M. Járó - L. Költő szerk.: Archaeometrical research in Hungary (Budapest, 1988)
Dating - CSAPÓ János, KÖLTŐ László , PAP Ildikó: Archaeological age determination based on the racemization and epimerization of amino acids
copper acetate they have been able to separate all the D- and L-enantiomers of all protein amino acids. In our opinion the method is very nicely applicable for the quantitative determination of amino acids. It is sensitive, quick and with further improvement it can become a routine method comparable to the amino acid analysis. Marfey (1984) has also invented a method for the separation of enantiomers which is also based on high pressure liquid chromatography. With the aid of l-fluoro2,4-dinitrophenyl-5-L-alanine-amide, which contains a very reactionable fluorine atom, he created diastereomer derivatives from the mixture of D- and L-amino acids. He succeeded in separating these derivatives with high pressure liquid chromatography by the application of triemylamino-phosphate and acetonitrile eluenses' gradient. This included their quantitative determination as well. In his publication he describes the separation of the mixture of D- and L-aspartic acids, glutamic acid, me mionine, alanine and phenylalanine but by altering the conditions in an appropriate manner the possibility of the separation of other amino acid enantiomers also exists. In order to check the optical cleanliness of biologically active materials Knabe (1984), Gübitz and Mihellyes (1984), Giibitz et al. (1982) have elaborated a direct method with high pressure liquid chromatography. It is based on the chiralic column, made up of chemically bonded L-hydroxylproline-Cu 2+ . The moving phase is a water solution containing Cu 2+ . With the application of the above stationary solution it is possible to check the optical cleanliness of all those chemical compounds which form a chelate complex with the Cu 2+ ions, such as the amino acids. The disadvantage of the method in relation to the aforementioned is that only one D and L form of a single amino acid can be determined in one measurement. Due to its very frequent use in geochronology, the analysis of D-alloisoleucine needs to be mentioned separately. Apart from hydroxyproline, isoleucine also contains two centres of asymmetry. The D-alloisoleucine formed with time from isoleucine, during the routine application of ion-exchange amino acid separation, appears on the chromatogram between the isoleucine and the methionine, by giving a separate, easily évaluable peak. The racemization of the carbon atom with an cvposition, and the formation of the D-alloisoleucine during the peptid-synthesis were examined in detail by Bodanszky and ConkJin (1967). Among other asp acts they considered the effect of hydrochloric hydrolysis and of the different tertiary amines on racemization. As acidic hydrolysis is the most important step in the separation of amino acids bound in protein, several researchers have examined the changes occurring during hydrolysis; in other words, what is the extent of acid-catalysed racemization? WÜtshire (1953) refluxed L-glutamic acid with 6 mole HCl for a period of 24 hours. He found that 3—5% of it will mm into D-glutamic acid. By hydrolysing horse myoglobin and cow insulin he obtained 6.6-4.6% of D-glutamic acid. Manning and Moore (1968) when examining the racemization of free and peptide-bonded amino acids found that for some amino acids the measured racemization during the acidic hydrolysis will depend on whether the amino acid in question is free or in a peptide chain, and what is the location among the given amino acid in the peptide chain. Manning (1971) definitely states that by using free L-amino acid as a control, the degree of racemization occurring during the hydrolysis of protein cannot be accurately predicted. In spite of this the hydrochloric acid treatment of free amino acids during the hydrolysis of protein is used by some as an assessment of the occurring racemization. Most authors have found a racemization between 0.1 and 3.7% in these experiments for the various amino acids. Bada and Protsch (1973) measured, for the ration of D- and L--aspartic acids, 0.07 following the method of acidic hydrolysis. They call attention to
