This presentation will impact the forensic science community by shedding new light on the thermal stability of bone collagen, allowing for an improved prediction of the success rate of collagen extraction for subsequent analyses such as...
moreThis presentation will impact the forensic science community by shedding new light on the thermal stability of bone collagen, allowing for an improved prediction of the success rate of collagen extraction for subsequent analyses such as dating or stable isotope analyses.
All amino acids found in proteins exist as one of two possible stereoisomers, known as L-amino acids and D-amino acids. With the exception of glycine, all amino acids are L-amino acids, which however have the ability to change into the D-form over time. The process of reaching an equilibrium between the L and the D form is called racemization. Amino Acid Racemization (AAR) is a time and temperature dependent process that has found application in the dating of materials, paleothermometry and age at death estimation. Currently there is no clear consensus in the literature regarding the thermal stability of collagen, an issue which this research sheds more light on. 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 demineralization samples were suspended in 0.5 M HCl, which was exchanged every 2 days. After 10 days the HCl was removed and replaced by distilled water until a solution 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 solution 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 composition was analyzed by reverse-phase HPLC using fluorescent detection. 2 μL of sample was injected and mixed with 2.2 μL derivitizing reagent. The amino acids were separated on a C18 HyperSil BDS column (5 mm * 250 mm) at 25 °C using a gradient elution 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 excitation wavelength of 230 nm and emission wavelength of 445 nm. The D and L isomers of 12 amino acids could be analyzed, namely serine, L-threonine, L-histidine, glycine, L-arginine, alanine, tyrosine, valine, phenylalanine, leucine, isoleucine as well as aspartic acid and glutamic acid. The amino acid concentration rapidly decreases from 250 °C onwards, being below reliable detection levels from 400 °C. Up to temperatures of 250 °C, solely aspartic acid racemizes, reaching a D/L ratio of 0.3 at 250 °C. From 300 °C onwards the other amino acids commence racemization. From 400 °C onwards, the total amino acid concentration is too low to accurately depict D/L ratios. The composition of amino acids was dominated by collagen up to 400 °C. The findings illustrate the thermal degradation of bone collagen. Up to 250 °C virtually no amino acid racemization with the exception of aspartic acid, which is one of the only amino acids which can racemize whilst still internally bound, takes place. Collagen, when heated, begins to locally unravel its triple helix, releasing the collagen stabilizing H-bonded water, which leads to a gradual collapse of the triple helical structure at around 150 °C. At temperatures from between 250-300 °C a sudden drop in total amino acid concentration as well as the commencing of racemization of all other amino acids can be observed indicating a catastrophic breakdown of collagen. The now free amino acids continue racemization up until their complete combustion from around 400 °C onwards. Successful collagen extraction is the basis for a multitude of forensically relevant analyses, such as stable isotope analysis, radio carbon dating or genetic profiling. Being able to accurately determine the point at which collagen denaturizes and amino acids are lost is therefore of paramount importance for forensic analyses.
