu) in foraminifer from two marine cores off NE Australia. The cores are located 80 km apart Depth (cm) Glu Asp D/L
11 V. Procedures A. Sampling Use multiple shells per horizon (>5 shells per "sample") Shell size is typically a 10 s of mg (although smaller will work) Typically 5-10% variability among shells from single stratigraphic unit Sample from at least 1 m below ground surface Samples sensitive to contamination; don t handle
12 V. Procedures B. Laboratory Shells are subsampled in same position, or entire shell is used Protein composition and therefore rates of racemization vary within a shell Samples are cleaned, dissolved, hydrolyzed in laboratory
13 V. Procedures C. Analytical Amino acids are separated on a highperformance liquid chromatograph (HPLC) using either ion exchange (IE) or reverse phase (RP) procedures
14 V. Procedures C. Analytical (cont.) IE is used most commonly Resolves individual amino acids, but not enantiomers (D from L), only diasteromers (isoleucine from alloisoleucine) GC separates D/L isomers for multiple amino acids Analytical procedure is more complex; difficult to automate Requires larger sample sizes
15 Limnocythere ceriotuberosa ~90 ka 0 Fluorescence (%) D-Ser D-Val D-Phe D-aIle D-Leu D-Glu L-Thr L-Phe L-Ile D-Ala L-Val L-Leu D-Asp L-Ser L-Ala L-hArg L-Asp L-Glu Gly + D-Thr pmol Time (min) Fluorescence (%) D-Ser L-Thr D-Val L-Phe L-Ile D-Phe D-Glu L-Ser L-Val L-Leu D-aIle D-Leu D-Ala D-Asp Gly + D-Thr L-Ala L-Asp L-Glu L-hArg V. Procedures C. Analytical (cont.) RP combines advantages of HPLC and GC Separates D/L ratios for multiple samples; simple; small samples A. B. 620 ka 40 pmol
16 V. Procedures C. Analytical (cont.) Sample solutions are typically analyzed more than once Values for interlaboratory standards are reported with results Analytical precision based on repeat measurements of same solution is usually 1-5%
17 VI. Challenges Protein diagenesis is infinitely complex Reaction rates are highly temperature sensitive Racemization rate depends on taxon Need to use multiple genera for splicing together a stratigraphy Analytical procedure is tricky; can lead to laboratory biases No convention for reporting uncertainties in age estimates
18 VII. Opportunities A. Reduced sample sizes Integrate amino acid geochronology into studies of lake and marine cores 1 mm Limnocythere cerituberosa
19 VII. Opportunities B. Improved screening criteria Aberrant samples can often be recognized by amino acid signatures Modern contamination exhibits abundant unstable amino acids Excluded Glu D/L Asp D/L Asp D/L
20 VII. Opportunities C. Intra-crystal fraction Extract and analyze the best-protected fraction of protein Preliminary results indicate that intra-crystal fraction behaves as a closed system
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