Download D-alloisoleucine

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
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
II. Principles
A. Amino acids
•
All living organisms contain amino acids (~20 common in protein)
•
Molecule contains an amino group (NH2) attached to central C
•
Amino acids link together by peptide bonds to form proteins
Decalcified eggshell
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)
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
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 Lisoleucine
• A/I ratio equilibrium = ~1.3
II. Principles
D. Temperature
•
Rate of racemization is highly dependent on temperature
•
Rate approximately doubles for each 10°C increase
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
E
(5e II)
20
A
(1)
15
C
(5a)
?
F
(5e I) (7)
I
(>13)
G/H
(9/11)
10
5
0
0.0
0.1
0.2
0.3
0.4
0.5
A/I
0.8
0.6
0.7
0.8
1.0
9/11
0.6
7
5eI
0.5
5eII
0.4
5a
0.3
1/mid Holocene
modern
0.2
0.1
0.0
20
0.9
correlated marine
oxygen-isotope stage
0.7
A/I
•
Number of samples
25
21
22
23
24
25
26
27
Latitude (°N)
From Hearty and Kaufman, 2000
III. Age determinations
A. Relative ages: aminostratigraphy (cont.)
0
Asp
100
Depth (cm)
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
Glu
200
300
400
0.0
0.1
0.2
D/L
0.3
0.4
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
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
V. Procedures
C. Analytical
•
Amino acids are
separated on a highperformance liquid
chromatograph (HPLC)
using either ion
exchange (IE) or reverse
phase (RP) procedures
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
Limnocythere ceriotuberosa
~90 ka
Gly +
D-Thr
L-Ala
L-hArg
L-Ser
0
60
B.
D-aIle
D-Leu
D-Phe
L-Phe
L-Ile
L-Val
D-Val
D-Ser
10
L-Thr
D-Glu
20
L-Leu
40 pmol
D-Ala
30
D-Asp
40
620 ka
50
L-hArg
L-Val
D-Val
L-Phe
L-Ile
D-Phe
D-Ser
L-Leu
D-aIle
D-Leu
D-Ala
L-Ala
Gly +
D-Thr
L-Glu
10
40 pmol
L-Thr
20
D-Glu
L-Ser
30
L-Asp
40
D-Asp
•
RP combines
advantages of HPLC
and GC
Separates D/L ratios
for multiple samples;
simple; small samples
Fluorescence (%)
•
50
Fluorescence (%)
C. Analytical (cont.)
L-Asp
V. Procedures
A.
L-Glu
60
0
20
30
40
50
Time (min)
60
70
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%
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
VII. Opportunities
A. Reduced sample sizes
• Integrate amino acid geochronology into studies of lake and
marine cores
1 mm
Limnocythere cerituberosa
VII. Opportunities
B. Improved screening criteria
•
Aberrant samples can
often be recognized by
amino acid signatures
Modern contamination
exhibits abundant
unstable amino acids
•
0.8
0.7
0.6
0.5
0.12
Glu D/L
0.4
Excluded
0.10
0.08
0.3
0.06
0.2
0.04
0.1
0.02
0.00
0.00
0.05
0.10
0.15
0.20
Asp D/L
0.25
0.30
0.35
0.0
0.00
0.20
0.40
Asp D/L
0.60
0.80
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