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

1. Hydrolysis of proteins performed at high temperatures and for short times with reduced racemization, in order to determine the enantiomers of D- and L-amino acids 1.1. Introduction The role of optical activity in living organisms has long been known. A large group of biologically active molecules, such as the amino acids, are all optically active. Thus in order to know their roles in living organisms, we should be able to separate and determine their enantiomers. Recently, considerable effort has been devoted as to separation and quantification of amino acid enantiomers. Among these is the archaeometric application whereby one can establish the age of archaeological relics based on the racemization of amino acids, specifically the epimerization of isoleucine (WEHMILLER AND HARE, 1971; WILLIAMS AND SMITH, 1977; MILLER AND HARE, 1980; CSAPÓ ET AL., 1988, 1990a). Another example of recent work is the study of the composition of extraterrestrial materials (CRONIN AND PIZZARELLO, 1983). When attempting to quantify amino acid enantiomers, it is not sufficient to separate these from each other. One also has to also pay attention to the separation of these from the other amino acids and their derivatives. The amino acid derivative on which we decide to depend should be detectable with good sensitivity. Lately, pre-column derivative formation has been used with a fluorescent reagent, followed by Reversed Phase Chromatography (RPC) of the derivatives. Using these methods, the detection limits for the amino acids of interest are extremely low. On the other hand, the flexibility of this analytical method provides outstanding advantages (LINDROTH AND MOPPER, 1979; TOPHUI ET AL., 1981; EINARSSON ET AL., 1987a). Thus, automatic methods have been developed for the simultaneous determination of optically inactive o-phthalic aldehyde/mercapto-ethanol (OPA) and a-amino acids (SMITH AND PANICO,1985), and of 9-fluorenyl-methyl chloroformate (FMOC-C1) in the presence of a-amino and imino acids (CUNICO ET AL., 1986; BETNER AND FÖLDI, 1988). The reaction of optically active (chiral) amino acids with chiral reagents yields dia-stereoisomercompounds. In theory, one should be able to separate these using a non-chiral column. If the chiral reagent is another amino acid, then the separation and determination of the diastereomer di-peptide may be achieved using ion-exchange column chromatography (HIRSCHMANN ET AL., 1967; MANNING AND MOORE, 1968; CSAPÓ ET AL., 1990b; CSAPÓ ET AL., 1991a). Following derivative formation with chiral reagents, the enantiomers of protein building block amino acids may be separated in a single run using RPC. Since the chromatographic separation takes 50-70 minutes, it is of paramount importance that the analytical method be adaptable to full automation. Another prerequisite is that the derivative formation should be simple, proceeding in a short time at room temperature. The reaction between the optically active thiols, the OPA and the amino acids to be determined has been used to separate and quantify amino acid enantiomers (ASWAD, 1984; BUCK AND KRUMMEN, 1987). The use of chiral l-(9-fluorenyl) ethyl chloroformate (FLEC) for the separation of enantiomers has the advantage of being able to form derivatives, not only with the a-amino acids, but also with the imino-acids (EINARSSON ET AL; 1987a). It is very important to know whether or not racemization occurs during protein hydrolysis. If so, the results of the determination will be influenced adversely. Various studies reported that the degree of racemization during hydrolysis of peptide is dependent on protein type and amino acid background. It was found (FRANK ET AL., 1981; 22