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

LIARDON AND LEDERMAN, 1986, LIARDON AND FRIEDMAN, 1987; SMITH AND REDDY, 1989) that amino acids bound in peptide racemize faster than free amino acids. Several reports appeared in the literature dealing with the use of microwave technology in protein hydrolysis. Some authors reported excellent results (CSAPÓ ET AL., 1994) using high temperatures and short times for the hydrolysis process. It appears that, during microwave promoted hydrolysis, significant racemization occurs, because microwaves have been purposely used to trigger racemization of amino acids. Reports have been published describing the increase of D-enantiomers in foods under the influence of microwave treatment. Racemization is no cause for concern if one does not wish to determine the enantiomers of amino acids. However, if our aim is the separation and determination of amino acid enantiomers, the protein hydrolysis procedure selected should be such that the accompanying racemization is as small as possible. This is necessary since, in the case of significant racemization, we are unable to distinguish between the amino acid enantiomers initially present in the sample and those that appear during the hydrolysis process. Several methods have been developed which restrict racemization occurring during hydrolysis. However, these proved to be lengthy and tedious. As a consequence, the objective was to develop a protein hydrolysis method having the lowest possible degree of racemization, by using high temperatures for a short time duration. 1.2. Experimental 1.2.1. Hydrolysis and processing of the hydrolysate Pyrex reusable hydrolysis tubes having 8 mm I.D. (Pierce Chemical Company, Rockford, IL, USA) were used for the hydrolysis of proteins or for treating free amino acids. Each tube can contain up to 8 cm-* of hydrolysing agent without making contact with the PTFE (polytetrafluoroethylene) sealing cup. One ml of 6M hydrochloric acid (HCl) was added to each tube for preparation of protein and peptide hydrolysate. Each tube had two PTFE sealing caps to get complete leak-free operation during heating at 160, 170 or 180 °C. Either 1 mg peptide, protein, or free amino acids or 20-50 mg bone samples were weighed into Pyrex tubes previously washed with hydrochloric acid and deionised water. 1-10 ml 6M HCl was added to each sample (HCl was obtained from Pierce Chemical Company, Rockford, IL, USA) and nitrogen was bubbled for five minutes through the hydrolysing agent by glass capillary. After bubbling with nitrogen, the Pyrex tubes were immediately closed, and put into the heating oven at 160, 170 and 180 °C for 15, 30, 45 or 60 minutes respectively. One sample of each examined material was hydrolysed at 110 °C for 24 h, according to the method of MORE AND STEIN (1963), with 6M HCl. Another sample was hydrolysed at 110 °C for 48 h using 4M barium-hydroxide for determination of tryptophan. After hydrolysis, the tubes were cooled at room temperature and HCl was evaporated by lyophylysation and the residue of the sample was dissolved in 0.01 M HCl. After the barium-hydroxide hydrolysis, the pH of the hydrolysate was set to neutral with IM HCl, and the barium was removed from the hydrolysate in the form of barium-sulphate. During neutralisation, the temperature was held below 30 °C with the help of a sodium chloride - ice mixture. Next all hydrolysates were filtered and stored at 25 °C until the analysis of D- and L-amino acid enantiomers by HPLC. 1.2.2. Materials tested The following materials were used for testing the racemization during hydrolysis: Bovine ribonuclease, lysozyme, citochrom C, fossil bone sample, and individual free amino 23