Achaeometrical Research in Hungary II., 1988

PROSPECTING and DATING - János CSAPÓ - Zsuzsanna CSAPÓ-KISS - János CSAPÓ JR.: How the amino acids and amino acid racemization can be used and with what limits for age determination of fossil materials in archaeometry

were able to demonstrate the presence of 50%» L- and 50% D-enantiomers. Because of this, barium hydroxide based hydrolysis may not be used to measure racemization of the tryptophan contained in proteins. 1.4. Conclusions and recommendations Racemization of free amino acids was considerably lower than that of amino acids bound in peptide. In the same experimental conditions, the degree of racemization of free amino acids was only 20 to 80% that of peptide bound amino acids. Traditional protein hydrolysis produced racemization is 1.5 times as high as that obtained at high temperatures (160-180 °C), under conditions ensuring total hydrolysis of the protein. This lower degree of racemization may be explained by the fact that, at high temperatures, the protein hydrolyses more rapidly into free amino acids. Racemization of free amino acids is considerably less that of amino acids bound in polypeptides. Therefore, high temperature hydrolysis promotes conversion to the free state in which amino acids are less subject to racemization. When hydrolysis is conducted at lower temperatures for longer times, the amino acids bound in the peptide chain are exposed for a longer time to the effects actually causing racemization. As a result, we may say that factors which speed up hydrolysis, will lower the degree of racemization. In the case of bone samples, racemization was higher than in the case of pure proteins. This may be explained by catalysis of racemization associated with the heavy metals present. After 48 hours at 110 °C and in presence of 4M barium hydroxide, all amino acids (whether free or bound in peptide) totally racemized. Therefore the racemization of tryptophan cannot be determined using barium hydroxide promoted protein hydrolysis. We recommend that protein samples be hydrolysed at high temperature for a short time (160 °C for 60 minutes and 170 °C for 45 minutes) for all those who would like to determine the degree of racemization occurring in the polypeptide chain, but do not wish to use enzyme hydrolysis. 2. Age determination based on amino acid racemization: a new possibility 2.1. Introduction Amino acid contents in fossil shell, bone and tooth samples from early ages were reported first by ABELSON in 1954. In 1967, HARE AND ABELSON reported that Damino acids in fossils resulted from conversion of L- amino acids of protein. It was found that the older the fossil the higher the D/L ratio and, after a certain age, amino acids occurred in racemic form. The ratio of D-allo isoleucine and L-isoleucine content in a fossilised shell sample was found to be 0.32 and the fossil was estimated to be 70,000 years old, as reported by HARE AND MITTERER (1968). It is considered the first application of amino acid racemization (AAR) - or rather epimerization - in geochronology. Subsequently, racemization of amino acids was used for age determination of various materials containing protein. Isoleucine and aspartic acid were given special attention because L-isoleucine can be easily separated from D-allo isoleucine by an amino acid analyser and aspartic acid, being the most acidic of amino acids, is the first to come off of the ion exchange column. However, some errors of age determination based on AAR were reported by WILLIAMS AND SMITH in 1977. Temperature, pH, soil composition and various contaminants should also be considered when estimating the age of fossil bone samples. Recently MARSHALL (1990) established that the bones are not reliable 50