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

Table 6 Racemization of free amino acids treated by protein hydrolysis with different temperatures for different times Amino acids 6 M HCl 110°C for24h 6 M HCl Amino acids 6 M HCl 110°C for24h 160°C for 170°C for 180°C for Amino acids 6 M HCl 110°C for24h 45min 60min 30min 45min 15min 30min Asp 1.42 0.79 0.93 0.73 1.04 0.94 1.12 Glu 1.07 0.54 0.82 0.68 0.89 0.91 1.03 Thr 0.83 0.31 0.38 0.32 0.41 0.42 0.61 Ala 0.69 0.22 0.29 0.27 0.35 0.37 0.52 Val 0.54 0.19 0.25 0.17 0.24 0.37 0.57 Phe 0.72 0.21 0.27 0.20 0.31 0.32 0.47 His 0.47 0.11 0.18 0.10 0.19 0.21 0.33 Data expressed as in Table 1. 1.3.3. Racemization of free amino acids during hydrolysis Racemization of free amino acids has been reported to be lower than that of amino acids bound in peptide. In order to test the hypothesis that there is difference between racemization of free amino acids and those bound in peptide, we have treated each free amino acid with 6M HCl for various times, and at various temperatures. The results of this investigation are shown in Table 6. When samples were treated for the same length of time, racemization increased with increased treatment temperature. Also, racemization increased with increased treatment times. The high temperature treatments yielded 2055% less racemization than was observed for traditional treatment of 110 °C for 24 h. Even in the case of the sample treated at 180 °C for 30 minutes, the racemization, except of valine was only 70-90% ofthat seen for traditional treatment. If we compare the racemization of free (Table 6) and peptide bound (Tables 1, 2, 3, 4, 5) amino acids, we find that the percentage racemization of peptide bound amino acids is 4 to 6 times as great as that of free amino acids at 110 °C for 24 h. When both free amino acids and peptide bound amino acids were subjected to the same high temperature, short time hydrolysis, the above ratio ranged from 5 to 7. 1.3.4. Racemization when using barium hydroxide promoted hydrolysis It was observed earlier in this report that, during hydrolysis with 6M HCl, tryptophan decomposed almost completely. If we wish to determine the degree of racemization for tryptophan, we must resort to a hydrolysis method which does not cause decomposition of Trp. Since we have been using the barium-hydroxide hydrolysis for the determination of Trp content of proteins, we decided to examine racemization associated with this process. We hydrolysed pure proteins (ribonuclease, lysozyme, cytochrome C), fossile bone and free amino acids with 4M barium hydroxide. We found that, in all materials examined (including free amino acids), all amino acids were fully racemized. In other words we