Ostrich eggshell (OES) is ubiquitous in archaeological sites in Africa and is a favoured substrate for amino acid racemisation (AAR) geochronology, yielding consistent results and thought to approximate a closed system with respect to protein diagenesis. It was found that the intra-crystalline fraction in OES can be isolated by oxidative pre-treatment and that these amino acids are resistant to leaching and unaffected by changes in the environmental pH (between pH 5 and 9). The intra-crystalline fraction in OES therefore effectively operates as a closed system with respect to protein diagenesis. This intra-crystalline fraction is likely to be a superior sample compared to whole-shell samples, removing uncertainty introduced through variable leaching rates, different ground water conditions and potentially exogenous contamination.
Kinetic studies on modern OES indicate that high temperature data should be treated with caution when used to extrapolate kinetic parameters at low temperatures. This reinforces previous work that indicates the underlying mechanisms driving protein diagenesis need to be better understood in order to estimate accurate kinetic parameters, as the pathways of diagenesis are different at high and low temperatures. Nonetheless, hydrolysis and racemization have been shown to follow predictable patterns of diagenesis, and are therefore useful for the application of relative dating in AAR geochronology. Therefore, a chronological framework for southern South Africa was built by AAR analysis of a suite of OES samples, with calibration from independent dating methods (optically stimulated luminescence and radiocarbon), up to 151 ka. Through extensive artificial heating studies on modern OES samples, a checklist of 6 markers has been developed to identify sub-fossil samples that have been exposed to high temperatures. Exclusion of these heated samples from AAR age models significantly increases the age resolution. Analysis of several amino acid chiral pairs allowed age resolution over the full time range studied (~0.3 ka – 151 ka), allowing differentiation of MIS 1, 2, 3/4, 5 and 6. It has been shown that OES AAR offers a useful additional relative dating technique for archaeological and palaeontological sites from the same regions as Pinnacle Point and Elands Bay Cave, up to at least 151 ka (MIS 6). Furthermore, as 151 ka samples have racemization for some amino acids (e.g. isoleucine) only half way to equilibrium, there is considerable potential for OES AAR geochronology in South Africa beyond 151 ka.
In addition, a preliminary study has demonstrated the potential of mass spectrometry in helping to unravel the complex nature of protein diagenesis within bio-minerals. Peptides from ~71 ka sub-fossil samples were successfully extracted and sequenced. It is believed this is the first time this has been done for eggshell samples of this age. This kind of study offers interesting insights into the patterns of protein preservation and degradation, with implications for understanding the role and function of proteins in the bio-minerals.
Finally, a new ultra-high pressure liquid chromatography (UHPLC) method for the chiral separation of amino acids has been developed, with an analysis time (60 min including flush time) approximately half that of the previous HPLC method, allowing a higher throughput of samples. This new method also separates additional analytes, previously unstudied in AAR geochronology, which may offer further insight into protein diagenesis within bio-minerals.
