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

In earlier studies (CSAPÓ ET AL., 1994), it was reported that, protein hydrolyses performed at 160 °C for 15-45 minutes were insufficient for complete hydrolyses of proteins, and especially for breakage of the bond adjacent to Val, He and Leu. Therefore, for hydrolysis made at 160 °C, only the 60 minute times have practical importance. If we compare the racemization obtained after 60 minutes hydrolysis with results of the traditional method, it is found that the racemization degree for the traditional method, on the average, is 1.5 times as high as that of brief hydrolysis performed at 160 °C. Table 1 D-amino acid content of bovine ribonuclease hydrolysed by 6 M hydrochloric acid at 160 °C for different times Amino acid 6 M HCl, 110 °c for 24 h 6 M HCl, 160 °C for Amino acid 6 M HCl, 110 °c for 24 h 15 min 30 min 45 min 60 min Asp 6.73 1.73 2.78 3.11 3.34 GIu 4.58 1.58 2.59 2.61 2.84 Thr 3.64 1.47 1.70 1.97 2.12 Ala 2.95 1.41 1.58 1.60 1.73 Val 2.34 1.22 1.29 1.51 1.54 Phe 3.28 2.13 2.47 2.93 3.21 His 1.96 0.92 1.41 1.52 1.64 Trp* The values refer to the percent of racemization expressed as the ratio [D/(D+L)]xl00. Each values is the mean of triplicate determinations. Hydrolysis conditions: 6 M HCL 110 °C for 24 h and 160 °C for different times using Pyrex No. 9826 tubes. * Almost totally decomposed during 6M HCl hydrolysis at 160 °C for 15-90 min. We reach similar conclusions when analysing the data featured in Tables 2 and 3. Performing the hydrolysis at 170 °C, the hydrolysis reaction practically concludes after 45 minutes and after 60 minutes, even the very stubborn bonds adjacent to Val are broken. At 180 °C, 30-45 minutes are sufficient for complete hydrolysis. Therefore, when comparing results obtained during traditional hydrolysis, it is advisable to make comparisons with data obtained at 170 °C for 45 minutes and 180 °C for 30 minutes. Hydrolysis made at 160 °C for 60-90 minutes yields racemization similar to hydrolysis performed at 170 °C for 45 minutes. Hydrolysis performed at 180 °C for 45 minutes yields a racemization ca. 1.5 times as high as that of hydrolysis carried out at a lower temperature which results in total breakage of bonds. Obviously, both increasing temperatures (from 160 to 180 °C) and increasing time (from 15 to 60 minutes), produced a higher degree of racemization. However, at all three temperatures, continuation of hydrolysis until total hydrolysis of the peptide bond (e.g. at 180 °C for 30 minutes), produced a degree of racemization which represented only ca. 50-70% ofthat observed in the case of traditional hydrolysis. 26