What Time was it Ask the Amino Acid Clock

The racemization of biological L-amino acids towards D-amino acids really starts when counteractive processes, e.g. based on D-amino acid oxidases, become inactive after the death of a living organism which over time results in complete racemization. The ongoing increase of the D-amino acid quantity can thus be applied for archaeological and geochemical dating by a method called "the amino acid clock". This method has thermodynamic roots because the racemic mixture of chiral molecules is thermodynamically favoured over its enantioenriched or homochiral form, due to the mixture's increased entropy. Therefore, enantiopure amino acids approach spontaneously the state of a racemic mixture. The questions are: what does "spontaneously" mean in this context and how long does this racemization reaction take? At 0° C, a-alanine in aqueous solution racemizes in about one hundred thousand years. Aspartic acid racemizes faster, isoleucine more slowly. At room temperature, some thousand years are sufficient for a-alanine racemization (Rein 1992). If amino acids are stored in solid state without any humidity, the half-life time can be increased to some million years. The racemization of an amino acid in a peptide chain, however, is more complicated. As illustrated in Fig. 3.1, it is not important whether we start the racemization reaction from the enantiopure L-enantiomer or from the enantiopure D-enantiomer. The racemic mixture is favoured in both cases.

Fig. 3.1 Thermodynamically favoured racemization of chiral molecules. The "amino acid clock" is based on the racemization of l-amino acids accompanied by a gain of entropy towards a racemic d ,l-mixture as depicted on the left side of this figure

When living organisms such as plants or animals die, the homochiral proteina-ceous L-amino acids immediately start to racemize by changing their handedness at a very slow but rather uniform rate. The controlled recording of the racemization process hence provides a way of dating ancient objects by determining the enantiomeric excess of one or more amino acids. This way of dating is called the "amino acid clock". It is complementary to the 14C-method of geochemical dating, since older objects can be dated. The half-life of 14C is limited to only about 6 000 years making dating possible for approx. up to ten half-life times, i.e., up to 50 000 years.

Bone samples are important constituents of many archaeological sites and often the only biogenic material available. Hartwig Elster and Steve Weiner have investigated the amino acid racemization of fossil bones in the team of Emanuel Gil-Av at the Weizmann Institute of Science in Rehovot. Enantioselective analyses of aspartic acid in different fractions of 51 fossil bones of various ages showed that the collagen-rich fractions do manifest increasing D-aspartic acid contents with increasing age. No such correlation existed for the fractions rich in non-collagenous proteins (Elster et al. 1991). The accurate application of "the amino acid clock" is therefore limited to archaeological dating of collagen-rich samples of bones.

Amino acid racemization was also used in the case of human specimens of known age for the determination of their temperature history. The German emperor Lothar I, who died on 5 December 1137 in Bavaria, was buried 500 km north of the place of his death at his castle in Königslutter (20 km west of Braunschweig). Using 20-30 km per day as the estimated transportation speed for his corpse, Bada et al. (1989) calculated that an interval of several weeks is likely to have occurred between the time of his death in Bavaria and his subsequent burial at the castle. This long period would have obviously presented problems of post-mortem decay. Bada et al. (1989) have thus carried out aspartic acid racemization analyses of Lothar, and two of his relatives, his wife Richenza and brother-in-law Duke Heinrich der Stolze, both of whom died at Konigslutter within a few years of Lothar's death and were buried in the castle along-side him. Enantioselective aspartic acid analyses of the obtained bones from the three individuals revealed that the corpse of Lothar I contained significantly higher amounts of D-aspartic acid than expected from the above calculations and was thus interpreted to be boiled in water for about 6 h before burial. Boiling was used to deflesh Lothar's corpse to prevent post-mortem decay during transit to his castle.

Despite its obviously wide range of applications, the "amino acid clock" has a considerable margin of error, because the rate of racemization of an amino acid is mostly not a simple linear function of time but also influenced by various parameters such as temperature, pH value, humidity, and position in a peptide linkage. Limitations of "the amino acid clock" became visible when Kunnas and Jauhiainen (1993) from the Joensuu University in Finland tried to estimate the age of a peat bog from the enantioselective determination of the D/L-ratios of free amino acids. Too many environmental factors influenced the D/L-ratios in natural conditions and the age of the selected peat samples was too young only by 9 000 years to allow the obtaining of reliable results on pure racemization without an additional source of interfering D-enantiomers, for example from the destruction of plant and microbial material.

Racemization reactions are not limited to amino acids. Sugar molecules such as glucose and ribose are also chiral and entropically favour their racemate. Since these molecules possess more than one stereogenic carbon atom, reactions that form a racemate are more complex. Here, the interconversion at one stereogenic center is called an epimerization instead of racemization since the molecule possesses more than one stereogenic center. Nevertheless, the epimerization of D-ribose can be accomplished within about one hundred years (Rein 1992) and is therewith too fast to be of interest for dating specific samples or other scientific applications in the context of archaeological research.

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