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. 2008 Feb;3(1-2):2-25.
doi: 10.1016/j.quageo.2007.07.001.

Closed-system behaviour of the intra-crystalline fraction of amino acids in mollusc shells

K E H Penkman  1 , D S Kaufman, D Maddy, M J Collins
Affiliations

Closed-system behaviour of the intra-crystalline fraction of amino acids in mollusc shells

K E H Penkman et al. Quat Geochronol. 2008 Feb.

Abstract

When mollusc shells are analysed conventionally for amino acid geochronology, the entire population of amino acids is included, both inter- and intra-crystalline. This study investigates the utility of removing the amino acids that are most susceptible to environmental effects by isolating the fraction of amino acids encapsulated within mineral crystals of mollusc shells (intra-crystalline fraction). Bleaching, heating and leaching (diffusive loss) experiments were undertaken on modern and fossil Corbicula fluminalis, Margaritifera falcata, Bithynia tentaculata and Valvata piscinalis shells. Exposure of powdered mollusc shells to concentrated NaOCl for 48 h effectively reduced the amino acid content of the four taxa to a residual level, assumed to represent the intra-crystalline fraction. When heated in water at 140 degrees C for 24 h, only 1% of amino acids were leached from the intra-crystalline fraction of modern shells compared with 40% from whole shell. Free amino acids were more effectively retained in the intra-crystalline fraction, comprising 55% (compared with 18%) of the whole shell after 24 h at 140 degrees C. For fossil gastropods, the inter-shell variability in D/L values for the intra-crystalline fraction of a single-age population was reduced by 50% compared with conventionally analysed shells. In contrast, analysis of the intra-crystalline fraction of C. fluminalis does not appear to improve the results for this taxon, possibly due to variability in shell ultrastructure. Nonetheless, the intra-crystalline fraction in gastropods approximates a closed system of amino acids and appears to provide a superior subset of amino acids for geochronological applications.

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Figures

Fig. 1
Fig. 1
Change in THAA concentration (6 h hydrolysis) of Asx with oxidation time in H2O2 for Corbicula (left) and Margaritifera (right).
Fig. 2
Fig. 2
Decline in the amino acid concentration with oxidation time using NaOCl. Upper: THAA concentration of Asx (6 h hydrolysis) for Corbicula (left) and Margaritifera (right). Other amino acids show similar trends (Fig. 4). The particle size of the powder does not appear to affect the extent of oxidation. Lower: THAA (24 h hydrolysis; left) and FAA (right) concentrations of Asx, Glx, and Ala for Bithynia.
Fig. 3
Fig. 3
Absolute abundance of 11 amino acids before and after oxidation with H2O2 and NaOCl for Corbicula (left) and Margaritifera (right). The concentration of each amino acid decreases on oxidation.
Fig. 4
Fig. 4
Amino acid composition in Corbicula, Margaritifera and Bithynia shells, unbleached (left) and after oxidation with NaOCl for 48 h (right). The colours for each amino acid follow the convention of RasMol. The concentration of the silk-like protein decreases relative to that of the acidic amino acids in bleached Margaritifera and Bithynia.
Fig. 5
Fig. 5
Change in Asx D/L in the THAA with oxidation time using NaOCl. Upper: Asx D/L for Corbicula (left) and Margaritifera (right). Lower: D/L in Asx, Glx and Ala for Bithynia.
Fig. 6
Fig. 6
Conceptual model of the effect of bleach on different amino acid fractions in a shell (upper figure modified from Sykes et al., 1995). The highly racemized FAA in the matrix (1) are removed first, leading to a small drop in concentration but a decrease in the D/L value. A rise in D/L coincides with a rapid drop in concentration as the intact proteins of the matrix (2) are removed. Prolonged bleaching may selectively remove amino acids in the intra-crystalline fraction or begins to etch the carbonate, thereby exposing intra-crystalline amino acids (3). This coincides with only a small drop in concentration. At this point the bleaching could induce further racemization, or it could oxidize the less-racemized molecules. The increase in racemization is minimal, however.
Fig. 7
Fig. 7
Unbleached, pre-heated bleached, and post-heated bleached concentration of Asx in the THAA under hydrous conditions for Corbicula (left) and Margaritifera (right) heated at 140 °C.
Fig. 8
Fig. 8
Concentration of Asx (left) and Ala (right) for FAA and THAA (6 h hydrolysis) for the bleached Corbicula powder during heating at 140 °C. The increase in abundance of THAA (except Asx) in the early time steps suggests incomplete preparatory hydrolysis.
Fig. 9
Fig. 9
Relative composition of Asx in each of the four fractions of Bithynia shell during heating at 140 °C: intra-crystalline free, intra-crystalline bound, inter-crystalline free, and inter-crystalline bound.
Fig. 10
Fig. 10
Percentage of THAA in supernatant water (uncorrected for procedural blank) compared to that within the shells, for unbleached and bleached Corbicula (left) and Bithynia (right) shell at 140 °C. Unbleached shells (u=closed symbols) leach a higher percentage of amino acids into the water compared to bleached shells (b=open symbols).
Fig. 11
Fig. 11
Racemization of bleached Bithynia tentaculata shell at 140 °C in the FAA (left) and THAA (right). The upper graphs show the measured D/L values; the lower graphs show the data plotted as ln{(1+D/L)/(1−D/L)} vs. time. If the reaction follows reversible first-order kinetics, the relation should be linear.
Fig. 12
Fig. 12
FAA D/L vs. THAA D/L for unbleached (u) and bleached (b) Asx and Glx (left) and Ala and Val (right) for Margaritifera (upper) and Bithynia (lower) shell heated at 140 °C under hydrous conditions.
Fig. 13
Fig. 13
Concentration vs. THAA D/L for Asx for bleached and unbleached fossil shells. Upper: [THAA] for Bithynia. Middle: [THAA] and [FAA] for unbleached (left) and bleached (right) Valvata. Lower: [THAA] and [FAA] for unbleached (left) and bleached (right) Corbicula. Insets show an expanded scale.
Fig. 13
Fig. 13
Concentration vs. THAA D/L for Asx for bleached and unbleached fossil shells. Upper: [THAA] for Bithynia. Middle: [THAA] and [FAA] for unbleached (left) and bleached (right) Valvata. Lower: [THAA] and [FAA] for unbleached (left) and bleached (right) Corbicula. Insets show an expanded scale.
Fig. 14
Fig. 14
Concentrations for unbleached (u) and bleached (b) FAA and THAA fractions for Valvata piscinalis and Bithynia tentaculata from Funthams Lane, and Bithynia tentaculata from Purfleet. Error bars are one standard deviation about the mean. Little change is observed upon bleaching for the FAA.
Fig. 15
Fig. 15
Average amino acid compositions for intra- and inter- free (F) and bound (B) amino acids within modern, MIS 7, and MIS 9 Bithynia tentaculata.
Fig. 16
Fig. 16
THAA concentration vs. D/L for Asx, Glx, Ala, and Val for bleached and unbleached Bithynia tentaculata from Funthams Lane.
Fig. 17
Fig. 17
Frequency distribution of THAA D/L for Ala and Val for unbleached (■) and bleached (□) Valvata piscinalis from Funthams Lane.
Fig. 18
Fig. 18
THAA vs. FAA D/L Asx for bleached Bithynia shells from Funthams Lane (MIS 7) and Purfleet (MIS 9). Each of the measurements is from a single shell. Note the much greater consistency in the measurements obtained from bleached shells.
Fig. 19
Fig. 19
Coefficient of variation (CV) of the concentrations and D/L values of THAA in unbleached and bleached fossil samples. Upper: 15 individual unbleached (■) and bleached (□) Valvata piscinalis shell powders from Funthams Lane. Lower: CVs of bleached samples as a percentage of the CVs for unbleached samples, for the THAA concentration and D/L values from Funthams Lane (FL) Valvata piscinalis, Bithynia tentaculata and from Purfleet (P) Bithynia tentaculata. Inter-shell variability is reduced in all measures of the amino acid composition, except for D/L Ala in shells from Purfleet, where the CVs are low.
Fig. 20
Fig. 20
THAA D/L for Inter-laboratory comparison samples C (ILC-C) and Z (ILC-Z), unbleached and bleached.
Fig. 21
Fig. 21
THAA D/L vs. FAA D/L for Ala for bleached (●) and unbleached (○) Bithynia (left) and Valvata (right). The trendlines are derived from kinetic experiments on the intra-crystalline fraction of the two species.
Fig. 22
Fig. 22
FAA to THAA D/L correlation for each amino acid for bleached and unbleached Valvata (left) and Corbicula (right). There is no improvement upon bleaching for Corbicula, unlike that observed for the fossil gastropods.
Fig. 23
Fig. 23
THAA D/L for Glx vs. Asx for Funthams Lane Bithynia tentaculata (left) and Valvata piscinalis (right). The results on bleached shells form a more coherent cluster, except for one outlier, which contains unexpectedly high amino acid concentration and is probably incompletely bleached.
Fig. 24
Fig. 24
Schematic of intra-crystalline amino acids entrapped within carbonate crystals. Unlike the proteins of the organic matrix between the crystallites, which leach from the shell with time, amino acids are entrapped in the closed intra-crystalline system. Thus the relation between the D/L values of different amino acids and between free (non-protein bound) and total hydrolysable (both free and originally protein-bound amino acids, released by acid hydrolysis) amino acids is predictable. Analysis of the whole shell results in lower-than-expected D/L values for the THAA, due to the loss of the more highly racemized FAA.
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