Wet degradation of keratin proteins: linking amino acid, elemental and isotopic composition.

RATIONALE: Archaeological keratin samples are increasingly the subject of palaeodietary, provenancing and dating studies. Keratin samples from wet archaeological contexts are microbiologically and chemically degraded, causing differential diagenesis of protein structures in hair fibres. The effects of these processes on the analytical parameters of interest are currently unknown. METHODS: This study examined the impact of degradation of wool fibres on isotopic (δ(13)C, δ(15)N, un-exchangeable δ(2)H and δ(18)O values) composition. It compared two models of archaeological protein degradation in wet burial environments: (1) short term (up to 8 years) experimental burial in three contrasting soil environments; and (2) laboratory wet conditions, in which elevated temperature (80 °C, 110 °C, and 140 °C) and pressure simulated longer exposure. Elemental and amino acid (AA) composition were also measured. RESULTS: In experimentally soil-buried samples, AA, elemental and isotopic composition changes were small, despite extensive macroscopic alteration. Isothermally heated samples showed preferential loss of hydrophilic AAs (Asx, Glx, Ser, Gly) from wool residues, with depletion in (2)H and (18)O at higher temperatures (up to -73‰ change in δ(2)H and -2.6‰ in δ(18)O values). The δ(13)C and δ(15)N values showed little change except in densely pigmented samples at low temperatures only. Samples dyed with madder/alum were better preserved than undyed samples. CONCLUSIONS: Diagenesis in experimentally soil-buried wool textiles was consistent with microbiological, non-protein-selective activity, in contrast to highly AA-selective hydrolytic behaviour under laboratory wet conditions. Changes in δ(2)H and δ(18)O values were correlated with degree of AA change, but the δ(13)C and δ(15)N values were not. The results contribute to a baseline for interpreting analytical data from archaeological hair samples preserved by burial in wet environments.

Intra-crystalline protein diagenesis (IcPD) in Patella vulgata. Part II: Breakdown and temperature sensitivity.

Artificial diagenesis of the intra-crystalline proteins isolated from Patella vulgata was induced by isothermal heating at 140 °C, 110 °C and 80 °C. Protein breakdown was quantified for multiple amino acids, measuring the extent of peptide bond hydrolysis, amino acid racemisation and decomposition. The patterns of diagenesis are complex; therefore the kinetic parameters of the main reactions were estimated by two different methods: 1) a well-established approach based on fitting mathematical expressions to the experimental data, e.g. first-order rate equations for hydrolysis and power-transformed first-order rate equations for racemisation; and 2) an alternative model-free approach, which was developed by estimating a "scaling" factor for the independent variable (time) which produces the best alignment of the experimental data. This method allows the calculation of the relative reaction rates for the different temperatures of isothermal heating. High-temperature data were compared with the extent of degradation detected in sub-fossil Patella specimens of known age, and we evaluated the ability of kinetic experiments to mimic diagenesis at burial temperature. The results highlighted a difference between patterns of degradation at low and high temperature and therefore we recommend caution for the extrapolation of protein breakdown rates to low burial temperatures for geochronological purposes when relying solely on kinetic data.

Intra-crystalline protein diagenesis (IcPD) in Patella vulgata. Part I: Isolation and testing of the closed system.

This study successfully isolates a fraction of intra-crystalline proteins from shells of the marine gastropod Patella vulgata and assesses the suitability of these proteins for IcPD (Intra-crystalline Protein Diagenesis) geochronology. We discuss the mineralogical composition of this gastropod, investigated for the first time by X-ray diffraction mapping, and use the results to inform our sampling strategy. The potential of the calcitic rim and of a bulk sample (containing both apex and rim) of the shell to act as stable repositories for the intra-crystalline proteins during diagenesis is examined. The composition and the diagenetic behaviour of the intra-crystalline proteins isolated from different locations within the shell are compared, highlighting the necessity of targeting consistent sampling positions. We induced artificial diagenesis of both intra-crystalline and whole-shell proteins by conducting high-temperature experiments in hydrous environment; this allowed us to quantify the loss of amino acids by leaching and therefore evaluate the open- or closed-system behaviour of the different fractions of proteins. The results obtained provide further confirmation that patterns of diagenesis vary according to the protein sequence, structure, and location within or outside the intra-crystalline fraction. As Patella is frequently found in the fossil record, both in archaeological and geological contexts, the application of IcPD geochronology to this biomineral opens up the possibility to obtain reliable age information from a range of sites in different areas of the world.

Protein and mineral characterisation of rendered meat and bone meal.

We report the characterisation of meat and bone meal (MBM) standards (Set B-EFPRA) derived from cattle, sheep, pig and chicken, each rendered at four different temperatures (133, 137, 141 and 145 °C). The standards, prepared for an EU programme STRATFEED (to develop new methodologies for the detection and quantification of illegal addition of mammalian tissues in feeding stuffs), have been widely circulated and used to assess a range of methods for identification of the species composition of MBM. The overall state of mineral alteration and protein preservation as a function of temperature was monitored using small angle X-ray diffraction (SAXS), amino acid composition and racemization analyses. Progressive increases in protein damage and mineral alteration in chicken and cattle standards was observed. In the case of sheep and pig, there was greater damage to the proteins and alteration of the minerals at the lowest treatment temperature (133 °C), suggesting that the thermal treatments must have been compromised in some way. This problem has probably impacted upon the numerous studies which tested methods against these heat treatments. We use protein mass spectrometric methods to explore if thermostable proteins could be used to identify rendered MBM. In more thermally altered samples, so-called 'thermostable' proteins such as osteocalcin which has been proposed as a ideal target to speciate MBM were no longer detectable, but the structural protein type I collagen could be used to differentiate all four species, even in the most thermally altered samples.

The impact of random natural variability on aspartic acid racemization ratios in enamel from different types of human teeth.

Previous research has indicated that the extent of amino acid racemization in enamel varies systematically between tooth types within the dentition. This phenomenon was suggested to be due to differences in temperature at various locations within the mouth. This paper presents an analysis of aspartic acid racemization in a fraction of the enamel proteins which should be particularly susceptible to deviations in temperature, in order to assess the impact of temperature on variability in racemization values. The acid soluble fraction of the enamel was analysed from 129 human teeth of different tooth types and from both living individuals and archaeological skeletal remains. Samples were collected by acid etching of the enamel to isolate proteins located a small distance below the enamel surface. For each population, the racemization values for different tooth types were compared to identify any possible systematic variation. Where multiple teeth were analysed from the one individual, the age estimates produced for the different teeth were compared to obtain an indication of the overall level of variability in racemization values. No systematic variation in the extent of racemization between different tooth types was observed in any of the populations analysed. There appeared instead to be a high level of random variability in the extent of racemization, with substantial differences observed between age estimates produced from multiple teeth from the one individual. The results of this study suggest that the differences in racemization values observed here are due to random variations and not the temperature at different locations within the mouth.

Amino acid geochronology of the type Cromerian of West Runton, Norfolk, UK.

Aminostratigraphic studies of continental deposits in the UK have hitherto relied almost exclusively on data from the aragonitic shells of non-marine molluscs for dating Pleistocene sequences. This is usually based on the d/l value of a single amino acid, d-alloisoleucine/l-isoleucine (A/I), in the total shell proteins. Two genera of freshwater gastropods (Valvata and Bithynia) are used to explore the value of using multiple amino acids from the intra-crystalline fraction, which should be more protected from the effects of diagenesis than the inter-crystalline component. Results are compared from both the aragonitic shells and opercula composed of calcite, a more stable form of calcium carbonate. In order to put the amino acid data from the West Runton Freshwater Bed into perspective, statistical analyses are used to compare them with results from the Hoxnian (MIS 11) site at Clacton-on-Sea, Essex. Twelve protein decomposition indicators revealed that the results from the shells were not as clear-cut as those from the opercula. Five indicators from the Valvata shell suggest that West Runton is older than Clacton (at a 95% significance level), but two actually suggested a younger age. Seven indicators show that the Bithynia shells from West Runton are older than congeneric shells from Clacton. In marked contrast, all 12 indicators isolated from the opercula demonstrate that West Runton is significantly older than Clacton. The data are also compared with results from Waverley Wood, an important archaeological site in the English Midlands falling within the 'Cromerian Complex'. Contrary to earlier interpretations, the new amino acid data from Bithynia opercula indicate that West Runton is older than Waverley Wood, a relationship now consistent with the available biostratigraphy.

Age estimation of archaeological remains using amino acid racemization in dental enamel: a comparison of morphological, biochemical, and known ages-at-death.

The poor accuracy of most current methods for estimating age-at-death in adult human skeletal remains is among the key problems facing palaeodemography. In forensic science, this problem has been solved for unburnt remains by the development of a chemical method for age estimation, using amino acid racemization in collagen extracted from dentine. Previous application of racemization methods to archaeological material has proven problematic. This study presents the application to archaeological human remains of a new age estimation method utilizing amino acid racemization in a potentially closed system-the dental enamel. The amino acid composition and extent of racemization in enamel from two Medieval cemeteries (Newcastle Blackgate and Grantham, England) and from a documented age-at-death sample from a 19th century cemetery (Spitalfriedhof St Johann, Switzerland) were determined. Alterations in the amino acid composition were detected in all populations, indicating that diagenetic change had taken place. However, in the Medieval populations, these changes did not appear to have substantially affected the relationship between racemization and age-at-death, with a strong relationship being retained between aspartic acid racemization and the morphological age estimates. In contrast, there was a poor relationship between racemization and age in the post-medieval documented age-at-death population from Switzerland. This appears to be due to leaching of amino acids post-mortem, indicating that enamel is not functioning as a perfectly closed system. Isolation of amino acids from a fraction of enamel which is less susceptible to leaching may improve the success of amino acid racemization for archaeological age estimation.

Closed-system behaviour of the intra-crystalline fraction of amino acids in mollusc shells.

When mollusc shells are analysed conventionally for amino acid geochronology, the entire population of amino acids is included, both inter- and intra-crystalline. This study investigates the utility of removing the amino acids that are most susceptible to environmental effects by isolating the fraction of amino acids encapsulated within mineral crystals of mollusc shells (intra-crystalline fraction). Bleaching, heating and leaching (diffusive loss) experiments were undertaken on modern and fossil Corbicula fluminalis, Margaritifera falcata, Bithynia tentaculata and Valvata piscinalis shells. Exposure of powdered mollusc shells to concentrated NaOCl for 48 h effectively reduced the amino acid content of the four taxa to a residual level, assumed to represent the intra-crystalline fraction. When heated in water at 140 degrees C for 24 h, only 1% of amino acids were leached from the intra-crystalline fraction of modern shells compared with 40% from whole shell. Free amino acids were more effectively retained in the intra-crystalline fraction, comprising 55% (compared with 18%) of the whole shell after 24 h at 140 degrees C. For fossil gastropods, the inter-shell variability in D/L values for the intra-crystalline fraction of a single-age population was reduced by 50% compared with conventionally analysed shells. In contrast, analysis of the intra-crystalline fraction of C. fluminalis does not appear to improve the results for this taxon, possibly due to variability in shell ultrastructure. Nonetheless, the intra-crystalline fraction in gastropods approximates a closed system of amino acids and appears to provide a superior subset of amino acids for geochronological applications.

The application of amino acid racemization in the acid soluble fraction of enamel to the estimation of the age of human teeth.

Estimation of age-at-death for skeletonised forensic remains is one of the most significant problems in forensic anthropology. The majority of existing morphological and histological techniques are highly inaccurate, and show a bias towards underestimating the age of older individuals. One technique which has been successful in forensic age estimation is amino acid racemization in dentine. However, this method cannot be used on remains where the post-mortem interval is greater than 20 years. An alternative approach is to measure amino acid racemization in dental enamel, which is believed to be more resistant to change post-mortem. The extent of amino acid racemization in the acid soluble fraction of the enamel proteins was determined for modern known age teeth. A strong correlation was observed between the age of the tooth and the extent of racemization. No systematic bias in the direction of age estimation errors was detected. For the majority of teeth analyzed, the presence of dental caries did not affect the results obtained. In a minority of cases, carious teeth showed a higher level of racemization than would be expected given the age of the individual. These results indicate that amino acid racemization in enamel has the potential to be used in age estimation of skeletal remains.

Testing the aminostratigraphy of fluvial archives: the evidence from intra-crystalline proteins within freshwater shells.

Until recently few studies of amino acid racemization of fossil bivalves and gastropods collected from river terrace deposits in Europe were based on the analysis of the intra-crystalline fraction. Instead they were based on the epimerization (racemization) of a single amino acid, isoleucine, and its inter-conversion to alloisoleucine. This paper presents data from the analysis of the intra-crystalline fraction of the shells, using a preparation technique of sample bleaching to remove the leachable matrix, thus leaving a component that exhibits closed-system behaviour. Reverse-phase HPLC separation with fluorescence detection allows the interpretation of four amino acids in detail: aspartic acid, glutamic acid, alanine and valine. The intra-crystalline fraction offers greater potential for improved resolution, especially when combined with the analysis of multiple amino acid d/l values, which racemize at different rates. This is explored using three species of freshwater gastropods (Bithynia tentaculata and troschelii, Valvata piscinalis) and the bivalve Corbicula. Sites of different ages within the Lower Thames river terrace sequence are used as a stratigraphical framework, with samples from other southern UK sites providing supplementary evidence. The results indicate better resolution using the intra-crystalline fraction over that obtained using unbleached shells, with differentiation possible at sites of up to MIS 7 age. However, for older sites, although values are always higher, the separation is less successful. A species effect has been identified between the gastropod shells. Despite the analysis of intra-crystalline protein, amino acid data from Corbicula remain problematical. Preliminary data on the opercula from Bithynia indicate that better resolution is possible, particularly at older sites.