Collagen Structural Changes and Decomposi5on in Burnt Bone and their Significance for Forensic Anthropology – New Insights through Amino Acid Racemiza5on Sarah Ellingham, PhD; Tim Thompson, PhD School of Science and Engineering, Teesside University, TS1 3BA Middlesbrough, UK Introduc*on All amino acids found in protein exist as one of two possible stereoisomers, known as L-amino acids and D-amino acids. With the excep*on of glycine, all amino acids are L-amino acids, which however have the ability to change into the D-form over *me. The process of reaching an equilibrium between the L and the D form is called racemiza*on. Amino Acid Racemiza*on (AAR) is a *me and temperature dependent process that has found applica*on in the da*ng of materials, paleothermometry and age at death es*ma*on. Currently there is no clear consensus in the literature regarding the thermal stability of collagen, an issue which this research sheds more light on. Materials and Methods Sheep (ovis aries) ribs were cut into 4 cm long pieces and burnt at temperatures between 100 and 1000 °C in 50 °C increments for 45 minutes. All samples were weighed pre and post burning. For demineraliza*on samples were suspended in 0.5 M HCl, which was exchanged every 2 days. ASer 10 days the HCl was removed and replaced by dis*lled water un*l a solu*on of pH 3 was obtained. Samples were heated at 70 °C for 48 hours and subsequently filtered. The extracted collagen was frozen at -20 °C. 250 μL of solu*on from each sample were placed in a sterile glass vial adding 100 μL 7 M HCl per sample. The vials were flushed with nitrogen, heated at 110 °C for 18 hours and subsequently dried under vacuum in a centrifugal evaporator. Samples were rehydrated for analysis. The sample’s amino acid composi*on was analyzed by reverse-phase HPLC using fluorescent detec*on. 2 μL of sample was injected and mixed with 2.2 μL derivi*zing reagent. The amino acids were separated on a C18 HyperSil BDS column (5 mm * 250 mm) at 25 °C using a gradient elu*on of 3 solvents: sodium acetate buffer, methanol and acetonitrile. The fluorescence detector uses a xenon-arc flash lamp at a frequency of 55 Hz with a 280 nm cut-off filter and an excita*on wavelength of 230 nm and emission wavelength of 445 nm. Results The D and L isomers of 13 amino acids could be analysed, namely serine (Ser), L-threonine (L-Thr), L- his*dine (L-Hist), glycine (Gly), L-arginine (L-Arg), alanine (Ala), tyrosine, (Tyr), valine (Val), phenylalanine (Phe), leucine (Leu) and isoleucine (Ile). Asparagine and glutamine undergo rapid irreversible deamina*on to aspar*c acid and glutamic acid respec*vely during the prepara*ve hydrolysis, and their deriva*ves are therefore reported together as Asx and Glx . The amino acid concentra*on rapidly decreases from 250 °C onwards (Fig. 1), being below reliable detec*on levels from 400 °C (Fig. 2). Up to temperatures of 250 °C, solely aspar*c acid racemizes, reaching a D/L ra*o of 0.3 at 250 °C. From 300 °C onwards the other amino acids commence racemiza*on. From 400 °C onwards, the total amino acid concentra*on is too low to accurately depict D/L ra*os (Fig. 3). The composi*on of amino acids was dominated by collagen up to temperatures of 400 °C. . Conclusion Amino acid racemiza*on analysis is a very useful tool to gain a more complete insight into the thermal behavior and stability of bone collagen. Although collagen can be detected in bone samples up to temperatures of 400 °C, its structural conforma*on undergoes several changes before reaching those temperatures. From temperatures slightly above body temperature onwards, the collagen’s triple helical structure gradually unravels, with only racemiza*on of Asx occurring at the telopep*de ends. At 250 °C the triple helix completely transforms to random coils, which allow for the racemiza*on of other amino acids, un*l at temperatures between 350-400 °C a catastrophic breakdown of all amino acids occurs. There are s*ll mul*ple factors which may influence the collagen preserva*on which have not been inves*gated within the framework of this research and which could become the focus of future research; nonetheless the findings presented in this chapter allow for an improved comprehension of the behavior of bone collagen when exposed to hea*ng and how this behavior links in to the overall decomposi*on and transforma*on of heated bone. References (1) Bozec, L. and Odlyha, M. (2011) 'Thermal denatura*on studies of collagen by microthermal analysis and atomic force microscopy', Biophysical Journal, 101(1), pp. 228-236. (2) Ellingham S., Thompson T., Islam M. Thermogravimetric Analysis of Property Changes and Weight Loss in Incinerated Bone. Palaeogeography, Palaeoclimatology, Palaeoecology 2015; 438: 293-244. Discussion These findings illustrate the thermal degrada*on of bone collagen. Up to 250 °C the amino acid concentra*on is very high, and virtually no amino acid racemiza*on with the excep*on of Asx, which is one of the only amino acids which can racemize whilst s*ll internally bound, takes place. Whilst the primary structure of collagen, the linear sequence of amino acids, is favorable to amino acid racemiza*on, the quaternary structure of the helix inhibits this process. Collagen, when heated, begins to locally unravel the triple helix while absorbing heat, a process which up to a point is s*ll reversible. If hea*ng con*nues beyond this point, which is at around 60 °C +/- 10 °C the locally unfolded structures experience irreversible mel*ng to denatured collagen. Once this denaturing sets in, the collagen fibrils experience several conforma*onal changes, caused by the breaking of cross links. As the bone is heated further, the collagen stabilizing H-bonded water is released, which leads to a gradual collapse of the triple helical structure at temperatures of around 150 °C (1). At temperatures from between 250-300 °C a sudden drop in total amino acid concentra*on as well as the commencing of racemiza*on of all other the amino acids can be observed, indica*ng a catastrophic breakdown of collagen. This temperature coincides with the change from an endothermic to an exothermic reac*on (2), the complete conforma*onal change from triple helix to random coil and the onset of the cleavage of individual amino acids, which occur from around 305 +/- 10 °C onwards. The now free amino acids con*nue racemiza*on up un*l their complete combus*on from around 400 °C onwards. The authors would like to thank Dr Kirsty Penkmann (University of York) for her invaluable assistance with the amino acid racemiza*on. The extrac*on methodology is based on the a methodology derived by the Max Planck ins*tute, Leipzig.